Proteins influencing the septal junctions in heterocystous cyanobacteria
Abstract
La multicelularidad es una forma de organización biológica que ha aparecido de forma independiente en muchos grupos filogenéticos a lo largo de la evolución. En los organismos multicelulares se dan universalmente procesos de adhesión, comunicación y diferenciación celular. La multicelularidad se observa en varios grupos bacterianos, estando las cianobacterias entre los organismos multicelulares más antiguos de la Tierra. Las cianobacterias se caracterizan por llevar a cabo la fotosíntesis oxigénica, mediante la cual se produce la liberación de oxígeno y la producción de “poder asimilatorio” (ATP y ferredoxina reducida/NADPH), el cual se usa en la fijación de CO2 atmosférico y en la asimilación de algunos otros nutrientes oxidados. Dentro de este filo se pueden encontrar tanto organismos unicelulares como multicelulares. Aquellas cianobacterias que se comportan como organismos multicelulares crecen formando filamentos, y en condiciones de deficiencia de nitrógeno algunas de ellas pueden dar lugar a un proceso de diferenciación celular produciendo un tipo celular llamado heterocisto. Este último lleva a cabo la fijación de nitrógeno atmosférico (N2), mientras que el resto de las células del filamento, denominadas células vegetativas, continúan con la fijación de CO2. Otras especies del filo tienen la capacidad de formar aquinetos, que son una forma celular de resistencia que aparece en algunas cianobacterias cuando las condiciones ambientales se vuelven adversas, y aún otras forman hormogonios, pequeños filamentos móviles con una función de dispersión. Este trabajo se ha realizado con la estirpe modelo Anabaena sp. PCC 7120, que es una cianobacteria filamentosa formadora de heterocistos. Las cianobacterias poseen una envuelta celular de tipo Gram negativa, y en las cianobacterias filamentosas la membrana plasmática envuelve individualmente a cada célula mientras que la membrana externa, situada por fuera de la capa de peptidoglicano, es continua definiendo por tanto un periplasma continuo. Cuando Anabaena crece en ausencia de nitrógeno combinado, se produce la diferenciación de heterocistos, formándose un patrón a lo largo del filamento de un heterocisto cada 10-15 células vegetativas. El crecimiento diazotrófico exige una comunicación intercelular que permita el intercambio de nutrientes y moléculas reguladoras de la diferenciación a lo largo del filamento, llevándose a cabo esta comunicación a través de los nexos septales. Estos son unas estructuras proteicas que comunican de forma directa las células adyacentes. Actualmente se conocen algunas proteínas que forman parte de los nexos septales como son FraC y FraD, y algunas otras que son esenciales para el correcto funcionamiento o ensamblaje de los mismos, como son SepJ, SepI, HglK (demostrada en esta Tesis) o FraE. Para el ensamblaje de los nexos septales es necesario una perforación del peptidoglicano existente entre las células adyacentes del filamento. Estas perforaciones reciben el nombre de nanoporos y se encuentran en el disco septal de peptidoglicano. En la primera parte de este trabajo se intentó establecer una relación entre los nanoporos y la comunicación intercelular mediada por los nexos septales, utilizando como herramientas un marcador fluorescente y diferentes mutantes de Anabaena. El marcador fluorescente utilizado fue la calceína, que posee la propiedad de difundir desde el exterior hasta el interior de la célula, donde sufre un proceso de hidrólisis que le impide volver a atravesar la membrana plasmática, quedando atrapada en el interior de la célula y emitiendo fluorescencia. Por medio de experimentos de FRAP (“Fluorescence Recovery After Photobleaching”) se pudo calcular un indicador cuantitativo de transferencia entre células adyacentes del filamento. Por otra parte, mediante el aislamiento del peptidoglicano y su visualización por microscopía electrónica de transmisión se pudo cuantificar el número de nanoporos de las estirpes estudiadas. Estos ensayos se realizaron en filamentos (cultivados en presencia o ausencia de nitrógeno combinado) de la estirpe silvestre y de mutantes tanto de proteínas que intervienen en la formación de los nexos septales como de proteínas que influyen en su ensamblaje y funcionamiento. Los resultados obtenidos permitieron establecer una correlación entre el número de nanoporos y la transferencia intercelular de calceína en filamentos que habían sido incubados en medio sin nitrógeno combinado, pero no en aquellos incubados en presencia de nitrógeno, en los que se observaba una elevada proporción de células no comunicantes. En la segunda parte de esta Tesis se procedió a la caracterización de la proteína HglK, que pertenece a la familia de las pentapeptide repeat proteins (PRP). HglK se compone de cuatro segmentos transmembrana en su mitad N-terminal y de una sección soluble de localización periplásmica que contiene el dominio PRP en su mitad C-terminal. Estudios previos de HglK mostraron que mutantes que carecían de ella eran incapaces de crecer diazotróficamente y tenían un espacio incrementado entre las células del filamento. En esta Tesis se abordó la localización de HglK en estirpes de Anabaena que producían HglK fusionada a la superfolder-GFP (sfGFP) o con una cola de histidinas o con la etiqueta molecular Strep II. Mediante microscopía confocal y de fluorescencia y ensayos de inmunolocalización, se pudo establecer que HglK poseía una localización preferentemente polar en las células de Anabaena. El estudio de mutantes hglK determinó que HglK es necesaria para la integridad del filamento y para observar unos niveles normales de intercambio molecular entre las células vegetativas del filamento. Sin embargo, los experimentos realizados con mutantes de HglK y de otras proteínas septales mostraron que ni la fragmentación de los filamentos ni la alteración en el intercambio molecular era mayor cuando se combinaba la inactivación de HglK con la de otras proteínas septales. En la caracterización de HglK también se pudo establecer que el fenotipo de crecimiento diazotrófico negativo del mutante hglK estaba determinado por su incapacidad de fijar nitrógeno atmosférico como consecuencia de un defecto en la expresión génica durante la diferenciación de los heterocistos, puesto de manifiesto como una expresión disminuida de los genes del operón cox2 (citocromo oxidasa del heterocisto) y nifHDK (nitrogenasa) en los mutantes hglK. Los avances obtenidos mediante la caracterización de HglK y el conocimiento disponible sobre la proteína SepJ, ponían de manifiesto la implicación de ambas proteínas en los nexos septales. SepJ es una proteína integral de membrana con una localización focalizada en el centro del septo intercelular. SepJ está compuesta por una permeasa en su parte C-terminal y por un linker (rico en el amino ácido prolina) y un dominio coiled-coil en su parte N-terminal, la cual parece ser periplásmica. En el tercer capítulo de esta Tesis se intentó identificar proteínas con las que pudieran tener algún tipo de interacción tanto HglK como SepJ. El abordaje experimental se hizo mediante el uso de estirpes de Anabaena que portaban HglK fusionada a la Strep II o SepJ fusionada a la Strep II o a la GFP. Estas fusiones permitieron la purificación de las proteínas, que se llevó a cabo bajo condiciones no desnaturalizantes, intentando evitar la disgregación de los posibles complejos que pudieran formar con otras proteínas. Las muestras obtenidas tras las purificaciones se analizaron por espectrometría de masas para identificar todas las proteínas presentes en ellas. El análisis de los resultados mostró varias proteínas que podrían interactuar con HglK o SepJ. El primer resultado destacable es la posible interacción entre ambas (SepJ y HglK), aunque la co-purificación de las dos proteínas solamente se observó en los aislados de SepJ. También destaca la presencia de dos proteínas comunes en los aislados de SepJ y HglK. Estas proteínas son Alr2937 y Tic22 (Alr0114), ambas periplásmicas, la segunda de las cuales con un papel importante en la formación de la membrana externa de Anabaena. Finalmente, mientras que no se ha encontrado ninguna proteína que interaccione solamente con HglK, para SepJ se detectó Alr1690, una proteína anclada a la membrana plasmática que presenta dominios periplásmicos de unión al peptidoglicano. Continuando con el estudio de las proteínas septales, se quiso profundizar en el conocimiento de las proteínas SepJ, FraC y FraD de otras cianobacterias filamentosas formadoras de heterocistos, concretamente de cianobacterias simbióticas con diatomeas marinas. En el cuarto capítulo de esta Tesis, se estudiaron estas proteínas septales codificadas por los genes de las cianobacterias Richelia intracellularis (RintHH01) y Calothrix rhizosoleniae (CalSC01). R. intracellularis es un endosimbionte obligado que vive en el citoplasma de la diatomea Hemiaulus hauckii, mientras que C. rhizosoleniae es un exosimbionte facultativo de la diatomea Chaetoceros compressus, pudiendo proliferar anclada extracelularmente a la diatomea o como un organismo de vida libre. En Anabaena, fraC y fraD forman parte del operón fraCDE que también se encuentra presente en CalSC01 y, sólo fraCD, en RintHH01. Respecto a SepJ, tanto RintHH01 como CalSC01 contienen genes que determinan proteínas homólogas a la de Anabaena. Los mutantes sepJ y fraC-fraD de Anabaena muestran un fuerte fenotipo de fragmentación e imposibilidad de crecer diazotróficamente, ambos fenotipos de fácil estudio, por lo que se intentó la complementación de dichos mutantes con los genes de las cianobacterias simbióticas. Los genes sepJ y fraCD de RintHH01 y los genes sepJ y fraCDE de CalSC01 se obtuvieron por síntesis química, en el caso de sepJ de CalSC01 con un uso de codones adaptado a Anabaena, para ser introducidos en mutantes de Anabaena que portaban las deleciones sepJ (estirpe CSVM34) o fraC-fraD (estirpe CSVT22), respectivamente. La complementación de CSVM34 con sepJ de RintHH01 y de CalSC01 se hizo de forma que la expresión de estos tenía lugar desde el promotor natural del sepJ de Anabaena. Sin embargo, ni la complementación con sepJ de RintHH01 ni la realizada con sepJ de CalSC01 consiguieron revertir el fenotipo mutante, ni siquiera parcialmente. En cambio, sí se consiguió revertir al fenotipo silvestre cuando el mutante se complementó con sepJ de la propia Anabaena utilizado como control positivo. Por otro lado, los genes fraC-fraD de RintHH01 se transfirieron a la estirpe CSVT22 con dos estrategias distintas: en una su expresión tenía lugar desde el promotor natural fraC de Anabaena, y en otra la expresión tenía lugar desde el promotor glnA (promotor de fuerte expresión en condiciones de deficiencia de nitrógeno) insertado en un plásmido replicativo de Anabaena. Por su parte, la complementación de CSVT22 con los genes fraC-fraD-fraE de CalSC01 solamente se intentó con expresión desde el promotor glnA en un plásmido replicativo. Nuevamente ninguna de las complementaciones abordadas tuvo éxito. Es más, las expresiones génicas realizadas bajo el promotor glnA causaban efectos perjudiciales para Anabaena, como pudimos comprobar con controles utilizando los propios genes de Anabaena, lo que sugiere que quizás no habíamos expresado los distintos genes en sus niveles óptimos de funcionalidad. La última parte de esta Tesis se dedicó a la realización de un breve estudio de la proteína FraE, siendo esta la otra proteína codificada en el operon fraC-fraD-fraE de Anabaena. FraE es una proteína de membrana necesaria para la integridad del filamento. En este capítulo se estudió su localización, para lo que se construyó una estirpe de Anabaena que producía FraE-sfGFP. Los ensayos de análisis de fluorescencia de la GFP llevados a cabo con esta estirpe pusieron de manifiesto que FraE se localiza principalmente en los polos de los heterocistos y que sus niveles disminuyen a las 48 horas de inducción en un medio sin nitrógeno combinado.
Full text
PROTEINS INFLUENCING THE SEPTAL JUNCTIONS IN HETEROCYSTOUS CYANOBACTERIA SERGIO ARÉVALO DÍAZ Sevilla, 2020 Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Cover: Micrograph of the peptidoglycan sacculus of part of a filament of Anabaena grown in BG110 medium. The peptidoglycan sacculus was stained with uranyl acetate and visualized by TEM. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Agradecimientos Esta tesis ha sido realizada en el Instituto de Bioquímica Vegetal y Fotosíntesis de Sevilla, Centro Mixto del Consejo Superior de Investigaciones Científicas y la Universidad de Sevilla, gracias a una beca de Formación de Personal Investigador (BES-2015-071939) asociada al proyecto de investigación BFU2014-56757-P, del que el Dr. Enrique Flores García ha sido investigador principal. Durante la ejecución de la tesis disfruté de dos estancias, una de cuatro meses y otra de mes y medio, en la Universidad Goethe (Frankfurt, Alemania), formando parte del grupo del profesor Enrico Schleiff y bajo la supervisión del Dr. Rafael Pernil García. Para comenzar me gustaría dar las gracias a mi director de tesis, el Dr. Enrique Flores, por darme la oportunidad de realizar la tesis en su grupo, su sabiduría a la hora de afrontar los experimentos e interpretación de sus resultados y por su infinita paciencia conmigo. De igual modo quería agradecer a la Dra. Antonia Herrero por su visión crítica y constructiva que, sumada a la de Enrique, ha hecho posible la realización de esta tesis. También quisiera expresar mi agradecimiento tanto a los miembros actuales del grupo de investigación como a todos aquellos que han formado parte del mismo durante esta etapa, aportando cada uno de ellos su granito de arena a la elaboración del trabajo. Especialmente quisiera agradecer a Gracia su ayuda esencial en los momentos de máximo agobio y saber siempre qué decir para animarme en los malos momentos. A Mireia, que pese a tener la tarea de cargar con el novato del grupo puso todo de su parte para explicarme cómo hacer las construcciones genéticas. A Sergio Camargo por su paciencia a la hora de introducirme en el mundo de la microscopía. A Vicente Mariscal e Ignacio Luque por tener siempre un momento para resolver dudas y no perder la sonrisa pese a lo básico de la pregunta. A Mercedes y Ana por su ayuda y consejos. A José Enrique por toda su ayuda científica (y por ser un gran compañero de debate futbolístico). Y finalmente, quisiera agradecer a Félix toda su ayuda a pesar de tener mil cosas que hacer y por ser el primero en acogerme en Sevilla con los brazos abiertos, siendo mi primer amigo aquí. En general, gracias a todos mis compañeros del grupo y a la gente del IBVF por ser como son y estar siempre dispuestos tanto a ayudarte en el uso de cualquier máquina como a beberse una caña anti-estrés. I would like to express my gratitude to Prof. Enrico Schleiff for giving me a warm welcome in his group and for all his help during my stays in Frankfurt. I also thank Enrico for his detailed analysis of the results obtained in his laboratory. To Rafa Pernil for all his scientific advises and help with administrative issues (since my first stay in Frankfurt, the term “administrative problems” took on a new meaning for me). To Dr. Roman Ladig for all his help in the analysis of my protein samples. Finally, I would like to thank the whole research group in Frankfurt, both the cyanobacteria line and the other lines, which made my stays there really enjoyable and made the idea that Germans are cold people just a myth. I would especially like to thank Nicklas who made me feel part of the group from the very first moment despite my language problems. I would also like to thank Drs. Rachel Foster, Martin Ekman and Mercedes Nieves from Stockholm University for providing me with DNA sequences and synthetic genes from symbiotic cyanobacteria. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
To finish acknowledging my colleagues, I would like to thank Dr. Félix Ramos, Prof. Enrico Schleiff, Dr. Ignacio Luque, Dr. Rachel Foster and Dr. Mercedes Nieves for making time in their busy schedules to read parts of this thesis. Your comments and suggestions have been really helpful to complete this work. Quisiera dar las gracias a toda mi familia. A mi madre por apoyarme en todas mis decisiones y ser para mí un auténtico ejemplo de valor y valentía. A mi tita Manoli, tita Mari, tito Paco y tito José por estar siempre presentes en mi vida y enseñarme lo que realmente significa la palabra familia. A mi prima Ana y mi primo Jesús por ser mucho más que mis primos, siendo como unos hermanos para mí. A mi primo Paco por ser un referente para mí, siendo el hermano mayor que te guía y te enseña que puedes conseguir más de lo que tú mismo creías. Finalmente, no quisiera olvidarme de mi Tía María por haber sido la “abuela” que toda persona necesita. Resumiendo, gracias a todos, sin vosotros no hubiera llegado hasta aquí. No quiero pasar la oportunidad de agradecer a todos mis amigos, especialmente a Carlos, Champion, Esteban, Jaime y Santi, por hacerme sentir de vuelta en casa cada vez que voy al pueblo, haciendo como si el tiempo no hubiera pasado para nosotros, aunque pasemos meses sin vernos. No hay nada más reconfortante que esas cañas en la barra del bar, en la cual, durante unas horas, todos somos expertos psicólogos, políticos, arquitectos, informáticos… siendo capaces de arreglar todos los problemas del mundo. A mi amiga Mari, que si he llegado hasta aquí en parte ha sido por ella, por nunca darse por vencida conmigo y explicarme mil veces las cosas para que pudiera aprobar los exámenes. A mi amiga Cristina, por ser un soporte para mí en los días malos de tesis (¿cuántos negocios alternativos a la ciencia habremos pensado, por Dios?) y por ser mi alojamiento en mis escapaditas a Granada. Y finalmente me gustaría agradecer a Rubén por transmitirme sus conocimientos tanto en ciencia como en beber cerveza (aunque lo que él beba no se considere cerveza), gran jefe y mejor amigo. Infine voglio ringraziare Bea, per avermi dato la pazienza e la sanità mentale di cui ho bisogno in quei brutti giorni in cui si vede tutto nero e soprattutto per avermi sopportato ogni giorno, anche dopo un'intera quarantena (questa donna merita un monumento). Senza i tuoi spaghetti alla carbonara e i tuoi gnocchi la stesura di questa tesi sarebbe stata molto più difficile. Ti amo Italianini. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
INDEX Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Index ______________________________________________________________________ iii INDEX iii FIGURES INDEX vii TABLE INDEX x RESUMEN EN ESPAÑOL xiii CONCLUSIONES EN ESPAÑOL xvi 1. INTRODUCTION 1 1.1 Multicellularity in bacteria 3 1.2 Cyanobacteria 3 1.2.1 Origin, diversity and classification of cyanobacteria 3 1.2.2 Physiology of cyanobacteria 7 1.2.3 Cell envelope 9 1.2.3.1 Outer membrane 9 1.2.3.2 The peptidoglycan layer 10 1.3 Heterocysts 11 1.3.1 Characteristics of the heterocyst 11 1.3.1.1 Structural characteristics 11 1.3.1.2 Metabolic characteristics 13 1.3.2 Heterocyst differentiation and pattern formation 15 1.3.3 Genes involved in the formation of heterocyst envelopes 16 1.4 Septal junctions and septal proteins 18 1.4.1 SepJ 20 1.4.2 Fra proteins 22 1.5 Intercellular communication 24 1.5.1 Continuous periplasm 24 1.5.2 Cell-cell communication by septal junctions 25 1.5.3 Possible different types of septal junctions 25 1.5.4 Possible regulation of the septal junctions 26 1.6 Pentapeptide repeat proteins 26 1.6.1 The HglK protein 28 1.7 Objectives 30 2. MATERIALS & METHODS 31 2.1 Organisms and Growth conditions 33 2.1.1 Escherichia coli 33 2.1.1.1 Strains 33 2.1.1.2 Culture media and conditions 33 2.1.1.3 Harvest methods 33 2.1.2 Cyanobacteria 34 2.1.2.1 Strain 34 2.1.2.2 Culture media and conditions 35 Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Table Index ______________________________________________________________________ x Table Index Materials & Methods Table 1. E. coli strains used in this work together with their genotypes and ………...references 33 Table 2. Anabaena strains previously available 34 Table 3. Cyanobacterial strains constructed in this work 34 Table 4. General and synthetic plasmids 37 Table 5. Plasmids constructed in this work 38 Table 6. Primers used in this work 40 Results & Discussion Chapter 1: Intercellular communication in Anabaena Table 7. Nanopores in septal peptidoglycan disks of selected sepJ mutants 60 Table 8. Analysis of non-communicating cells in each replicate 64 Chapter 2: The HglK protein Table 9. HglK, PatL, HetL and the Fra proteins in cyanobacteria 75 Table 10. Expression of the hglK gene in Anabaena 78 Table 11. Intercellular transfer of fluorescent markers in Anabaena with ………….different genetic backgrounds 90 Chapter 3: HglK and SepJ proteomics Table 12. Interaction partners identified with SepJ-GFP as bait -Experiment 1 107 Table 13. Interaction partners identified with SepJ-GFP as bait -Experiment 2 107 Table 14. Interaction partners identified with SepJ-Strep-tag II as bait 108 Table 15. Interaction partners identified with HglK-Strep-tag II as bait - ………….Experiment 1 108 Table 16. Interaction partners identified with HglK-Strep-tag II as bait - ………….Experiment 2 109 Chapter 4: The SepJ and Fra proteins from marine symbiotic Richelia …………...and Calothrix Table 17. Nitrogenase activity in strain CSSA15 and control strains 128 Table 18. Nitrogenase activity in strains carrying the fra gene constructs in a . . replicative plasmid 131 Chapter 5: Subcellular localization of FraE Table 19. Relative fluorescence from FraE-sfGFP at different subcellular ………….locations 142 Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Resumen y Conclusiones Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Resumen ______________________________________________________________________ xiii Resumen La multicelularidad es una forma de organización biológica que ha aparecido de forma independiente en muchos grupos filogenéticos a lo largo de la evolución. En los organismos multicelulares se dan universalmente procesos de adhesión, comunicación y diferenciación celular. La multicelularidad se observa en varios grupos bacterianos, estando las cianobacterias entre los organismos multicelulares más antiguos de la Tierra. Las cianobacterias se caracterizan por llevar a cabo la fotosíntesis oxigénica, mediante la cual se produce la liberación de oxígeno y la producción de “poder asimilatorio” (ATP y ferredoxina reducida/NADPH), el cual se usa en la fijación de CO2 atmosférico y en la asimilación de algunos otros nutrientes oxidados. Dentro de este filo se pueden encontrar tanto organismos unicelulares como multicelulares. Aquellas cianobacterias que se comportan como organismos multicelulares crecen formando filamentos, y en condiciones de deficiencia de nitrógeno algunas de ellas pueden dar lugar a un proceso de diferenciación celular produciendo un tipo celular llamado heterocisto. Este último lleva a cabo la fijación de nitrógeno atmosférico (N2), mientras que el resto de las células del filamento, denominadas células vegetativas, continúan con la fijación de CO2. Otras especies del filo tienen la capacidad de formar aquinetos, que son una forma celular de resistencia que aparece en algunas cianobacterias cuando las condiciones ambientales se vuelven adversas, y aún otras forman hormogonios, pequeños filamentos móviles con una función de dispersión. Este trabajo se ha realizado con la estirpe modelo Anabaena sp. PCC 7120, que es una cianobacteria filamentosa formadora de heterocistos. Las cianobacterias poseen una envuelta celular de tipo Gram negativa, y en las cianobacterias filamentosas la membrana plasmática envuelve individualmente a cada célula mientras que la membrana externa, situada por fuera de la capa de peptidoglicano, es continua definiendo por tanto un periplasma continuo. Cuando Anabaena crece en ausencia de nitrógeno combinado, se produce la diferenciación de heterocistos, formándose un patrón a lo largo del filamento de un heterocisto cada 10-15 células vegetativas. El crecimiento diazotrófico exige una comunicación intercelular que permita el intercambio de nutrientes y moléculas reguladoras de la diferenciación a lo largo del filamento, llevándose a cabo esta comunicación a través de los nexos septales. Estos son unas estructuras proteicas que comunican de forma directa las células adyacentes. Actualmente se conocen algunas proteínas que forman parte de los nexos septales como son FraC y FraD, y algunas otras que son esenciales para el correcto funcionamiento o ensamblaje de los mismos, como son SepJ, SepI, HglK (demostrada en esta Tesis) o FraE. Para el ensamblaje de los nexos septales es necesario una perforación del peptidoglicano existente entre las células adyacentes del filamento. Estas perforaciones reciben el nombre de nanoporos y se encuentran en el disco septal de peptidoglicano. En la primera parte de este trabajo se intentó establecer una relación entre los nanoporos y la comunicación intercelular mediada por los nexos septales, utilizando como herramientas un marcador fluorescente y diferentes mutantes de Anabaena. El marcador fluorescente utilizado fue la calceína, que posee la propiedad de difundir desde el exterior hasta el interior de la célula, donde sufre un proceso de hidrólisis que le impide volver a atravesar la membrana plasmática, quedando atrapada en el interior de la célula y emitiendo fluorescencia. Por medio de experimentos de FRAP (“Fluorescence Recovery After Photobleaching”) se pudo calcular un indicador cuantitativo de transferencia entre células adyacentes del filamento. Por otra parte, mediante el aislamiento del peptidoglicano y su visualización por microscopía electrónica de Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Resumen ______________________________________________________________________ xiv transmisión se pudo cuantificar el número de nanoporos de las estirpes estudiadas. Estos ensayos se realizaron en filamentos (cultivados en presencia o ausencia de nitrógeno combinado) de la estirpe silvestre y de mutantes tanto de proteínas que intervienen en la formación de los nexos septales como de proteínas que influyen en su ensamblaje y funcionamiento. Los resultados obtenidos permitieron establecer una correlación entre el número de nanoporos y la transferencia intercelular de calceína en filamentos que habían sido incubados en medio sin nitrógeno combinado, pero no en aquellos incubados en presencia de nitrógeno, en los que se observaba una elevada proporción de células no comunicantes. En la segunda parte de esta Tesis se procedió a la caracterización de la proteína HglK, que pertenece a la familia de las pentapeptide repeat proteins (PRP). HglK se compone de cuatro segmentos transmembrana en su mitad N-terminal y de una sección soluble de localización periplásmica que contiene el dominio PRP en su mitad C-terminal. Estudios previos de HglK mostraron que mutantes que carecían de ella eran incapaces de crecer diazotróficamente y tenían un espacio incrementado entre las células del filamento. En esta Tesis se abordó la localización de HglK en estirpes de Anabaena que producían HglK fusionada a la superfolder-GFP (sfGFP) o con una cola de histidinas o con la etiqueta molecular Strep II. Mediante microscopía confocal y de fluorescencia y ensayos de inmunolocalización, se pudo establecer que HglK poseía una localización preferentemente polar en las células de Anabaena. El estudio de mutantes hglK determinó que HglK es necesaria para la integridad del filamento y para observar unos niveles normales de intercambio molecular entre las células vegetativas del filamento. Sin embargo, los experimentos realizados con mutantes de HglK y de otras proteínas septales mostraron que ni la fragmentación de los filamentos ni la alteración en el intercambio molecular era mayor cuando se combinaba la inactivación de HglK con la de otras proteínas septales. En la caracterización de HglK también se pudo establecer que el fenotipo de crecimiento diazotrófico negativo del mutante hglK estaba determinado por su incapacidad de fijar nitrógeno atmosférico como consecuencia de un defecto en la expresión génica durante la diferenciación de los heterocistos, puesto de manifiesto como una expresión disminuida de los genes del operón cox2 (citocromo oxidasa del heterocisto) y nifHDK (nitrogenasa) en los mutantes hglK. Los avances obtenidos mediante la caracterización de HglK y el conocimiento disponible sobre la proteína SepJ, ponían de manifiesto la implicación de ambas proteínas en los nexos septales. SepJ es una proteína integral de membrana con una localización focalizada en el centro del septo intercelular. SepJ está compuesta por una permeasa en su parte C-terminal y por un linker (rico en el amino ácido prolina) y un dominio coiled-coil en su parte N-terminal, la cual parece ser periplásmica. En el tercer capítulo de esta Tesis se intentó identificar proteínas con las que pudieran tener algún tipo de interacción tanto HglK como SepJ. El abordaje experimental se hizo mediante el uso de estirpes de Anabaena que portaban HglK fusionada a la Strep II o SepJ fusionada a la Strep II o a la GFP. Estas fusiones permitieron la purificación de las proteínas, que se llevó a cabo bajo condiciones no desnaturalizantes, intentando evitar la disgregación de los posibles complejos que pudieran formar con otras proteínas. Las muestras obtenidas tras las purificaciones se analizaron por espectrometría de masas para identificar todas las proteínas presentes en ellas. El análisis de los resultados mostró varias proteínas que podrían interactuar con HglK o SepJ. El primer resultado destacable es la posible interacción entre ambas (SepJ y HglK), aunque la co-purificación de las dos proteínas solamente se observó en los aislados de SepJ. También destaca la presencia de dos proteínas comunes en los aislados de SepJ y HglK. Estas proteínas son Alr2937 y Tic22 (Alr0114), ambas periplásmicas, la segunda de las cuales Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Resumen ______________________________________________________________________ xv con un papel importante en la formación de la membrana externa de Anabaena. Finalmente, mientras que no se ha encontrado ninguna proteína que interaccione solamente con HglK, para SepJ se detectó Alr1690, una proteína anclada a la membrana plasmática que presenta dominios periplásmicos de unión al peptidoglicano. Continuando con el estudio de las proteínas septales, se quiso profundizar en el conocimiento de las proteínas SepJ, FraC y FraD de otras cianobacterias filamentosas formadoras de heterocistos, concretamente de cianobacterias simbióticas con diatomeas marinas. En el cuarto capítulo de esta Tesis, se estudiaron estas proteínas septales codificadas por los genes de las cianobacterias Richelia intracellularis (RintHH01) y Calothrix rhizosoleniae (CalSC01). R. intracellularis es un endosimbionte obligado que vive en el citoplasma de la diatomea Hemiaulus hauckii, mientras que C. rhizosoleniae es un exosimbionte facultativo de la diatomea Chaetoceros compressus, pudiendo proliferar anclada extracelularmente a la diatomea o como un organismo de vida libre. En Anabaena, fraC y fraD forman parte del operón fraCDE que también se encuentra presente en CalSC01 y, sólo fraCD, en RintHH01. Respecto a SepJ, tanto RintHH01 como CalSC01 contienen genes que determinan proteínas homólogas a la de Anabaena. Los mutantes sepJ y fraC-fraD de Anabaena muestran un fuerte fenotipo de fragmentación e imposibilidad de crecer diazotróficamente, ambos fenotipos de fácil estudio, por lo que se intentó la complementación de dichos mutantes con los genes de las cianobacterias simbióticas. Los genes sepJ y fraCD de RintHH01 y los genes sepJ y fraCDE de CalSC01 se obtuvieron por síntesis química, en el caso de sepJ de CalSC01 con un uso de codones adaptado a Anabaena, para ser introducidos en mutantes de Anabaena que portaban las deleciones sepJ (estirpe CSVM34) o fraC-fraD (estirpe CSVT22), respectivamente. La complementación de CSVM34 con sepJ de RintHH01 y de CalSC01 se hizo de forma que la expresión de estos tenía lugar desde el promotor natural del sepJ de Anabaena. Sin embargo, ni la complementación con sepJ de RintHH01 ni la realizada con sepJ de CalSC01 consiguieron revertir el fenotipo mutante, ni siquiera parcialmente. En cambio, sí se consiguió revertir al fenotipo silvestre cuando el mutante se complementó con sepJ de la propia Anabaena utilizado como control positivo. Por otro lado, los genes fraC-fraD de RintHH01 se transfirieron a la estirpe CSVT22 con dos estrategias distintas: en una su expresión tenía lugar desde el promotor natural fraC de Anabaena, y en otra la expresión tenía lugar desde el promotor glnA (promotor de fuerte expresión en condiciones de deficiencia de nitrógeno) insertado en un plásmido replicativo de Anabaena. Por su parte, la complementación de CSVT22 con los genes fraC-fraD-fraE de CalSC01 solamente se intentó con expresión desde el promotor glnA en un plásmido replicativo. Nuevamente ninguna de las complementaciones abordadas tuvo éxito. Es más, las expresiones génicas realizadas bajo el promotor glnA causaban efectos perjudiciales para Anabaena, como pudimos comprobar con controles utilizando los propios genes de Anabaena, lo que sugiere que quizás no habíamos expresado los distintos genes en sus niveles óptimos de funcionalidad. La última parte de esta Tesis se dedicó a la realización de un breve estudio de la proteína FraE, siendo esta la otra proteína codificada en el operon fraC-fraD-fraE de Anabaena. FraE es una proteína de membrana necesaria para la integridad del filamento. En este capítulo se estudió su localización, para lo que se construyó una estirpe de Anabaena que producía FraE-sfGFP. Los ensayos de análisis de fluorescencia de la GFP llevados a cabo con esta estirpe pusieron de manifiesto que FraE se localiza principalmente en los polos de los heterocistos y que sus niveles disminuyen a las 48 horas de inducción en un medio sin nitrógeno combinado. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Conclusiones ______________________________________________________________________ xvi Conclusiones 1. En la cianobacteria filamentosa formadora de heterocistos Anabaena sp. PCC 7120 existe una fuerte correlación entre el número de nanoporos septales y la transferencia intercelular de calceína cuando los filamentos son incubados en un medio sin nitrógeno combinado. Esta observación apoya la idea de que los nanoporos albergan los nexos septales que median la transferencia molecular intercelular, que es esencial para el crecimiento diazotrófico. 2. HglK es una pentapeptide repeat protein necesaria para el crecimiento diazotrófico de Anabaena. Los mutantes hglK muestran una expresión disminuida de algunos genes que se expresan durante la diferenciación de los heterocistos y están afectados tanto en la integridad del filamento como en la formación de nanoporos y en la transferencia molecular intercelular. La proteína HglK presenta una localización preferente en los polos celulares. Concluimos que HglK contribuye a la formación de estructuras septales funcionales necesarias para la diferenciación de los heterocistos maduros en Anabaena. 3. El análisis por espectrometría de masas de preparaciones de las proteínas HglK y SepJ de Anabaena marcadas con etiquetas moleculares ha identificado varias proteínas que podrían interaccionar con aquéllas. Para SepJ destacamos la propia HglK y la proteína de unión de peptidoglicano Alr1690. Además, se detectaron dos proteínas periplásmicas que podrían interaccionar tanto con SepJ como con HglK: Alr2947 y la chaperona Tic22 (Alr0114). Así, SepJ y HglK son dos proteínas septales que pueden interaccionar entre sí y con algunas otras proteínas periplásmicas. 4. Las cianobacterias filamentosas formadoras de heterocistos Richelia intracellularis y Calothrix rhizosoleniae, simbióticas de diatomeas marinas, contienen genes codificantes de SepJ y de FraC, FraD y, en el caso de C. rhizosoleniae, FraE. Sin embargo, no hemos tenido éxito en la complementación de mutantes sepJ y fraC-fraD de Anabaena con los genes de estas cianobacterias simbióticas. 5. La proteína FraE presenta una localización preferente en los polos de los heterocistos de Anabaena, lo que es consistente con el hecho de que los mutantes fraE de esta cianobacteria producen heterocistos que carecen de la estructura polar característica conocida como heterocyst neck. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
1. Introduction Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Introduction _______________________________________________________________________ 3 1.1 Multicellularity in bacteria Multicellularity is a form of biological organization in which individual cells aggregate, working together as a single organism and showing greater complexity than when they are separated (Herrero et al., 2016). Multicellularity marks a new form of evolutionary innovation, in which the advantages of aggregate life must be greater than the disadvantages that may appear such as competition for local resources, reduced mobility or the existence of non-cooperating cells that lead to the failure of such cooperation (Bonner, 1998; Szathmary & Smith, 1995; West et al., 2006). This form of organization has appeared several times throughout evolution and is not a characteristic just of eukaryotes (Bonner, 1998; Claessen et al., 2014). All forms of multicellularity have some common characteristics such as some mechanism of cell-cell adhesion, intercellular communication and coordination (Claessen et al., 2014). There are different forms of bacterial multicellularity including the formation of a biofilm based on the production of an extracellular matrix that surrounds the cells, and where some of them can differentiate to carry out different functions, such as the production of spores in biofilms of Bacillus subtillis. Another example of multicellular bacteria is the swarming bacteria such as myxobacteria, which are soil bacteria wrapped in a matrix allowing them to move as a coordinated group through the soil and to produce spores in the absence of nutrients (Muñoz-Dorado et al., 2016). Organisms that grow as chains of cells represent another type of multicellularity. Within filamentous organisms we can find very different structures (Herrero et al., 2016). A concrete example can be the Streptomycetes which is a group of Gram-positive bacteria belonging to the phylum of Actinobacteria. Under adverse conditions Streptomycetes can produce spores that, when those conditions revert, can germinate giving rise to vegetative hyphae divided into compartments by cross-walls (Jakimowicz & van Wesel, 2012). Other different phylogenetic groups such as cyanobacteria, Desulfobulbaceae (Pfeffer et al., 2012) and Lachenospiraceae (Thompson et al., 2012) contain filamentous forms, while bacteria of the genera Chloroflexus (Pierson & Castenholz, 1974), Beggiatoa (Strohl & Larkin, 1978) and Entotheonella (Wilson et al., 2014) are filamentous. In filamentous cyanobacteria, the separation between daughter cells is incomplete after cell division, so that the outer membrane is continuous along the filament (Wilk et al., 2011). 1.2 Cyanobacteria 1.2.1 Origin, diversity and classification of cyanobacteria Cyanobacteria are among the oldest multicellular organisms on Earth. They constitute a monophyletic group of Gram-negative bacteria, are considered the ancestors of plastids (Giovannoni et al., 1988), and are of great ecological importance since they are the only bacteria that can perform oxygenic photosynthesis (Herrero et al., 2016). Cyanobacteria were responsible for the accumulation of oxygen in the atmosphere that led to the Great Oxygenation Event (GEO) 2.4-2.3 billion years ago, which provoked major changes in the life forms on our planet (Lyons et al., 2014). Oxygenic photosynthesis affects the carbon cycle since it efficiently removes atmospheric CO2 by fixing it to produce organic matter, thus contributing to primary production which is basic in the biosphere's food chain. In addition, cyanobacteria also intervene in the nitrogen cycle since some of them can fix atmospheric nitrogen, which can be assimilated by other organisms (Knoll, 2004, 2008). Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Introduction _______________________________________________________________________ 10 composition in sugars and length is variable between bacteria but specific to bacterial strains (Sperandeo et al., 2019; Xu et al., 1997). 1.2.3.2 The peptidoglycan layer The peptidoglycan layer is found in the periplasm, the space between the outer and cytoplasmic membranes. This periplasmic zone provides the oxidative environment necessary for some enzymes present there to catalyze the formation of disulfide bonds (Nakamoto & Bardwell, 2004). The periplasm is a zone in which there is no source of energy in the form of ATP but, in spite of this, there are many factors that modulate the position of the polymers of the envelope. In addition, some proteins that transport small molecules can be found in the periplasm (Ruiz et al., 2006). The peptidoglycan is basically composed by a repetition of a disaccharide made of β-1-4-linked N-acetylmuranic acid (MurNAc) and N-acetylglucosamine (GlcNAc) that is linked by peptide side chains (Ruiz et al., 2016), acting all of this structure as a huge single molecule (the murein sacculus) (see Fig. 5 below). This sacculus determines the cellular form, provides osmotic resistance, and serves as an anchor for many components of the cellular envelope, as for example occur in Gram-positive bacteria in which it serves as an anchor to teichoic acids (Vollmer et al., 2008). Although the basic composition of PG is as indicated above, there are many chemical, structural and architectural differences between different bacterial strains (Turner et al., 2014). Hence, it is interesting to indicate that in cyanobacteria the degree of density and cross-linking between the different chains of peptidoglycan is more similar to that of Gram-positive than to that of Gram-negative bacteria (Hoiczyk & Hansel, 2000). Specifically, in the case of Anabaena, the PG has two or three layers and is found wrapping each cell individually, being also present between the cells at the intercellular septa (Flores et al., 2006; Wilk et al., 2011). The murein sacculi of some filamentous cyanobacteria, such as Anabaena or Nostoc punctiforme, can be isolated as a single structure encompassing several cell units, allowing their study by transmission electron microscopy (TEM). Their study has shown the presence of a rigid diskshaped septal structure composed of the PG from the adjacent cells. These disks bear perforations in their central part that are 15-20 nm in diameter and have been called "nanopores" (Lehner et al., 2013; Nürnberg et al., 2015). It has been proposed that nanopores are openings in the peptidoglycan disk that hold protein structures that connect adjacent cells (Mariscal et al., 2014). The size of nanopores appears to be regulated, since a protein called SjcF1 that is conserved in filamentous cyanobacteria and could be involved in nanopore size determination has been described in Anabaena (Rudolf et al., 2015). SjcF1 is a membrane-anchored, mostly periplasmic protein that contains three functional domains, two peptidoglycan-binding domains and a proteinprotein interaction SH3 domain. Anabaena mutants of this protein present nanopores wider and of more variable diameter than the wild-type strain (Rudolf et al., 2015). PG is a dynamic structure since it has to be hydrolyzed to allow the incorporation of new peptidoglycan subunits and, subsequently, covalent bonds are re-established, so that the structure of PG is not altered during cell growth. The proteins that perform this hydrolysis are called autolysins, which are found in the periplasm and not only play an essential role in cell growth but are also important to determine the shape, size and density of the PG, as well as for the separation of the daughter cells after cell division (Typas et al., 2012). Although genes encoding autolysins have been studied mainly in model organisms such as E. coli, their homologues have also been Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Introduction _______________________________________________________________________ 11 found in cyanobacteria (Hahn & Schleiff, 2014). Autolysines are generally PG amidases (Vollmer et al., 2008; Typas et al., 2012). In E. coli, there are three known amidases: AmiA, AmiB and AmiC. Amidase AmiC has two variants in heterocystous cyanobacteria, AmiC1 and AmiC2 (Zhu et al., 2001). However, the role of AmiC amidases in filamentous cyanobacteria is complex because there is no hydrolysis of the peptidoglycan and invagination of the outer membrane at cell division; instead, it is known that AmiC1 in Anabaena and AmiC2 in Nostoc punctiforme are necessary for the differentiation of heterocysts and formation of nanopores (Lehner et al., 2011; Berendt et al., 2012). In heterocystous cyanobacteria, amiC1 and amiC2 are found within a gene cluster with murI, which codes for a glutamate racemase (amiC1-amiC2-murI). In some strains, a gene named amiC3 has also been found, but it is not located within that cluster (Bornikoel et al., 2017; Zheng et al., 2017). 1.3 Heterocysts Heterocysts are a differentiated type of cell in which the conditions necessary for the fixation of atmospheric dinitrogen occur. Heterocysts are present in strains of filamentous cyanobacteria that belong to taxonomic sections IV and V (Rippka et al., 1979). They are easily recognized along the filament, because they have a shape and size different from the rest of the cells. Regarding to their localization in the filament, this can be intercalary (between vegetative cells) or terminal (at the end of the filament). 1.3.1 Characteristics of the heterocyst In order to facilitate the description of the different characteristics of the heterocyst, this section will be divided into two sub-sections, one of which will refer to structural characteristics and the other one to metabolic aspects. 1.3.1.1 Structural characteristics Beyond size and shape, there are certain structures (or processes that take place during heterocyst differentiation) that have a role in providing adequate conditions for nitrogen fixation in the heterocysts and that clearly differ from those found in the vegetative cells. (i) A reorganization of intracellular membranes places the thylakoid membranes arranged near the heterocyst cell poles in the so-called "honeycomb" structure (Lang & Fay, 1971); a high respiratory rate takes place in the honeycomb membranes that allows the elimination of oxygen thus contributing to avoid the inactivation of nitrogenase (Valladares et al., 2007). (ii) A loss from the cytoplasm of carboxysomes and glycogen granules can be seen, as well as a narrowing of the cytoplasm in the heterocyst poles giving rise to a structure known as the "heterocyst neck", which makes the septum between heterocysts and vegetative cells narrower than between vegetative cells; this could help to control the entry of gases in the heterocyst (Fig. 3) (Walsby, 2007). (iii) Granules of cyanophycin also appear at the heterocyst poles; these granules are composed of a polymer of arginine and aspartic acid of non-ribosomal synthesis that is a nitrogen reservoir (Fig. 3) (Lang et al., 1972). Finally, (iv) the heterocysts have a thick envelope that provides protection against gas diffusion and consists of heterocyst-specific glycolipids (HGL) and polysaccharides (HEP) deposited outside of the outer membrane (see Fig. 3) (Cardemil & Wolk, 1979; Flores & Herrero, 2010). The layer closest to the outer membrane is the glycolipid layer that functions as a barrier against gases (Fay, 1992), whereas the polysaccharide layer protects the glycolipid layer from physical damage (Xu et al., 2008). These extra wrappers are important because the lack or harm Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Introduction _______________________________________________________________________ 12 of any of them impair the diazotrophic growth of the filament (Fan et al., 2005; Huang et al., 2005). 10 𝞵m A BC Honeycomb Heterocyst neck CGP HEP HGL 10 !m 10 !m Fig. 3. Heterocyst morphology. (A and B) Filaments of Anabaena sp. PCC 7120 grown without combined nitrogen photographed with the optical microscope with the 20x and 40x objectives respectively. The white arrows show terminal heterocysts and the black arrows show intercalary heterocysts. (C) Transmission electron micrograph of a fragment of an Anabaena filament showing a vegetative cell (top cell) and a terminal heterocyst (bottom cell). The image shows the place that the cyanophycin granule normally occupies (CGP), the neck of the heterocyst, the honeycomb membranes, the heterocyst glycolipid layer (HGL) and the heterocyst polysaccharide layer (HEP). Image C by Victoria Merino-Puerto (CSIC, Seville) and Iris Maldener (Universität Tübingen, Germany). Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Introduction _______________________________________________________________________ 13 1.3.1.2 Metabolic characteristics From a metabolic point of view, heterocysts also have some characteristics that differentiate them from the rest of the cells of the filament. Among these characteristics, we can highlight: (i) the inability to carry out oxygenic photosynthesis due to the loss of PSII activity; (ii) the inability to fix atmospheric CO2 as a consequence of repression of Rubisco; (iii) the increase in respiratory rate to reduce oxygen levels; and (iv) the expression of all the machinery necessary to fix atmospheric dinitrogen (Golden & Yoon, 2003; Herrero et al., 2004). Although not a metabolic characteristic itself, it should be noted that the heterocysts are terminally differentiated cells, so they are unable to divide once they have completed cell differentiation and cannot revert to vegetative cells (Wang & Xu, 2005; Kuhn et al., 2000). Due to the inability of heterocysts to perform certain functions (photosynthesis) and their capacity to perform others (N2 fixation), there must be an exchange of molecules between the heterocysts and the vegetative cells. For example, the heterocysts transfer glutamine and a dipeptide (baspartyl-arginine) to the vegetative cells and receive sucrose and some amino acids (glutamate, alanine) from the vegetative cells. In order to explain more clearly the importance of this exchange of metabolites, its involvement in carbon and nitrogen metabolism will be presented below. As mentioned above, the nitrogen-fixing machinery is very sensitive to oxygen, and the heterocysts have several ways of maintaining a micro-oxic environment. The oxygen that enters the heterocyst is eliminated by the high respiratory rate in the honeycomb membranes and the action of flavodiiron proteins that absorb O2 (Valladares et al., 2003, 2007; Ermakova et al., 2014). The synthesis of ATP necessary for nitrogen fixation depends on respiration as well as on PSI-dependent photophosphorylation (Herrero et al., 2013). Because the heterocysts are unable to fix atmospheric CO2, they need a contribution of reduced carbon from the vegetative cells. The vegetative cells transfer reduced carbon to the heterocysts mainly in the form of sucrose, which is split into glucose and fructose by heterocyst-specific invertases (López-Igual et al., 2010; Vargas et al., 2011). In relation to nitrogen metabolism, the oxidation of sugars received from vegetative cells produces the necessary reducing power for nitrogenase. Nitrogenase is formed by a tetramer of NifD and NifK that contains the active site of the enzyme, and a dimer of NifH acts as an electron donor to reduce N2 to ammonium (Rubio & Ludden, 2008). The resulting ammonium is incorporated into glutamate forming glutamine, which is one of the amino acids transferred to the vegetative cells (Wolk et al., 1976; Meeks et al., 1977). The glutamate needed to form glutamine is received from the vegetative cells because the heterocysts possess GS but not GOGAT, making them unable to produce enough glutamate for the incorporation of ammonium (Thomas et al., 1977; Martín- Figueroa et al., 2000). Finally, the heterocysts also transfer to the vegetative cells b-aspartyl- arginine dipeptide, which results from the cyanophycinase-catalyzed degradation of cyanophycin (previously synthesized by cyanophycin synthetase). Once the dipeptide is in the vegetative cells, it is hydrolyzed by an aspartyl-arginine dipeptidase releasing L-arginine and L-aspartic acid, which are used as sources of nitrogen (Burnat et al., 2014) (Fig. 4). Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Introduction _______________________________________________________________________ 14 Fig. 4. Diagram of the main carbon and nitrogen flows between vegetative cells and heterocysts. (A) Heterocyst metabolism. The OPPP (oxidative pentose phosphate pathway) and the Krebs cycle are the major routes used by heterocysts to generate reducing equivalents, while glycolysis has a smaller contribution. Metalloenzymes (red circles) and enzymes catalyzing reactions that generate (yellow circles) or require (green circles) NAD(P)H are indicated. Abbreviations for metabolites: Suc, sucrose; G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; F1,6P, fructose 1,6-bisphosphate; G3P, glyceraldehyde 3-phopshate; PEP, phosphoenolpyruvate; Ac-coA, acetyl-coA; Mal, malate; OA, oxaloacetate; Cit, citrate; Isocit, isocitrate; 2-OG, 2-oxoglutarate. Abbreviations for enzymes: G6PD, glucose 6-phosphate dehydrogenase; 6PGD, 6-phosphogluconate dehydrogenase; FBA, class-II fructose bisphosphate aldolase; FBP, fructose bisphosphatase; G3PD, glyceraldehyde 3-phosphate dehydrogenase; PK, pyruvate kinase; PEPS, PEP synthase; PDH, pyruvate dehydrogenase; PFOR, pyruvate:ferredoxin oxidoreductase; MDH, malate dehydrogenase; CS, citrate synthase; AcnB, aconitase; IDH, isocitrate dehydrogenase; GS, glutamine synthetase. Picture and legend from Pernil & Schleiff (2019). (B) Simplified diagram of the exchange of compounds rich in nitrogen and compounds rich in carbon between the heterocyst and the vegetative cell. It includes the synthesis and degradation of cyanophycin (polymer of Asp + Arg that serves as a nitrogen reservoir in the heterocyst). Asp, aspartic acid; Arg, arginine; CS, cyanophycin synthetase; Gln, glutamine; Glu, glutamic acid; GOGAT, glutamine-oxoglutarate amido transferase (glutamate synthase); GS, glutamine synthetase; N2ase, nitrogenase. Asp + Arg Cyanophycin !-Asp-Arg !-Asp-Arg Asp + Arg Glu Gln Gln Glu Sucrose CO2 Glucose + fructose Sucrose N2 NH4+ GS GOGAT Cyanophycinase Invertase Dipeptidase N2ase CS A B Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Introduction _______________________________________________________________________ 15 1.3.2 Heterocyst differentiation and pattern formation When some filamentous strains of cyanobacteria are under nitrogen deprivation, a process of cell differentiation begins in which about 495 genes are induced and about 196 genes are inhibited producing structural and metabolic changes that result in heterocyst differentiation (Ehira & Ohmori, 2006a; data re-evaluated in Xu et al., 2008). The appearance of multiple non-coding RNA transcripts suggests that this type of nucleic acid may also play an important role in the differentiation process (Mitschke et al., 2011; Flaherty et al., 2011). The response to nitrogen deprivation is mainly regulated by two transcriptional regulators. On one hand, the ntcA gene is early expressed when 2-OG levels increase, and the NtcA protein is in charge of governing the expression of genes that respond to the cellular need for nitrogen (Picossi et al., 2014; Li et al., 2003). On the other hand, hetR is also early induced in differentiation, and HetR acts by binding to DNA as a homodimer (Kim et al., 2011) or a tetramer (Valladares et al., 2016) and regulates the expression of some genes related to the development, function and pattern of the heterocysts (Black et al., 1993; Buikema & Haselkorn, 1991). To maintain high levels of NtcA and HetR during the first steps of differentiation, both genes are self-induced and also mutually induced (Frías et al., 1993; Muro-Pastor et al., 2002). Another regulator called NrrA has been identified whose involvement in cell differentiation is not entirely clear (Flores & Herrero, 2010), but it does appear to act as a regulatory link between NtcA and HetR (Ehira & Ohmori, 2006b). After 6-12 hours of nitrogen deprivation (intermediate differentiation phase), the induction of genes involved in the synthesis of the heterocyst envelope begins (Maldener et al., 2014). Finally, genes encoding different oxidases and nitrogen assimilation proteins are induced in a late differentiation period (Herrero et al., 2013; Flores et al., 2019). Together with the previously mentioned genes, during cell differentiation the expression of patS and hetN is also induced. These genes encode a peptide (PatS) or a protein (HetN) that, after being processed (at least in the case of PatS, see below), play a fundamental role in the establishment of the pattern of heterocysts along the filament, which in Anabaena consists of one heterocyst for 10-15 vegetative cells (reviewed in Herrero et al., 2016). If the conditions of deprivation of combined nitrogen continue, the filament will begin to grow diazotrophically as a consequence of the division of the vegetative cells, making new heterocysts to appear in the center of the increasing interval, maintaining in this way the described pattern (Herrero et al., 2016). In Anabaena, the patS gene is constitutively expressed in the presence of combined nitrogen and induced in small cell clusters in the initial phases of cell differentiation in response to nitrogen deficiency. Mutants in which patS has been eliminated have a phenotype in which multiple contiguous heterocysts (Mch) are observed (Yoon & Golden 1998, 2001; Corrales-Guerrero et al., 2013; 2015). The expression of patS produces a peptide of 17 amino acids that is processed to produce a peptide that corresponds to the C-terminal part of the original peptide, has the sequence ERGSGR, and behaves as a morphogen that is transferred to neighboring cells and inhibits their differentiation (Corrales-Guerrero et al., 2013; Hu et al. 2015; Yoon & Golden 1998, 2001; Zhang et al., 2017). HetN also has the sequence ERGSGR, but in this case internal to the protein; HetN or a derivative of it has a negative effect on heterocyst formation and, as it happens with PatS, its inactivation produces multiple contiguous heterocysts, although this phenotype is delayed compared to the one of inactivation of patS (Callahan & Buikema, 2001; Corrales- Guerrero et al., 2013, 2014a; Higa et al., 2012). Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Introduction _______________________________________________________________________ 16 Finally, HetR, in addition to be regulated at the level of gene expression, can be regulated posttranslationally by phosphorylation, preventing the accumulation of HetR tetramers, which are the active form of the protein (Valladares et al., 2016). HetR binds, and is inhibited by, the PatS morphogen and HetN (or a HetN-derived factor), and has been suggested to be regulated by the HetF protease and by PatA, which has similarity with CheY (Risser & Callahan, 2008). Away from the heterocyst, the concentration of HetR increases making it possible to stimulate further heterocyst differentiation (Risser & Callahan, 2009). 1.3.3 Genes involved in the formation of heterocyst envelopes The formation of the heterocyst envelope requires enzymes that carry out the synthesis and export of the materials that make up HEP and HGL, and is also regulated by various proteins. In Anabaena, HEP formation is one of the first morphological changes that occur after nitrogen deprivation, and many of the genes responsible for this process are found in a cluster called "HEP island" (alr2825-alr2841) (Huang et al., 2005). Although some genes encoding proteins involved in this process have been described, such as hepA (similar to an ABC-type exporter of lipid A of lipopolysaccharide; Huang et al., 2005), hepB, alr3699 (both glysosyl transferases), alr4388 (polysaccharide export protein) and hepP (predicted membrane protein), the detailed process of production and deposition of the HEP layer remains unknown (Maldener et al., 2003; Wang et al., 2007; Lechno-Yossef et al., 2011; López-Igual et al., 2012). As with hep genes, several genes encoding proteins for HGL layer formation are found in a cluster (all5343-alr5347) within the Anabanea genome (Fan et al., 2005). The HGL layer is formed by fatty alcohols glycosidically linked to sugar residues, and within the HGL cluster there are several genes encoding fatty acid synthases and polyketide synthases, examples of which are hglB, hglC, hglD and hglE (Fan et al., 2005; Maldener et al., 2014). Some genes have also been identified that are necessary for the correct deposition of the HGL layer, some of which constitute the devBCA operon that encodes an ABC-type exporter (Fiedler et al., 1998) and hgdD that encodes an outer membrane TolC-like protein (see Fig. 5) (Fan et al., 2005; Moslavac et al., 2007). Finally, an example of a protein that regulates the process of synthesis and deposition of the HGL layer is the DNA-binding protein DevH (Ramírez et al., 2005), however, a very complex regulation appears to be operative that is not yet fully understood (reviewed in Flores et al., 2019). Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Introduction _______________________________________________________________________ 17 OMF ABC HgdD DevB DevC HepK HepN HK-I HK-II DevR DevA PG layer Outer membrane Cytoplasmic membrane HGL HEP Cytoplasm Periplasm Environment Fig. 5. General structure of the heterocyst envelope in Anabaena. The figure shows the cytoplasmic membrane formed by a bilayer of phospholipids. As an example of cell wall biosynthesis regulation, there are two histidine kinase complexes in the cytoplasmic membrane that are necessary for the activation of the synthesis of heterocyst envelope polysaccharide (HEP), although they do not intervene in the transport of polysaccharides. In the histidine kinase complex known as HK-I, HepN acts as a phosphorylation regulator, whereas HK-II has HepK as a phosphorylation regulator and phosphorylates DevR, which must be another intermediate regulatory factor since it does not have a transcription activation domain (for further details see Flores et al., 2019). The cytoplasmic membrane also assembles part of the DevBCA-HgdD secretion complex that crosses the peptidoglycan layer (PG layer) and the outer membrane and is essential for the export of the glycolipids responsible for forming the heterocyst-specific glycolipid (HGL) layer, although the mechanism by which they are assembled is unknown. The DevBCA complex is an ABC-transporter while HgdD forms a tunnel that crosses the outer membrane. Between the cytoplasmic membrane and the outer membrane, there is an aqueous space called periplasm with the peptidoglycan layer (represented by hexagons, where one color represents N-acetyl glucosamine and the other represents N-acetyl muramic acid). The outer membrane is an asymmetric lipid bilayer composed of phospholipids in its inner layer and lipopolysaccharides in its outer layer. Outside the outer membrane is the HGL layer (composed of glycolipids) and the HEP layer (formed by polysaccharides). Both layers together protect the heterocyst from the entry of ambient oxygen. Although they have not been drawn to simplify the scheme, the cytoplasmic membrane and the outer membrane also have many other proteins including lipoproteins and, in the outer membrane, porins. The elements represented in the scheme are not to scale. The figure is adapted from Fig. 2 of Nicolaisen et al. (2009a). Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Introduction _______________________________________________________________________ 18 1.4 Septal junctions and septal proteins As it has been commented above, filamentous cyanobacteria grow as chains of cells in which differentiated cells are observed if the environmental conditions are appropriate. This type of growth made microscopists focus their attention on the septal areas between two adjacent cells, being Wildon & Mercer (1963) among the first to show them. Thin structures perpendicular to the cytoplasmic membrane were observed that received the name "microplasmodesmata" (Lang & Fay, 1971). Later, by freeze-fracture electron microscopy, Giddings & Steahelin (1978) observed these microplasmodesmata as a pit and a protrusion on the exoplasmic and protoplasmic fracture faces, respectively, of each adjacent cell membrane. They also observed that the microplasmodesmata had an outer diameter of less than 20 mn and occupied a circular area in the center of the septum (Giddings & Steahelin, 1978). In the last decade, the septum between two adjacent vegetative cells of Anabaena has been studied by electron tomography (Wilk et al., 2011). For this purpose, the samples were chemically fixed and stained, either with positive staining (potassium permanganate) showing protein structures communicating the two cells and having a diameter of 5.5 nm and a length of 18 nm, or with negative staining (osmium tetroxide) showing holes in the septal PG that had a diameter of 14 nm and a length of 18 nm. The structures connecting the two adjacent cells were called “septosomes” (Wilk et al., 2011). Studies on the septa between vegetative cells and on those between vegetative cells and heterocysts in filaments of Anabaena prepared by cryo-fixation, subjected to negative staining (osmium tetroxide) and visualized by electron tomography, showed the presence of structures, termed "channels", that cross the septal PG (Omairi-Nasser et al., 2014). The size of the channels varies from 12 nm long and 14 nm in diameter in the septum between vegetative cells to 21 nm long and 14 nm in diameter between vegetative cells and heterocysts. This suggests a remodeling of the septa during the development of the heterocysts, and that the septal channels are larger between heterocysts and vegetative cells than between vegetative cells (Omairi-Nasser et al., 2014, 2015). These septal structures, now termed “septal junctions” (Mariscal, 2014; Flores et al., 2016), have been recently visualized by cryo-electron tomography (Weiss et al., 2019). In the cytoplasmic membrane, in the part that is in contact with the cytoplasm, the septal junctions have a cap-like structure and just below it a plug-like density (see Fig. 6). These cytoplasmic membrane structures from adjacent cells are connected by a 11-nm wide tube (lumen, 7 nm). The channels present in the septal disks (Wilk et al., 2011; Omairi-Nasser et al., 2015) would correspond to the perforations described as nanopores (Lehner et al., 2013) and could be the places through which the septal junction traverse the septal PG (Flores et al., 2016). The number and diameter of nanopores per septal disk is not the same for all strains of cyanobacteria, since for example in septa between vegetative cells the number of nanopores in Nostoc punctiforme is about 155 with a diameter of about 20 nm (Lehner et al., 2013), while in Anabaena the number has been described to be about 75 with a diameter of about 15 nm (Nümberg et al., 2015). As mentioned earlier, in the septum between adjacent cells there are proteinaceous structures that appear to join the cells in the filament. Some proteins have been identified whose localization is septal and their absence results in filament fragmentation, which led to think that they could have a role in the septal junctions (Bauer et al., 1995; Flores et al., 2007; Nayar et al., 2007; Merino- Puerto et al., 2010). Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Introduction _______________________________________________________________________ 19 A B C D E F G H H Fig. 6. Septal junctions in Anabaena. (A) Electron micrograph of a portion of a filament of Anabaena where the white arrows indicate structures perpendicular to the cytoplasmic membrane located in the septum between two vegetative cells, known as septal junctions. (B) Electron micrograph of the septal zone between a vegetative cell (bottom) and a heterocyst (top) where the septal junctions are visible (white arrows). Het neck, the narrowed region located at the pole of the heterocyst. (C and D) Cryo-electron- tomograms of an Anabaena filament and magnified areas in each box (same size bars). The multiple septal junctions present in the septum between two vegetative cells are marked with arrowheads. CM, cytoplasmic membrane; OM, outer membrane; PG, septal peptidoglycan; TM, thylakoid membranes; PB, phycobilisomes. (E, F) Subtomogram of the terminal modules of the septal junctions and their schematic representation showing tube, cap and plug. (G) Oblique view of the septal junction. (H) View from above of the septal junction scheme. A and B from Flores et al. (2016); C-H from Weiss et al. (2019). Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Introduction _______________________________________________________________________ 26 study of Anabaena has only identified, however, one type of septal junctions, which include FraD and need FraC for full assembly (Weiss et al., 2019). 1.5.4 Possible regulation of the septal junctions A study of esculin transfer in diazotrophically-grown Anabaena filaments showed that, in contrast to heterocysts that had the same level of labeling as their neighboring vegetative cells, some heterocysts showed a lower level of labeling, defining the existence of non-communicating heterocysts (Nürnberg et al., 2015). These results suggested that the septal junctions in a heterocyst could be regulated in some way, so that they were open or closed allowing or not, respectively, the passage of esculin (Flores et al., 2018). The same conclusion, that the septal junctions can be regulated in a cell, was reached after re-evaluation of data in which the fluorescent marker was calcein or 5-CF (Flores et al., 2018). These data showed that the recovery rate constants in the wild type conformed to a normal distribution of the data, but when mutants of the glsC and glsD genes (encoding components of an ABC transporter for glucosides) were analyzed, there was a clear increase in the number of cells with a low recovery rate constant (< 0.01 s-1), defining them as non-communicating cells (Flores et al., 2018). It was hypothesized that GlsC and GlsD could intervene in the opening or closing of the septal junctions, perhaps transmitting information on the availability of sucrose to regulate the septal junctions (Flores et al., 2018). Finally, in a mutant of gene hepP (which encodes an MFS glucoside transporter), it was observed that the transfer of esculin between vegetative cells is decreased, but transfer from vegetative cells to heterocyts is increased (Nieves-Morión et al., 2017a). The hypothesis has been considered that HepP could inhibit specifically the heterocyst septal junctions under conditions of sucrose scarcity (Flores et al., 2018). In addition, FRAP experiments performed under different stresses showed the presence of a high number of non-communicating cells, but if the filaments were transferred and incubated without stress conditions the number of non-communicating cells decreased (Weiss et al., 2019). These data support the hypothesis that the septal junctions in a cell can be opened or closed to allow or not, respectively, the passage of molecules. 1.6 Pentapeptide repeat proteins Proteins of the pentapeptide repeat protein (PRP) family are defined as proteins that have at least eight tandem repeats of a five-amino acid sequence, which could be framed within the consensus (STAV)(D/N)(L/F)(S/T/R)(X) (Bateman et al., 1998, Vetting et al., 2006; Ni et al., 2009). More than 3,500 PRPs have been identified through computer platforms (Pfam database) (Bateman et al., 2000; El-Gebali et al., 2019). Approximately 10% of PRPs are found in eukaryotes and 90% in prokaryotes, both archaea and bacteria, and they are also present in some bacteriophages (Vetting et al., 2006; Zhang et al., 2019). Specifically, in Anabaena, 32 PRPs have been identified (Ni et al., 2009). The PRPs present a structure that can be described as a collection of type II and IV beta turns, because the conformation that they usually adopt is that of four-sided, right-handed beta helical structure (Buchko et al., 2006, 2008; Vetting et al., 2006, 2007). However, beyond the fact that their general structure is known, their biochemical function is still unknown in most cases (Ni et al., 2009). Recently, a new PRP type with a type I beta turn structure has been discovered in Anabaena (termed Nostoc sp. PCC 7120 by the authors) (Zhang et al., 2019), making it clear that other types of structures could appear as the study of PRPs deepens. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Introduction _______________________________________________________________________ 27 In cyanobacteria, several PRPs have been described, but only in a few cases a function could be envisaged. For example, RfrA isolated from Synechocystis sp. PCC 6803 is involved in the absorption of manganese (Chandler et al., 2003), although its structure and precise biochemical function are unknown (Ni et al., 2009). HetL described in Anabaena is fundamental for the establishment of the pattern of heterocysts along the filament, its structure has been determined (Fig. 9) but its mechanism of action is unknown (Ni et al., 2009). Other PRPs have also been described in Anabaena, such as PatL, which is also related to the establishment of the heterocyst pattern (Liu & Wolk, 2011), or HglK, whose inactivation prevents diazotrophic growth apparently affecting Hgl deposition (see below; Black et al., 1995). Apart from cyanobacteria, other PRPs have also been described such as MfpA from Mycobacterium tuberculosis that has been determined to perform a DNA gyrase inhibitor function, likely because the DNA gyrase could bind to the DNA-mimicking PRP (Hegde et al., 2005; Ni et al., 2009). Finally, to give an example in eukaryotes, a PRP has been described that is related to the tetramerization of potassium channels in the membrane (Vetting et al., 2006). Although the pentapeptide repeat sequence described above is the major pentapeptide found in PRPs, a second pentapeptide repeat has been described in the Pfam database as "peptapetide_2 repeat”, which responds to the consensus sequence (N)(T,L,I)(G)(S,N)(G), with 61 examples in prokaryotes and two examples in eukaryotes (Vetting et al., 2006). To date, not much is known about the structure of the pentapeptide_2 repeat, but it is thought that its structure should not be the same as that of the original pentapeptide (Vetting et al., 2006). Both types of PRPs are associated with other types of domains, including, for example, transmembrane segments, WD40 repeats, Ser/Thr protein kinase and PPE (Pro-Pro-Glu) domains (Vetting et al., 2006). However, there is no protein where both pentapeptide repeat types occur at the same time, nor is there a domain that has been associated with one type of PRP and is present in the other type of PRP. Therefore, a certain type of domain appears to be associated only with one PRP or the other (Vetting et al., 2006). C- -N Fig 9. Diagram of HetL. The image shows the a -helix at the N-terminal part and the b -sheets that constitute most of HetL (Ni et al., 2009). Strucrure PDB 3DU1. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Introduction _______________________________________________________________________ 28 1.6.1 The HglK protein More than half of the sequences found in the Pfam database belonging to the PRP family are within the phylum of cyanobacteria (Zhang et al., 2019), so it is not surprising that it was in a cyanobacterium that this type of proteins was first discovered and characterized. The first PRP described was HglK (Black et al., 1995), ORF all0813 from the Anabaena genome. HglK is composed of several parts since it has an N-terminal domain with four TMSs and a C-terminal domain in which the pantapeptide repeat with 36 repetitions of consensus sequence ADLSG is found (Black et al., 1995) (Fig. 10). Fig. 10. HglK sequence. Amino acid sequence with indication of transmembrane segments (magenta) and pentapeptide repeats (alternatively shown in blue and red color), as described by Black et al. (1995). The hglK mutant was unable of diazotrophic growth. This mutant also presented some structural alterations in the heterocysts as observed by TEM. Among these alterations, the following stand out: the lack of the HGL layer, the lack of reorganization of the intracellular membranes and the lack of a polar cyanophycin granule (Fig. 11b) (Black et al., 1995). The lack of the glycolipid layer is not due to the inability of the heterocysts to synthesize Hgl, since the images show lipid vesicles inside the cells; these observations suggested that HglK could be involved in glycolipid transport (Black et al., 1995). Additionally, the microscopy images also showed that the intercellular septum between vegetative cells (Fig. 11a) or between vegetative cells and heterocysts (Fig. 11b) was much wider than in the wild type (Black et al., 1995). MTTPIVKKSNNAPSKNLVKPNSLPLATRRLAAWATEITLLATTGLVPFGLGVYINSRSDIN REPLNPALVVTERAIARPLALPADYGIRNVAWPTNYLWMLALLAPTALSWWQLYLLAKTGS TLPKRWFGVKVLNEEGTPPGLATVVVREGIGRWTVPMSVAYILWRYSFAFPNLGLFTSLAV LMVIGEALALPARRGRKALHDWLAGTYVVDANRPVPSPDVALNGRGLSGVSPQPEEGNATL ATTAMAMSYPQGEVITTDNSSLISLWRRMQQNPSLTLFGVALTSMTAVLATLIGTQVYIQT QQGNRESQKINSQQFLEFVKQLSPESGASIEDRQRTILALGSLKDFQSIQFLTDMMVKETN PILIDTIQQALTSVGTAAIPELQNKNQFLATELDSVGSASPEREARQKRLQINQQTINKIL NVYSGKTLGLDLSRTQLGQSGTVGGSFFNLILDNIDLSGIKFKSANLNQASFKGSRFRSVG DDGRWDTYDDAIADLSQAQMKQANFTDANLSRVLMTRSDLSRATLNRANLSNARLIGANLS SAQLVGADLRGTVLENASLTGADLGDAKLQEANLYGARLSRVIAIGAQLSFANLTKTDWQS SDLSGADLERANLSNADLSATRMTGAILRSAQLENANLRNADLSLVDLRGANVAGADFKDT ILTPSRQDPADQFVQTPELGSVSAVVKGVDFSQAKNLDGKQLAYICTQGGVHPRCP A Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Introduction _______________________________________________________________________ 29 Fig. 11. Phenotype of the hglK mutant. All the images are from Black el al. (1995). (A) TEM micrographs of Anabaena filaments grown with nitrate, where it is shown that the hglK mutant (bottom) has a wider distance between vegetative cells than the wild type. (B) TEM of filaments of Anabaena incubated in the absence of combined nitrogen shows that the heterocyst (H) of mutant (right) does not form the honeycomb or the glycolipid layer (g) that are observed in the wild type (left). 2 𝞵m 2 𝞵m A 1 µm B 1 µm Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Introduction _______________________________________________________________________ 30 1.7 Objectives In this work, we addressed some aspects of the intercellular septa in filamentous cyanobacteria, mainly the Anabaena sp. PCC 7120 model strain, by carrying out further studies of mutants of the septal proteins SepJ, FraC and FraD and a characterization of the cytoplasmic-membrane, pentapeptide-repeat protein HglK, previously thought to be involved in the export of glycolipids. The main objectives of this Thesis were: 1. To study the possible relationship between intercellular molecular transfer through septal junctions and nanopores under different culture conditions. 2. To characterize the HglK protein from Anabaena by the study of the phenotype of hglK mutants and the study of HglK protein fusions. 3. To search for possible partners of HglK and SepJ in protein complexes. 4. To study the SepJ and Fra proteins from the marine symbiotic cyanobacteria Richelia intracellularis and Calothrix rhizosoleniae. 5. To study the subcellular localization of the FraE protein in Anabaena. Part of the results of this thesis have been included in the following articles: • Arévalo S & Flores E (2020) Pentapeptide-repeat, cytoplasmic-membrane protein HglK influences the septal junctions in the heterocystous cyanobacterium Anabaena. Mol Microbiol 113: 794-806. • Ramos-León F, Arévalo S, Mariscal V & Flores E (2018) Specific mutations in the permease domain of septal protein SepJ differentially affect functions related to multicellularity in the filamentous cyanobacterium Anabaena. Microb Cell 5: 555-65. • Springstein BL, Arévalo S, Helbig AO, Herrero A, Stucken K, Flores E & Dagan T (2020) A novel septal protein of multicellular heterocystous cyanobacterial is associated with the divisome. Mol Microbiol (doi: 10.1111/mmi/14483). Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
2. Materials & Methods Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Materials & Methods ______________________________________________________________________ 33 2.1 Organisms and Growth conditions The living organisms used in this work were two types of gram-negative bacteria, Escherichia coli and Anabaena sp., whose growth conditions are detailed below. 2.1.1 Escherichia coli 2.1.1.1 Strains The Escherichia coli strains used in this work, together with their genotypic characteristics, are described in Table 1. Table 1. E. coli strains used in this work together with their genotypes and references. The DH5a strains were obtained from the Servicio de Cultivos Biológicos del Centro de Investigaciones Científicas Isla de la Cartuja, whereas the ED8654 and HB101 strains are stored in the strains bank of our research group. Strain Genotype Uses Reference DH5 a F- supE44 hsdR17(rk-mk+) recA1 girA96 (NaIr) endA1 thi-1 relA1 D(lacaya-argF) (Ø80lacZDM15) U59 Gene cloning and plasmid construction Hanahan, 1983 ED8654 Lac-3 o LacY1 supE44 supF58 hsdR514 (rk-mk-) recA56mcrA1 metB1 lacY galk2 galT22 trpR55 Anabaena conjugation Murray et al., 1977 HB101 F- hdsS20 (rB-mB-) leu supE44 ara14 galK2 lacY1 proA2 rpsL20 xyl-5 mtl-1 recA13 mcrB Anabaena conjugation Boyer & Roulland- Dussoix, 1969 2.1.1.2 Culture media and conditions E. coli strains were grown axenically in rich Luria-Bertani (LB) medium consisting of 10 g/L NaCl, 10 g/L tryptone and 5 g/L yeast extract (Sambrook & Russell, 2001). For preparing a solid medium, it was supplemented with 1.5% (w/v) agar (Panreac) before autoclaving. Both types of medium (solid or liquid) were sterilized in autoclave. The liquid media were incubated in orbital shakers at 200 rpm and 37°C, while the solid media were grown in Petri dishes in a 37°C incubator. If necessary, the media were supplemented with antibiotics that were sterilized by filtration and used at a final concentration of: ampicillin (Ap) 50 µg/ml; chloramphenicol (Cm) 30 µg/ml; streptomycin sulfate (Sm) 25 µg/ml; spectinomycin dihydrochloride pentahydrate (Sp) 100 µg/ml; and kanamycin sulfate (Km) 50 µg/ml. The antibiotic stocks were prepared in ultrapure water (milliQ) with the exception of chloramphenicol, which was dissolved in absolute ethanol. 2.1.1.3 Harvest methods For volumes of less than 3 mL, cells were collected by centrifugation using 1.5-mL eppendorf tubes in an Eppendof microcentrifuge model miniSpind plus at 13,000 rpm for 1 minute. For volumes between 5 and 50 mL, a Haraeus Megafuge 1.0 centrifuge was used at 4000 rpm for 5 minutes. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Materials & Methods ______________________________________________________________________ 34 2.1.2 Cyanobacteria 2.1.2.1 Strain The prokaryotic organism on which this work has been focused is the filamentous heterocystforming cyanobacterium Anabaena sp. (also known as Nostoc sp.) strain PCC 7120 (ATCC 27893), which is in section IV of the taxonomic classification of Rippka et al. (1979). All Anabaena strains used during the development of this Thesis are described in the following tables. Table 2. Anabaena strains previously available. Strain Genotype Resistance Reference PCC 7120 Wild type None Rippka et al., 1979 CSAM137 sepJ-gfpmut2::pCSV3 Sm Sp Flores et al., 2007 CSVM34 DsepJ None Mariscal et al., 2011 CSVM141 DsepJ DfraC DfraD None Nürnberg et al., 2015 CSVT22 DfraC DfraD None Merino-Puerto et al., 2011b Table 3. Cyanobacterial strains constructed in this work. Strain Genotype Description Resistance CSSA1 DhglK::C.K1 Strain obtained by double homologous recombination (DHR) in which hlgK is inactivated by deletion of an internal fragment and insertion of the C.K1 cassette Nm CSSA2 DfraC DfraD DhglK::C.K1 Strain obtained by DHR in which hlgK is inactivated by deletion/ insertion of C.K1 in strain CSVT22 (DfraC DfraD) Nm CSSA3 DsepJ DhglK::C.K1 Strain obtained by DHR in which hlgK is inactivated by deletion/ insertion of C.K1 in strain CSVM34 (DsepJ) Nm CSSA4 hglK::sf-gfp (+pCSV3) Strain obtained by single homologous recombination (SHR) in which hglK lacks the stop codon and bears sf-gfp fused at the 3’ end (pCSV3 vector also integrated) Sm Sp CSSA7 hglK::6xHis (+pCSV3) Strain obtained by SHR in which hglK lacks the stop codon and bears a 3’ sequence encoding a 6xHis tag (+ pCSV3) Sm Sp Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Materials & Methods ______________________________________________________________________ 35 CSSA12 DsepJ DfraC DfraD DhglK::C.K1 Strain obtained by DHR in which hlgK is inactivated by deletion/ insertion of C.K1 strain CSVM141 (DsepJ DfraC DfraD) Nm CSSA14 DsepJ::sepJRintHH01 (+pCSV3) Strain obtained by SHR to complement Anabaena DsepJ with sepJ from Richelia intracellularis HH01 (+ pCSV3) Sm Sp CSSA15 DfraC-DfraD::fraC-fraDRintHH01 Strain obtained by SHR to complement Anabaena DfraC- DfraD with fraC fraD from Richelia intracellularis HH01 Sm Sp CSSA17 sepJ::Strep-tag II (+pCSV3) Strain obtained by SHR in which sepJ lacks the stop codon and bears a 3’ sequence encoding the Streptag II is his C-terminal (+ pCSV3) Sm Sp CSSA18 hglK::Strep-tag II (+pCSV3) Strain obtained by SHR in which hglK lacks the stop codon and bears a 3’ sequence encoding Strep-tag II (+ pCSV3) Sm Sp CSSA28 DsepJ::sepJCalSC01 (+pCSV3) Strain obtained by SHR to complement Anabaena DsepJ with sepJ from Calothrix rhizosoleniae SC01 (pCSV3 vector integrated as well) Sm Sp CSSA29 DsepJ::sepJPCC7120 (+pCSV3) Strain obtained by SHR to complement Anabaena DsepJ strain with sepJ from Anabaena, used as a positive control (pCSV3 vector integrated as well) Sm Sp CSSA30 DfraC-DfraD [pCSSA48] Strain with replicative plasmid pCSSA48 to complement Anabaena DfraC-DfraD with fraC fraD from R. intracellularis HH01 cloned in pRL3845 Em CSSA31 DfraC-DfraD [pCSSA49] Strain with replicative plasmid pCSSA49 to complement Anabaena DfraC DfraD with fraC fraD from Anabaena cloned in pRL3845 (positive control) Em CSSA31 DfraC-DfraD [pCSSA50] Strain with replicative plasmid pCSSA50 to complement Anabaena DfraC DfraD with fraC fraD fraE from C. rhizosoleniae SC01 cloned in pRL3845 Em DHR, double homologous recombination; SHR, single homologous recombination. 2.1.2.2 Culture media and conditions The different strains of Anabaena were grown in axenic culture under photoautotrophic conditions in a modified BG11 medium (Rippka et al., 1979), which consists of: 0.2 mM Na2CO3, Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Materials & Methods ______________________________________________________________________ 42 two types, T4 ligase from Dominion MBL or T4 ligase that was part of the pSpark DNA Cloning System kit. Both ligases were provided with the necessary buffers. Finally, the ligation mixture was incubated for 1 to 24 hours at a temperature of 21ºC. 2.2.7.3 Dephosphorylation To avoid recirculation, the 5' protruding extremes of linearized vectors were dephosphorylated. DNA from the vector was incubated with rAPid alkaline phosphatase (Roche) following the manufacturer’s instructions. 2.2.8 RNA radioactive probes DNA fragments that had been previously amplified by PCR were used as a template for radioactively labeling the DNA. 40-50 ng of two-stranded DNA were mixed with 10 pmol of oligonucleotides in a final volume of 34.5 µL. The mixture was denatured for 3 min by heating to 100°C. A mixture containing 0.03 mM dATP, 0.03 mM dGTP, 0.03 mM dTT, 1 U Klenow enzyme (Fermentas), 5 µl Klenow buffer 10X (Fermentas) and 5 µCi of (a-32P)-dCTP (10 µCi/µL)-dCTP was then added. After incubation at 37°C for 30 min, the reaction was terminated by addition of 2 µL of 1 mM dCTP and incubation for 5 min at 37°C. Finally, the unincorporated (a-32P)-dCTP was removed by gel filtration. After quantifying the incorporated radioactivity by scintillation counting (in a model LS 6000 Beckman counter), the labeled DNA fragment was used as a radioactive probe in northern blot experiments. 2.3 Genetic methods 2.3.1 Transformation of E. coli by heat shock Exogenous DNA in the form of a plasmid was introduced into E. coli DH5a and HB101 strains by means of thermal shock transformation. To perform the transformation in an Eppendof tube, 100 µL of competent cells were mixed with either 10 µL of a ligation product or 2 µL of purified plasmid. These mixtures were incubated for 10 minutes on ice, then subjected to a thermal shock at 42°C for 90 seconds and finally incubated on ice for 5 minutes. After this last incubation, 1 mL of liquid LB medium was added, and the mix was incubated in a shaker for one hour at 37ºC to allow expression of resistance genes. The cells from the transformation were collected by centrifugation and the pellet (resuspended in approximately 20 µL of LB medium) was spread on a Petri dish with LB medium supplemented with the corresponding antibiotic. For pSpark-derived constructs, the plates were supplemented with the antibiotic and 40 µg/mL of X-gal, which facilitates the selection of positive colonies (a complementation assay) (Sambrook & Russell, 2001). 2.3.2 Conjugation to Anabaena The conjugation protocol is described in Elhai & Wolk (1988b). Two strains of E. coli are needed to perform the conjugation. Strain ED8654 that bears plasmid pRL443 which provides conjugation functions, and strain HB101 that carrries the cargo plasmid and the helper plasmid pRL623. The latter bears genes that encode methylases for AvaI, AvaII and AvaIII restriction Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Materials & Methods ______________________________________________________________________ 43 sites (Elhai el al., 1997), which protects the cargo plasmid from digestion by the Anabaena restriction endonucleases, as well as the mob gene. In a first step, pRL433 is transferred from ED8654 to HB101, and in a second step, the cargo plasmid is transferred from HB101 to Anabaena. Once HB101 [pRL623] is transformed with the construct to be sent to Anabaena, the conjugation is performed following the next steps. Two independent pre-inocula each in 3 mL of liquid LB medium supplemented with the antibiotics corresponding to the strains of E. coli, HB101 [pRL623 (30 µgCm/mL) and cargo plasmid (antibiotic depending on particular plasmid)] and ED8654 [pRL443 (50 µg Ap/mL)], are incubated at 37ºC with shaking. When the cultures reach their stationary phase, 250 µL of the ED8654 strain and 350 µL of the HB101 strain are taken, 10 mL of fresh medium with the appropriate antibiotics are added, and the cultures are incubated at 37ºC for 2.5 hours in a shaker. Then, the cells are collected by centrifugation, washed 3 times with fresh LB to remove the antibiotics and resuspended together to approximately 0.5 mL. The cell suspension of the E. coli strains is incubated at room temperature for at least 2 hours, and an amount of Anabaena (wild type or a mutant used as parental strain) containing 10 µg of Chl (5 µg Chl if the resistance cassette is C.K1) is added, everything is gently homogenized, and finally spread on a nitrocellulose filter (Nucleopore) on a Petri dish with BG11 medium + 5% LB (v/v) without antibiotics. The Petri dish is incubated at 30°C under low white light intensity (10 µE/(s·m2)) for at least 3 hours and then incubated at standard light conditions (30 µE/(s·m2)). Twenty-four hours after inoculation, the nitrocellulose filter is transferred to a new BG11 plate where it is incubated for a further 24 hours before transferring the filter to a plate of BG11 medium supplemented with the corresponding antibiotic(s). The filter is transferred to a new plate with fresh medium and antibiotic(s) every 48 hours until antibiotic-resistant colonies appear. When the transferred plasmid has not a replication origin for Anabaena, two types of homologous recombination can take place depending on the type of construct used: • Single cross-over: in this case, the plasmid only bears a fragment that is homologous with a region of the chromosome. Here, the final result is the integration of the plasmid in a specific place of the Anabaena chromosome through homologous recombination. To ensure that the conjugation was performed correctly, isolated colonies are checked by PCR (Fig. 12). Fig. 12. Schematic representation of integration through single cross-over. Target Gene Homologous Region Resistance Cassette Resistance Cassette Plasmid Integrated Plasmid Integrated Plasmid Chromosome Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Materials & Methods ______________________________________________________________________ 44 • Double cross-over: the plasmid has two fragments homologous to sequences in a region of the chromosome. The final result will be the integration of a DNA fragment in a specific region of the chromosome in a process of gene replacement (in this case the whole plasmid is not inserted). The process takes place in two steps and includes a step of positive selection through sucrose sensitivity provided by the sacB gene (encoding the Bacillus subtilis levansucrase; this enzyme converts sucrose into levan, which accumulates in the periplasm and is toxic). After the first cross-over, the whole plasmid will be inserted, which is checked by PCR. In case of a positive result (ideally, presence only of chromosomes with the insertion of the whole plasmid), the strain is transferred to a plate of BG110 medium suplementated with ammonium (NH4+) (as nitrogen source) + 5% sucrose. Exconjugants that can grow in sucrose-supplemented medium should have lost the plasmid vector by a second cross-over event. In this work, when the double recombination was tried, a neomycin resistance gene cassette (C.K1) was introduced, so exconjugants that can grow in sucrose can also grow in the presence of neomycin (Fig. 13). Finally, to verify that the segregation was complete and that the mutation is correct, the selected clones are tested by PCR. In some occasions, clones with the correct mutation but still not segregated (i.e., containing some wild-type chromosomes) are obtained. To facilitate the segregation, some clones that can grow in the presence of sucrose are incubated in liquid BG11 medium with antibiotic (Nm); once grown, they are subjected to sonication in a cleaning bath to fragment the filaments to a size of between 3-5 cells per filament. After sonication, serial dilutions are plated in BG11 medium with antibiotic (Nm). The exconjugants obtained should originate from a short filament, increasing the probability that all the cells in the filament are genetically homogeneous. Fig. 13. Schematic representation of gene replacement through double cross-over. Chromosome Target Gene Resistance sacB Homologous Region Plasmid Chromosome Homologous Region Resistance Insertion in the chromosome Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Materials & Methods ______________________________________________________________________ 45 2.4 Molecular techniques based on RNA manipulation 2.4.1 RNA isolation from cyanobacteria Isolation of total RNA from cyanobacteria was performed following a modified protocol based on the hot phenol method of Mohamed & Jansson (1989). Cells from 50 mL of culture were collected by centrifugation (see 2.1.2.3), washed with 5 mL T50E100 buffer (50 mM Tris-HCl, 100 mM EDTA-Na2, pH 8) and resuspended in 2 mL of the same buffer. This cell suspension was transferred to a 2-mL Eppendorf tube where it was centrifuged. After centrifugation, the supernatant was removed and the pellet was frozen with liquid nitrogen and stored at -20°C until use. The cell pellet was resuspended in 300 µL of resuspension buffer (300 mM sucrose, 10 mM sodium acetate pH 4.5) supplemented with 100 µL of 250 mM EDTA-Na2 and 400 µL of lysis buffer (2% SDS, 10 mM sodium acetate pH 4.5). When the pellet was well homogenized in this mixture, 1 mL of acid phenol (pH 4.5) preheated to 65ºC was added, and the mixture was shaken in a vortex for 30 s and incubated at 65ºC for 2 minutes. The process of shaking and incubation was repeated 3 times. Then the mixture was centrifuged for 5 minutes at 4ºC and 15,000 x g. The aqueous phase was transferred to a clean Eppendorf tube and treated successively with 1 mL of acid phenol preheated to 65ºC, then with a mixture of acid phenol:chloroform (1:1 (v/v)), and finally with 1 mL of chloroform (Merck). Before adding the solvents previously indicated, the mixture was vortexed for 30 seconds, incubated at 65ºC for 2 minutes and centrifuged for 5 minutes (15,000 x g, 4ºC), always collecting the aqueous phase. After treatment with chloroform, the aqueous phase was again transferred to a clean Eppendorf tube, 1 mL of isopropanol was added and the mixture was incubated at -20°C for at least 1 hour to precipitate the nucleic acids. Then, the sample was centrifuged for 30 minutes, the supernatant was discarded, and the pellet was washed with 70% ethanol (v/v) and dried at room temperature. Finally, the nucleic acid precipitate was resuspended in 90 µL of water (previously treated with diethylpyrocarbonate [DEPC]) and treated with 10 units of I-turbo DNase (Ambion) at 37ºC for 30 minutes in the buffer supplied by the manufacturer. After 30 minutes, the DNase was removed using the DNAse inactivating reagent that is supplied with the DNase. 2.4.2 RNA quantification The concentration of RNA was quantified in the same way as the concentration of DNA, using the Nanodrop Specrophotometer ND-1000. The measurement was performed at an absorbance of 260 nm (extinction coefficient ε = 0.020 mL/(mg·cm)). 2.4.3 RNA electrophoresis in agarose gels RNA electrophoresis was performed in 1% (w/v) agarose gels in MOPS 1x buffer (40 mM MOPS, 10 mM sodium acetate, 1 mM EDTA pH 7.2) to which formaldehyde 2% (v/v) was added. The samples were prepared to load a final volume of 20 µL, which was composed of 10 µg total RNA and 15 µL loading buffer consisting of 60% (v/v) deionized formamide, 6% (v/v) formaldehyde, MOPS 1x buffer, 0.25% (w/v) bromophenol blue, 0.25% (w/v) xylenecyanol FF, 0.1% (v/v) ethidium bromide, and 10% (v/v) glycerol. Before loading the samples, they were incubated for Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Materials & Methods ______________________________________________________________________ 46 3 minutes at 80ºC and immediately cooled in ice until they were loaded into the gel. Once all the samples were loaded, a commercial Riboruler High Range RNA Ladder (Thermo Scientific) was also loaded, and finally the gel was run for 1-2 hours at 100 V. 2.4.4 RNA transfer to nylon filters To perform this step, after the electrophoresis the gel is washed three times for 5 minutes each time with abundant deionized water and then balanced with 10x SCC buffer (1.5 M NaCl and 150 mM sodium citrate, pH 7) for 10 minutes. The RNA was then transferred to a nylon filter GeneScreen Plus (PerkinElmer) by capillarity for 16 hours. When the transfer was completed, the filter was briefly washed with 2x SSC buffer and the RNA was fixed to the nylon filter by irradiating it with UV light for 2 minutes and allowing it to dry for 1-2 hours in an oven at 80°C. 2.4.5 Hybridization with radioactive probes Before hybridizing the RNA-carrying nylon membrane with radioactive DNA probes, the filter was incubated with a pre-hybridization solution (0.3 M phosphate buffer [pH 6.9], 7% SDS and 1 mM EDTA [pH 8]) for 1-2 hours in a hybridization oven at 65ºC. After pre-hybridization of the filter, the radioactively marked DNA probe (section 2.2.8) was denatured for 3 minutes at 95ºC and added to the filter, which was incubated for 12-16 hours in the hybridization oven at 65ºC. After the incubation, the filter was washed with a first washing solution (0.3 N NaCl, 0.3 M trisodium citrate dihydrate and 0.1% SDS) for 10 minutes at 65ºC and then with a second solution (0.15 M NaCl, 0.015 M trisodium citrate dihydrate and 0.1% SDS) at 65ºC for 10 minutes again. Although the agarose gel was loaded with the same amount of RNA in each well, the filters were hybridized with a probe of rnpB gene (Vioque, 1997) as a control for the amount of RNA loading and transfer. 2.4.6 Detection of radioactivity Both for detection of hybridization signals in the GeneScreen Plus nylon membranes (PerkinElmer) and for analyzing those signals, electronic auto-radiography with the Cyclone Storage Phosphor System (PerkinElmer) and image analysis with the program Optiquant (Packard) were carried out. 2.5 Protein purification from Anabaena 2.5.1 Induction and cellular lysis The strains from which the purification of proteins was performed were grown in 1.5 L of BG11C medium (supplemented with antibiotics if necessary) with bubbling (see section 2.1.2.2). Once grown, they were harvested by centrifugation at 6,000 x g for 10 minutes, the pellet was resuspended in 1.5 L of liquid BG110C without antibiotics, and an incubation was performed for 48 hours with bubbling (induction under nitrogen deprivation). After induction, the cells were collected again by centrifugation until a single pellet was obtained in a Falcon tube. The pellet was resuspended in 10 mL of lysis buffer (20 mM Tris/HCl pH 8, 150 mM e-amino-n-caproic acid, 5 mM β-mercaptoethanol and one pill of protease inhibitor cocktail [Roche]), which was Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Materials & Methods ______________________________________________________________________ 47 poured dropwise with the aid of a pipette into a mortar containing liquid nitrogen. Once poured into the mortar, the sample was grinded until all the liquid nitrogen was evaporated to give a cyanobacterial powder. The powder was collected with a spoon and poured into a clean Falcon tube to which a volume of 2 mL of lysis buffer was added; when the sample was thawed, it was supplemented with 1 mg/mL lysozyme and 1% (w/v) n-dodecyl-b-D-maltoside (DDM), and this mixture was incubated on a rotary shaker at 4ºC for 1 hour. The sample was then centrifuged at low speed (1,000 x g) for 5 minutes at 4°C to remove any unbroken cells; the supernatant was passed into a clean Falcon tube and placed on ice to begin the purification of the sample. 2.5.2 Complex purification using the Strep-tag II The supernatant obtained in the above-described process (approximately 12 mL) was supplemented with 2 µg of avidin and incubated with 120 µL (v/v/) MagStrep "type3" XT Beads (IBA) (previously equilibrated with Wash Buffer 1 –described below– following the manufacturer’s instructions) for 1.5 h at 4ºC on a rotary shaker. After incubation, the sample was transferred to an Eppendorf tube in a magnetic rack that allowed us to separate the magnetic beads from the rest of the sample. After collecting all the magnetic beads, they were resuspended in 1.2 mL of Wash Buffer 1 (20 mM Tris/HCl [pH 8], 100 mM e-amino-n-caproic acid, 5 mM βmercaptoethanol and 1 mM EDTA), separating again the supernatant from the magnetic beads using the magnetic rack and freezing the supernatant in liquid nitrogen before being stored at -80ºC. This step was repeated using Wash Buffer 2 (20 mM Tris/HCl [pH 8], 150 mM e-amino- n-caproic acid, 5 mM β-mercaptoethanol and 1 mM EDTA) and Wash Buffer 3 (20 mM Tris/HCl [pH 8], 150 mM e-amino-n-caproic acid, 5 mM β-mercaptoethanol, 1 mM EDTA and 0.5% NP- 40). After using the latter buffer, the magnetic beads pellet was resupplied again in 120 µL of Wash Buffer 3, where a small aliquot (20 µl) of the magnetic beads were incubated with biotin elution buffer (20 mM Tris/HCl [pH 8], 150 mM e-amino-n-caproic acid, 5 mM βmercaptoethanol, 1 mM EDTA and 50 mM D-biotin) at room temperature for 10 min with occasional vortexing to bring beads into suspension. To ensure that no proteins remained attached to the magnetic beads, they were incubated for 5 minutes at 95ºC with sample buffer. Finally, the rest of magnetic beads (~100 µL) that were resuspended in Wash Buffer 3 were frozen in liquid nitrogen and stored at -80°C until further analysis in the mass spectrometer. All aliquots (from each of the washes and elution buffer) were checked by Western blot analysis to see if the purification had come out correctly. 2.5.3 Complex purification using the GFP The supernatant obtained in section 2.5.1 was mixed with 75 µL (v/v) of GFP-Trap®_MA bead (Choromotek) previously equilibrated in dilution buffer as indicated in the manufacturer's instructions. This mixture of supernatant and magnetic beads was incubated for 1 hour at 4°C and shaking. After incubation, the magnetic beads were separated from the supernatant by passing the supernatant to a 1.5-mL Eppendorf tube on a magnetic rack. The beads were washed twice with 500 µL of washing buffer (10 mM Tris/Cl [pH 7.5], 150 mM NaCl and 0.5 mM EDTA); after the last washing the beads were resuspended in 75 µL of washing buffer, frozen with liquid nitrogen and stored frozen at -80ºC until processing. Before freezing the magnetic beads (75 µL suspension), 25 µL were taken for elution and analysis by Western Blot to check if the isolation had gone correctly. To this aliquot, 50 µL of glycine-elution buffer (200 mM glycine, pH 2.5) was added and incubated for 30 seconds in constant agitation. After incubation, the supernatant Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Materials & Methods ______________________________________________________________________ 48 was transferred to a clean Eppendorf tube containing 5 µL of 1 M Tris (pH 10.4). This step was repeated again to improve protein elution. Finally, to elute proteins attached to the magnetic beads, these were resuspended in 100 µl of sample buffer and incubated for 10 min at 95°C. Aliquots of each of the washes and the starting supernatant were kept to be checked by Western blot. 2.5.4 Protein biochemical methods 2.5.4.1 SDS-PAGE For protein separation by polyacrylamide gel electrophoresis, the protocol of Laemmli (1970), which is described in Sambrook & Russell (2001), was followed. The polyacrylamide gels are composed of two gels, a running gel that is prepared with acrylamide:bisacrylamide (29:1) at 12% in 375 mM Tris-HCl (pH 8.8) and the stacking gel (where the wells are located) prepared with acrylamide:bisacrylamide (29:1) at 12% in 125 mM Tris-HCl (pH 6.8). Both gels are supplemented with 0.1% (w/v) SDS, which facilitates the denaturation of the proteins allowing them to migrate better through the gel. To make the polymeric gels, it is necessary to add 0.05% APS (ammonium persurfate) and 0.1% TEMED just before pouring the gels between the crystals. Before loading into the stacking gel wells, the samples are supplemented with 2x protein sample buffer consisting of 125 mM Tris/HCl (pH 6.8), 20% (v/v) glycerol, 4% SDS (v/v), 10% (v/v) 2- mercaptoethanol and 0.0025% (w/v) bromophenol blue. Finally, to ensure that all proteins are denatured, samples are heated for 5 minutes at 95°C just before loading into the gel. To carry out the electrophoresis, the gels were immersed in an electrophoresis buffer consisting of 25 mM Trizma base, 192 mM glycine and 0.1% SDS (pH 8.3). In addition to the above mentioned, a protein size marker (gTPbio) was loaded into the gels. 2.5.4.2 Transference of proteins to PVDF membranes In order to perform protein immunodetection, proteins separated by size in a polyacrylamide gel are transferred to a polyvinylidene difluoride (PVDF) Hybond-P 0.45-µm pore size membrane (GE Healthcare) using a GE Healthcare Amersham semi-Dry Transfer system. Prior to the transfer, the PVDF membranes were activated by soaking them in methanol for a few seconds, then washed in ultrapure water for about 10 minutes (changing the water at least twice) and finally soaked in transference buffer (478 mM Trizma base pH 8, 0.03% (w/v) SDS, 39 mM glycine and 5% (v/v) methanol) for 10 minutes. Regardless of the solution where the membrane was immersed, it was always shaken in a rotatory shaker to prevent the membrane surface from drying out. The gels were also immersed for 10 minutes in transference buffer. Once impregnated by the transference buffer, the transfer components were assembled as a "sandwich" consisting of (from bottom to up): three Whatman papers previously wetted in transference buffer, the PVDF membrane, the acrylamide gel and three other Whatman papers also wetted in transference buffer. The electrical current applied for transfer was calculated using the following formula: I = S-t; where I is the current in mA, S the surface of the gel in cm2 and t is the thickness of the gel in mm. The transfer was carried out for 70 min at a continuous current intensity. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Materials & Methods ______________________________________________________________________ 49 2.5.4.3 Staining PVDF membranes The membranes were incubated with DB71 solution (0.008% (v/v) (DB71 in 40% (v/v) ethanol and 10% (v/v) acetic acid) for 5 minutes with shaking. After staining, the membrane was rinsed with double distilled water and the protein size marker was marked with a pen. Then, the membrane was distained with DB71 distaining solution (50% (v/v) ethanol and 150 mM NaHCO3) for 5 minutes at room temperature on a shaker. Finally, the membrane was incubated once with PBS and twice with PBS-T (each time for 5 minutes at room temperature and shaking). (This procedure was dispensable when a prestained protein size marker was used.) 2.5.4.4 Western blotting for Strep-tag II detection The membranes were incubated with blocking buffer containing PBS buffer (5.5 mM Na2HPO4, 1.8 mM KH2HPO4, 137 mM NaCl and 2.7 mM KCl [pH 7.4]), 3% BSA (w/v) and 0.5% v/v Tween 20, for 1 hour at room temperature with gentle shaking or overnight at 4°C (with shaking too). Then they were washed 3 times with 20 mL of PBS-Tween buffer (PBS buffer with 0.1% v/v Tween 20) per membrane for 5 min with shaking. After the washings, the membrane was immersed in 10 mL of PBS-Tween buffer and 10 µL Biotin Blocking Buffer was added, and then it was incubated for 20 min at room temperature and gentle shaking. For the detection of the Strep-tag II, the membrane in 10 mL of PBS-Tween buffer was supplemented with 10 µL of Strep-Tactin horseradish peroxidase conjugate diluted 1:100 in enzyme dilution buffer (PBS with 0.2 % w/v BSA and 0.1% v/v Tween 20) and incubated during 60 min at room temperature and gentle shaking. After the incubation, the membrane was washed twice with PBS-Tween buffer for 5 min and then washed twice with PBS-buffer for 5 min (all washes were done at room temperature and with gentle shaking). Finally, WesternBright ECL (Advansta) reagents were used for Strep-Tactin detection and exposure was made with Hyperfilm (GE Healthcare). 2.5.4.5 Western blotting for GFP detection Western Blot analysis was carried out on membranes with transferred proteins. The membrane was incubated with a blocking buffer consisting of PBS-T (5.5 mM Na2HPO4, 1.8 mM KH2HPO4, 137 mM NaCl and 2.7 mM KCl [pH 7.4] and 1% (v/v) Tween-20) with 5% (w/v) milk powder for 1 hour at room temperature (or overnight at 4ºC) and shaking. After the blocking step, the membrane was washed three times with PBS-T for 5 minutes and then incubated with the primary antibody dissolved in 5% milk PBS-T (GFP monoclonal antibody, a 1:1,000 dilution was used) for 1 hour at room temperature and shaking. Then the membrane was washed three times with PBS-T for 10 minutes with shaking, and after the last washing the secondary antibody (anti-rabbit antibody) was added to a 1:10,000 dilution, and the membrane was incubated overnight at 4ºC and with shaking. The next step was to wash the membrane twice with PBS-T and then 3 times with PBS for 5 min with shaking and room temperature. Finally, WesternBright ECL (Advansta) chemiluminescent substrate was added and radiographic film (GE Halthcare) was used for the detection of the secondary antibody. 2.5.5 Mass spectrometry analysis The identification of peptides from the samples isolated using the magnetic beads against Streptag II and GFP, respectively, was done by mass spectrometry performed by Dr. Roman Ladig Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Materials & Methods ______________________________________________________________________ 50 from Prof. Enrico Schleiff's group at Goethe University of Frankfurt (Germany). The samples were processed as described in Brouwer et al. (2019). The label-free quantification (LFQ) values were calculated with MaxQuant quantitative proteomics software for all experiments. The LFQ values for the experiments for SepJ-GFP (three with baits and three control experiments with same affinity column material), for SepJ-Strep-tag II (three with baits and three control experiments with same affinity column material) and for HglK-Strep-tag II (five with baits and three control experiments with same affinity column material) were extracted from the list. All proteins for which no LFQ value was calculated for any of the experiments with bait were removed. Subsequently, all zero LFQ values were set to the obtained minimal LFQ value in each of the three sets. This simulates a high false negative discovery rate. Subsequently, the p-value for the distribution of the ratio was calculated by t-test. Two different cut offs were used, namely p < 0.05 (95% confidence) and p < 0.1 (90% confidence), the latter considering the low abundance of the bait. 2.6 Peptidoglycan isolation The isolation of the murein sacculi (peptidoglycan, PG) was carried out following the protocol of Lehner et al. (2011) with some modifications. The strains were grown in 50 mL of liquid BG11 medium to a concentration of 3-4 µg Chl/mL (supplemented with antibiotics if necessary) and collected by centrifugation. To isolate peptidoglycan from induced cultures, the BG11-grown cells were washed, resuspended in 50 mL of liquid BG110 medium (without antibiotics) and incubated for 24 or 48 hours (section 2.1.2.2). After harvesting, the pellet was resuspended in 1 mL of PBS (5.5 mM Na2HPO4, 1.8 mM KH2HPO4, 137 mM NaCl and 2.7 mM KCl [pH 7.4]) and the filaments were fragmented by sonication in a cleaning bath to obtain individualized cells or filaments with a length of between 2-3 cells per filament. The cell suspension was poured dropwise into 10 ml of a boiling 6% (w/v) SDS solution, and the mixture was boiled for at least 2 hours with strong stirring. After this, the samples were incubated overnight at 37ºC with gentle stirring. Then, the samples were centrifuged at 320,000 x g at 25ºC for 35 minutes in a Beckman Coulter Optima XPN-100 ultracentrifuge using rotor 90Ti. After centrifugation, the supernatant was removed and the pellet was resuspended in 3 ml of 3% SDS (w/v) and boiled for 2.5 hours. The samples were then sedimented by centrifugation (320,000 x g at 25°C for 35 minutes). Again, the supernatant was removed and the pellet was resuspended in 2 mL of 0.05% (w/v) SDS and boiled for 2.5 hours (always maintaining stirring). After boiling, the samples were sedimented as indicated above, the supernatant was removed and the pellet was resuspended in 1.5 mL of 50 mM sodium phosphate buffer (pH 6.8) supplemented with 50 µg (corresponding to 2U) of achymotrypsin (from bovine pancreas, Sigma). The mixture of sample and enzyme was incubated at 37ºC overnight with gentle stirring. After treatment with the enzyme, 0.5 mL of double distilled water and 750 µL of 6% (w/v) SDS were added to the samples, which were boiled for 2.5 hours and centrifuged at 320,000 x g at 25ºC for 35 minutes. The supernatant was removed and the pellet was washed three times with double distilled water to remove the remaining SDS. In each wash step the pellet was resuspended in a volume of 2 mL and centrifuged at 320,000 x g at 25ºC for 20 minutes. Finally, after the last centrifugation the pellet was resuspended in 100 µL of double distilled water. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Materials & Methods ______________________________________________________________________ 51 2.7 Microscopy 2.7.1 Optical microscopy In order to visualize the Anabaena filaments, bright-field microscopy was performed using an Olimpus BX60 microscope. The following objectives were used: UplanFI 20x/0.5, UplanFI 40x/0.75Ph2 and UplanFI 100x/1.30 oil. In addition, the microscope had attached a Leica DFC 300FX camera that allowed the samples to be photographed using the PhotoED (Microsoft) software. 2.7.2 Immunofluorescence localization of proteins The strains on which immunolocalization was to be performed were grown in liquid BG11 cultures (supplemented with antibiotics if necessary). When grown, the cells were collected and resuspended in liquid BG11 or BG110 medium (without antibiotics), at a concentration of 1 µg Chl/ml, and incubated for 24 or 48 hours. Samples of 1.5 mL of the cultures were centrifuged in an Eppendorf tube. After centrifugation, a small volume of the sample was placed atop a poly-L- lysine pre-coated microscope slide (Sigma) and covered with a 45-µm pore-size Millipore filter. After a few minutes, the filter was removed and the slide was allowed to dry at room temperature, then it was placed in cold 70% ethanol and left at -20°C for 45 minutes. After this time, the slide was removed from the ethanol and allowed to dry at room temperature for 20 minutes. The area of the holder containing the sample was delimited with a marker (liquid blocker) creating a border that allowed us to add various solutions. The sample was washed twice by covering the slide with PBS-T (5.5 mM Na2HPO4, 1.8 mM KH2HPO4, 137 mM NaCl, 2.7 mM KCl [pH 7.4]) and 0.1% (v/v) Tween-20 for 2 minutes at room temperature, and the liquid was removed with a vacuum pump. Afterwards, the slide was treated with blocking buffer (5% milk power in PBS-T) for 30 minutes at room temperature. After removing the blocking buffer, the primary antibody was added. For His- or Strep II-tagged proteins, the primary antibody was a fluorophore-conjugated commercial antibody against His-tag (Penta-His™ Alexa Fluor® 488 conjugate from Qiagen GmBH, Germany) or against Strep-tag II (StrepMAB-Clasic, specific for Strep-tag II, conjugated to Cromeo™ 488 from IBA GmBH, Germany). The primary antibody was diluted 1:200 and 1:250 for His-tag II and Strep-tag II, respectively, in blocking buffer and incubated in the dark for 45 min at room temperature. After incubation, the slide was washed 3 times with 2 ml of PBS-T for 2 min at room temperature. Finally, 2 drops of FluorSave (Calbiochem) were placed on the sample, a cover was placed on top, and the samples were visualized with a Leica DM6000B fluorescence microscope, using the FITC L5 filter (excitation, band-pass (BP) 480/40 filter; emission, BP 527/30 filter) to detect the fluorescence. The microscope had attached an ORCAER camera (Hamamatsu) that allowed the samples to be photographed. Images were analyzed using the ImageJ software (http://imagej.nih.gov/ij). To detect SepJ or FraD, anti-SepJ-CC or anti-FraD antibody was used as the primary antibody at a 1:200 dilution in blocking buffer and incubated for 105 minutes at room temperature. After incubation, the samples were washed 3 times with PBS-T for 2 minutes at room temperature to remove all primary antibody. Secondary anti-rabbit antibody conjugated to fluorescein isothiocyanate (FITC) (Sigma) was then added at a 1:500 dilution in PBS-T buffer and incubated Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 1 | Intercellular communication in Anabaena _______________________________________________________________________ 58 Figure 15A shows the frequency distribution of nanopore numbers, showing decreased numbers in strains CSVM34, CSVT22 and CSVM141. This trend of decrease in nanopore numbers is Fig. 14. Nanopores in septal peptidoglycan disks of Anabaena. (A) Peptidoglycan (murein) sacculus of part of a filament of Anabaena grown in BG110 medium. The PG was isolated as described in section 2.6 of Materials & Methols, stained with uranyl acetate and visualized by TEM. Note the thickness of septal PG, the presence of one heterocyst, and a septal PG disk with nanopores. (B) Nanopores in disks from Anabaena mutants CSVM34, CSVT22 and CSVM141. (C) Genotypes of the strains analyzed and mean and standard deviation of nanopore number and diameter. n, number of septal disks (nanopore counting) or nanopores (diameter) analyzed. Size bars, 500 nm. Septal peptidoglycan (PG) disk Heterocyst Vegetative cells Septal PG Murein (PG) sacculus Nanopores PCC 7120 A CSVM34 CSVT22 CSVM141 B C Strain Genotype Nanopores/disk mean ±SD (n) Nanopore diameter mean ±SD nm (n) This work Nürnberg et al . (2015) This work Nürnberg et al . (2015) Anabaena sp. PCC 7120 (WT) 42.14 ±21.52 (22) 59.25 ±34.90 17.95 ±11.01 (132) 14.98 ±2.94 CSVM34 DsepJ 17.59 ±11.54 (22) 14.00 ±7.52 18.83 ±9.75 (156) 16.84 ±5.01 CSVT22 DfraC DfraD 7.27 ±4.92 (11) 10.91 ±4.06 19.29 ±13.26 (48) 20.12 ±5.74 CSVM141 DsepJ DfraC DfraD 4.40 ±2.81 (10) 6.60 ±2.97 19.81 ±6.40 (40) 24.13 ±8.76 Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 1 | Intercellular communication in Anabaena _______________________________________________________________________ 59 similar to that described by Nürnberg et al. (2015) except for CSVM34 that had about 42% of the nanopore number in the wild type, in contrast to 24% described previously (Fig. 14C). Figure 15B shows the frequency distribution of nanopore diameter for the wild type and the three mutants. In contrast to Nürnberg et al. (2015), in the current analysis we did not find any significant difference in nanopore diameters between any of mutants and the wild type (Fig. 14C). Thus, analysis the results with the Student’s t test (each mutant vs. WT) showed P > 0.15 values in all three cases. Fig. 15. Frequency distribution of nanopore numbers and diameters in septal PG disks. Samples studied were from wild-type Anabaena and DsepJ (strain CSVM34), DfraC DfraD (CSVT22) and DsepJ DfraC DfraD (CSVM141) mutants. (A) The number of nanopores is organized in groups of 5 (from 0 to 5; from 5.01 to 10; etc.). (B) Nanopore diameter is organized in groups of 2 nm (from 0 to 2; from 2.01 to 4; etc.) Given the central role of SepJ in the construction of intercellular septa in Anabaena, we investigated Anabaena mutants that produce a SepJ protein that lacks most of its permease domain (strain CSVM90) or have a short internal deletion (CSFR12) or an amino acid substitution (CSFR14). Strains CSFR12 and CSFR14 were constructed by the reconstruction of the sepJ gene 0 10 20 30 40 50 60 0 - 5 5 - 10 10 - 15 15 - 20 20 - 25 25 - 30 30 - 35 35 - 40 40 - 45 45 - 50 50 - 55 55 - 60 60 - 65 70 - 75 75 - 80 > 80 WT CSVT22 CSVM34 CSVM141 Nanopores (number/disk) Percentage A 0 5 10 15 20 25 0 - 2 2 - 4 4 - 6 6 - 8 8 - 10 10 - 12 12 - 14 12 - 16 16 - 18 18 - 20 20 - 22 22 - 24 24 - 26 26 - 28 28 - 30 30 - 32 32 - 34 34 - 36 36 - 38 38 - 40 > 40 WT CSVT22 CSVM34 CSVM141 Nanopore diameter (nm) Percentage B Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 1 | Intercellular communication in Anabaena _______________________________________________________________________ 60 in strain the CSVM90, lacking most of the permease domain, and we have compared these mutants to control strain CSFR11, which produces a reconstituted wild-type SepJ protein. Nanopore parameters are summarized in Table 7. Nanopore number in strain CSVM90 was similar to that of the sepJ deletion mutant CSVM34 (see Fig. 14C), which is consistent with the fact that the SepJ protein produced in strain CSVM90 has null activity (Ramos-León et al., 2018); however, nanopore diameter was somewhat smaller in CSVM90 as compared to CSVM34 (Fig. 14C and Table 7). Strains CSFR12 and CSFR14 showed a 40% decrease and a normal number of nanopores, respectively, which is consistent with the observation that they have somewhat altered and normal SepJ-related phenotypes, respectively (Ramos-León et al., 2018). Table 7. Nanopores in septal peptidoglycan disks of selected sepJ mutants. The table shows the genotypes of the strains analyzed and the mean and standard deviation of nanopore number and diameter. n, number of septal disks (nanopore counting) or nanopores (diameter) analyzed. These data were included in Ramos-León et al. (2018). SepI is a recently identified septal protein that interacts with divisome proteins and may also interact with SepJ (Springstein et al., 2020). We observed that the ΔsepI mutant shows two different types of perforations in the septal PG (Fig. 16A), one type with a tiny diameter (in “sealed disks”) and another with a very large diameter (defining “unsealed disks”). Although we cannot exclude that unsealed disks correspond to not yet fully divided cells and would eventually become normal septal disks, it should be noted that we rarely observed such large perforations in the wild type. The septal disks with those large perforations were not included in the measurement of nanopore numbers and diameter. The number of nanopores per septal PG disk was strongly reduced in the ΔsepI mutant and we never observed more than 4 nanopores in any of the isolated sacculi, regardless of the availability of combined nitrogen. This observation should be compared to about 39 and 37 nanopores per disk in the wild type grown under the same conditions, BG11 or BG110 medium respectively (Fig. 16B). Nanopore diameter was unaffected in the ΔsepI mutant in BG11 medium but significantly reduced by about 35 % in the ΔsepI mutant under nitrogen deprivation (BG110 medium; Fig. 16B). Strain Genotype Nanopores/disk mean ± SD nm (n) Nanopore diameter mean ± SD nm (n) CSVM90 sepJ SepJD463-748 16.3 ± 9.5 (15) 13.4 ± 2.9 (132) CSFR11* Wild-type sepJ 44.8 ± 6.3 (9) 15.4 ± 3.5 (57) CSFR12 sepJ (SepJD498-507)26.4 ± 11.5 (9) 17.6 ± 3.1 (128) CSFR14 sepJ (SepJE663A)41.6 ± 11.5 (11) 17.4 ± 3.3 (175) Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 1 | Intercellular communication in Anabaena _______________________________________________________________________ 61 3.1.2 Intercellular calcein transfer Intercellular molecular transfer in cyanobacteria has been studied using FRAP (Fluorescence Recovery After Photobleaching) analysis, which basically consists in loading the cyanobacterial filaments with a fluorescent marker, bleaching a specific cell of the filament with the confocal microscope laser, and measuring the fluorescence as it recovers over time (Mullineaux et al., 2008). This method (explained in detail in Materials & Metthods, section 2.7.3) allows us to calculate the exchange coefficient (E) and the recovery rate constant (R) (Mullineaux et al., 2008; Merino-Puerto et al., 2010). The results of these parameters have generally been presented as the mean and standard deviation of the data, assuming that the data fit a normal distribution. However, Fig. 16. Nanopores of the Anabaena sepI mutant. (A) TEM micrographs of peptidoglycan isolated from Anabaena wild-type strain and ΔsepI mutant from BG11 medium (top) and BG110 medium (bottom). (B) Genotypes of the strains analyzed and mean and standard deviation of nanopore number and diameter. n, number of septal disks (nanopore counting) or nanopores (diameter) analyzed. Size bars, 0.5 µm. A B A B Strain Genotype Nanopores/disk mean ±SD nm (n) Nanopore diameter mean ±SD nm (n) BG11 BG11 0 BG11 BG11 0 Anabaena sp. PCC 7120 (WT) 39.45 ±13.41 (11) 37.00 ±16.93 (9) 19.07 ±12.32.01 (100) 21.25 ±4.77 (108) D sepI Dalr3364 2.00 ±0.91 (13) 2.33 ±2.31 (3) 23.37 ±11.08 (26) 13.82 ±5.14 (7) Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 1 | Intercellular communication in Anabaena _______________________________________________________________________ 62 it has recently been observed that some Anabaena mutants present a substantial number of cells that do not recover after bleaching, presenting an R value lower than 0.01 s-1; these cells have been defined as non-communicating cells (Flores et al., 2018). The presence of noncommunicating cells may not be restricted to some Anabaena mutants, but it may happen in the wild type as well. Therefore, we decided to reevaluate the transfer of calcein in different Anabaena strains. Those selected for this study were the wild type and mutants CSVM34 (DsepJ), CSVT22 (DfraC DfraD) and CSVM141 (DsepJ DfraC DfraD). All these strains were grown in liquid BG11 medium, washed and re-inoculated at a concentration of 1 µg Chl/mL in flasks containing BG11 or BG110 medium respectively, and then incubated for 48 hours under culture conditions. By re-inoculation of the stains, we induce heterocyst differentiation in BG110 medium (without combined nitrogen) and ensure the same growth rate in all strains growing in BG11 medium. After incubation, a sample (1 mL) from each strain and condition was taken and incubated with calcein as described in Materials & Methods (section 2.7.3). Then, drops of each treated sample were placed onto plates of BG11 or BG110 medium depending on the treatment that the sample had received, and small pieces of agar carrying the filaments were prepared to be visualized under the confocal microscope. The samples were excited at a wavelength of 488 nm to visualize the filaments that had been loaded with calcein, and in homogeneously marked filaments a cell was bleached by increasing the intensity of the laser. After the bleaching, 10 photographs were taken with an interval between photographs of one to two seconds, and the recovery rate constant (R) was calculated (Nieves-Morión et al., 2017a). The BG11-grown cultures of the wild type had a substantial number of non-communicating cells, accounting for about 58% of the total number of cells subjected to analysis (Fig. 17). On the other hand, a few cells showed extremely high R values, for example cells with R values > 0.3 s-1. Because these high R values are similar to the recovery times expected for free diffusion (Rudolf et al., 2015), in these cells the cytoplasm might be not fully divided from that of neighboring cells after cell division, and they were not included in further analysis. In contrast to BG11-grown cells, the cultures incubated in BG110 medium had less non-communicating cells (about 16%). The estimate of non-communicating cells was also performed individually in each experimental replicate, observing the presence of non-communicating cells in most cultures, especially in BG11 medium (Table 8). When the R values of cells grown in BG11 medium or incubated in BG110 medium were calculated excluding non-communication cells (R < 0.01 s-1) and cells that might have not yet divided (those showing R > 0.3 s-1), very similar recovery constants are obtained (Fig. 17, lower panel). The increase in the number of communicating cells in cultures incubated in BG110 medium can represent a significant aspect of the acclimation of Anabaena to diazotrophic growth. It should be noted, nonetheless, that the fraction of non-communicating cells observed in filaments grown in BG11 or incubated in BG110 medium may depend on other growth parameters (temperature, light intensity, CO2 supply), which could explain why they have not been noticed previously. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 1 | Intercellular communication in Anabaena _______________________________________________________________________ 63 Fig. 17. Intercellular transfer of calcein between vegetative cells of Anabaena. Filaments grown in BG11 medium (blue) or incubated for 48 h in BG110 medium (orange). (Upper panel) Filaments/cells analyzed by FRAP are organized by increasing value of recovery rate constant. 90 filaments from BG11 medium and 103 filaments from BG110 medium were analyzed. Five filaments from BG11 medium giving R values of 0.247, 0.266, 0.312, 0.436 and 0.505 s-1, respectively, and three filaments from BG110 medium giving R values of 0.227, 0.327 and 0.861 s-1, respectively, are not included to facilitate the visibility of the graph. R values > 0.3 s-1 were not considered in further analysis because they may correspond to not fully divided cells (see main text). (Lower panel) Data from the upper panel organized to show frequency distributions (R data organized in groups of 0.01 s-1). The mean and SD are shown for R values between 0.010 and 0.300 s-1. The data used in this analysis combine data obtained for the wildtype controls in the studies of the DsepI mutant (Fig. 21 below) and of the hglK gene (section 3.2 of this Thesis). 0 10 20 30 40 50 60 70 0 - 0,01 0,011 - 0,02 0,021 - 0,03 0,031 - 0,04 0,041 - 0,05 0,051 - 0,06 0,061 - 0,07 0,071 - 0,08 0,081 - 0,09 0,091 - 0,1 0,101 - 0,11 0,111 - 0,12 0,121 - 0,13 0,131 - 0,14 0,141 - 0,15 0,151 - 0,16 0,161 - 0,17 0,171 - 0,18 0,181 - 0,19 0,191 - 0,2 > 0,2 Recovery rate constant R (s-1) Percentage BG11, R = 0.093 ±0.057 s-1 BG110, R = 0.091 ±0.040 s-1 0.011 -0.02 0.021 -0.03 0.031 -0.04 0.041 -0.05 0.051 -0.06 0.061 -0.07 0.071 -0.08 0.081 -0.09 0.111 -0.12 0.101 -0.11 0.121 -0.13 0.131 -0.14 0.141 -0.15 0.151 -0.16 0.161 -0.17 0.171 -0.18 0.181 -0.19 0.191 -0.2 > 0.2 0.091 -0.1 0 -0.01 Filament # Recovery rate constant R (s-1) 0 0,02 0,04 0,06 0,08 0,1 0,12 0,14 0,16 0,18 0,2 020 40 60 80 100 120 BG11 BG110 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 1 | Intercellular communication in Anabaena _______________________________________________________________________ 64 We then addressed calcein FRAP in the deletion mutants of septal protein-encoding genes sepJ, fraC and fraD. As shown in Fig. 18, non-communicating cells were observed in all the mutants not only in BG11-grown filaments but also in filaments that had been incubated under nitrogen deprivation (BG110 medium). As the results do not follow a parametric distribution, to compare these data with those for the wild type, we represented the data as box plots and used the Mann- Whitney U test to assess whether the differences observed are significant or not (Fig. 19). This analysis shows that in BG11 medium there were hardly differences between any of the mutants and the wild type, whereas in BG110 medium the three mutants were significantly different from the wild type. Thus, perhaps because of the high number of non-communicating cells found in the wild type, results differ from those previously reported for the mutants grown in BG11 medium (Mullineaux et al., 2008; Merino-Puerto et al., 2010). Nonetheless, our current results confirm that the sepJ and fraC-fraD mutants are impaired in calcein transfer as assessed in filaments that had been incubated in the absence of combined nitrogen. Percentage of non-communicating cells (R< 0.01 s -1 ) Medium Replicate 12345678 BG11 79 91 45 70 54 30 75 - BG11 0 31 29 9 27 0 0 0 17 Table 8. Analysis of non-communicating cells in each replicate. The table shows the percentage of non-communicating cells (recovery rate constant R < 0.01 s-1) in filaments of Anabaena that had been grown in a medium with combined nitrogen (BG11) or in a medium in absence of nitrogen (BG110). Each value corresponds to the percentage of non-communicating cells in a data set from an individual culture after performing the FRAP assay using calcein as a fluorescent marker. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 1 | Intercellular communication in Anabaena _______________________________________________________________________ 65 Recovery rate constant R (s -1 ) Percentage 0 10 20 30 40 50 60 CSVM34 BG11 CSVM34 BG11o 0 10 20 30 40 50 60 CSVT22 BG11 CSVT22 BG11o 0 5 10 15 20 25 30 35 40 45 CSVM141 BG11 CSVM141 BG11o .01 .02 .03 .04 .05 .06 .07 .08 .09 .10 .11 .12 .13 .14 .15 .16 .17 .18 .19 .2 >.2 .01 .02 .03 .04 .05 .06 .07 .08 .09 .10 .11 .12 .13 .14 .15 .16 .17 .18 .19 .2 >.2 .01 .02 .03 .04 .05 .06 .07 .08 .09 .10 .11 .12 .13 .14 .15 .16 .17 .18 .19 .2 >.2 Fig. 18. Frequency distribution of calcein transfer values in different Anabaena septal protein mutants. Intercellular transfer of calcein between vegetative cells of Anabaena mutant strains CSVT22 (DfraC DfraD), CSVM34 (DsepJ) and CSVM141 (DfraC DfraD DsepJ) grown in BG11 medium (left bars, blue) or incubated for 48 h in BG110 medium (right bars, orange). Filaments/cells analyzed by FRAP are organized in groups of R = 0.01 s-1. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 1 | Intercellular communication in Anabaena _______________________________________________________________________ 66 To address the specific sepJ mutants for which nanopores are presented above, we have reevaluated data previously obtained in this laboratory (F. Ramos-León, Ph. Thesis, 2017; summarized in Ramos-León et al., 2018). BG11-grown filaments of strain CSVM90 had a substantial number of non-communicating cells, as well as CSFR12 and CSFR14 (Fig. 20A). In contrast, strain CSFR11, which expresses a reconstituted wild-type SepJ protein, showed a lower number of non-communication cells. Figure 20B shows box plots of these data and the results of Mann-Whitney U tests, which indicate that the recovery rate constant (R) was significantly lower in strains CSVM90 and CSVM12 than in strain CSFR11. On the other hand, there was no significant difference between the CSFR14 mutant and the strain that has the reconstituted sepJ gene (CSFR11). Fig. 19. Box-plot representation and statistical analysis of intercellular transfer of calcein between vegetative cells. The graph shows the results of tests performed in Anabaena and mutant strains CSVT22 (DfraC DfraD), CSVM34 (DsepJ) and CSVM141 (DfraC DfraD D sepJ) grown in BG11 medium or incubated for 48 h in BG110 medium. Grey, quartile group 2 (from Q1 to median); yellow, quartile group 3 (from median to Q3). The results of the non-parametric Mann-Whitney U test are included on the top of the figure (P values). PCC 7120 CSVM34 CSVT22 CSVM141 PCC 7120 CSVM34 CSVT22 CSVM141 BG11 BG11o Recovery rate constant R (s -1 ) P = 0.733 P = 0.054 P = 0.270 P = 0.000 P = 0.000 P = 0.000 Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 1 | Intercellular communication in Anabaena _______________________________________________________________________ 67 As shown above, the sepI mutant presents two types of septal disks that we have identified as sealed and unsealed. This observation made it of especial interest to study intercellular molecular transfer in this mutant. Figure 21 shows the data for calcein transfer obtained with the sepI mutant grown in BG11 or BG110 medium. Cultures from both media contained a substantial number of non-communicating cells. However, whereas the percentage on non-communicating cells was not very different from that of the wild type in filaments from BG11 media (65% for DsepI, 64% for the WT), it was notably increased as compared to the wild type in filaments from BG110 media 0,00 0,02 0,04 0,06 0,08 0,10 0,12 0,14 0,16 CSVM90 CSFR11 CSFR12 CSFR14 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 Recovery rate constant R (s -1 ) P = 0.001 P = 0.034 P = 0.211 Recovery rate constant R (s -1 ) Percentage 0 10 20 30 40 50 60 70 80 0 - 0,01 0,011 - 0,02 0,021 - 0,03 0,031 - 0,04 0,041 - 0,05 0,051 - 0,06 0,061 - 0,07 0,071 - 0,08 0,081 - 0,09 0,091 - 0,1 0,101 - 0,11 0,111 - 0,12 0,121 - 0,13 0,131 - 0,14 0,141 - 0,15 0,151 - 0,16 0,161 - 0,17 0,171 - 0,18 0,181 - 0,19 0,191 - 0,2 > 0,2 CSVM90 CSFR11 CSFR12 CSFR14 0.011 -0.02 0.021 -0.03 0.031 -0.04 0.041 -0.05 0.051 -0.06 0.061 -0.07 0.071 -0.08 0.081 -0.09 0.111 -0.12 0.101 -0.11 0.121 -0.13 0.131 -0.14 0.141 -0.15 0.151 -0.16 0.161 -0.17 0.171 -0.18 0.181 -0.19 0.191 -0.2 >0.2 0.091 -0.1 0 -0.01 Recovery rate constant R (s -1 ) Percentage A B Fig. 20. Analysis of intercellular calcein transfer between vegetative cells in different sepJ mutants. (A) Frequency distributions of the recovery rate constant (R, data organized in groups of 0.01 s-1) in the different sepJ mutants grown in BG11 medium. The number of recorded bleached cells were: 18 for CSFR11 (wild-type SepJ) and CSVM90 (SepJD463-748); 23 for CSFR12 (SepJD498-507); and 22 for CSFR14 (SepJE663A). (B) Box-plot representation of the data in panel A and its statistical analysis by means of the Mann-Whitney U test in which the mutant that has the sepJ gene reconstructed (CSFR11) was compared with the rest of the mutants. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 2: The HglK protein _______________________________________________________________________ 75 3.2 The HglK protein The pentapeptide-repeat HglK protein was initially described by Black et al. (1995) as a protein that could be involved in the transport of glycolipids in the heterocysts of Anabaena. This hypothesis was proposed based on a TEM study of a ΔhglK mutant, which showed that the heterocysts did not possess the glycolipid envelope characteristic of this cell type, but lipid vesicles could be seen inside the heterocysts (Black et al., 1995). Along with this observation, the miscroscopic images of the mutant also showed other structural anomalies such as the presence of intercellular septa between vegetative cells and between vegetative cells and heterocysts that were wider than those observed in the wild type (Black et al., 1995). The hglK gene is found in all cyanobacteria belonging to clade B1 (Shih et al., 2013), which includes heterocystous cyanobacteria and other phylogenetically related strains that do not produce heterocysts (Table 9). The phenotype of wide septa presented by the hglK mutant and the general presence of this gene in heterocystous cyanobacteria led us to ask whether HglK could have a role in the septal junctions. Table 9. HglK, PatL, HetL and the Fra proteins in cyanobacteria. The cyanobacteria included in the phylogenetic analysis are those of clade B1 from Shih et al. (2013). This clade includes heterocystous cyanobacteria and phylogenetically related strains that do not produce heterocysts (†). The indicated proteins from Anabaena sp. strain PCC 7120 were used as query in BLAST analysis. The presence of a Strain HglK PatL HetL FraCDE-F Fischerella sp. PCC 9605 ✓ ✓ ✓✓ Fischerella sp. PCC 9431 ✓ ✓ ✓ ✓✓ Fischerella sp. PCC 9339 ✓ ✓ ✓ ✓✓ Fischerella sp. JSC-11 ✓ ✓ ✓ ✓✓ Chlorogloeopsis sp. PCC 7702 ✓ ✓✓ Mastigocladopsis repens PCC 10914 ✓ ✓ ✓✓ Calothrix sp. PCC 7103 ✓ ✓ ✓ ✓✓ Calothrix sp. PCC 6303 ✓ ✓ ✓ Nostoc sp. PCC 7524 ✓ ✓ ✓✓ Anabaena sp. PCC 7120 ✓ ✓ ✓ ✓✓ Anabaena variabilis ATCC 29413 ✓ ✓ ✓ ✓✓ Nostoc sp. PCC 7107 ✓ ✓ ✓✓ * Cylindrospermum stagnale PCC 7417 ✓ ✓ ✓✓ Anabaena cylindrica PCC 7122 ✓ ✓ ✓ ✓ Anabaena sp. PCC 7108 ✓ ✓ ✓ ✓ Raphidiopsis brookii D9 † ✓ ✓ Cylindrospermopsis raciborskii CS-505 ✓ ✓ ‘Nostoc azollae’ 0708 ✓ ✓ ✓ Nodularia spumignea CCY9414 ✓ ✓ ✓ Calothrix sp. PCC 7507 ✓ ✓ ✓✓ Microchaete sp. PCC 7126 ✓ ✓ ✓✓ Nostoc puntiforme PCC 73102 ✓ ✓ ✓✓ * Scytomena hofmanni UTEX 2349 ✓ ✓ ✓ Rivularia sp. PCC 7116 ✓ ✓ ✓ Synechocystis sp. PCC 7509 † ✓ Gloeocapsa sp. PCC 7428 † ✓ Chroococcidiopsis thermalis PCC 7203 † ✓ Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 2: The HglK protein _______________________________________________________________________ 76 protein in a strain is indicated by a check mark (✓). For the Fra proteins, the presence of a fraC-fraD-fraE operon is indicated by one check mark, and the additional presence of the adjacent (reverse) fraF gene is indicated by a second check mark. *In these strains, the fraF gene is about 1.6 (PCC 7107) and 5.7 (PCC 73102) kb downstream of the fraCDE operon. 3.2.1 Subcellular localization of HglK To address the subcellular localization of HglK, two approaches were tried, one by fusing a fluorescent protein (superfolder GFP) to HglK and the other by fusing HglK with different tags for immunolocalization analysis. With the use of different bioinformatic programs it was predicted that HglK has four transmembrane segments with both the C- and N-termini in the periplasm (Fig. 23). For convenience in the genetic constructs and to avoid as much as possible effects on the targeting of the protein to the membrane, we decided to add the sfGFP and the different tags to the carboxyl end of HglK. Because of its predicted periplasmic localization, we used the superfolder GFP (sfGFP) that can fold efficiently in the periplasm (Dinh & Bernhardt, 2011). Fig. 23. Topology of the HglK (All0813) protein. (A) Transmembrane segments predicted with the TMHMM program (http://www.cbs.dtu.dk/services/TMHMM-2.0/). (B) Protter representation based on prediction with Phobius (http://phobius.sbc.su.se). CM, cytoplasmic membrane. 24/4/17 9:29TMHMM result Página 1 de 1http://www.cbs.dtu.dk/cgi-bin/webface2.fcgi?jobid=58FDA9410000576EADF01A8D&wait=20 decodeanhmm 1.1g Copyright (C) 1998 by Anders Krogh Mon Apr 24 09:29:07 2017 Model in file "/usr/cbs/bio/src/tmhmm-2.0d/lib/TMHMM2.0.model" parsed successfully. TMHMM result HELP with output formats # all0813 Length: 727 # all0813 Number of predicted TMHs: 4 # all0813 Exp number of AAs in TMHs: 87.7959000000000001 # all0813 Exp number, first 60 AAs: 21.5702 # all0813 Total prob of N-in: 0.06216 # all0813 POSSIBLE N-term signal sequence all0813 TMHMM2.0 outside 1 29 all0813 TMHMM2.0 TMhelix 30 52 all0813 TMHMM2.0 inside 53 95 all0813 TMHMM2.0 TMhelix 96 118 all0813 TMHMM2.0 outside 119 170 all0813 TMHMM2.0 TMhelix 171 193 all0813 TMHMM2.0 inside 194 280 all0813 TMHMM2.0 TMhelix 281 303 all0813 TMHMM2.0 outside 304 727 ppmtogif: computing colormap... ppmtogif: 5 colors found # plot in postscript, script for making the plot in gnuplot, data for plot TMHMM posterior probabilities for All0813 A Periplasm Cytoplasm CM B Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 2: The HglK protein _______________________________________________________________________ 77 Strain CSSA4 was first constructed that expressed HglK fused to the sfGFP. To create this strain, a plasmid that carried a 671-bp 3' fragment of hglK fused through a sequence encoding a fourglycine linker to sf-gfp was prepared using vector pCSV3 (which contains the C.S3 cassette that provides resistance to Sm and Sp; Valladares et al., 2011). This plasmid was transferred to Anabaena wild type by conjugation (Elhai et al., 1997) with selection for resistance to Sm and Sp. The insertion into hglK and segregation of chromosomes carrying the fusion was tested by PCR using primers all0813-10 and gfp-8 to check the insertion of sf-gfp, and all0813-16 and all0813-10 to check segregation of the mutated chromosomes (Fig. 24). asl0814 all0813-15 asl0815 hglK 5’3’ alr0812 1 kb all0813-16 all0813-10 all0813-14 C.S3 pCSSA13 GGGG hglK 5’3’ 671 bp KpnI KpnI 3’ 5’ GGGG C.S3 asl0814 asl0815 pCSV3 alr0812 sf-gfp sf-gfp alr0812 asl0814 asl0815 hglK all0813-16 1.7 kb all0813-10gfp-8 671 BsaI KpnI-HindIIIG GGGG sf-GFP Km KpnIKpnI 671 GGGG sf-GFP Km HindIIIBsaIKpnI C.S3 KpnIKpnI pCSSA13 pCSSA12 pCSV3 pCSAL39 + + A A Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 2: The HglK protein _______________________________________________________________________ 78 Fig. 24. Construction of an Anabaena strain that produces a fusion of the superfolder GFP to the C- terminus of HglK. (A) Scheme of the construction of plasmid pCSSA13 and of the recombination between this plasmid and the Anabaena chromosome. (B) PCR analysis with the indicated oligodeoxynucleotide primers. The sf-gfp fusion was detected in the two exconjugants analyzed but the not in the WT, and wildtype chromosomes were not detected in the exconjugants. The hglK gene shows low levels of expression (Black et al., 1995) even after incubation without combined nitrogen, which induces its expression (Table 10). The HglK-sfGFP strain and the wild type (negative control) were grown for 4 days in liquid BG11C medium (supplemented with Sm+Sp for the HglK-sfGFP strain, CSSA4) under bubbling conditions. The cells were then collected, washed and re-inoculated at a concentration of 3 µg Chl/mL in liquid BG110C medium (without combined nitrogen or antibiotics) for induction during 24 hours with bubbling. After the incubation period, HglK-sfGFP could be detected by confocal microscopy in the periphery of the cells (outside of the thylakoid's red fluorescence), mainly in the intercellular septa, indicating that HglK is a cytoplasmic membrane protein with a polar location (Fig. 25). In most cases, the GFP was observed only as one fluorescence spot, but in some cells two spots could be observed. Because the test was performed under nitrogen deprivation, the filaments developed heterocysts, in which HglK-sfGFP could be observed mainly at the poles. Table 10. Expression of the hglK gene in Anabaena. *Appreciable expression was observed in the presence of ammonium. Reference Fold increase after N step-down Time after N step-down 3 h 8 h 12 h 21 h 24 h Ehira & Omori (2006a) 2.3 x 3.4 x Flaherty et al., 2011* 4.7 x 3.3 x 1.7 kb 1.0 kb 1. PCC 7120 (WT) 2. CSSA4 exconjugant 1 (hglK-sf-gfp) 3. CSSA4 exconjugant 2 (hglK-sf-gfp) M, size markers 1 2 3 M 1 2 3 all0813-10 + gfp-8 all0813-10 + all0813-16 B 1.7 kb 1.0 kb 1. PCC 7120 (WT) 2. CSSA4 exconjugant 1 (hglK-sf-gfp) 3. CSSA4 exconjugant 2 (hglK-sf-gfp) M, size markers 1 2 3 M 1 2 3 all0813-10 + gfp-8 all0813-10 + all0813-16 B Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 2: The HglK protein _______________________________________________________________________ 79 Fig. 25. Subcellular localization of HglK-sfGFP. Confocal microscopy of Anabaena wild type (PCC 7120) and strain CSSA4 that produces a protein with a fusion of superfolder GFP to the C-terminus of HglK (hglK-sf-gfp). Filaments are from cultures incubated for 24 h in bubbled BG110C medium. GFP fluorescence (green) and cyanobacterial autofluorescence (red) are shown for each strain. Some intercellular septa in which the GFP fluorescence in clearly observed are indicated by arrows; doubleopposite arrows indicate some intercellular septa in which two spots are observed. het, heterocyst. Size bars, 5 µm. To study whether the added GFP could alter the functionality of HglK, growth tests were performed. The strains were grown in liquid BG11 medium (adding antibiotics for the mutant), washed and resuspended in liquid BG110 medium at a concentration of 1 µg Chl/mL, from which serial dilutions were made, and drops were placed on plates of BG11 and BG110 media (no plate contained antibiotics since the wild type was also inoculated as a negative control). After 14 days, it was observed that the strain that produced HglK-sfGFP did not grow (or grew very weakly) under diazotrophic growth conditions (Fig. 26). These results indicate that Hglk-sfGFP is hardly functional, making it necessary to take another approach to corroborate HglK localization. Fig. 26. Growth test of Anabaena wild type (PCC 7120) and strains CSSA4 (hglK-sf-gfp), CSSA7 (hglK-6xHis) and CSSA18 (hglK-Strep-tag II). Filaments grown in BG11 medium (with antibiotics for the mutants) were inoculated with the indicated amounts of Chl in BG11 or BG110 medium (without antibiotics) and incubated under culture conditions for 14 days. PCC 7120 CSSA4 (hglK-sf-gfp) het het het het Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 2: The HglK protein _______________________________________________________________________ 80 As an alternative approach to address the subcellular localization of HglK, two strains were prepared, one producing HglK with a 6xHis tag and another with the Strep-tag II. Both tags were fused to the C-terminus and allowed us to perform immunolocalization analysis. To construct strain CSSA7 that carries the 6xHis tag, we cloned the 3’ 519 bp of hglK (omitting the stop codon) adding a sequence for 6 histidines. On the other hand, to construct strain CSSA18 that carries the Strep-tag II, we cloned the 3’ 698 bp of hglK (omitting the stop codon) adding the sequence encoding Strep-tag II. Both fragments were introduced into vector pCSV3 generating plasmids pCSSA11 and pCSSA30, respectively. These plasmids were corroborated by sequencing and transferred to Anabaena by conjugation with selection for Sm+Sp (Fig. 27). In order to test whether these tags affect HglK functionality, growth tests were performed (as described above). It was observed that the strain carrying the 6xHis tag did not grow diazotrophically but, on the other hand, the strain carrying the Strep-tag II did grow, indicating that HglK was functional in this case (Fig. 26). Fig. 27. Construction of Anabaena strains that produce an HglK protein with a 6 x His tag or a Streptag II at the C-terminus. (A) Scheme of the construction of plasmid pCSSA1 and of recombination between this plasmid and the Anabaena chromosome. (B) Scheme of the construction of plasmid pCSSA30 and of recombination between this plasmid and the Anabaena chromosome. (C) PCR analysis of pCSSA11 exconjugants and the WT with the indicated oligodeoxynucleotide primers. The correct construct was found in all but one (# 6) of the tested exconjugants, one of which was chosen and termed CSSA7. (D) PCR hglK alr0812 5’3’ asl0814 asl0815 all0813-13 all0813-12 C.S3 pCSSA11 6 x His hglK 5’3’ EcoRI EcoRI alr0812 asl0814 asl0815 519 bp 3’ 5’ 6 x His C.S3 asl0814 asl0815 pCSV3 alr0812 hglK all0813-18 all0813-10 1 kb C.S3 EcoRIEcoRI pCSV3 EcoRI EcoRI 6 x His 619 bp + pCSSA11 C.S3 pCSSA30 Strep-tag II hglK 5’3’ EcoRI EcoRI alr0812 asl0814 asl0815 698 bp all0813-10 3’ 5’ C.S3 asl0814 asl0815 pCSV3 alr0812 hglK Strep-tag all0813-10 1 kb Strep-tag II 914 pb all0813-17 hglK 5’3’ alr0812 asl0814 asl0815 all0813-25 all0813-24 EcoRI EcoRI Strep-tagII 619 bp C.S3 EcoRIEcoRI pCSV3 + pCSSA30 M: size markers 1 to 12: independent clones 13: PCC 7120 (WT) all0813-18 + all0813-10 M 1 2 3 4 5 6 7 8 9 10 11 12 13 1 kb 1 kb M 1 2 3 4 M: size markers 1, 3: CSSA18 2, 4: PCC 7120 (WT) 1, 2: Strep-tag + all0813-10 3, 4: all0813-17 + all0813-10 A B C D Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 2: The HglK protein _______________________________________________________________________ 81 analysis of pCSSA30 exconjugant CSSA18 and the WT with the indicated oligodeoxynucleotide primers (3, 4: positive controls of DNA loading). The strains were grown in liquid BG11 medium (with antibiotics, Sm+Sp, for the mutants), and filaments were collected, washed and incubated in liquid BG110 medium (without combined nitrogen or antibiotics) for the time indicated in each case. Immunolocalization was performed on those filaments with commercial antibodies that carried a fluorophore and were specific for the 6xHis-tag and the Strep-tag II, respectively. Samples were visualized by fluorescence microscopy with excitation at 488 nm. Strain CSSA7 (HglK-6xHis) produced small fluorescent foci mainly in the intercellular septa and in the poles of the heterocysts (Fig. 28A), and strain CSSA18 (HglK-Strep-tag II) also showed fluorescent foci in or near the intercellular septa (Fig. 28B). With both tagged proteins, the presence of double spots of fluorescence was frequently observed (double arrows in Fig. 28A and B), resembling the double spots occasionally observed with HglK-sfGFP (double arrows in Fig. 25). To confirm that the subcellular location of HglK was mainly septal, the spots observed in the immunolocalization analysis were quantified using the wild type as a control of non-specific binding of antibodies to biological samples (Fig. 28, lower panels). About one septal spot and 0.2 non-septal spots per cell were counted for both HglK- 6xHis and HglK-Step-tag II, whereas the wild-type control showed in both cases about 0.1 septal and 0.1 non-septal spots per cell. If the wild-type controls are subtracted, the number of immunoflourescence spots is about 7.5-fold higher in the septal region than in the rest of the cell for both HglK-6xHis and HglK-Strep-tag II. In summary, in spite of a low expression of the hglK gene (Black et al., 1995), these results together with those obtained with HglK-sfGFP indicate that HglK is a protein of predominant localization at the intercellular septa in Anabaena. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 2: The HglK protein _______________________________________________________________________ 82 Fig. 28. Subcellular localization of 6xHis- and Strep II-tagged HglK. (A) Immunofluorescence analysis of Anabaena wild type (PCC 7120, negative control) or strain CSSA7 that produces to HglK protein with a C-terminal 6xHis tag. Filaments incubated for 48 h in BG110 medium were prepared and subjected to immunofluorescence analysis with anti-His-tag antibodies as described in section 2.7.2 of Materials & Methods. No signal is observed at the vegetative cells in the bottom panel because the filament was focused to see the signal at the heterocyst (het). (B) Immunofluorescence analysis of Anabaena wild type (PCC 7120, negative control) or strain CSSA18 that produces the HglK protein with C-terminal Strep II tag. Filaments incubated for 24 h in BG110 medium were prepared and subjected to immunofluorescence analysis with anti-Strep-tag II antibodies as described in in section 2.7.2 of Materials & Methods. Solid single arrows point to some immunofluorescence spots localized at intercellular septa; double-opposite arrows indicate paired spots frequently observed in the regions of the intercellular septa; the dotted arrow indicates an example of an immunofluorescence spot localized outside of the septal region. Histograms in the lower part show the number of immunofluorescence spots counted in the septal regions (right columns, orange) or outside the septal region (left columns, blue) in the strains producing the HglK tagged proteins or the wild type (PCC 7120) as a negative control. The number of cells analyzed were: for the His-tag antibodies, 115 (CSSA7) and 99 (PCC 7120); for the Strep-tag antibodies, 111 (CSSA18) and 136 (PCC 7120). Size bars, 10 µm. B aStrep II tag 0,000 0,200 0,400 0,600 0,800 1,000 1,200 HglK-Strep WT Non-septal Septal 1.0 0.8 0.6 0.4 0.2 0.0 Number of spots per cell CSSA18 PCC 7120 CSSA18 PCC 7120 PCC 7120 CSSA7 Vegetative cells het CSSA7 Aa6xHis tag 0,000 0,200 0,400 0,600 0,800 1,000 1,200 HglK-His WT Non-septal Septal 1.0 0.8 0.6 0.4 0.2 0.0 Number of spots per cell CSSA7 PCC 7120 Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 2: The HglK protein _______________________________________________________________________ 83 3.2.2 Inactivation of hglK in different genetic backgrounds To investigate some aspects of the phenotype of the hglK mutant that were not addressed in the first description made by Black et al. (1995), a plasmid was constructed that produced the mutation of hglK by deletion of an internal fragment and insertion of a gene cassette. This plasmid was transferred by conjugation to Anabaena with different genetic backgrounds. For creation of the plasmid, 713 bp of the 5’ region of hglK and 548 bp of the 3’ region were cloned, linked and corroborated by sequencing. Then, gene cassette C.K1 (which bears the npt gene that confers resistance to neomycin and its own natural promoter; Elhai & Wolk, 1988a) was introduced, and the whole fragment was transferred to plasmid vector pCSRO (Merino-Puerto et al., 2013) that bears cassette C.S3 and the sacB gene for positive selection through sensitivity to sucrose (Cai & Wolk, 1990), thus obtaining plasmid pCSSA6 that carries everything necessary to obtain the hglK mutant by deletion/insertion (Fig. 29A). The plasmid was transferred by conjugation to different genetic backgrounds with the objective of producing the replacement of hglK by the mutated DhglK::C.K1 construct. The genetic backgrounds used and the strains produced were: Anabaena wild type generating mutant CSSA1 (ΔhglK::C.K1); the double ΔfraC ΔfraD mutant (CSVT22) that gave rise to strain CSSA2 (ΔfraC ΔfraD ΔhglK::C.K1); the ΔsepJ mutant (CSVM34) that generated CSSA3 (ΔsepJ ΔhglK::C.K1); and, finally, a triple mutant of the genes ΔfraC, ΔfraD and ΔsepJ (CSVM141) generating CSSA12 (ΔfraC ΔfraD ΔsepJ ΔhglK::C.K1). All these exconjugants were initially selected for their resistance to neomycin and for their ability to grow in a medium supplemented with sucrose, and finally checked to corroborate their genetic structure (Fig. 29B). Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 2: The HglK protein _______________________________________________________________________ 90 that had been incubated in BG110 medium. Filaments of strains CSSA1, CSSA2, CSSA3, CSSA12, CSCT22, CSVM34, CSVM141 and the wild type were grown in liquid BG11 medium (supplemented with neomycin for the strains carrying the ΔhglK::C.K1 mutation), collected, washed, re-inoculated in liquid BG110 medium at a concentration of 1 µg Chl/mL and incubated for 48 h under culture conditions. One mL of each culture was incubated with calcein or 5- carboxyfluorescein (5-CF) and FRAP analysis was carried out as described in Materials and Methods (section 2.7.3). The results obtained are presented in Table 11 and visually in Fig. 35, and the non-parametric Mann-Whitney U test was performed to assess whether the differences observed between strains were significant. The results indicate that transfer of calcein between vegetative cells is decreased by inactivation of sepJ, fraC and fraD, as previously described (Merino-Puerto et al., 2010; Mullineaux et al., 2008). Additionally, transfer of calcein was significantly decreased by inactivation of hglK. However, inactivation of hglK did not decrease significantly transfer in the fraC fraD (CSVT22), sepJ (CSVM34) or fraC fraD sepJ (CSVM141) backgrounds beyond the decrease caused by these mutations (Fig. 35A, Table 11). The negative effect of inactivation of hglK on the molecular exchange between vegetative cells was corroborated by analyzing the transfer of 5-CF, which was also significantly decreased in the mutant (Fig. 35B, Table 11). Thus, hglK is required for the normal function of the septal junctions, but its effect is not additive to that of the removal of SepJ or FraC-FraD. In contrast to its negative effect on calcein transfer between vegetative cells, inactivation of hglK resulted in a significant increase of calcein transfer from vegetative cells to heterocysts (Fig. 35C, Table 11). This implies the presence of functional junctions in the septa between vegetative cells and heterocysts of the hglK mutant. Table 11. Intercellular transfer of fluorescent markers in Anabaena with different genetic backgrounds. Cells were grown in BG11 medium (in the presence of Nm for strains carrying the hglK mutation), incubated for 48 h in BG110 medium without antibiotics, and used in FRAP analysis as described in section 2.7.3 of Materials & Methods. Results of the parental strains and strains with the hglK mutation added are shown. M, median; IQP, interquartile range. Each hglK mutant was compared to its parental strain by the Mann-Whitney U test (p indicated in each case). Fluorescence marker, cell type and genotype R (s-1) Mann- Whitney (p) Parental + hglK M IQR M IQR Calcein transfer between vegetative cells Wild type 0.0901 0.0529 0.0670 0.0469 0.008 DfraC DfraD 0.0025 0.0386 0.0126 0.0271 0.600 DsepJ 0.0298 0.0700 0.0227 0.0595 0.509 DfraC DfraD DsepJ 0.0131 0.0331 0.0028 0.0198 0.771 5-CF transfer between vegetative cells Wild type 0.1133 0.0567 0.0681 0.0800 0.003 Calcein transfer to heterocysts Wild type 0.021 0.0243 0.077 0.0497 0.000 Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 2: The HglK protein _______________________________________________________________________ 91 Fig 35. Effect of the D hglK::C.K1 mutation on the intercellular transfer of fluorescent markers in different genetic backgrounds (box-plot representations of the data in Table 11). (A) Calcein transfer between vegetative cells of the hglK mutant in the indicted genetic background and of the corresponding parental strains. (B) 5-carboxyfluorescein (5-CF) transfer between vegetative cells. (C) Calcein transfer from vegetative cells to heterocysts. Data are presented as the recovery rate constant, R. The number of filaments analyzed is indicated in parenthesis for each strain. Mann-Whitney U test analysis of hglK versus indicated genetic background was performed and p is indicated in each case. 3.2.4 Subcellular localization of SepJ and FraD The results shown in the previous section suggest a role of HglK in the septal junctions. We then investigated by immunofluorescence whether the ΔhglK::C.K1 mutation affected the subcellular localization of septal proteins SepJ and FraD. The strains used for this study were grown in liquid BG11 medium (supplemented with neomycin for the strains carrying the hglK mutation: CSSA1, CSSA2 and CSSA3), collected, washed and resuspended at a concentration of 1 µg Chl/mL in 5-CF transfer between vegetative cells p = 0.004 Recovery constant R (s -1 ) 0.25 0.20 0.15 0.10 0.05 0.00 Calcein transfer to heterocysts Recovery constant R (s -1 ) 0.25 0.20 0.15 0.10 0.05 0.00 p = 0.000 PCC 7120 CSSA1 PCC 7120 CSSA1 DhglK (20) WT (15) DhglK (20) WT (15) B C Calcein transfer between vegetative cells 0,0000 0,0500 0,1000 0,1500 0,2000 0,2500 PCC 7120 CSSA1 CSVT22 CSSA2 CSVM34 CSSA3 CSVM141 CSSA12 Recovery constant R (s-1) 0.25 0.20 0.15 0.10 0.05 0.00 WT (45) DhglK (49) DfraCD (16) DsepJ (38) DsepJ DhglK (41) DfraCD DsepJ (21) DfraCD DhglK (45) DfraCD DsepJ DhglK (19) p = 0.008 p = 0.600 p = 0.509 p = 0.771 A Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 2: The HglK protein _______________________________________________________________________ 92 liquid BG110 medium (without the antibiotic) for 24 hours. For immunolocalization, two types of primary antibodies were used to specifically detect SepJ or FraD: an antibody that recognizes the coiled-coil domain of SepJ (Mariscal et al, 2011) on the CSSA1, CSSA3 and wild-type samples; and an antibody against an extramembrane (periplasmic) fragment of FraD (Merino-Puerto et al., 2011b) on the CSSA1, CSSA2 and wild-type samples. An anti-rabbit antibody carrying a fluorophore was used as a secondary antibody, and the samples were inspected by fluorescence microscopy. For SepJ, septal signals were evident in the wild type and were missing from a negative control strain that lacks the sepJ gene, strain CSSA3 (ΔsepJ ΔhglK::C.K1) (Fig. 36A). In the hglK mutant (CSSA1), SepJ was localized at the intercellular septa like in the wild type. Labeling of FraD was unfortunately not as clear as that of SepJ. Nonetheless, FraD spots localized at the intercellular septa could be observed in the wild type that were missing from a fraD mutant control, strain CSSA2 (ΔfraC ΔfraD ΔhglK::C.K1) (Fig. 36B). In the hglK mutant, FraD spots could be observed at the intercellular septa but also out of the septa (Fig. 36B). Quantification of immunofluorescence spots did not show, however, any significant difference between the hlgK mutant and the wild type in the septal signals of FraD. Fig. 36. Subcellular localization of SepJ and FraD in the hglK mutant. Filaments from the indicated strains incubated for 24 h in BG110 medium were used in immunofluorescence analysis with antibodies raised against the coiled-coil domain of SepJ (A) or against the extramembrane fragment of FraD (B). White arrows point to some SepJ (A) or FraD (B) spots at intercellular septa; yellow arrows point to some FraD spots outside of the intercellular septa in the hglK mutant. Size bars, 10 µm. PCC 7120 (WT) CSSA1 (hglK) CSSA3 (hglK sepJ) a-SepJ-CCA PCC 7120 (WT) CSSA1 (hglK) CSSA2 (hglK fraC fraD) a-FraD B Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 2: The HglK protein _______________________________________________________________________ 93 3.2.5 Discussion In this Chapter, we have shown that HglK is a protein predominantly localized in the septal regions of the filaments of Anabaena, and that inactivation of the hglK gene results in filament fragmentation, a decreased number of septal PG nanopores and decreased activity of intercellular molecular transfer between vegetative cells. However, inactivation of hglK does not increase the effects on these parameters that result from inactivation of sepJ or fraC and fraD. Furthermore, inactivation of sepJ or fraC and fraD has a stronger effect on those parameters than inactivation of hglK (Nürnberg et al., 2015). Hence, HglK might influence the formation of septal junctions rather than constitute an independent type of junctions. The recently visualized FraD-containing septal junctions evidently comprise additional proteins (Weiss et al., 2019). Whether HglK could be a component of the septal junctions or solely contribute to their assembly remains to be investigated. In any case, HglK could be a structural component of the intercellular septa affecting the formation of septal junctions. In this context, the frequent observation of HglK double spots that are laterally, rather than centrally, localized in the septa (Figs. 25 and 28) is of interest. A role of HglK in the construction of mature septa is consistent with the original observation that inactivation of hglK results in the formation of broad intercellular septa (Black et al., 1995). Our results have shown that inactivation of hglK impairs calcein and 5-CF transfer between vegetative cells. In contrast, calcein transfer to heterocysts was increased significantly in the hglK mutant. Calcein transfer to the heterocysts appears to be specifically related to SepJ (Mariscal et al., 2016), whose localization is unaltered in the hglK mutant (Fig. 36A). The positive effect of inactivation of hglK on calcein transfer to the heterocysts can be related to the lack, in the mutant, of cyanophycin plugs (Black et al., 1995), which have been shown to restrict transfer of calcein into the heterocysts (Mullineaux et al., 2008). Whether, additionally, HglK has a regulatory effect on the septal junctions that are functional between vegetative cells and heterocysts is unknown. As described in the introduction of this Chapter, HglK is a membrane protein with a predicted long pentapeptide-repeat fragment that is likely localized in the periplasm (Fig. 23). As predicted by Phyre2 (www.sbg.bio.ic.ac.uk/phyre2/), the pentapeptide-repeat fragment of HglK has a structure very similar to that of HetL (Ni et al., 2009). Pentapeptide-repeat proteins adopt a highly regular four-sided, right-handed β helical structure (Fig. 37) that might perform a structural function. Fig. 37. Three-dimensional structure of the pentapeptide-repeat region of HglK (All0813). The three-dimensional structure as predicted by Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/) is shown. There are as many as 32 pentapeptide-repeat proteins in Anabaena, and at least five of them bear signal peptides that may direct those proteins to the periplasm (Ni et al., 2009). The limited effect Phyre2 prediction from aa 40 to aa 664 of All0813 Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 2: The HglK protein _______________________________________________________________________ 94 of inactivation of hglK on the parameters that we have studied may reflect that, in Anabaena, other proteins have a role redundant with that of HglK. Indeed, inactivation or altered expression of some genes encoding pentapeptide-repeat proteins have been shown to affect filamentation or heterocyst formation related to intercellular molecular transfer in Anabaena. Thus, FraF is a pentapentide-repeat protein encoded in a cluster of genes (fraC-fraD-fraE ß fraF, where the arrow indicates a change of orientation) that affect filament length (Merino-Puerto et al., 2013), and HetL and PatL are pentapeptide-repeat proteins that affect heterocyst pattern formation (Liu & Golden, 2002; Liu & Wolk, 2011). Interestingly, whereas PatL and FraF are widely distributed in heterocystous cyanobacteria and HetL seems to have a more restricted distribution, HglK is universally present in these organisms suggesting an essential function as mentioned earlier (Table 9). Of further interest, PatL is a predicted membrane-anchored protein that likely has a topology very similar to that of HglK (Fig. 38, compare to Figs. 23 and 37). Whether PatL and HglK have somewhat associated or redundant functions remains to be investigated Fig. 38. Overview of the PatL (All3305) protein. (A) Amino acid sequence with indication of transmembrane segments (magenta) and pentapeptide repeats (alternatively shown in blue and red color), as described by Liu & Wolk (2011). (B) Protter representation based on prediction with the Phobius B PRF (aa 243 to 467) Periplasm Cytoplasm CM Phyre2 prediction from aa 230 to aa 461 of All3305 MVRNYTIMPANKTDKLWRWLIGITVIFAVTIAVILFASSNPQGLTIQQKLQ SRNQALTITAIIFLGLGVMINAYYAAKRVDVISKSALAAEKNLEISIQNAK LSEDRLISERLMTAVTQLGHDKIETRTGAIYVLEKVAQDFPQEHWTVIEIL TAFIRENAAVREVEEQKEDESQPTSLSRYKQGERRTPQLPLNVHEESPKIR RDIQVALTVVGRRNYLQEEADKRLDLRNINLRRVELLGANLQRADLRGCDF RGADLRGCDFSGANLEGAELAGSILFEANFLKANLQGANIQGANLNRAELG GANLRSANLMGASLRAANLPGANLYKVNLQHAILKLANLCGAKLFLANLQG AKLGKANLSLCGLIGANLQGANLNGANLAEANLNAAKLQQAEMLFVDFTEA SLTEADLCKANLMGANLQRASLYQANLFQCNLVGANLSDTNFCDVKLAGAI LTGVKNLKPQQVSIAIGDRTTRLPDNVEIPIHWRQSS A C Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 2: The HglK protein _______________________________________________________________________ 95 program (http://phobius.sbc.su.se). The approximate localization of the pentapeptide-repeat fragment (PRF) is indicated. CM, cytoplasmic membrane. (C) Three-dimensional structure of the pentapeptide repeat region as predicted by Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/). Because the hglK mutant was shown to synthesize heterocyst-specific glycolipids (Hgl) but not to deposit an Hgl layer, HglK was suggested to be involved in the export of Hgl or deposition of the Hgl layer (Black et al., 1995). However, the transporter of Hgl to the outside of the outer membrane has now been identified as the ATP-driven efflux pump DevBCA/HgdD (Staron et al., 2011). Here we have shown, on the other hand, that the hglK mutant is impaired in heterocyst differentiation, failing to express at normal levels some genes activated at intermediate and late steps of the differentiation process. It is possible, therefore, that rather than being specifically involved in Hgl export or deposition, HglK has a more general role in heterocyst differentiation, its inactivation resulting in a deficiency of several differentiation-related processes. Because HglK appears to influence the formation of the septal junctions, impairment, in the hglK mutant, in the intercellular transfer of some compounds could be responsible for the phenotype of deficient differentiation. This would be similar to the situation in the sepJ mutant, in which heterocyst differentiation is aborted after synthesis of the heterocyst polysaccharide layer but before synthesis of Hgl (Flores et al., 2007; Nayar et al., 2007). SepJ appears to be involved in the intercellular transfer of the PatS morphogen and of a HetN-dependent regulator of heterocyst differentiation (Mariscal et al., 2016; Rivers et al., 2014), but the physiological compounds whose intercellular transfer may be affected by inactivation of hglK are currently unknown. In summary, our results have shown that the pentapeptide-repeat protein HglK is involved in the formation of fully functional septal junctions and is needed for the differentiation of functional heterocysts in Anabaena. The function of a number of pentapeptide-repeat proteins in different aspects of heterocyst differentiation is intriguing and raise the possibility that some of those proteins are (at least partially) redundant for the formation of the septal junctions, which are complex entities that appear to include a number of different proteins. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
3. Results & Discussion Chapter 3 HglK and SepJ proteomics Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 3 | HglK and SepJ proteomics _______________________________________________________________________ 99 3.3 HglK and SepJ proteomics The pentapeptide-repeat protein HglK from Anabaena is needed for the formation of mature heterocysts (Black et al., 1995). According to bioinformatics predictions, HglK has four transmembrane segments with its amino and carboxyl termini located in the periplasm (Fig. 23). It is the product of the hglK gene, which is low-expressed under normal conditions and induced after N-stepdown (Ehira & Ohmori, 2006a; Flaherty et al., 2011). As shown in Results & Discussion Chapter 2, HglK is predominantly located in the cell poles, and it is necessary for the integrity of the filament and for intercellular molecular transfer as probed by fluorescent markers. SepJ is also a membrane integral protein. Our current model, based on bioinformatics predictions and experimental evidence (Rudolf et al., 2015; Herrero et al., 2016; Ramos-León et at., 2018), considers that SepJ from Anabaena contains 11 transmembrane segments leaving the amino terminal part (about 410 amino acid residues) in the periplasm and the carboxyl terminus in the cytoplasm (Fig. 7). SepJ is located in the septum between adjacent cells and, as observed for hglK mutants, sepJ mutants are impaired in diazotrophic growth, exhibit filament fragmentation and show decreased intercellular transfer of fluorescent markers (Flores et al., 2007; Mullineaux et al., 2008; Mariscal et al., 2011; Ramos-León et al., 2018). SepJ has coiled-coil domains, which are usually involved in protein-protein interactions, and engages in protein complexes that have been observed by blue native (BN)-PAGE (Ramos-León et al., 2017). Because of the importance of the two proteins for the integrity and function of the cyanobacterial filament, it is possible that they are part or participate in the formation of the protein complexes that constitute the septal junctions. In this Chapter, the objective was to isolate these proteins avoiding denaturation in order to detect possible purification partners by mass spectrometry. 3.3.1 Isolation the HglK and SepJ proteins To facilitate the isolation HglK and SepJ, we prepared Anabaena strains producing these proteins fused to Strep II or GFP tags. For the isolation using the Strep-tag II, Anabaena strain CSSA18 that produces the C-terminally Strep II-tagged HglK (described in Fig. 27) and strain CSSA17 that produces the C-terminally Strep II-tagged SepJ (to be described in the next section) were used. The isolation using GFP antibodies was carried out with Anabaena strain CSAM137 (described in Table 2 of Materials & Methods) that produces a SepJ with a deletion of the last nine amino acids at the C-terminus and a C-terminal GFP fusion. An Anabaena strain bearing this construct has been shown to exhibit normal SepJ activity (Ramos-León et al., 2017). 3.3.1.1 Isolation using the Strep-tag II For isolation of proteins with Strep-tag II, the two strains CSSA17 and CSSA18 were used. Strain CSSA17 was constructed by cloning 420 bp from the 3' end of the sepJ gene to facilitate recombination of an appropriate construct into the Anabaena genome. The amplification was carried out by PCR with primers alr2338-57 and alr2338-58 producing a DNA fragment of 432 bp that includes the 3’ part of the sepJ gene (except the stop codon) and sequences encoding the Strep-tag II and the target for EcoRI at its 3' end. This fragment was inserted into the pSPARK cloning vector forming plasmid pCSSA25, which was corroborated by sequencing. The construct was extracted from pCSSA25 by EcoRI restriction and introduced into plasmid pCSV3 (which Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 3 | HglK and SepJ proteomics _______________________________________________________________________ 106 Fig. 42. Analysis of the proteomics results. Volcano plots for the log2-value of the ratio between the labelfree quantification (LFQ) value of protein detection using the strain expressing the bait compared to the wild-type strain as negative control, with the calculated p-value. (A) Shows the results for SepJ-GFP experiment 1 (left) and 2 (right), (B) SepJ-Strep-tag II and (D) HglK-Strep-tag II experiment 1 (left) and 2 (right). Black dots show all identified proteins with 0.15 < p < 0.815; green dots all identified proteins with p < 0.05, orange dots with 0.05 < p < 0.1 and yellow dots with 0.1 < p < 0.15, the latter for proteins that have been identified in one of the other experiments. The p-value 0.1 was selected because the bait proteins are low-abundant. (C) The overlap of the three SepJ experiments is shown (left). The proteins that are found in at least two experiments and with a p < 0.1 in at least one experiment are indicated. In D, the overlap of the two experiments is shown on the right and the proteins identified with p < 0.1 in the overlap are indicated. GFP SepJ HglK ApcD ApcA1 All5255 KaiC SecA IlvA CpcG2 FtsH Alr1744InfB Alr2450 / Alr3277 MoeB / All1076 / Alr4534 Tic22 HglK Trypsin Alr3659 AtpA AtpD Alr2947 A Trypsin SepJ Alr2051 Tic22 Alr2947 Alr1690 AtpG AtpA HglK B D HglK ChlP Streptavidin GroL Alr3301 GroS Alr0834 Alr3277 FusA Alr4550 Exp1 Exp2 48129 15 SepJ HglK Alr2947 CpcD Streptavidin Keratin GFP GFP1 GFP2 Strep 29 64 110 816 11 8 C SpeJ HglK Alr2947 Tic22 AtpG AtpA Alr1690 CpcD ApcD CpcG Alr3277 HglK AtpA AtpD SepJ Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 3 | HglK and SepJ proteomics _______________________________________________________________________ 107 Acc. ORF Protein Bait/Control Further information log2p-value Q8YSY2 alr2947 8.63 8.4*10-5 Signal peptide (SPI) containing protein Q44230 all0813 HglK 7.65 0.04 Chapter 2 Q8YUK6 alr2338 SepJ 5.63 5.8*10-8 Bait Streptavidin 4.32 3.6*10-3 Streptavidin P13647 Krt5 2.12 2.0*10-3 Keratin, type II cytoskeletal 5 P07124 asr0531 OpcD 0.99 7.3*10-5 Phycobilisome 8.5-kDa linker polypeptide Q9U6Y5 GFP 6.92 0.09 Table 12. Interaction partners identified with SepJ-GFP as bait —Experiment 1. Three independent repetitions of SepJ-GFP isolation and three control experiments were analyzed by MaxQuant to calculate the LFQ value for analysis. The accession number (column 1), the ORF (column 2), the protein name (column 3), the log2 value and the p-value for the LFQ ratio between bait and control (column 4 & 5), and the assigned function (when appropriate) is presented for the proteins identified with p < 0.05 (top proteins) and one with 0.05 < p < 0.1 (bottom protein). SPI, signal peptidase I-type of signal peptide. Table 13. Interaction partners identified with SepJ-GFP as bait —Experiment 2. Three independent repetitions of SepJ-GFP isolation and three control experiments were analyzed by MaxQuant to calculate the LFQ value for analysis. The accession number (column 1), the ORF (column 2), the protein name (column 3), the log2 value and the p-value for the LFQ ratio between bait and control (column 4 & 5), and the assigned function (when appropriate) is presented for the proteins identified with p ≤ 0.05 (top proteins) and 0.05 < p < 0.1 (four lower proteins). Acc. ORF Protein Bait/Control Further information log 2 p-value Q9U6Y5 GFP 17.77 0.00 Bait tag Q8YUK6 alr2338 SepJ 14.43 0.00 Bait Q44230 all0813 HglK 2.10 1.2*10 -22 Chapter 2 P80556 all3653 ApcD 7.04 3.3*10 -22 Allophycocyanin subunit alpha-B P80555 alr0021 ApcA1 1.21 1.3*10 -15 Allophycocyanin subunit alpha 1 Q8YLP1 all5255 1.23 6.0*10 -7 Polyphenol oxidase Q8YT40 alr2886 KaiC 1.02 4.4*10 -4 Circadian clock protein kinase KaiC Q8YMS8 alr4851 SecA 1.19 3.8*10 -3 Protein translocase subunit SecA Q8YPG2 alr4232 IlvA 0.95 0.01 L-threonine dehydratase P29987 alr0535 CpcG2 0.63 0.02 Phycobilisome rod-core linker polypeptide Q8YXF2 alr1261 FtsH 0.44 0.02 ATP-dependent zinc metalloprotease with TMD Q8YW72 alr1744 1.75 0.04 Sulfolipid biosynthesis protein Q8YQJ1 alr3832 InfB 5.49 0.05 Translation initiation factor IF-2 Q8YUA2 alr2450 1.48 0.05 1,4-alpha-glucan branching enzyme activity Q8YS15 alr3277 5.77 0.06 ATP-dependent RNA/DNA helicases Q8YT21 all2906 MoeB 1.48 0.06 Molybdopterin biosynthesis protein Q8YXY0 all1076 1.19 0.08 DNA-binding domain containing protein Q8YNN0 alr4534 6,76 0.08 Coiled-coil domain containing protein with TMD Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 3 | HglK and SepJ proteomics _______________________________________________________________________ 108 Acc. ORF Protein Bait/Control Further information log2p-value Q8YVC7 alr2051 6.11 0.00 Gamma-glutamyl-transpeptidase with transmembrane domain Q8YUK6 alr2338 SepJ 13.73 0.00 Bait P00761 Trypsin 12.28 7.9*10-110 Q8Z0I2 alr0114 Tic22 6.12 4.5*10-22 Periplasmic protein translocase Q8YSY2 alr2947 1.13 8.8*10-4 Signal peptide (SPI) containing protein Q8YWC3 alr1690 1.60 3.2*10-3 Cell wall-binding protein P12410 all0008 AtpG 4.03 8.6*10-3 ATP synthase subunit b P12405 all0005 AtpA 1.59 0.02 ATP synthase subunit alpha Q44230 all0813 HglK 2.83 0.11 Chapter 2 Table 14. Interaction partners identified with SepJ-Strep-tag II as bait. Three independent repetitions of SepJ-Strep-tag II isolation and three control experiments were analyzed by MaxQuant to calculate the LFQ value for analysis. The accession number (column 1), the ORF (column 2), the protein name (column 3), the log2 value and the p-value for the LFQ ratio between bait and control (column 4 & 5), and the assigned function (when appropriate) is presented for the proteins identified with p < 0.05 (top proteins) and one with p = 0.11 (bottom protein). SPI, signal peptidase I-type of signal peptide. Acc. ORF Protein Bait/Control Further information log 2 p-value Q44230 all0813 HglK 12.05 7.6*10 -14 Bait Q8Z0G8 alr0128 ChlP 3.15 4.5*10 -5 Geranyl-geranyl hydrogenase Streptavidin 2.94 9.6*10 -3 Streptavidin Q8YRZ1 alr3301 2.63 1.2*10 -4 Coiled-coil domain containing protein Q8YQZ8 alr3661 GroL 1.39 3.8*10 -3 60-kDa chaperonin Q8YQZ9 alr3661 GroS 0.57 9.7*10 -3 10-kDa chaperonin Q8YYL4 alr0834 4.16 0.06 Porin —major outer membrane protein Q8YS15 alr3277 3.76 0.10 ATP-dependent RNA/DNA helicases Q8YP62 all4338 FusA 0.86 0.06 Elongation factor G Q8YNL5 alr4550 0.58 0.08 Porin —major outer membrane protein Table 15. Interaction partners identified with HglK-Strep-tag II as bait —Experiment 1. Four independent repetitions of HglK-Strep-tag II isolation and three control experiments were analyzed by MaxQuant to calculate the LFQ value for analysis. The accession number (column 1), the ORF (column 2), the protein name (column 3), the log2 value and the p-value for the LFQ ratio between bait and control (column 4 & 5) and the assigned function (when appropriate) is presented for the identified proteins with p < 0.05 (top proteins) and 0.05 < p ≤ 0.1 (four lower proteins). Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 3 | HglK and SepJ proteomics _______________________________________________________________________ 109 3.3.3 Discussion SepJ and HglK are proteins exclusively or predominantly located at the cell poles in the intercellular septa of Anabaena and whose mutation cause alterations both in the integrity of the filament and in the intercellular exchange of fluorescent markers (Flores et al., 2007; Mullineaux et al., 2008; see also Results & Discussion Chapter 2). Another feature that is altered in the mutants is the number of nanopores through which septal junctions appear to traverse the septal peptidoglycan (Results & Discussion Chapter 1). SepJ and HglK are therefore needed in Anabaena to produce mature septa, and they could interact with other proteins forming protein complexes involved in septal junction formation or function. In order to search for these possible partners, SepJ and HglK were isolated under non-denaturing conditions with the help of one (HglK) or two (SepJ) of two different tags, the Strep-tag II and the GFP. The different protein isolates were subjected to mass spectrometry with the aim of detecting the proteins that could have co-purified with SepJ and HglK. The mass spectrometry results provided us with a large number of potential partners that were submitted to statistical analysis, using the results of the negative control (wild-type extracts) to correct for unspecific precipitation. To enhance the reliability of the detection of a protein, we concentrated on proteins that were repeatedly detected in different samples (see Fig. 42C). Some of these proteins were probably not SepJ or HglK physiological partners, since they likely are proteins very abundant in the cell that could bind nonspecifically to the protein used as bait in the purification processes. Such proteins that might have bound to SepJ or HglK non-specifically include phycobilisome or ATPase subunits such as ApcD, CpcD, CpcG, AtpA, AtpD and AtpG. On the other hand, some other proteins show characteristics that make us to consider them as candidate partners as discussed below. HglK was detected in the three SepJ isolations, in two of them with p < 0.05 (Tables 12 and 13; Fig. 42A, B), strongly suggesting that both proteins interact in vivo. In contrast, SepJ was not detected in the HglK isolations. However, we noted that the precipitation of HglK was less effective when compared to the precipitation of SepJ. This might reflect a lower abundance of Acc. ORF Protein Bait/Control Further information log2p-value Q44230 all0813 HglK 14.91 0.00 Bait P00761 Trypsin 12.47 3.8*10-173 P12405 all0005 AtpA 2.63 1.2*10-4 ATP synthase subunit Q8YSY2 alr2947 0.95 0.03 Signal peptide (SPI) containing protein Q8YR01 alr3659 5.59 0.07 Hemolysin-typ Ca-binding domain P06540 all5093 AtpD 3.18 0.09 ATP synthase subunit beta Q8Z0I2 alr0114 Tic22 3.98 0.11 Periplasmic protein translocase Table 16. Interaction partners identified with HglK-Strep-tag II as bait —Experiment 2. Five independent repetitions of HglK-Strep-tag II isolation and three control experiments were analyzed by MaxQuant to calculate the LFQ value for analysis. The accession number (column 1), the ORF (column 2), the protein name (column 3), the log2 value and the p-value for the LFQ ratio between bait and control (column 4 & 5) and the assigned function (when appropriate) is presented for the identified proteins with p < 0.05 (top proteins) and 0.05 < p ≤ 0.11 (three lower proteins). SPI, signal peptidase I-type of signal peptide. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 3 | HglK and SepJ proteomics _______________________________________________________________________ 110 HglK than of SepJ in Anabaena cells, which can be deduced from the difficulty of detection of HglK-GFP fusions as compared to SepJ-GFP fusions (Chapter 2, compare to Flores et al., 2007). Also, the sepJ transcript is detected more easily than the hglK transcript (Black et al., 1995; Flores et al., 2007). Additionally, the selection of the tag might also interfere with the mode of interaction. For example, HglK was precipitated more efficiently with SepJ-GFP than with SepJ- Strep II (Fig. 42A vs. Fig 42B). Thus, it is possible that the Strep-tag II of HglK might have had an influence on the affinity of the HglK-SepJ complex resulting in loss of SepJ during the wash steps of the column material. The observed interaction of SepJ and HglK deduced from the presented proteomic analysis is consistent with the phenotypic analysis of sepJ and hglK mutants. The two mutants show a number of similar characteristics that have led to the suggestion that SepJ and HglK contribute to the maturation of intercellular septal structures in Anabaena (Flores et al., 2018 and Results & Discussion Chapter 2). The nature and structural basis of the SepJ-HglK interaction remain to be investigated. We note, nonetheless, that both proteins contain integral-membrane and putative periplasmic domains that could mediate the interaction. On the other hand, SepJ is localized very focused in the center of the septa (Flores et al., 2007), whereas HglK shows a preferential localization in the intercellular septa with possible double spots that are laterally, rather than centrally, localized in the septa (Chapter 2). Perhaps the interaction of the two proteins is limited to the places that delimit their localization. In addition to HglK, Alr2947 was identified with statistical significance in two SepJ isolations (Tables 12 and 14) and one of the HglK isolations (Table 16). This suggests that Alr2947 could be a common partner for SepJ and HglK, and even a three-part complex involving the three proteins might be considered for future research. Alr2947 is predicted to bear a signal peptidase I-type of signal peptide (Fig. 43A), implying that it is a soluble periplasmic protein. It is a very basic protein (pI, 9.74), and it seems to be rather unstructured (Fig. 43B). Interestingly, it is currently thought that proteins that are unstructured in solution become structured in complexes (Mohan et al., 2006). It is tempting to hypothesize that Alr2947 interacts with periplasmic domains of both SepJ and HglK. Alr0114 was repeatedly identified in our analyses, with very good statistical significance in one of the SepJ isolations (Table 14) and limited significance in one of the HglK isolations (Table 16). This is the well-known protein Tic22, a periplasmic holdase (chaperons that assist the noncovalent, ATP-independent folding of proteins) that is important in outer membrane biogenesis in Anabaena (Brouwer et al., 2019; Tripp et al., 2012). Considering that secretion system protein SecA was also found to be significantly enriched in one of the SepJ-based isolates (Fig. 43A, right panel; Table 13), it is tempting to hypothesize that these proteins interact with the translocating protein in the cytoplasm (SecA) or periplasm (Tic22). Here, Tic22 might be involved in the folding of periplasmic domains of both SepJ and HglK. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 3 | HglK and SepJ proteomics _______________________________________________________________________ 111 Another protein that was found to interact with SepJ is Alr1690 (Table 14). This is a cytoplasmic membrane-anchored protein that is largely periplasmic (Fig. 44A). It bears four predicted peptidoglycan-binding domains and, consistently (Fig. 44A), it is predicted to form structures that are found in PG-binding and PG-metabolism proteins (Fig. 44B). Because the possible periplasmic domain of SepJ also shows PG-binding activity (Ramos-León et al., 2017), Alr1690 and SepJ might interact as PG-binding proteins. Further, it has been previously shown that SepJ interacts with All1861, which is a PG-binding protein called SjcF1 that is involved in the regulation of nanopore size in Anabaena (Rudolf et al., 2015). Fig. 43. Properties of putative SepJ- and HglK-interacting protein Alr2947. (A) Phobius prediction of a signal peptide and periplasmic location of Alr2947. Signal peptidase I-signal peptide predicted by SignalP-5.0. Note that signal peptidase I releases the protein to the periplasm. Alr2947 is a 143-residue protein, and the processed protein is predicted to have 116 residues. (B) Phyre2 analysis revealed no highly predicted 3D model, showing instead a largely unstructured protein. For the two 3D model examples depicted, the top bar shows in blue the Alr2947 fragments that align with the target sequences. Note the low confidence of the predictions. 4/5/20 13:03Phobius prediction Página 1 de 1http://phobius.sbc.su.se/cgi-bin/predict.pl Phobius prediction Prediction of tr|Q8YSY2|Q8YSY2_NOSS1 ID tr|Q8YSY2|Q8YSY2_NOSS1 FT SIGNAL 1 27 FT REGION 1 7 N-REGION. FT REGION 8 19 H-REGION. FT REGION 20 27 C-REGION. FT TOPO_DOM 28 143 NON CYTOPLASMIC. // The probability data used in the plot is found here, and the gnuplot script is here. SignalpeptidaseI signalpeptide Phobius posterior probabilities A B Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 3 | HglK and SepJ proteomics _______________________________________________________________________ 112 4/5/20 14:27Phobius prediction Página 1 de 1http://phobius.sbc.su.se/cgi-bin/predict.pl Phobius prediction Prediction of tr|Q8YWC3|Q8YWC3_NOSS1 ID tr|Q8YWC3|Q8YWC3_NOSS1 FT TOPO_DOM 1 46 CYTOPLASMIC. FT TRANSMEM 47 66 FT TOPO_DOM 67 424 NON CYTOPLASMIC. // The probability data used in the plot is found here, and the gnuplot script is here. Phobius posterior PGb PGb PGb PGb D/P A B Fig. 44. Properties of putative SepJ-interacting protein Alr1690. (A) Phobius prediction of a transmembrane segment and periplasmic location of Alr1690. The upper scheme shows the approximate location of peptidoglycan-binding motifs (PGb, orange) and a disordered/polar region (D/P, yellow; taken from information in Uniprot). (B) Phyre2 analysis of Alr1690 predicts structural motifs found in PG- binding proteins and PG-metabolism proteins. The left part shows the Alr1690 fragments that align with the target sequences (red lines) and their predicted structures. The right part indicates the proteins to which those fragments show highest structural similarity. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 3 | HglK and SepJ proteomics _______________________________________________________________________ 113 Finally, we note Alr3277, detected with limited statistical significance in isolations of SepJ (Table 13) and HglK (Table 15). Alr3277 is a 179-amino acid residue protein of predicted cytoplasmic localization that shows a domain similar in structure to the late competence protein ComFB from Bacillus subtilis (Fig. 45). We do not know the significance that this finding may have. In summary, this proteomic analysis has provided us with independent evidence for a possible SepJ-HglK interaction. Additionally, it has identified three periplasmic proteins that may interact with SepJ (Alr1690) or with both SepJ and HglK (Alr2947 and Alr0114/Tic22). Although these possible interactions need further study, the predominance of periplasmic proteins in this analysis is consistent with the important role that SepJ and HglK have for the maturation of intercellular septal structures in Anabaena. Fig. 45. Properties of putative SepJ- and HglK-interacting protein Alr3277. Phyre2 analysis predicts a domain with a structure similar to that of ComFB from Bacillus subtilis. The left part shows the Alr3277 fragment that aligns with the target sequence (red line) and its predicted structure, and the right part indicates the protein to which that fragment shows highest structural similarity. Alr3277 is composed of 179 amino acid residues and ComFB is a small, 98-residue protein. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
3. Results & Discussion Chapter 4 The SepJ and Fra proteins from marine symbiotic Richelia and Calothrix Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 4 | The SepJ and Fra proteins from marine symbiotic Richelia and Calothrix _______________________________________________________________________ 122 A B C D E F M 1 2 3000 2500 750 500 M 1 2 3 3000 2500 2000 sepJ mutant of Anabaena (CSVM34) Plasmid with the sepJ gene from Richelia Wild type genotype sepJ pCSSA21 EcoRIEcoRI C.S3 sepJ + EcoRI EcoRI RINTHH17990_sepJ EcoR I EcoRI sepJ C.S3 pCSV3 pCSSA21 5' 3' 5' 3' C.S3 sepJ SepJ_Rich-3 SepJ_Rich-2≈ 2500 bp Ap sepJ mutant of Anabaena (CSVM34) Plasmid with the sepJ gene from Anabaena Wild type genotype sepJ pCSSA47 SacISacI C.S3 sepJ + pCSSA47 5' 3' 5' 3' C.S3 sepJ Alr2336-62 Alr2336-63≈ 2853 bp SacISacI sepJ Alr2336-62 Alr2336-63 SacI SacI pCSV3 C.S3 Alr2336-62 Alr2336-63≈ 700 bp sepJ mutant of Anabaena (CSVM34) Plasmid with the sepJ gene from Calothrix Wild type genotype sepJ pCSSA48 EcoRIEcoRI C.S3 sepJ + EcoRI EcoRIEcoR I EcoRI C.S3 pCSV3 pCSSA48 5' 3' Ap sepJ pEX-A258-sepJ SC01 5' 3' C.S3 sepJ SepJ Calothrix_5 SepJ Calothrix_6≈ 2605 bp Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 4 | The SepJ and Fra proteins from marine symbiotic Richelia and Calothrix _______________________________________________________________________ 123 To study whether the heterologous genes were expressed and resulted into a properly located protein, we carried out immunofluorescence analysis with antibodies against the coiled-coil domain of Anabaena SepJ (Mariscal et al., 2011; details in Materials & Methods section 2.7.2). The antibodies detected the SepJ protein at the intercellular septa in Anabaena and in the selfcomplemented strain CSSA29, but did not detect any material in the negative control CSVM34 (DsepJ), as expected (Fig. 51). In strain CSSA14 (Anabaena::sepJRintHH01), some spots were observed that however were not localized at the intercellular septa, and in strain CSSA28 (Anabaena::sepJCalSC01) no spots were observed. These negative results provide no evidence for the proper expression and location of the heterologous proteins in Anabaena, although some expression is possible at least for HH01 SepJ. Nonetheless, we carried out growth tests and determined filament length to enquire on any possible function of the heterologous genes in Anabaena if, for example, they were expressed at low levels. Growth tests showed that, as is was the case for the parental strain CSVM34, neither strain CSSA14 (Anabaena::sepJRintHH01) nor CSSA28 (Anabaena::sepJCalSC01) could grow diazotrophically (BG110 medium; Fig. 52). In contrast, the self-complemented strain CSSA29 grew diazotrophically as the wild type (PCC 7120), and all strains could grow with combined nitrogen (BG11 medium; Fig. 52). Additionally, we tested filament length in BG11 medium to test whether the symbiont genes could provide filament function in spite of lack of growth without combined nitrogen. Fig. 50. Constructs for expression of symbiont sepJ genes in Anabaena. The figure shows how the CSSA14, CSSA28 and CSSA29 strains were obtained by single recombination to express SepJ of different cyanobacteria under the natural Anabaena sepJ promoter, together with their corresponding genotypic tests by PCR. (A) Construction of Anabaena strain CSSA14 in which sepJ from RintHH01 is cloned at the natural sepJ locus in Anabaena strain CSVM34 (DsepJ). (B) PCR verification of two independent clones of strain CSSA14 (lanes 1 and 2) where it can be seen the amplification of Richelia sepJ, while that amplification did not occur with Anabaena wild-type DNA (lane 3). (C) Construction of Anabaena strain CSSA29 (positive control) that expresses sepJ from Anabaena cloned at the Anabaena's natural sepJ site in strain CSVM34 (DsepJ). (D) PCR test of strain CSSA29 (lane 1) where a band of 2853 bp is seen corresponding to the amplification of the sepJ gene plus about 500 bp upstream from the start of the gene. This band of approximately 2853 bp can also be seen in wild-type Anabaena (lane 2). In strain CSSA29 (lane 1) there is also a band of approximately 700 bp corresponding to the size of the region including deleted sepJ, indicating that this strain is not completely segregated. (E) Construction of Anabaena strain CSSA28 in which sepJ from CalSC01 (modified to have an Anabaena–adapted codon usage) is expressed under the natural sepJ promoter of Anabaena in strain CSVM34 (DsepJ). (F) PCR verification of strain CSSA29 (lane 1) where it can be seen the amplification of sepJ from SC01, while that amplification did not occur with Anabaena wild-type DNA (lane 2). M, base pair markers. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 4 | The SepJ and Fra proteins from marine symbiotic Richelia and Calothrix _______________________________________________________________________ 124 PCC 7120 CSVM34 CSSA14 CSSA29 CSSA28 A B C D E 10 !m 10 !m 10 !m 10 !m 10 !m 10 !m 10 !m Fig. 51. Immunolocalization of SepJ. Localization of SepJ using antibodies against the Anabaena SepJ coiled-coil domain in the different strains supplemented with sepJ from different cyanobacteria. The white arrows indicate the septal zones where the antibody bound, while yellow arrows indicate the lateral zones. (A) Anabaena wild type; (B) strain CSVM34 (DsepJ) used as negative control; (C) CSSA14 (DsepJ::sepJRintHH01); (D) CSSA29 (DsepJ::sepJPCC7120); (E) CSSA28 (DsepJ::sepJCalSC01). 1 2 3 4 5 BG11 PCC 7120 CSVM34 CSSA14 CSSA28 CSSA29 Ng Chl 8 4 2 1 0.5 1 2 3 4 5 BG11 0 8 4 2 1 0.5 1 2 3 4 5 BG11 PCC 7120 CSVM34 CSSA14 CSSA28 CSSA29 Ng Chl 8 4 2 1 0.5 1 2 3 4 5 BG11 0 8 4 2 1 0.5 ng Chl Fig. 52. Growth test on solid medium. The figure shows the growth of Anabaena wild type (PCC 7120), CSVM34 (DsepJ), CSSA14 (DsepJ::sepJRintHH01), CSSA29 (D sepJ::sepJPCC7120) and CSSA28 (D sepJ::sepJCalSC01) in solid medium with a combined nitrogen source (BG11) or without combined nitrogen (BG110). The spots were inoculated with the amounts of Chl indicated below (ng) and the plates were incubated under standard growth conditions for 7-10 days. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 4 | The SepJ and Fra proteins from marine symbiotic Richelia and Calothrix _______________________________________________________________________ 125 The parental strain CSVM34 (DsepJ) produced a very low percentage of long filaments, as previously described (Mariscal et al., 2011), and similar filament lengths were observed with strains CSSA14 and CSSA28 that carried the symbiont genes, whereas only the selfcomplemented strain CSSA29 showed a distribution of filament lengths similar to the wild type (Fig. 53). In conclusion, we did not find any indication of successful expression and function of the sepJ genes from the symbionts in Anabaena. 3.4.3 Expression in Anabaena of symbiont fra genes To clone the fra genes from the symbionts in Anabaena, we chose to try complementation of Anabaena strain CSVT22 that has a deletion of the fraC and fraD genes (Merino-Puerto et al., 2011b). We first tried complementation with fraC-fraD from RintHH01 and, for that, we followed a strategy similar to one described above for sepJ, preparing a construct that contained 500 bp of the Anabaena fraC upstream sequence followed by the symbiont custom-synthesized fraC-fraD genes from RintHH01, all cloned in plasmid vector pCSV3. As in the previous section, for the cloning of the RintHH01 fraC-fraD genes from a custom-synthesized plasmid into plasmid pCSV3, both plasmids were digested with EcoRI, and the insert of the plasmid generated was corroborated by sequencing. This construct allows the integration of the whole plasmid just upstream of the Anabaena fraC locus by a single recombination event, resulting in a genomic A B C 0 20 40 60 80 100 120 PCC 71 20 CSVM34 CSSA14 CSSA14 #5 CSSA28 CSSA29 BG11 48 hours 0 20 40 60 80 100 120 PCC 71 20 CSVM34 CSSA14 CSSA14 #5 CSSA28 CSSA29 BG11 48 hours 0 20 40 60 80 100 120 PCC 71 20 CSVM34 CSSA14 CSSA14 #5 CSSA28 CSSA29 BG11 48 hours 0 20 40 60 80 100 120 PCC7120 CSVM34 CSSA14 CSSA14 #5 CSSA28 CSSA29 BG11 24 hours 0 20 40 60 80 100 120 PCC7120 CSVM34 CSSA14 CSSA14 #5 CSSA28 CSSA29 BG11 24 hours 0 20 40 60 80 100 120 PCC7120 CSVM34 CSSA14 CSSA14 #5 CSSA28 CSSA29 BG11 24 hours 0 20 40 60 80 100 120 PCC712 0 CSVM34 CSSA14 CSSA14 #5 CSSA28 CSSA29 BG11 72 hours 0 20 40 60 80 100 120 PCC712 0 CSVM34 CSSA14 CSSA14 #5 CSSA28 CSSA29 BG11 72 hours 0 20 40 60 80 100 120 PCC712 0 CSVM34 CSSA14 CSSA14 #5 CSSA28 CSSA29 BG11 72 hours 0 20 40 60 80 100 120 PCC712 0 CSVM34 CSSA14 CSSA14 #5 CSSA28 CSSA29 BG11 72 hours 0 - 5 5.01 - 10 10.01 - 15 15.01 - 20 20.01 - 25 25.01 - 30 30.01 - 35 35.01 - 40 40.01 - 45 45.01 - 50 50.01 - 55 55.01 - 60 > 6 0 BG11 72 hours PCC7120 CSVM34 CSSA14 CSSA28 CSSA29 Fig. 53. Filament length in Anabaena strains bearing sepJ from the symbionts. The figure shows the distribution of filaments with the indicated numbers of cells for strains Anabaena wild type (PCC 7120), CSVM34 (DsepJ), CSSA14 (DsepJ::sepJRintHH01), CSSA28 (DsepJ::sepJCalSC01) and CSSA29 (DsepJ::sepJPCC7120). Filaments were inoculated at 1 µg Chl/mL in liquid medium with a source of combined nitrogen (NaNO3) and incubated for 24 h (A), 48 h (B) and 72 h (C) under standard growth conditions. Filaments were taken with great care to determine filament length in micrographs taken under the light microscope. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 4 | The SepJ and Fra proteins from marine symbiotic Richelia and Calothrix _______________________________________________________________________ 126 structure that should allow expression of the heterologous fraC-fraD genes from the native fraC promoter, as well as expression of the last gene in the operon, fraE, from the fraC promoter (Fig. 54A). Following this procedure, Anabaena strain CSSA15 that carries the RintHH01 fraC-fraD genes as shown by PCR analysis was obtained (Fig. 54B). To study whether the heterologous genes were expressed and could result into properly located proteins, we carried out immunofluorescence analysis specifically with antibodies against the extramembrane domain of Anabaena FraD (Merino-Puerto et al., 2011b), since no antibodies for FraC are available. The antibodies detected the FraD protein at the intercellular septa in Anabaena and not in the negative control, the double DfraC DfraD mutant CSVT22 (Fig. 55A and B). Localization was also obtained, partially at the intercellular septa, in two clones of CSSA15 (Anabaena::fraCDRintHH01) that were analyzed, indicating that at least the FraD gene from RintHH01 was produced in Anabaena (Fig. 55C and D). EcoRI EcoRI C.S3 fraD fraC fraE fraF pCSSA22 fraC fraD mutant of Anabaena (CSVT22) Plasmid with the fraC and fraD genes from Richelia Wild type genotype fraDfraC fraE fraF + EcoRI EcoRI C.S3 pCSV3 pCSSA22 EcoRIEcoRI fraDfraC RINTHH13270_FraCD Ap fraDfraC C.S3 fraE fraF FraCD_Rich-2FraCD_Rich-3 2014 bp 5' 3' 5' 3' M 1 2 3 4 2000 1000 1500 A B Fig. 54. Constructs for expression of the fraCD genes from RintHH01 in Anabaena. (A) Steps carried out for the insertion of the fraCD genes from strain HH01 by single recombination with the genome of Anabaena strain CSVT22 (D fraCD). The strain generated, CSSA15, should allow the expression of the Richelia fraCD genes using the natural fraCDE operon promoter of Anabaena. (B) Verification by PCR of some exconjugants. Lanes 1, 2 and 3: DNA from three different clones of CSSA15 which have the insertion of the Richelia fraCD genes as shown by the band of 2014 bp, while lane 4 corresponds to the wild-type DNA with which no amplification was obtained. M, base pair markers. Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 4 | The SepJ and Fra proteins from marine symbiotic Richelia and Calothrix _______________________________________________________________________ 127 As is parental strain CSVT22 (Merino-Puerto et al., 2011b), CSSA15 was however unable to grow diazotrophically (several independent clones tested, see Fig. 56). Additionally, nitrogenase activity under oxic conditions was tested in several clones of CSSA15, but undetectable or very low activity was found (Table 17). These results show that, in spite of detectable production (at least of FraD), FraC and FraD from RintHH01 did not functionally complement the DfraC DfraD mutant of Anabaena. However, we are not certain if the construct strategy was appropriate. For example, in spite of using the native Anabaena promoter, the expression level of RintHH01 fraCD genes could be poor and/or incompatible with fraE of Anabaena. A B C D PCC 7120 CSVT22 CSSA15 Fig. 55. Immunolocalization of FraD. The figure shows immunolocalization analysis carried out with antibodies against FraD. White arrows indicate those polar places where the antibody was attached while yellow arrows show lateral places. (A) Immunolocalization carried out in (A) Anabaena wild type (PCC 7120), (B) strain CSVT22 (DfraC DfraD), (C and D) two clones of strain CSSA15, in which the fraC fraD genes from RintHH01 were inserted into strain CSVT22. Fig. 56. Growth tests on solid medium with different nitrogen sources. (A) Solid BG110 medium supplemented with ammonium (NH4+) as a nitrogen source. (B) Solid BG11 medium (which has NaNO3 as a nitrogen source). In A and B, samples of the wild-type Anabaena (PCC 7120), and strains CSVT22 (DfraC D fraD) and CSSA15 (CSVT22 with the RintHH01 fraC-fraD genes) were inoculated with drops. (C) Solid BG110 medium (no source of combined nitrogen) in which twelve CSSA15 clones were inoculated by streaking. CSVT22 CSSA15 PCC 7120 1 2 3 4 5 6 CSVT22 CSSA15 PCC 7120 1 2 3 4 5 6 A C B 8 4 2 1 0.50.25 8 4 2 1 0.50.25 ng Chl ng Chl Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 4 | The SepJ and Fra proteins from marine symbiotic Richelia and Calothrix _______________________________________________________________________ 128 Strain Nitrogenase activity (nmol ethylene [µg Chl]-1 h-1) Mean ± SD (n) PCC 7120 8.628 ± 5.562 (3) CSVT22 0.048 ± 0.046 (3) CSSA15 #1 0.000 ± 0.008 (3) CSSA15 #2 0.282 ± 0.302 (3) CSSA15 #6 0.012 ; 0.000 (2) Based on these results, we attempted another strategy that consisted in expression of the fraC- fraD genes from RintHH01 or the whole fraC-fraD-fraE cluster from CalSC01 cloned downstream from a strong glnA promoter in a replicative plasmid (pRL3845, based on the pDU1 replicon of heterocystous cyanobacteria). For the construction of plasmid pCSSA50 composed of the custom-synthesized fraC-fraD-fraE cluster from CalSC01 (cloned in a plasmid) and plasmid pRL3845, both plasmids were digested with the same restriction enzymes (SmaI and BamHI) and ligated. On the other hand, for construction of plasmid pCSSA48, it was previously necessary to amplify the fraCD genes from RintHH01 (available in plasmids described above) using primers FraCD_Rich-4 and FraCD_Rich-5 that provided the restriction sequences necessary to perform the ligation between them and the plasmid pRL3845. This same process was performed with the fraCD genes from Anabaena, for which primers alr2392-93-1 and alr2392-93-2 were used. Before performing the conjugation, the inserts of all the synthesized plasmids were corroborated by sequencing. The construction of derivatives of Anabaena mutant CSVT22 (DfraC DfraD) carrying the custom-synthesized symbiont genes, as well as the strain carrying the PCR-amplified Anabaena fraC and fraD genes as positive control, is described in Figure 57. PCR analysis of the exconjugants showed that the following Anabaena strains bore the appropriate constructs: CSSA30, RintHH01 fraC-fraD; CSSA31, Anabaena fraC-fraD (self-complemented strain); CSSA32, CalSC01 fraC-fraD-fraE (Fig. 57). Table. 17. Nitrogenase activity in strain CSSA15 and control strains. Nitrogenase activity was determined under oxic conditions as described in Materials & Methods (section 2.8.3). Mean nitrogenase activity, standard deviation and number of replicates (n) for each strain are shown. The strains studied were: wild-type Anabaena (PCC 7120), CSVT22 (DfraC DfraD) and three clones of CSSA15 (CSVT22::fraC-fraD RintHH01). Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 4 | The SepJ and Fra proteins from marine symbiotic Richelia and Calothrix _______________________________________________________________________ 129 A B C D E F fraDfraC fraE fraF SmaI BamHI Km/Nm pCSSA48 FraCD_Rich-4 FraCD_Rich-5 ≈ 1638 bp SmaI BamHI Km/Nm pRL3845 5' 3' fraDfraC SmaI BamHI + pCSSA48CSVT22 + EcoRIEcoRI fraDfraC RINTHH13270_FraCD Ap FraCD_Rich-4 FraCD_Rich-5 5' 3' fraDfraC fraE fraF 5' 3' Wild type genotype CSSA30 SmaI BamHI Km/Nm pRL3845 fraDfraC fraE fraF 5' 3' Wild type genotype Alr2392-93-1 Alr2392-93-2 fraDfraC SmaI BamHI + pCSSA49CSVT22 + fraE fraF SmaI BamHI Km/Nm pCSSA49 Alr2392-93-1 Alr2392-93-2 ≈ 1619 bp fraDfraC CSSA31 5' 3' SmaI BamHI Km/Nm pRL3845 + pCSSA50CSVT22 BamHISmaI CalSC01_fraCDE Ap 5' 3' fraDfraC fraE + fraE fraF SmaI BamHI Km/Nm pCSSA50 FraC calothrix FraE calothrix ≈ 2192 bp fraDfraC fraE fraDfraC fraE fraF 5' 3' Wild type genotype CSSA32 5' 3' M 1 2 3 1500 2000 2500 M 1 2 3 2000 1000 1500 M 1 2 3 2000 1000 1500 250 Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 4 | The SepJ and Fra proteins from marine symbiotic Richelia and Calothrix _______________________________________________________________________ 130 To study whether the heterologous genes were expressed and could result into properly located proteins, we carried out immunofluorescence analysis specifically with antibodies against the extramembrane domain of Anabaena FraD (Merino-Puerto et al., 2011b). The positive (wild type) and negative (strain CSVT22) controls have been shown in Fig. 55 above. With the new constructs, the antibodies labeled material in the three test strains, CSSA30, CSSA31 and CSSA32, but the labeled material was only occasionally observed at the intercellular septa, even in the self-complemented strain CSSA31 (Fig. 58). Thus, whereas in the wild type 68% of the fluorescence spots were located in septal areas, in the compplemented strains only 27% (CSSA30, CSSA31) and 17% (CSSA32) of the fluorescence spots were located in septal areas. These results show that the constructs are effectively expressed producing substantial amounts of protein, at least in the case of FraD, but perhaps the high expression expected from the glnA promoter produces an excessive amount of protein that affects expected localization. Fig. 57. Constructs for expression of fraCD genes from different cyanobacteria using the glnA promoter in a replicative plasmid in Anabaena. Figures A, C and E show the construction of strains CSSA30, CSSA31 and CSSA32 that express the fraCD genes (fraCDE in strain CSSA32) from different cyanobacteria. These genes are expressed from the Anabaena glnA promoter, represented in the figures with a small red box-arrow, in a cyanobacterial replicative plasmid (pRL4835) in Anabaena strain CSVT22 (DfraCD). (A) Construction of strain CSSA30 using fraCD from RintHH01. Plasmid pCSSA48 was composed by fraCD genes from RintHH01 cloned in plasmid pRL4835. Once the plasmid was made, it was transferred to strain CSVT22. (B) Verification by PCR strain CSSA30 that has Richelia fraCD as shown by the band of 1638 bp in lanes 1 and 3, corresponding to DNA of strain CSSA30 and plasmid RINTHH13270_FraCD respectively. Lane 2 corresponds to Anabaena wild-type DNA where no amplification is seen. (C) Construction of strain CSSA31 using fraCD of Anabaena itself to reconstruct the wild-type genotype. The fraCD genes from Anabaena were cloned in pRL4835 generating plasmid pCSSA49 which was transferred to strain CSVT22. (D) PCR verification with DNA from (1) strain CSSA31, (2) wild-type Anabaena, and (3) pCSSA49, in which a correct band of 1619 bp was detected. In lane 1, there is a band with a size lower than 250 bp that corresponds to the region of the fraCD genes present in the parental strain (CSVT22). (E) Construction of strain CSSA32 using fraCDE of CalSC01. The fraCDE genes were extracted from plasmid CalSC01_fraCDE and introduced into pRL4835, producing plasmid pCSSA50 that was transferred to strain CSVT22. (F) Verification by PCR of strain CSSA32 that has the fraCDE genes of Calothix as shown by the band of 2192 bp in lanes 1 and 3 corresponding to DNA of CSSA32 strain and plasmid CalSC01_fraCDE respectively. Lane 2 corresponds to DNA of wild-type Anabaena where no amplification is seen, as expected. M, base pairs marker. A B C CSSA30 CSSA31 CSSA32 Fig. 58. Immunolocation of FraD in strains carrying the fra gene constructs in a replicative plasmid. Immunolocalization analysis carried out with antibodies against FraD. White arrows indicate those septal places where the antibody was attached while yellow arrows indicate lateral places. (A) CSSA30 (DfraC- DfraD [pCSSA48], with RintHH01 fraC fraD); (B) CSSA31 (DfraC-D fraD [pCSSA49], with Anabaena fraC-fraD); (C) CSSA32 (DfraC-DfraD [pCSSA50], with SC01 fraC-fraD-fraE). Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1
Chapter 4 | The SepJ and Fra proteins from marine symbiotic Richelia and Calothrix _______________________________________________________________________ 131 Growth tests were then performed, showing that the strains grew well in the presence of combined nitrogen (BG11 medium) but that neither CSVT22 nor any of its derivatives could grow diazotrophically (BG110 medium) (Fig. 59). Nonetheless, we note that at the beginning of the incubation in BG110 medium some growth that failed with time was observed in the complemented strains compared to CSVT22 (not shown). We then tested nitrogenase activity under both oxic and anoxic conditions and found that the complemented strains did not show improved nitrogenase activity but, rather, showed decreased activity as compared to strain CSVT22 under anoxic conditions (Table 18). These results suggest that the fra genes cloned under the glnA promoter in the replicative plasmid were indeed expressed, producing a deleterious effect on the physiology of Anabaena, even in the case of the Anabaena genes themselves. PCC 7120 CSVT22 CSSA30 CSSA31 CSSA32 1 2 3 4 5 1 2 3 4 5 BG11 BG11 0 Ng Chl 10 52.5 1.25 0.625 10 52.5 1.25 0.625 PCC 7120 CSVT22 CSSA30 CSSA31 CSSA32 1 2 3 4 5 1 2 3 4 5 BG11 BG11 0 Ng Chl 10 52.5 1.25 0.625 10 52.5 1.25 0.625 ng Chl Fig. 59. Growth test in solid medium with strains carrying the fra gene constructs in a replicative plasmid. The figure shows the growth of Anabaena wild type (PCC 7120) and strains CSVT22 (DfraCDfraD), CSSA30 (DfraC-DfraD [pCSSA48], with RintHH01 fraC fraD), CSSA31 (DfraC-D fraD [pCSSA49], with Anabaena fraC-fraD), and CSSA32 (DfraC-DfraD [pCSSA50], with SC01 fraC-fraD- fraE), in solid medium with a combined nitrogen source (BG11) and in solid medium without combined nitrogen (BG110). The spots were inoculated with the amounts of Chl indicated below and the plates were incubated under standard growth conditions for 10 days. Table. 18. Nitrogenase activity in strains carrying the fra gene constructs in a replicative plasmid. Nitrogenase activity was determined under oxic or anoxic conditions as described in Materials & Methods (section 2.8.3). Mean nitrogenase activity, standard deviation and number of replicates (n) for each strain are shown. The strains studied were: wild-type Anabaena (PCC 7120), CSVT22 (DfraC-D fraD), CSSA30 (DfraC-DfraD [pCSSA48], with RintHH01 fraC fraD), CSSA31 (DfraC-D fraD [pCSSA49], with Anabaena fraC-fraD) and CSSA32 (DfraC-DfraD [pCSSA50], with SC01 fraC fraD fraE). Strain Nitrogenase activity (nmol ethylene [µg Chl] -1 h -1 ) Mean ± SEM (n) Oxic condition Anoxic condition PCC 7120 0.750 ± 0.606 (3) 2.400 ± 1.282 (3) CSVT22 0.026 ± 0.043 (3) 1.448 ± 0.738 (3) CSSA30 0.000 ± 0.000 (3) 0.004 ± 0.008 (3) CSSA31 0.000 ± 0.000 (3) 0.094 ± 0.054 (3) CSSA32 0.000 ± 0.006 (3) 0.018 ± 0.016 (3) SD Código seguro de Verificación : GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 | Puede verificar la integridad de este documento en la siguiente dirección : https://sede.administracionespublicas.gob.es/valida ÁMBITOPREFIJO CSV FECHA Y HORA DEL DOCUMENTO GEISER GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1 12/06/2020 08:18:06 Horario peninsular Nº registro DIRECCIÓN DE VALIDACIÓN O00008745e2000022470 https://sede.administracionespublicas.gob.es/valida GEISER-42b6-8a54-06c6-41ac-9516-66b9-50c5-9ad1