Overexpression of SepJ alters septal morphology and heterocyst pattern regulated by diffusible signals in Anabaena

For Peer Review Overexpression of SepJ alters septal morphology and heterocyst pattern regulated by diffusible signals in Anabaena Journal: Molecular Microbiology Manuscript ID MMI-2016-15872.R1 Manuscript Type: Research Article Date Submitted by the Author: n/a Complete List of Authors: Mariscal, Vicente; CSIC, Instituto de Bioquimica Vegetal y Fotosintesis Nuernberg, Dennis; Queen Mary University of London, School of Biological and Chemical Sciences; Imperial College London, Department of Life Sciences Herrero, Antonia; Consejo Superior de Investigaciones Científicas, Institto de Bioquímica Vegetal y Fotosíntesis; Mullineaux, Conrad; Queen Mary University of London, School of Biological and Chemical Sciences Flores, Enrique; CSIC, Instituto de Bioquimica Vegetal y Fotosintesis Key Words: Bacterial development, Cyanobacteria, Heterocyst differentiation, Intercellular communication, Morphogen Molecular Microbiology For Peer Review 1 Overexpression of SepJ alters septal morphology and heterocyst 1 pattern regulated by diffusible signals in Anabaena 2 3 Vicente Mariscal 1 , Dennis J. Nürnberg 2# , Antonia Herrero 1 , Conrad W. 4 Mullineaux 2 and Enrique Flores 1,* 5 1 Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones 6 Científicas and Universidad de Sevilla, Américo Vespucio 49, E-41092 Seville, Spain, 7 and 2 School of Biological and Chemical Sciences, Queen Mary University of London, 8 London E1 4NS United Kingdom. 9 10 # Current address: Department of Life Sciences, Imperial College London, London SW7 11 2AZ, United Kingdom 12 13 *For correspondence. Tel.: +34954489523; E-mail: [email protected]. 14 15 Running title: SepJ overexpression in Anabaena 16 17 Key words: Bacterial development; Cyanobacteria; Heterocyst differentiation; 18 Intercellular communication. 19 20 21 Page 1 of 44 Molecular Microbiology For Peer Review 2 Summary 22 Filamentous, N 2 -fixing, heterocyst-forming cyanobacteria grow as chains of cells that 23 are connected by septal junctions. In the model organism Anabaena sp. strain PCC 24 7120, the septal protein SepJ is required for filament integrity, normal intercellular 25 molecular exchange, heterocyst differentiation and diazotrophic growth. An Anabaena 26 strain overexpressing SepJ made wider septa between vegetative cells than the wild 27 type, which correlated with a more spread location of SepJ in the septa as observed with 28 a SepJ-GFP fusion, and contained an increased number of nanopores, the septal 29 peptidoglycan perforations that likely accommodate septal junctions. The septa between 30 heterocysts and vegetative cells, which are narrow in wild-type Anabaena, were notably 31 enlarged in the SepJ-overexpressing mutant. Intercellular molecular exchange tested 32 with fluorescent tracers was increased for the SepJ-overexpressing strain specifically in 33 the case of calcein transfer between vegetative cells and heterocysts. These results 34 support an association between calcein transfer, SepJ-related septal junctions and septal 35 peptidoglycan nanopores. Under nitrogen deprivation, the SepJ-overexpressing strain 36 produced an increased number of contiguous heterocysts but a decreased percentage of 37 total heterocysts. These effects were lost or altered in patS and hetN mutant 38 backgrounds, supporting a role of SepJ in the intercellular transfer of regulatory signals 39 for heterocyst differentiation. 40 41 Introduction 42 Filamentous cyanobacteria of the order Nostocales fix dinitrogen in specialized cells 43 called heterocysts, which are found with a spaced pattern of about one heterocyst per 44 ten vegetative photosynthetic cells along the filament (Flores and Herrero, 2010). 45 Heterocysts are formed from vegetative cells when the organism has no source of 46 Page 2 of 44Molecular Microbiology For Peer Review 3 combined nitrogen available (Wolk, 1996). It was proposed that, to attain such spacing, 47 heterocysts or prospective heterocysts (proheterocysts) produce a diffusible inhibitor to 48 prevent the differentiation of neighboring cells into heterocysts (Wilcox et al., 1973; 49 Wolk and Quine, 1976). The patS gene encoding a polypeptide of 17 amino acids that 50 appears to be processed to produce a smaller peptide that inhibits heterocyst 51 differentiation (Yoon and Golden, 1998, 2001; Corrales-Guerrero et al., 2013) has been 52 identified in the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 53 7120 (hereafter Anabaena or wild type [WT]). A synthetic peptide, known as PatS-5, 54 corresponding to the five C-terminal amino acids of PatS (RGSGR) inhibits heterocyst 55 differentiation when added to cultures of Anabaena, suggesting that it could be the 56 inhibitor (Yoon and Golden, 1998, 2001). More recent work has shown, however, that 57 the active inhibitor could correspond to a product consisting of the eight C-terminal 58 amino acids of PatS (Corrales-Guerrero et al., 2013). In Anabaena, the hetN gene 59 encodes a 287-amino acid protein that somehow inhibits heterocyst differentiation and 60 contains an internal ERGSGR sequence (amino acid residues 131 to 136), which is 61 common to the six C-terminal residues of PatS (Callahan and Buikema, 2001; Higa et 62 al., 2012; Corrales-Guerrero et al., 2014a). This internal sequence is needed for HetN to 63 produce inhibition of hetererocyst differentiation, implying that in Anabaena HetN 64 might produce a regulator similar to PatS (Higa et al., 2012; Corrales-Guerrero et al., 65 2014a). Nonetheless, the PatS-like internal sequence is found only in HetN from a few 66 heterocyst-forming cyanobacteria related to Anabaena, and HetN appears to have an 67 additional role in heterocyst biology (Corrales-Guerrero et al., 2014a). 68 Heterocyst differentiation in response to nitrogen deficiency requires two main 69 transcription factors, the global regulator NtcA, which promotes gene expression under 70 high carbon-to-nitrogen balance cellular conditions, and the differentiation-specific 71 Page 3 of 44 Molecular Microbiology For Peer Review 4 HetR protein (Kumar et al., 2010; Herrero et al., 2013). Inhibition of heterocyst 72 differentiation by PatS- and possible HetN-derived products appears to involve 73 inhibition of HetR (Khudyakov and Golden, 2004), and the binding to HetR of different 74 peptides corresponding to C-terminal sequences of PatS has been described (Feldmann 75 et al., 2011, 2012). Indeed, PatS-5 inhibits a DNA-binding activity of HetR (Huang et 76 al., 2004; Valladares et al., 2016). The PatS- and possible HetN-derived products active 77 in inhibition of heterocyst differentiation would be transferred from the producing cells 78 to their neighbors (Yoon and Golden, 1998), where they have been suggested to 79 promote degradation of HetR (Risser and Callahan, 2009). Material reacting with 80 antibodies raised against PatS-5 has been detected in cells proximal to patS-producing 81 proheterocysts, but not in the proheterocysts, consistent with the intercellular transfer of 82 a PatS-derived morphogen (Corrales-Guerrero et al., 2013). The mechanism of transfer 83 is however unknown, although recent data has suggested the involvement of the ABC 84 transporter HetC in export of PatS from the heterocysts (Corrales-Guerrero et al., 85 2014b; Videau et al., 2015). 86 Cyanobacteria bear a Gram-negative type of cell envelope (Han and Schleiff, 87 2014). In filamentous cyanobacteria, whereas each cell is surrounded by its cytoplasmic 88 membrane and peptidoglycan layer(s), the outer membrane is continuous along the 89 filament (reviewed in Han and Schleiff, 2014). Cells in the filament appear to be joined 90 by septal junctions (Mariscal, 2014; Mullineaux and Nürnberg, 2014; Flores et al., 91 2016), previously known as microplasmodesmata or septosomes (Lang and Fay, 1971; 92 Giddings and Staehelin, 1978, 1981; Wilk et al., 2011). These structures traverse the 93 septal peptidoglycan layers through perforations (termed nanopores or channels) that 94 are visible by transmission electron microscopy of whole filaments (Wilk et al., 2011) 95 or isolated murein sacculi (Lehner et al., 2013), or by electron tomography of whole 96 Page 4 of 44Molecular Microbiology For Peer Review 5 filaments (Omairi-Nasser et al., 2014). Proteins that contribute to the formation of 97 septal junctions in Anabaena include SepJ (also known as FraG; Nayar et al., 2007), 98 FraC and FraD (Flores et al., 2007; Merino-Puerto et al., 2010; Nürnberg et al., 2015). 99 Fluorescent tracers, calcein, 5-carboxyfluorescein (5-CF) and the sucrose analogue 100 esculin, have been used to probe intercellular molecular exchange in heterocyst-forming 101 cyanobacteria (Mullineaux et al., 2008; Lehner et al., 2011; Mariscal et al., 2011; 102 Nürnberg et al., 2015). The molecular mass of the largest of the tested fluorescent 103 tracers, calcein, is 622 Da, similar to that of the PatS-5 peptide (532 Da) or of a peptide 104 made of the last eight amino acids of PatS (879 Da). However, calcein is different from 105 these peptides regarding chemical structure and charge, calcein being negatively 106 charged and the PatS peptides being positively charged. Anabaena mutants with sepJ 107 and fraC-fraD deleted show a decreased number of septal peptidoglycan perforations 108 (Nürnberg et al., 2015) and are impaired in the intercellular transfer of those tracers, but 109 whereas fraC and fraD deletion mutants are generally impaired in the transfer of the 110 three tracers, a sepJ deletion mutant is significantly more affected in the transfer of 111 calcein than of 5-CF (Mariscal et al., 2011; Merino-Puerto et al., 2011; Nürnberg et al., 112 2015). Two types of septal junctions may therefore be present in Anabaena, one related 113 to SepJ and another related to FraC and FraD (Merino-Puerto et al., 2011). 114 Based on expression of patS or hetN from a plasmid in vegetative cells and its 115 effect on HetR stability in some mutant backgrounds, it has been suggested that SepJ is 116 involved in intercellular transfer of a HetN-derived signal but not of a PatS-derived 117 regulator (Rivers et al., 2014). On the other hand, in a study of the expression of a hetR-118 gfp fusion along the cells in Anabaena filaments, the pattern of expression (in 119 ammonium-grown filaments or in filaments incubated for short times in the absence of 120 combined nitrogen) was observed to be altered in patS and sepJ deletion mutants 121 Page 5 of 44 Molecular Microbiology For Peer Review 6 suggesting a relation between the products of these genes in the regulation of hetR 122 expression (Corrales-Guerrero et al., 2015). In contrast, no evidence was found in that 123 study for a specific relation of HetN to SepJ. To explore further the possible roles of 124 SepJ in heterocyst-forming cyanobacteria, we introduced a sepJ overexpression 125 construct in several Anabaena mutant backgrounds. The results obtained support a role 126 of SepJ in the formation of septal peptidoglycan nanopores and in septal morphology, as 127 well as in the distribution of the PatS morphogen and, possibly, of a HetN-dependent 128 regulator of heterocyst differentiation along the Anabaena filament. 129 130 Results 131 Anabaena strains overexpressing SepJ 132 Mutation of sepJ in Anabaena causes a pleiotropic phenotype including filament 133 fragmentation, impaired intercellular transfer of calcein, a decrease in the number of 134 septal peptidoglycan nanopores, and an early arrest of heterocyst differentiation (Nayar 135 et al., 2007; Flores et al., 2007; Mullineaux et al., 2008; Mariscal et al., 2011; Nürnberg 136 et al., 2015). To gain further insights into the role of SepJ in multicellularity and 137 heterocyst differentiation, we sought the construction of Anabaena strains with an 138 increased expression of the sepJ gene as compared to the wild-type. For this purpose, 139 the sepJ open reading frame was cloned in a replicative plasmid next to the C.K1 gene 140 cassette, which can be used to promote gene expression (see e.g. Vázquez-Bermúdez et 141 al., 2000). The gene cassette was inserted in both orientations with respect to sepJ (Fig. 142 1A), and the SepJ protein was detected by western blot analysis in Anabaena and strains 143 carrying these plasmids (Fig. 1B). Strain CSDN2a carrying the plasmid bearing the 144 C.K1 gene cassette and the sepJ gene in the same orientation showed 4.1 ± 1.4 (mean ± 145 Page 6 of 44Molecular Microbiology For Peer Review 7 SEM; n = 3)-fold higher levels of SepJ than the wild type. Strain CSDN2b carrying the 146 plasmid bearing the C.K1 gene cassette in opposite orientation to sepJ showed about 147 2.3-fold higher levels of SepJ than the wild type (Fig. 1B). The increased SepJ levels in 148 strain CSDN2b may correspond to the added expression of the sepJ chromosomal copy 149 and of the sepJ copy in the plasmid (which however lacked a specific promoter). The 150 increased SepJ levels in strain CSDN2a should result from the added expression of the 151 chromosomal and plasmid copies of sepJ, the latter expressed from the npt gene 152 promoter in the C.K1 gene cassette. Strain CSDN2a (which is Anabaena carrying the 153 replicative plasmid pCSDN2a that bears the C.K1-sepJ construct) was further 154 characterized. For simplicity, it will be referred to as WT+pSepJ. 155 Strain WT+pSepJ could grow on solid medium with (BG11) or without (BG11 0 ) 156 combined nitrogen, although some impairment of growth was observed in BG11 0 157 medium (Fig. 2A). Growth in liquid medium was also tested as described in 158 Experimental procedures. In BG11 medium, similar growth rate constants were 159 determined for Anabaena and WT+pSepJ, µ = 0.62 ± 0.06 day -1 and 0.63 ± 0.02 day -1 , 160 respectively (mean ± SD, three independent cultures in each case). In BG11 0 medium, 161 the growth rate of the SepJ overexpressing strain was lower than that of the wild type, µ 162 = 0.39 ± 0.02 day -1 and 0.17 ± 0.03 day -1 for Anabaena and WT+pSepJ, respectively 163 (mean ± SD, n = 3; Student’s t test p = 0.001). Microscopic inspection showed the 164 presence of normal, long filaments in the liquid cultures of WT+pSepJ (Fig. 2B). 165 Nitrogenase activity (acetylene reduction) was determined in filaments that had been 166 incubated for 48 in liquid BG11 0 medium. Acetylene reduction assays were carried out 167 under oxic and anoxic conditions as previously described (Burnat et al., 2014). Under 168 oxic conditions, the activity of WT+pSepJ was about 7 % the wild-type activity (0.64 ± 169 0.36 vs. 8.67 ± 1.45 µmol ethylene produced (mg Chl) -1 h -1 ). Assay under anoxic 170 Page 7 of 44 Molecular Microbiology For Peer Review 8 conditions increased nitrogenase activity about twice both in the mutant and in the wild 171 type (1.30 ± 0.37 and 19.69 ± 1.47 µmol (mg Chl) -1 h -1 , respectively). 172 173 Filament structure in strain WT+pSepJ 174 The structure of WT+pSepJ was studied by electron microscopy and compared to wild-175 type Anabaena. Heterocysts from WT+pSepJ showed an unusually wide heterocyst 176 neck, which was observed in all the heterocysts that could be visualized (three 177 specimens shown in Fig. 3; compare with the neck of heterocysts from Anabaena in 178 Fig. 3 and in, e.g., Flores et al., 2007, 2016; Kumar et al., 2010; Omairi-Nasser et al., 179 2014). The wide heterocyst neck implies the presence of a wide heterocyst-vegetative 180 cell septum (Fig. 3). Although not observed in specimens A and B, the cyanophycin 181 plug present in the heterocyst necks in Anabaena could also be found in some 182 heterocysts from WT+pSepJ (Fig. 3, specimen C). In contrast, no alterations could be 183 noted in the septa between vegetative cells. 184 To look further into the septa between vegetative cells and between heterocysts 185 and vegetative cells, we performed labeling with a fluorescently labeled derivative of 186 vancomycin, Van-FL, which binds to regions of the cell that are actively synthesizing 187 peptidoglycan (Daniel and Errington, 2003; Tiyanont et al., 2006; Lehner et al., 2013) 188 and labels well the intercellular septa in Anabaena (see, e.g., Burnat et al., 2014). Van-189 FL fluorescence was readily observed between vegetative cells of both Anabaena and 190 WT+pSepJ (Fig. 4A). Quantification of septal width based on Van-FL labeling revealed 191 the presence of wider septa in WT+pSepJ than in the wild type for BG11-grown 192 filaments (about 1.18-fold wider) and filaments incubated in BG11 0 medium (about 193 1.24- and 1.36-fold wider at 24 and 48 h after nitrogen deprivation, respectively; Fig. 5). 194 It should be noted that the increase in septal width observed under the microscope 195 Page 8 of 44Molecular Microbiology For Peer Review 15 septal peptidoglycan disk of the SepJ-overproducing strain (Fig. 7). Thus, SepJ could be 342 a primer promoting nanopore formation by septal AmiC-type amidases. 343 We have previously suggested the presence in Anabaena of two types of septal 344 junction complexes, one related to SepJ with preference for calcein and another related 345 to FraCD with preference for 5-CF (Merino-Puerto et al., 2011; Nürnberg et al., 2015). 346 Calcein is larger and more negatively charged than 5-CF (Nürnberg et al., 2015). The 347 SepJ-overexpressing strain exhibits an increased intercellular transfer of calcein 348 between vegetative cells and heterocysts, whereas transfer between vegetative cells is 349 not apparently affected (Table 1). Interestingly, transfer of 5-CF is not significantly 350 affected in any case. This implies that SepJ-related septal junctions do not limit calcein 351 or 5-CF transfer between vegetative cells, whereas transfer of calcein between 352 vegetative cells and heterocysts appears limited in the wild type so that it is increased by 353 increasing SepJ levels. This is consistent with the fact that calcein transfer is higher 354 between vegetative cells than between vegetative cells and heterocysts in Anabaena 355 (Mullineaux et al., 2008). The effect of SepJ overexpression specifically on calcein 356 transfer is also consistent with the idea that the SepJ-related septal junction complexes 357 have some preference for calcein. Because sepJ inactivation mutants do not produce 358 mature heterocysts, a role of SepJ in calcein transfer between vegetative cells and 359 heterocysts could not be studied before. Thus, this is the first report showing that SepJ 360 is involved in the molecular exchange between vegetative cells and heterocysts that can 361 be probed with calcein. 362 Overexpression of SepJ also alters heterocyst pattern and leads to a lower level 363 of heterocyst differentiation in WT+pSepJ than in the wild type. Because the 364 intercellular transfer of PatS- (Corrales-Guerrero et al., 2015) or HetN-dependent 365 (Rivers et al., 2014) regulators has been associated with SepJ, we performed epistasis 366 Page 15 of 44 Molecular Microbiology For Peer Review 16 analysis by testing the effect of overexpression of SepJ in ∆patS or ∆hetN genetic 367 backgrounds. The inhibitory effect of SepJ overexpression on the production of 368 heterocysts is not observed in the ∆patS mutant, implying that PatS is needed for this 369 effect to be manifest. Additionally, overexpression of SepJ in the ∆hetN background is 370 accompanied by a decrease in the percentage of heterocysts and an increase in the mean 371 interval size and percentage of heterocyst doublets that was only observed 24 h after N 372 step-down. These observations are consistent with a role of SepJ in the intercellular 373 transfer of the inhibitory PatS morphogen. On the other hand, the decreased mean 374 interval size and increased percentage of heterocyst doublets observed in ∆patS+pSepJ 375 as compared to ∆patS, which were larger at 48 h than at 24 h, imply a role of SepJ in the 376 transfer of a factor other than PatS influencing heterocyst pattern. This factor could be a 377 HetN-dependent regulatory signal (Rivers et al., 2014), which is thought to act later 378 than PatS (Callahan and Buikema, 2001). Consistently, no effects of overexpression of 379 SepJ are observed in the ∆hetN background at 48 h after N step-down. Taken together, 380 the results of our epistasis analysis imply a role of SepJ in the intercellular transfer of 381 the PatS morphogen and, possibly, of the HetN-dependent regulatory signal. 382 Overexpression of SepJ resulted in an increased frequency of heterocyst doublets 383 in the different genetic backgrounds tested, except in ∆hetN+pSepJ at 48 h. This implies 384 a decreased accumulation of the inhibitory signals in the cells contiguous to 385 (pro)heterocysts, which could result from increased transfer among vegetative cells. The 386 increased transfer of calcein specifically between vegetative cells and heterocysts in the 387 SepJ overexpression strain discussed above might be taken to imply that the step of 388 transfer of regulatory signals from the heterocyst to its neighboring vegetative cells is 389 also stimulated by overexpression of SepJ. However, the possibility remains that, in the 390 wild type, the amount of SepJ limits the transfer of calcein between vegetative cells and 391 Page 16 of 44Molecular Microbiology For Peer Review 17 heterocysts but not of the heterocyst regulators between (pro)heterocysts and vegetative 392 cells. In this case, the effect of SepJ overexpression on the heterocyst pattern could 393 result only from an increased transfer of the regulators between vegetative cells. 394 Epistasis analysis similar to that done here for sepJ and patS or hetN has been 395 recently performed for hetC and patS or hetN. The results of these analyses are 396 consistent with the idea that the HetC ABC exporter participates in export from 397 proheterocysts of the PatS morphogen (Corrales-Guerrero et al., 2014b) or of the PatS 398 morphogen and a HetN-dependent signal (Videau et al., 2015). The existence of a 399 transporter specific for export of the regulators from the (pro)heterocysts to their 400 neighboring vegetative cells could make transfer not limited to a SepJ-dependent 401 pathway. Nonetheless, if SepJ participates in the transfer of regulators from the 402 (pro)heterocysts, the activity of a transporter such as HetC could be combined with 403 transfer through SepJ-related septal junctions. Interestingly, a HetC-GFP fusion protein 404 shows a preferential localization at the heterocyst poles (the region known as heterocyst 405 neck), where a SepJ-GFP fusion is also located (Flores et al., 2007; Flores and Herrero, 406 2010; Omairi-Nasser et al., 2015). 407 Based on expression of a P hetR -gfp transcriptional fusion in a sepJ (fraG) 408 background, it has been suggested that SepJ is not needed for proper heterocyst pattern 409 formation (Nayar et al., 2007). However, the results described in this work and those of 410 other recent studies (Corrales-Guerrero et al., 2014b, 2015; Videau et al., 2015) have 411 unraveled possible relations between SepJ and PatS and HetN, as well as between HetC 412 and PatS and HetN, affecting heterocyst pattern formation. Heterocyst differentiation 413 aborts early in sepJ (Flores et al., 2007; Nayar et al., 2007) and hetC (Khudyakov and 414 Wolk, 1997; Xu and Wolk, 2001) inactivation mutants, an effect that could result from 415 accumulation of inhibitory PatS that is not exported from proheterocysts. 416 Page 17 of 44 Molecular Microbiology For Peer Review 18 417 Experimental procedures 418 Strains and growth conditions 419 Anabaena sp. strain PCC 7120 and derived strains were grown in BG11 (containing 420 NaNO 3 ) or BG11 0 (lacking nitrate) medium at 30 ºC in the light (25 µE m -2 s -1 from 421 fluorescent lamps), in shaken (100 r.p.m.) liquid cultures or in medium solidified with 1 422 % (w/v) Difco agar. Antibiotics were used at the following concentrations: Sm, 5 µg 423 ml -1 ; Sp, 5 µg ml -1 and Nm, 50 µg ml -1 . Heterocyst formation was induced by growth in 424 BG11 0 medium. For transfer between media, cells were harvested by centrifugation 425 (3,000 x g) and washed three times with the appropriate medium. 426 Escherichia coli DH5α was used for plasmid constructions, and strains HB101 427 and ED8654 for conjugations with Anabaena strains. They were grown in LB medium, 428 supplemented when appropriate with antibiotics at standard concentrations (Ausubel et 429 al., 2014). 430 431 Construction of mutants 432 The genomic sequence of Anabaena is available (Kaneko et al., 2001). For construction 433 of pCSDN2a and pCSDN2b, genomic DNA from Anabaena was amplified by PCR 434 with primers Alr2338-7 (chromosome coordinates 2818355 - 2818375) and Alr2338-10 435 (chromosome coordinates: 2820781 - 2820764) and cloned in plasmid pMBL-T 436 (Canvax) producing pCSVM58. After verification by sequencing that the amplification 437 product did not contain mutations, the C.K1 resistance cassette (obtained from pRL161 438 digested with BamHI and filled with Klenow enzyme) was inserted into SmaI-ended 439 pCSVM58 plasmid, and clones bearing the two possible orientations of C.K1 with 440 Page 18 of 44Molecular Microbiology For Peer Review 19 respect to sepJ were selected. pCSVM59a bears C.K1 in the same orientation as sepJ, 441 and pCSVM59b bears C.K1 in opposite orientation. C.K1-sepJ was extracted from 442 plasmids pCSVM58a and pCSVM58b and inserted into the self-replicating vector 443 pCSAM200 (Ionescu et al., 2010) using EcoRI restriction sites, producing pCSDN2a 444 and pCSDN2b, respectively. Cassette C.K1 bears the npt gene and its own natural 445 promoter (Elhai and Wolk, 1988). 446 Plasmid pCSDN11 was generated by amplifying sepJ-gfp from genomic DNA 447 of strain CSAM137 (Flores et al., 2007) using primers Alr2338-7 and pRL500-1 (5’-448 ATAGGCGTATCACGAGGC-3’) and inserting the fragment into pSpark (Canvax). 449 Correctness of the inserted sequence was verified by sequencing. The resulting plasmid 450 was digested with SalI and the fragment was inserted into XhoI-digested plasmid 451 pRL278, which cannot replicate in Anabaena (Black et al., 1993). 452 Plasmids were transferred to Anabaena strains by conjugation as previously 453 described (Elhai et al., 1997). pCSDN2a was transferred to Anabaena wild type and 454 strains CSVT20 (∆patS) and CSL7 (∆hetN) with selection for resistance to Sm and Sp 455 producing strains CSDN2a, CSVM20 and CSVM21, respectively. pCSDN2b was 456 transferred to Anabaena with selection for resistance to Sm and Sp producing strain 457 CSDN2b. Strain CSDN11 was generated by transferring pCSDN11 to Anabaena with 458 selection for resistance to Nm. 459 460 Labeling with dyes 461 Staining with vancomycin-FL (Van-FL) was used to visualize septa between cells 462 (Lehner et al., 2013), and was performed in filaments grown in BG11 medium or 463 incubated for 24 or 48 hours in BG11 0 medium as previously reported (Nürnberg et al., 464 Page 19 of 44 Molecular Microbiology For Peer Review 20 2015). Staining was performed mixing 0.1 ml of a filament suspension with 2 µl of a 465 stock solution containing 100 µg Van-FL/ml dimethyl sulfoxide (DMSO) (Van-FL was 466 purchased from Invitrogen). 467 Staining with FM 1-43 was used to visualize the inner and outer cytoplasmic 468 membrane of cyanobacteria (Schneider et al., 2007). For labeling, 2.5 µl FM 1-43 (0.1 469 mg/ml DMSO; Molecular Probes) was added to 0.1 ml of culture. The suspension was 470 incubated for 10 min at room temperature and washed twice with growth medium prior 471 visualization. 472 Alcian Blue was used to visualize the polysaccharide layer of (pro)heterocysts 473 (Corrales-Guerrero et al., 2014). For staining, cell suspensions were mixed (1:2) with a 474 filtered 1 % (w/v) Alcian blue (Sigma) solution in water and visualized under standard 475 light microscopy. 476 477 Growth rates 478 The growth rate constant (µ=[ln2]/t d , where t d is the doubling time) was calculated from 479 the increase in OD 750 nm of shaken liquid cultures. Cultures were inoculated with an 480 amount of cells giving an OD 750 nm of about 0.05 (light path, 1 cm) and grew 481 logarithmically until reaching an OD 750 nm of about 0.8-0.9. The suspensions of 482 filaments were carefully homogenized with a pipette before taking the samples. 483 484 Standard, confocal and fluorescence microscopy 485 For standard light microscopy, filaments grown in BG11 medium (in the presence of 486 Sm and Sp for the mutants) were harvested, washed three times with nitrogen-free 487 (BG11 0 ) medium and resuspended in the same medium without antibiotics. After 24 h 488 Page 20 of 44Molecular Microbiology For Peer Review 21 of incubation at 30ºC in the light, samples were visualized by standard light 489 microscopy. For cell counting, dividing cells were counted as two cells. 490 For confocal microscopy, samples were visualized using a 63X 1.4 NA oil 491 immersion objective attached to a Leica TCS SP2 or Leica SP5 confocal laser-scanning 492 microscope. GFP was excited using 488-nm irradiation from an argon ion laser. 493 Fluorescent emission was monitored by collection across windows of 500-540 nm (GFP 494 imaging) and 630-700 nm or 670-720 nm (cyanobacterial autofluorescence). 495 For fluorescence microscopy, samples were visualized in a Leica DM6000B 496 fluorescence microscope with an ORCA-ER camera (Hamamatsu). Fluorescence was 497 monitored using a FITC L5 filter (excitation, band-pass [BP] 480/40 filter; emission, BP 498 527/30 filter). Images were treated with the Leica Application Suite Advanced 499 Fluorescence software and merged with ImageJ 1.47i software 500 (http://imagej.nih.gov/ij). 501 To correct for the point spread in the analysis of SepJ-GFP (Fig. 6), images were 502 taken with the same settings using Anabaena cells stained with FM 1-43. The full width 503 at half maximum (FWHM) fluorescence was determined in 36 septa and the corrected 504 FWHM of SepJ fluorescence was calculated using the formula FWHM’ = (FWHM 2 - 505 PSF 2 ) -0.5 , where FWHM is the initially observed FWHM of the SepJ fluorescence and 506 PSF is the FWHM of the FM 1-43 fluorescence. 507 508 Electron microscopy 509 Filaments incubated in BG11 0 for 24 h were harvested by gentle centrifugation and 510 fixed with 2.5 % (w/v) glutaraldehyde and 1 % (w/v) OsO 4 , dehydrated with increasing 511 Page 21 of 44 Molecular Microbiology For Peer Review 22 concentrations of acetone, embedded in Spurr resin, and ultra-thin sections were 512 prepared and post-stained with 2 % (w/v) uranyl acetate. The sacculi were isolated and 513 analyzed as described previously (Lehner et al., 2013; Nurnberg et al., 2015). The 514 purified sacculi were deposited on formvar/carbon film coated copper grids, and stained 515 with 1% (w/v) uranyl acetate. All the samples were examined with a ZEISS LIBRA 120 516 PLUS electron microscope at 120 kV. 517 518 Intercellular transfer of calcein and 5-CF 519 For calcein and 5-CF transfer assays, calcein and 5-CF staining and FRAP analysis 520 were performed as previously reported (Mullineaux et al., 2008; Merino-Puerto et al., 521 2011; Nürnberg et al., 2014). Cell suspensions were spotted onto BG11 or BG11 0 agar 522 and placed in a custom-built temperature-controlled sample holder with a glass cover 523 slip on top. All measurements were carried out at 30°C. For both calcein and 5-CF, cells 524 were imaged with a Leica TCS SP5 confocal laser-scanning microscope using a x63 oil-525 immersion objective (NA 1.4) and a 488 nm line argon laser as the excitation source. 526 Fluorescence emission was monitored by collection across windows of 500-527 nm. A 527 maximally opened pinhole of 600 µm, corresponding to an optical section thickness of 528 4.2 µm was used. After an initial image was recorded (pre-bleach), the bleach was 529 carried out by increasing the laser intensity and zooming into the region of interest, and 530 the recovery was recorded. 531 532 533 534 535 Page 22 of 44Molecular Microbiology For Peer Review 23 Acknowledgements 536 Work in Seville was supported by grant BFU2014-56757-P from Plan Nacional de 537 Investigación, Spain, co-financed by the European Regional Development Fund. DJN 538 was supported by a Queen Mary University of London College Studentship and the 539 University of London Central Research Fund. 540 541 Conflict of interest 542 The authors declare that they have no conflict of interest. 543 544 Author Contributions 545 All authors designed research, interpreted data and corrected the manuscript; VM and 546 DJN constructed mutants and acquired and analyzed data; EF wrote the manuscript. 547 548 Page 23 of 44 Molecular Microbiology For Peer Review 24 References 549 Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (2014) 550 Current Protocols in Molecular Biology. New York: Greene Publishing and Wiley-Interscience. 551 Black, T.A., Cai, Y., and Wolk, C.P. (1993) Spatial expression and autoregulation of hetR, a gene 552 involved in the control of heterocyst development in Anabaena. Mol Microbiol 9: 77-84. 553 Burnat, M., Schleiff, E., and Flores, E. (2014) Cell envelope components influencing filament length in 554 the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol 196: 4026-4035. 555 Callahan, S.M., and Buikema, W.J. (2001) The role of HetN in maintenance of the heterocyst pattern in 556 Anabaena sp. PCC 7120. Mol Microbiol 40: 941-950. 557 Corrales-Guerrero, L., Mariscal, V., Flores, E., and Herrero, A. (2013) Functional dissection and evidence 558 for intercellular transfer of the heterocyst-differentiation PatS morphogen. Mol Microbiol 88: 1093-1105. 559 Corrales-Guerrero, L., Mariscal, V., Nürnberg, D.J., Elhai, J., Mullineaux, C.W., Flores, E., and Herrero, 560 A. (2014a) Subcellular localization and clues for the function of the HetN factor influencing heterocyst 561 distribution in Anabaena sp. strain PCC 7120. J Bacteriol 196: 3452-3460. 562 Corrales-Guerrero, L., Flores, E., and Herrero, A. (2014b) Relationships between the ABC-exporter HetC 563 and peptides that regulate the spatiotemporal pattern of heterocyst distribution in Anabaena. PLoS One 9: 564 e104571. 565 Corrales-Guerrero, L., Tal, A., Arbel-Goren, R., Mariscal, V., Flores, E., Herrero, A., and Stavans, J. 566 (2015) Spatial fluctuations in expression of the heterocyst differentiation regulatory gene hetR in 567 Anabaena filaments. PLoS Genet 11: e1005031. 568 Daniel RA, and Errington J. (2003) Control of cell morphogenesis in bacteria: two distinct ways to make 569 a rod-shaped cell. Cell 113: 767-776. 570 Dunn, J.H., and Wolk, C.P. (1970) Composition of the cellular envelopes of Anabaena cylindrica. J 571 Bacteriol 103: 153-8. 572 Elhai, J., and Wolk, C.P. (1988) A versatile class of positive-selection vectors based on the nonviability 573 of palindrome-containing plasmids that allows cloning into long polylinkers. Gene 68: 119-138. 574 Elhai, J., Vepritskiy, A., Muro-Pastor, A.M., Flores, E., and Wolk, C.P. (1997) Reduction of conjugal 575 transfer efficiency by three restriction activities of Anabaena sp. strain PCC 7120. J Bacteriol 179: 1998- 576 2005. 577 Feldmann EA, Ni S, Sahu ID, Mishler CH, Risser DD, Murakami JL, Tom SK, McCarrick, RM, Lorigan, 578 GA, Tolbert BS, Callahan SM, Kennedy MA (2011) Evidence for direct binding between HetR from 579 Anabaena sp. PCC 7120 and PatS-5. Biochemistry 50: 9212-9224. 580 Feldmann, E.A., Ni, S., Sahu, I.D., Mishler, C.H., Levengood, J.D., Kushnir, Y., McCarrick, R.M., 581 Lorigan, G.A., Tolbert, B.S., Callahan, S.M., and Kennedy, M.A. (2012) Differential binding between 582 PatS C-terminal peptide fragments and HetR from Anabaena sp. PCC 7120. Biochemistry 51:2436-2442. 583 Flores, E., and Herrero, A. (2010) Compartmentalized function through cell differentiation in filamentous 584 cyanobacteria. Nat Rev Microbiol 8: 39-50. 585 Flores, E., Pernil, R., Muro-Pastor, A.M., Mariscal, V., Maldener, I., Lechno-Yossef, S., Fan, Q., Wolk, 586 C.P., and Herrero, A. (2007) Septum-localized protein required for filament integrity and diazotrophy in 587 the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol 189: 3884-3890. 588 Flores, E., Herrero, A., Forchhammer, K., and Maldener, I. (2016) Septal junctions in filamentous 589 heterocyst-forming cyanobacteria. Trends Microbiol 24: 79-82. 590 Frías, J.E., Flores, E., and Herrero, A. (2000) Activation of the Anabaena nir operon promoter requires 591 both NtcA (CAP family) and NtcB (LysR family) transcription factors. Mol Microbiol 38: 613-625. 592 Page 24 of 44Molecular Microbiology For Peer Review 31 Figure 8. Heterocyst pattern in wild-type Anabaena and mutant strains. 787 Filaments of the indicated strains from BG11 medium were washed with BG11 0 788 medium, resuspended in BG11 0 medium and incubated for 24 or 48 hours, as indicated. 789 Cells were counted after staining with Alcian Blue. 117 to 252 intervals (except for 790 CSVM21 at 24 h, 20 intervals) were counted. Interval zero corresponds to contiguous 791 heterocysts. 792 793 Page 31 of 44 Molecular Microbiology For Peer Review 32 Table 1. Intercellular transfer of calcein and 5-carboxyfluorescein (5-CF) in WT+pSepJ 794 and Anabaena (WT). 795 796 Student's t-tests show that R is significantly different for calcein between (3a) and (3b) 797 (p = 0.021), marked with an asterisk. No significant differences were detected for 798 calcein or 5-CF between (1a) and (1b) or (2a) and (2b), as well as for 5-CF between (3a) 799 and (3b). 800 801 Measurement (number of replicates) Calcein 5-CF R [s - 1 ] (± SD) R [s - 1 ] (± SD) 1. Vegetative cells in presence of nitrate a) WT (35/41) 0.083 ± 0.032 0.133 ± 0.049 b) WT+pSepJ (34/18) 0.075 ± 0.031 0.110 ± 0.052 2. Vegetative cells 48 h after nitrogen deprivation a) WT (21/19) 0.102 ± 0.025 0.101 ± 0.052 b) WT+pSepJ (24/18) 0.109 ± 0.039 0.094 ± 0.033 3. Heterocysts 48 h after nitrogen deprivation a) WT (15/5) 0.022 ± 0.013 0.027 ± 0.012 b) WT+pSepJ (10/10) * 0.038 ± 0.021 0.030 ± 0.024 Page 32 of 44Molecular Microbiology For Peer Review 33 Table 2. Heterocyst pattern in wild-type Anabaena and mutant strains. 802 803 Filaments from BG11-grown cultures were incubated in BG11 0 medium (without 804 combined N) for 24 or 48 h, and samples were taken and stained with Alcian blue to 805 facilitate the identification of heterocysts. Heterocysts and vegetative cells from two to 806 four different cultures from each strain and time of incubation were counted. % Hets, 807 percentage of heterocysts ± SD of the mean in 4 to 14 different microscopy fields; 106 808 to 576 cells, including heterocysts that were separated from filaments, were counted per 809 field. Interval size refers to the number of vegetative cells between heterocysts, which 810 could be determined only in filaments containing at least two heterocysts (117 to 252 811 intervals were counted for each strain and incubation time, except for ∆hetN+pSepJ at 812 24 h, 20 intervals). Mean interval size (± SD) corresponds to intervals ≥ 1 vegetative 813 cell, and percentage of heterocyst doublets corresponds to the percentage of interval 814 sizes with a value of 0 (both parameters were deduced from the same data represented 815 in Fig. 8). For the percentage of heterocysts and mean interval size, the Student’s t test p 816 value for the comparison of each SepJ-overproducing strain and its parental strain is 817 indicated in parenthesis. For the percentage of heterocyst doublets, the actual figures 818 were classified into two categories (interval = 0; interval > 0), and the distribution in 819 each SepJ-overproducing strain was compared to that in its parental strain using the X 2 820 test (p given in parenthesis). N.A., not applicable because of a zero value in the wild 821 type. 822 823 Strain Genotype 24 h without combined N 48 h without combined N Heterocysts (%) Mean interval size (cells) Heterocyst doublets (%) Heterocysts (%) Mean interval size (cells) Heterocyst doublets (%) PCC 7120 WT 7.4 ± 2.0 13.2 ± 3.9 0 12.1 ± 2.4 13.2 ± 4.7 1.5 WT+pSepJ PCC 7120 (pCSAM200::C.K1 -sepJ) 5.0 ± 1.6 (0.002) 18.0 ± 7.8 (<10 -3 ) 3.8 (N.A.) 8.3 ± 1.6 (0.001) 11.0 ± 4.1 (<10 -3 ) 8.4 (<10 -3 ) CSVT20 ∆ patS 15.4 ± 2.3 9.4 ± 3.8 13.4 17.9 ± 2.2 9.5 ± 4.3 13.2 ∆ patS+pSepJ CSVT20 (pCSAM200::C.K1 -sepJ) 16.3 ± 3.3 (0.482) 8.8 ± 3.9 (0.206) 19.8 (<10 -3 ) 22.3 ± 8.0 (0.213) 8.3 ± 5.1 (0.028) 23.4 (<10 -3 ) CSL7 ∆ hetN 11.5 ± 1.3 10.8 ± 3.3 6.8 13.5 ± 1.9 10.2 ± 4.6 16.2 ∆ hetN+pSepJ CSL7 (pCSAM200::C.K1 -sepJ) 2.9 ± 1.4 (<10 -3 ) 15.4 ± 5.0 (<10 -3 ) 10.0 (<10 -3 ) 10.2 ± 3.1 (0.077) 10.1 ± 4.0 (0.887) 14.0 (0.348) Page 33 of 44 Molecular Microbiology For Peer Review A C.K1 sepJ C.K1 promoter sepJ C.K1 promoter C.K1 WT+pSepJ, ΔpatS+pSepJ, ΔhetN+pSepJ CSDN2b B WT WT+pSepJ CSDN2b SepJ CΔpatS ΔpatS+ pSepJ ΔhetN+ pSepJ ΔhetN Page 34 of 44Molecular Microbiology For Peer Review BG11 BG110 WT WT+pSepJ ΔpatS+pSepJ ΔpatS ΔhetN+pSepJ ΔhetN WT+pSepJWT A B Page 35 of 44 Molecular Microbiology For Peer Review Sec_2 A Sec_1 A Sec_1 Sec_2 B C C CG CG WT CG Het Het Het Het Het Het Page 36 of 44Molecular Microbiology For Peer Review WT, BG110 WT, BG110 WT+pSepJ, BG110 WT+pSepJ, BG110 WT, BG11 WT+pSepJ, BG11 A B Het Het Het Het Het Het Page 37 of 44 Molecular Microbiology For Peer Review Septal length (µm) Percentage of counted intervals 0,00 0,50 1,00 1,50 2,00 0 5 10 15 20 25 0,1 0,4 0,7 11,3 1,6 1,9 2,2 2,5 2,8 3,1 3,4 3,7 BG11 CSDN2a 7120 1.92 ±0.44 2.27 ±0.51 2 1.6 1.2 0.8 0.4 0 0,00 0,50 1,00 1,50 2,00 0 5 10 15 20 25 0,1 0,4 0,7 11,3 1,6 1,9 2,2 2,5 2,8 3,1 3,4 3,7 BG11024 h 1.55 ±0.42 1.93 ±0.33 2 1.6 1.2 0.8 0.4 0 0,00 0,50 1,00 1,50 2,00 0 5 10 15 20 25 0,1 0,4 0,7 11,3 1,6 1,9 2,2 2,5 2,8 3,1 3,4 3,7 BG11048 h 1.42 ±0.30 1.94 ±0.46 2 1.6 1.2 0.8 0.4 0 022222222222222222222122222222222222222222222222222222222222222222322222222222222224222222222222222222222 022222222222222222222122222222222222222222222222222222222222222222322222222222222224222222222222222222222 022222222222222222222122222222222222222222222222222222222222222222322222222222222224222222222222222222222 WT+pSepJ WT Page 38 of 44Molecular Microbiology For Peer Review CSDN11 (SepJ-GFP, C.K3-sepJ) CSAM137 (SepJ-GFP) BC D A C.K3 C.K3 promoter sepJ pCSDN11 sepJ-gfp width [µm] Fluorescenceintensity [a.u.] Page 39 of 44 Molecular Microbiology For Peer Review A B C WT+pSepJ WT Page 40 of 44Molecular Microbiology