Microbiological study of the Añana Salt Valley. Diversity analysis, isolation and screening of bioactivecompounds producers

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Immunology, Mic obiology, and Pa asi ology Depa men
Immunologia, Mik obiologia e a Pa asi ologia Saila
Doc o al hesis/Dok o ego esia
Maia Azpiazu Muniozgu en
2025
Mic obiological s udy o he Añana Sal Valley.
Di e si y analysis, isola ion and sc eening o bioac i e
compounds p oduce s
Añanako Ga z Ha ana en az e ke a mik obiologikoa.
Dibe si a ea en analisia, isolamendua e a konposa u
bioak iboen baheke a
Supe iso s/Zuzenda iak:
D . Ila gi Ma ínez Balles e os
D . Ja ie Ga aiza Candina
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(cc) 2025 Maia Azpiazu Muniozgu en (cc by-nc-nd 4.0)

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FUNDING / FINANTZIAZIOA
The p esen Doc o al hesis has been ca ied ou hanks o he ollowing
unding:
Dok o ego esi hau ondo engo lagun zei eske egin da:
Ag eemen /Hi za mena. Seguimien o espacio- empo al de a iables
ambien ales en el espacio del Valle Salado. Funding En i y/ E akunde
inan za zailea: Fundación Valle Salado. IP: Iñaki An iguedad. 01/01/2017-
31/12/2025.
P ojec / P oiek ua. Es udio de la di e sidad mic obiana en el seguimien o
espacio- empo al de a iables ambien ales en el espacio del Valle Salado
de Añana (US19/01). Funding En i y/ E akunde inan za zailea: Uni e si y
o he Basque Coun y UPV/EHU and Biogene ics. IP: Ila gi Ma inez
Balles e os. 29/11/2019-28/11/2021.
P ojec / P oiek ua. Mik oIke : Mic obial genomics and cha ac e isa ion
(GIU21/021). Funding En i y/ E akunde inan za zailea: Uni e si y o he
Basque Coun y UPV/EHU. IPs: I a i Ma ínez Malax-e xeba ia, Ila gi
Ma ínez Balles e os. 01/01/2022-31/12/2025.
P ojec / P oiek ua. P oducción y ca ac e ización de biosu ac an es po
p oca io as haló ilas del Valle Salado de Añana (PIBA23/06). Funding
En i y/ E akunde inan za zailea: Basque Go e nmen /Eusko Jau la i za.
IP: I a i Ma ínez Malax-e xeba ia. 26/07/2023-30/06/2025.
PhD ellowship om he Uni e si y o The Basque Coun y UPV/EHU /
UPV/EHUn ike zaileak p es a zeko kon a azio deialdia (PIF22/12).
A esea ch s ay a he Fundación MEDINA (G anada, Spain) was suppo ed
by a complemen a y g an om he Uni e si y o he Basque Coun y
UPV/EHU / UPV/EHUn mugiko asuna sus a zeko e a ike ke a en
emai zak heda zeko lagun za deialdia.
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This esea ch p ojec has been app o ed by he E hics Commi ee o Resea ch
on Biological Agen s and Gene ically Modi ied O ganisms o he UPV/EHU
(ABIEB-UPV/EHU) (M30/2020/181).
Ike ke a p oiek u honek UPV/EHUko Agen e biologikoen e a gene ikoki
e alda u ako o ganismoen ike ke e a ako E ika Ba zodea en (ABIEB-
UPV/EHU) aldeko xos ena dauka (M30/2020/181).
The au ho s would like o hank he edi o s o pe mission o include he
published a icles in his Doc o al hesis.
Egileek eske ak eman nahi dizkie e edi o eei a gi a a u ako a ikuluak
Dok o ego esi hone an sa zeko baimena ema eaga ik.
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SCIENTIFIC CONTRIBUTIONS / EKARPEN ZIENTIFIKOAK
Publica ions / A gi alpenak
 Azpiazu-Muniozgu en M, Valgañón-Pé ez E, Ga cía‑Ma ínez M,
Rod iguez-Paniagua A, Poppy Cla k H, Jus icia C, Ma ín J, de la C uz
Mo eno M, Reyes F, Lao den L, Ma inez-Malaxe xeba ia I, Ma inez-
Balles e os I. Isola ion and sc eening o bioac i e compounds p oduced by
halophiles and cha ac e izing compounds p oduced by Pseudoal e omonas
spp. ASV78. Unde e iew.
 Azpiazu-Muniozgu en M, Ga cía-Ma ínez M, Zabale a A, An iguedad I,
Ga aiza J, Lao den L, Ma inez-Malaxe xeba ia I, Ma inez-Balles e os I.
P oka yo ic Di e si y and Communi y Dis ibu ion in he Complex
Hyd ogeological Sys em o he Añana Con inen al Sal e n. Mic obial
Ecology. 2024;87(1):171. DOI: h ps://doi.o g/10.1007/s00248-025-
02488-2.
 Azpiazu-Muniozgu en M, Ga cía M, Lao den L, Ma inez-Malaxe xeba ia
I, Seoane S, Bikandi J, Ga aiza J, Ma ínez-Balles e os I. Anianabac e
salinae gen. no ., sp. no . ASV31T, a acul a i e alkaliphilic and ex emely
halo ole an bac e ium isola ed om b ine o a millennial con inen al
sal e n. Di e si y. 2022;14(11):1009.
DOI:h ps://doi.o g/10.3390/d14111009.
 Ma inez-Balles e os I, Azpiazu-Muniozgu en M, Ma ínez Malax-
e xeba ia I, Lao den L, Ga aiza J. Ca ac e ización de la di e sidad
mic obiana del agua de Salinas de Añana median e cul i o y genómica
(Spain). Book / Libu ua: Se en millennia o sal making. Edi o : Albe o
Pla a. Fundación Valle Salado. ISBN: 978-84-7821-936-0.
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La pe sona ha de ene la su icien e e en sí misma pa a emp ende
a en u as, y duda de sí misma lo su icien e pa a dis u a las.
G. K. Ches e on
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ESKER ONAK / AGRADECIMIENTOS
Guz iak dauka hasie a ba e a amaie a ba . Hi z hauek idaz e akoan a ze a z
begi a u e a pe sona, momen u zein bizipen mo do ba eki opo egi en du , e a
nola ez, posez be e zen naiz. Ezinezkoa bada e e le o gu xi haue an ni e eske
ona demos a zea, jakin ezazue momen u hone a a i is eko zuek guz iok izan
za e ela ga an zi suak. Be az, eske ik asko denoi.
Lehenik e a behin eske ak eman nahi dizkie ni e esi zuzenda iei, Ila gi e a
Ja i, lan hau au e a e ama eko nigan ja i zenu en kon ian za e a bide hone an
zeha eskaini didazuen lagun zaga ik. Be eziki, eske ik asko Ila gi, denbo a
guz i hone an zeha ge u ego ea en, mila oz opo i au e egi en e a pe sona
zein zien ziala i e edu iza en. E a nola ez Ja i, eske ik asko hasie a ik alde
gi oa sus a zea en, ilusioa emanez ike la i gaz eoi “bu u bela i equipo” behin
e a be i o esanez.
Eske ik asko bai a Mik oIke ike ke a aldea osoa i e e, lanean e a es o suan
oina i u a ilusioz be e iko ike ke a alde ba en so e a en pa e iza ea
ahalbide u duzuelako. Be i izango zai uz e gogoan labo a egian momen u ik
poli enak psa zea posible egin duzuen guz iok. Asko baza e e e e eske ik asko
bene an zue ako bakoi za ezagu zeko e a zuek gaindik ikas eko eman didazuen
auke aga ik. Modu be ean, Immunologia, Mik obiologia e a Pa asi ologiako
sail osoa i, emandako baliabide, a u e a ge uko asunaga ik. Azkenik, nola ez,
Añanako Ga z Ha ana Fundazioa i, pa e ha u dudan ike ke a p oiek uan
egindako apus uaga ik e a zuekgandik jaso ako p es u asunaga ik.
Po o o lado, cómo no dedica les unas palab as de o al ag adecimien o a odo
el equipo de la Fundación MEDINA. En pa icula muchas g acias Fe nando,
po habe me dado la opo unidad i y de ap ende de oso os. G acias a odo
el equipo, po habe me acogido an bien, empezando po mic o, pasando po
química has a sc eening me habéis ayudado en odo momen o y de e dad que
gua do un muy buen ecue do de odos oso os. Me cedes, cómo no
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ag adece e po esas cha las y isas, momen os ue a del labo a o io y bueno
simplemen e po se asi como e es. ¡Muchísimas g acias a odos!
Mila eske , ama e a ai a p oiek u honi au e egi eko auke a, medioak e a
animoak ema eaga ik. Bai a Txiki i e e, hi zik gabe konek a zeko e a be i
animoa al xa zeko gai asuna iza ea en. E a nola ez, Jon, zu gabe ez nin zen
p oiek u honen hasie a a i i siko, be az eske ik asko bide hone an zeha
ikasle-i akasle olak pa eka uz be i ge u ego ea en. Eske ik asko neskak,
be iko lagunak, denpo a hone an zeha izan duzuen ule pena e a
pazien ziaga ik, deskonek a zeko eman didazuen auke a bakoi zaga ik e a
zuekgandik jaso dudan be o asunaga ik. Amai zeko, guz ion gaine ik dagoen
Jaun Goikoa i, eske ik asko, denbo a e a pe sonez balia uz bidea ma ka u e a
be i sos engu iza eaga ik.
Mila eske .
Muchas g acias.
Maia Azpiazu Muniozgu en
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LIST OF ABBREVIATIONS / LABURDUREN ZERRENDA
English
Euske a
AAI
A e age Amino Acid Iden i y
Aminoazidoen ba ez bes eko
iden i a ea
ANI
A e age Nucleo ide Iden i y
Nukleo idoa en ba ez bes eko
iden i a ea
ASV
Amplicon Sequence Va ian
Anplikoien sekuen zien aldae a
BGCs
Biosyn he ic gene clus e s
Gene-mul zo biosin e ikoak
BPM
Biosu ac an /bioemulsi ie
p oduc ion medium
Biosu ak an ea/bioemul si ika-
zailea ekoiz eko medioa
CDSs
P o ein coding genes
P o einak kode zen di uz en
geneak
CECT
Spanish Type Cul u e Collec ion
Espainiako mik oo ganismo
ipo-en bilduma
CFS
Cell- ee supe na an
Zelula ik gabeko gainjalkina
dDDH
Digi al DNA-DNA hyb idiza ion
DNA-DNA hib idazio digi ala
DSMZ
Deu sche Sammlung on
Mik oo ganismen und
Zellkul u en
Alemaniako mik oo ganismoen
e a zelula-kul u en bilduma
EI24
Emulsi ica ion index (%)
Emul si ikazio indizea (%)
EPS
Exopolysaccha ides
Exopolisaka idoak
EUCAST
Eu opean Commi ee on
An imic obial Suscep ibili y
Tes ing
Mik obioen au kako
sen iko asun-p oben Eu opako
Ba zo dea
FAO
Food and Ag icul u e
O ganiza ion
Elikadu a e a Nekaza i za
E akundea
FDA
Food and D ug Adminis a ion
Elikagaien e a medikamen uen
adminiz azioa
GIAHS
Globally Impo an Ag icul u al
He i age Sys em
Mundu mailan ga an zi sua den
Nekaza i za Onda e Sis ema
HPLC
High-Pe o mance Liquid
Ch oma og aphy
Be eizmen al uko likido
k oma og a ia
ICNP
In e na ional Code o
Nomencla u e o P oka yo es
P oka io oen Nomenkla u a en
Nazioa eko Kodea
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IR
In a ed spec oscopy
Espek oskopia in ago ia
iTOL
In e ac i e T ee O Li e
Bizi za en zuhai z in e ak iboa
ITS
Ribosomal in e nal ansc ibed
space
T ansk iba u ako
ba ne-espazia zaile
e ibosomikoa
LB
Lu ia-Be ani cul u e media
Lu ia-Be ani hazkun za medioa
LC-HRMS
Liquid ch oma og aphy high
esolu ion mass spec ome y
K oma og a ia likidoa be eizmen
handiko masa-espek ome ia
MA
Ma ine aga cul u e media
I sas aga a hazkun za medioa
MB
Ma ine b o h cul u e media
I sas salda hazkun za medioa
MDM
"Mic obial Da k Ma e "
Mik obio-ma e ia iluna
EOR
Enhanced oil eco e y
Lagundu ako olioa en
be esku apena
MH
Mülle -Hin on cul u e media
Mülle -Hin on hazkun za medioa
MIC
Minimum Inhibi o y
Concen a ion
Kon zen azio minimo
inhibi zailea
MRSA
Me hicillin- esis an
S aphylococcus au eus
S aphylococcus au eus
me ilzilina e esis en ea
MS
Mass spec a
Masa espek oa
NCBI
Na ional Cen e o
Bio echnology In o ma ion
Bio eknologiako In o mazio
Zen o Nazionala
NGS
Nex Gene a ion Sequencing
Hu engo belaunaldiko
sekuen ziazioa
NMR
Nuclea Magne ic Resonance
E esonan zia magne iko
nuklea a
OGRI
O e all Genomic Rela edness
Index
E lazio-indize genomiko
o oko a
PCR
Polyme ase chain eac ion
Polime asa en ka e-e eakzioa
PHA
Polyhyd oxyalkanoa e
Polihid oxialkanoa oa
PICRUS 2
Phylogene ic In es iga ion o
Communi ies by Recons uc ion
o Unobse ed S a es
Komuni a een ike ke a
ilogene ikoa beha u gabeko
egoe ak be e aikiz
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POCP
Pe cen age O Conse ed
P o eins
Kon se ba u ako p o einen
ehunekoa
QIIME2
Quan i a i e Insigh s In o
Mic obial Ecology
Mik obio-ekologia i bu uzko
ikuspegi kuan i a iboak
RAST
Rapid Anno a ion wi h
Subsys ems Technology
Ano azio azka a azpisis emen
eknologia ekin
R
Re en ion ime
E e en zio denbo a
SDA
Sabou aud dex ose aga
Sabou aud dex osa aga a
SDS
Sodium dodecyl sulpha e
Sodio dodezil sul a oa
SGIke
Ad anced Resea ch Facili ies
Ike kun za ako Ze bi zu
O oko ak
TLC
Thin Laye Ch oma og aphy
Ge uza ineko k oma og a ia
TYGS
Type S ain Genome Se e
Andui ipoen genoma ze bi za ia
UV
Ul a iole
Ul amo ea
WGS
Whole genome sequencing
Genoma osoa en sekuen ziazioa
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TABLE OF CONTENTS / EDUKIEN TAULA
ENGLISH VERSION
Lis o igu es ................................................................................ I
Lis o ables ................................................................................. V
Abs ac ..................................................................................... VII
Sec ion 1 - O e iew .................................................................................... 1
GENERAL INTRODUCTION ................................................................ 3
THEORETICAL FRAMEWORK ........................................................... 5
HYPOTHESES AND AIM OF THE STUDY ....................................... 26
METHODOLOGY ................................................................................. 28
SUMMARY OF THE RESULTS AND DISCUSSION ........................ 51
REFERENCES ..................................................................................... 105
Sec ion 2 - Conclusions ........................................................................... 127
EUSKARAZKO BERTSIOA
I udien ze enda .......................................................................... XI
Taulen ze enda ......................................................................... XV
Labu pena ............................................................................... XVII
1. a ala - Ikuspegi o oko a .................................................................... 131
SARRERA OROKORRA .................................................................... 133
ESPARRU TEORIKOA ...................................................................... 135
HIPOTESIAK ETA HELBURUA ....................................................... 157
METODOLOGIA ................................................................................ 159
EMAITZEN LABURPENA ETA EZTABAIDA ................................ 183
ERREFERENTZIA ITURRIAK .......................................................... 238
2. a ala - Ondo ioak ................................................................................ 239
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Abs ac
VII
Abs ac
The Añana Sal Valley, in no he n Spain, is a con inen al sal e n loca ed on a
diapi ic s uc u e and ed by na u al hype saline sp ings ha ha e been
p oducing sal o mo e han 7,000 yea s. Despi e i s ecognised his o ical and
geological impo ance, he mic obial inhabi an s o his habi a ha e emained
la gely unde s udied.
In his Doc o al hesis, 16S RNA gene and ITS amplicon sequencing was
conduc ed o s udy he p oka yo ic and ungal communi ies p esen in di e en
wa e sou ces in he alley. In pa allel, halophilic and halo ole an bac e ia we e
isola ed and iden i ied o es ablish a s ain collec ion. A polyphasic
cha ac e isa ion was conduc ed on a numbe o he isola es ha showed
po en ial o be new axa, wi h he aim o p o iding a desc ip ion o hem.
Finally, he an imic obial ac i i y, he educ ion o su ace ension and he
emulsi ying capaci y o he isola es we e sc eened in o de o iden i y hose ha
we e capable o p oducing he bioac i e compounds o in e es . In addi ion, a
mo e de ailed cha ac e isa ion o he compounds p oduced by he ASV78 s ain
was conduc ed.
The genomic app oach e ealed he exis ence o wo dis inc mic obial
communi ies, one om sal y wa e s (∼200 g/L) and one om b ackish wa e s
(≤20 g/L). Salini y was shown o be he key en i onmen al ac o de e mining
mic obial composi ion, al hough niche-speci ic a ia ions we e also obse ed.
A chaeal axa, in pa icula he phylum Me hanobac e io a, la gely ep esen ed
by he genus Halo ub um, p edomina ed in sal y wa e s, whe eas bac e ial axa,
including Pseudomonado a, we e widesp ead, especially in b ackish wa e s.
Fungal di e si y, s udied o he i s ime in his con inen al sal e n, e ealed
he ubiqui y o he genus Saccha omyces, linked o en i onmen al ac o s such
as p oximi y o ag icul u al land.
In addi ion, a collec ion o 150 halophilic and halo ole an isola es was
gene a ed, wi h Pseudomonado a being he phylum wi h he highes numbe o
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Abs ac
VIII
isola es. Among hese, wo po en ial new bac e ial isola es we e inally ound
o ha e di e en genomic, pheno ypic and chemo axonomic cha ac e is ics
compa ed o p e iously desc ibed axa. One new species,
Al e ipon ixan hobac e mu iae sp. no . ( ype s ain SALINAS58T), was ound
o be a new membe o he E y h obac e aceae amily. On he o he hand, a
new genus, Anianabac e salinae gen. no . sp. no . ( ype s ain ASV31T), was
ound o be a new membe o he amily Pa acoccaceae.
Finally, bioassays iden i ied 20 an imic obial-p oducing isola es, 26 e ec i e
biosu ac an -p oducing isola es and 19 bioemulsi ie -p oducing isola es.
Membe s o he genus Pseudoal e omonas we e ound o be mainly esponsible
o he an ibac e ial ac i i y, and membe s o he genus Halomonas o su ace
ension educ ion and emulsi ying ac i i ies. One isola e, Pseudoal e omonas
sp. ASV78, was ound o syn hesizing bo h, an imic obial and su ace-ac i e
compounds. Fu he analysis o he s ain ASV78 showed ha he an ibac e ial
ac i i y agains G am-posi i e bac e ia was ela ed o he p oduc ion o
pen ab omopseudilin and b omophene, and ha he an ibac e ial ac i i y
agains G am-nega i e bac e ia was exclusi ely ela ed o he p oduc ion o
pen ab omopseudilin. O pa icula in e es was low concen a ion o
pen ab omopseudilin a which i is e ec i e agains S aphylococcus au eus
ATCC 29213. Rega ding he su ace-ac i e compounds p oduced by s ain
ASV78, i was ound ha educ ion in su ace ension was associa ed wi h
glycolipid- ype biosu ac an p oduc ion, and he abili y o emulsi y
hyd ophobic subs a es was associa ed wi h lipopep ide- ype bioemulsi ie
p oduc ion.
Taken oge he , hese indings emphasise he ole o he Añana Sal Valley as
a ese oi o mic obial di e si y o ex emophiles, as well as a p omising
sou ce o new axa and bioac i e compounds o in e es . This unde sco es he
necessi y o u he s udy in his a ea.
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Sec ion 1 - O e iew
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GENERAL INTRODUCTION
Hype saline ecosys ems a e en i onmen s whe e he concen a ion o sal in
wa e o soil is abou 10 imes ha o seawa e . These ecosys ems can be coas al
o con inen al sys ems and can include sal lakes o lagoons, sal e ns (coas al
o con inen al), sal e ns and sal diapi s, among o he s (1). The sal e ns
ep esen an en i onmen o g ea ecological impo ance. So much so ha in
2017, he Añana Sal Valley (Ála a, Spain) was ecognised by he Food and
Ag icul u e O ganiza ion o he Uni ed Na ions (FAO) as he i s Eu opean
Globally Impo an Ag icul u al He i age Sys em (GIAHS) (2), o being a
saline habi a wi h excep ional biodi e si y, ypical o we lands associa ed wi h
saline en i onmen s. The Añana Sal Valley is one o he mos emblema ic
examples o he alo isa ion o con inen al sal e ns. The sal p esen in his
sal e n is due o a la ge sal diapi , he esul o he d ying up o an ancien sea
200 million yea s ago. F esh ainwa e seeps h ough and dissol es he deepe
laye s o hali e o ock sal and eme ges in he alley as hype saline sp ings,
p o iding a habi a o a wide a ie y o halophilic and halo ole an
mic oo ganisms (3).
The s udy o mic obial biodi e si y in hype saline ecosys ems is an a ea o
g ea in e es wo ldwide. This in e es is mainly based on he ac ha many o
he mic oo ganisms ha inhabi hese en i onmen s ha e special cha ac e is ics
ha allow hem o li e in he p esence o high sal concen a ions. Thei
me abolism and ce ain molecules hey syn hesise a e he e o e o g ea in e es
o indus ial p ocesses and bio echnological applica ions (p oduc ion o
bioplas ics, pigmen s, an ibio ics, elimina ion o pollu an s, e c.) (4).
Du ing he 20 h cen u y, s udies o halophilic mic oo ganisms elied on
cul u e-based app oaches un il he ad en o new molecula and genomic
sequencing echniques ha allowed he de elopmen o gene ic and
cul u e-independen me hods. Inno a i e genome-based echniques opened up
a new way o unde s anding na u e and showed ha mic obial di e si y was
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much g ea e han o iginally hough (5). Hence, halophiles ha e been ound in
all h ee domains o li e: A chaea, Bac e ia and Euka ya, al hough he sal
ole ance and s a egies o cope wi h sal s ess a e qui e di e en in each g oup.
While ecen ly de eloped genome- esol ed me agenomics and single-cell
genomics echniques ha e e ealed he immense gene ic di e si y and
me abolic po en ial o uncul i a ed halophiles, cul u e o hese mic oo ganisms
is equi ed o desc ibe new axa, s udy hei physiology, con i m p edic ed
biochemical pa hways and exploi hei bio echnological po en ial (6).
E en hough con inen al sal e ns ha a e s ill ac i ely p oducing sal a e
excep ional (7) and despi e he ac ha a chaeological, hyd ogeological and
sal s udies ha e been ca ied ou in he Añana Sal Valley, he e is li le
mic obiological da a a ailable. Fu he mo e, i s dis inc i e sal - ela ed
a chi ec u e, encompassing sp ings, wells and pans buil o s one, wood and
clay, ende s i a aluable subjec o s udy, pa icula ly wi h ega d o he
a ia ions in he mic obial communi y a di e en si es wi hin he sal e n.
Con e sely, no cul u e-dependen s udies ha e ye been ca ied ou on his
sal e n. This, in u n, inc eases he likelihood o disco e ing p e iously
undesc ibed axa, as well as halophiles ha p oduce biomolecules wi h
po en ial applica ions in cu en heal h needs.
The knowledge gained could, in he u u e, con ibu e o he eco e y and
alo isa ion o he Añana Sal Valley, as well as o he knowledge o he
mic obial popula ion li ing in hype saline en i onmen s o he po en ial
bio echnological use o halophilic and halo ole an mic oo ganisms o he
bene i o human and en i onmen al heal h.
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THEORETICAL FRAMEWORK
Hype saline en i onmen s: he Añana Sal Valley
Hype saline en i onmen s a e conside ed ex eme en i onmen s, whe he
e es ial o aqua ic and a e de ined as hose wi h a high concen a ion o sal s.
These en i onmen s a e also cha ac e ised by ele a ed empe a u es and/o low
oxygen le els (5). Aqua ic hype saline en i onmen s a e hose whe e he sal
concen a ion is highe han ha o seawa e (a ound 3.5% (w/ ) in seawa e
s. up o 35% (w/ ) in b ines). This ype o en i onmen includes ecosys ems
such as con inen al lakes, lagoons, sal e ns and coas al o con inen al sal e ns.
All hese en i onmen s, om seawa e salini y o NaCl-sa u a ed b ines, a e
po en ial habi a s o mic obial li e. Rep esen a i e na u al examples include
he Dead Sea on he bo de be ween Is ael and Jo dan (346 g/L dissol ed sal s),
he G ea Sal Lake in U ah (270-300 g/L dissol ed sal s) and many o he s (4,5).
In gene al, hype saline aqua ic en i onmen s can be di ided in o wo g oups
acco ding o he ionic composi ion o he wa e : halassohaline and
a halassohaline (5). The b ines p oduced by he e apo a ion o seawa e a e
called halassohaline and e lec he ionic composi ion o he sea.
Thalassohaline b ines a e low in calcium and, o a lesse ex en , sulpha e. As
NaCl p ecipi a es as hali e, he ionic composi ion changes and he ela i e
concen a ions o po assium and magnesium inc ease. The G ea Sal Lake o
U ah, al hough long sepa a ed om he wo ld's oceans, s ill e lec s in i s ionic
composi ion he seawa e ha p o ided i s sal , so i s wa e s can s ill be
classi ied as halassohaline. Thalassohaline b ines a e cha ac e ised by neu al
o sligh ly alkaline pH alues. In o he hype saline en i onmen s, he ionic
composi ion can a y signi ican ly om ha o seawa e ; hese a e called
a halassohaline en i onmen s. The Dead Sea is a good example o an
a halassohaline lake. In his en i onmen , di alen ca ions p edomina e, wi h
magnesium and calcium concen a ions exceeding hose o sodium and
po assium. The pH o he Dead Sea b ine is ela i ely low a a ound 6.0 (5).
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On he o he hand, sal e ns a e e y di e se and can be classi ied acco ding o
a ious c i e ia, such as he loca ion o he sal deposi , he hyd ogeological
o igin o he deposi , he me hod o sal p oduc ion o he p esen s a e o he
sal e ns (8). Based on a classi ica ion acco ding o he loca ion o he esou ce,
h ee main g oups o sal e ns can be desc ibed, wi h some examples in Spain:
coas al sal e ns (Sanlúca de Ba ameda, Cádiz o Fuencalien e Sal e n, La
Palma), sal mines (Remolinos, Za agoza o Léniz, Guipúzcoa) and con inen al
sal ens (La Malahá, G anada o he Añana Sal Valley, Ála a (Figu e 1)).
Figu e 1. View o pa o he Sal Valley wi h he illage o Salinas de Añana in he backg ound.
The h eshing loo s o sal p oduc ion can be seen. Pho og aph aken in his s udy.
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The a ie y o con inen al sal ens in he Ibe ian Peninsula is e y la ge, due o
he a ie y o o og aphies, clima ological condi ions and geological
componen s ha make hem up (8). The bulk o hem da e om he La e
T iassic, mo e han 200 million yea s ago. This e en is based on he heo y ha
a la ge pa o he eas e n end o Spain was a ha ime subme ged in he
p esen -day Medi e anean Sea, which was hen known as he Te hys Sea, as
shown in Figu e 2A. Repea ed cycles o e apo a ion and looding c ea ed a
sal y laye , which was ac u ed by ec onic pla e mo emen s and deposi ed on
he su ace o , in o he cases, bu ied unde o he mo e mode n edaphic laye s,
o ming unde g ound b ine deposi s. These unique condi ions make he
con inen al sal e ns o he Ibe ian Peninsula an exclusi ely Ibe ian
phenomenon wi hin he Eu opean con inen . Howe e , less han 10% o hese
sal e ns emain ac i e. The es a e in dange o disappea ing o a a ie y o
easons, including economic abandonmen , pollu ion, changes in land use and,
abo e all, igno ance o hei his o ical and biological he i age (7,9).
The Añana Sal Valley (Ála a, Basque Coun y, Spain) is one o he bes
p ese ed con inen al sal e n in Spain. The unde g ound e apo i ic ocks we e
o med du ing he ea ly s ages o he agmen a ion o he supe con inen
Pangaea (abou 200 million yea s ago) and a e composed o sal , gypsum, clay
and o he s. These ocks a e now pa o a geologically complex diapi ic
s uc u e (Figu e 2B) ha c ea es an ellipsoidal s uc u e o app oxima ely
19 km2 (10). The hyd ogeological sys em a ound he Añana sal diapi is
ex emely complex and compa men alised. Acco ding o he hyd ochemical
in o ma ion, he di e en wa e s ha eme ge in he alley a e he esul o
mix u es, in di e en p opo ions and dep hs, o a leas wo ypes o wa e . On
he one hand, hose om he eas e n sec o o he diapi , which a e clea ly
sulpha ed and shallowe . On he o he hand, hose om he sou he n sec o o
he diapi , clea ly chlo ina ed and deepe , which condi ion he sal y sp ings
(11). The exis ence o hese sal y sp ings has enabled sal o be ac i ely
p oduced o a leas 7,000 yea s.
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S udies o he phylogeny o halophiles ha e shown ha he bes -known g oup
o ex eme halophiles is ep esen ed by he a chaeal phylum
Me hanobac e io a, which includes he widesp ead gene a Halobac e ium and
Haloquad a um. The phylum Me hanobac e io a also includes
me hane-p oducing anae obic halophiles belonging o he gene a
Me hanohalophilus and Me hanona ona chaeum. I is also wo h men ioning
he a chaea halophiles in he “Candida us” phylum Nanohala chaeo a. The
analysis o he highly conse ed ATP syn hase subuni s indica es an a ilia ion
wi h he Me hanobac e io a. Howe e , a his ime, no membe s o
“Candida us Nanohala chaeo a” ha e been cul i a ed in pu e cul u e (21,25).
On he o he hand, he la ges and bes -known g oup o mode a ely halophilic
bac e ia is he amily Halomonadaceae (phylum Pseudomonado a) (21).
Halophilic bac e ia a e a di e se g oup which include G am-posi i e and
G am-nega i e, pho o ophic and he e o ophic and ae obic and anae obic
bac e ia. Mos halophilic bac e ial species a e included in he ollowing phyla:
Ac inomyce o a, Bac e oido a, Cyanobac e io a, Bacillo a, Pseudomonado a
and Mycoplasma o a (26,27). Some o he ep esen a i e halophilic and
halo ole an gene a o hese phyla a e shown in he Figu e 4. Halophilic
membe s o he phylum Ac inomyce o a belong o he o de Ac inomyce ales,
while halophilic bac e ial species o he phylum Bac e oido a a e dis ibu ed
among he h ee classes. The phylum Cyanobac e io a, also known as blue-
g een algae, domina es he plank onic biomass and plays an ecological ole as
a majo con ibu o o pho osyn hesis in many hype saline lakes. Nume ous
halophilic species ha e been epo ed as membe s o he phyla Bacillo a and
Pseudomonado a, dis ibu ed in di e en amilies, while he e a e ew
mode a ely halophilic species belonging o he phyla Mycoplasma o a (26,27).
Al hough i is conside ed ha mos o he halophilic bac e ia a e mode a e
halophiles, he e a e examples o ex emely halophilic bac e ia, such as
Salinibac e ube (phylum Rhodo he mo a), which is he majo bac e ia o
hype saline aqua ic ecosys ems wo ldwide, he pho osyn he ic pu ple bac e ia
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belonging o he genus Halo hodospi a (phylum Pseudomonado a) o
Ac inopolyspo a halophile (phylum Ac inomyce o a) (21,28).
Wi hin he Euka ya domain, halophiles a e uncommon. The bes knowns a e
he unicellula g een algal genus Dunaliella, which is ound in mos high sal
aqua ic sys ems, he ungi Ho aea we neckii (phylum Ascomyco a) and
Wallemia ich hyophaga (phylum Basidiomyco a) and he mac oo ganism b ine
sh imp A emia (1,21). In gene al, yeas s and ilamen ous ungi ole a e
hype saline en i onmen s, g owing be e unde ae obic condi ions a mode a e
empe a u es and acidic o neu al pH. Among he p o ozoa in hype saline
en i onmen s, he mo e widely s udied is Fab ea salina, al hough o he
ep esen a i es a e also p esen (19,29).
Conce ning he halo ole an mic oo ganisms, he as majo i y o hem belong
o he Bac e ia and Euka ya domains. Examples include he bac e ia o he
gene a Al e omonas, Lac obacillus, Bacillus, Myxococcus and Pediococcus, as
well as many cyanobac e ia and ungi such as Deba omyces, Hansenula,
Cladospo ium and Saccha omyces (22).
Hype saline en i onmen s as a sou ce o no el mic obial axa
E en hough he e has been g ea p og ess in he isola ion and cul u e o
ex emophiles, mos o hese mic oo ganisms canno be cul u ed and do no
g ow in con en ional labo a o y condi ions. Recen s udies ha e es ima ed ha
80% o mic obial axa emain uncul u ed and ha he 85% o he phylogene ic
di e si y o p oka yo es consis s o uncul u ed axa (30). The e m used o name
he as di e si y o uncul i a ed mic oo ganisms ha can only be iden i ied
and s udied using non-cul u e-based me hods is "Mic obial Da k Ma e "
(MDM). I is es ima ed ha MDM domina e he as majo i y o en i onmen s
on Ea h (31). In pa icula , he ex emobiosphe e ha includes en i onmen s
whe e he physicochemical condi ions ep esen he known limi s o li e, is
ecognised as a ho spo o hese mic obes. These mic oo ganisms possess
special cha ac e is ics acqui ed du ing hei adap a ion o he en i onmen al
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niches in which hey li e. A signi ican numbe o p e iously uniden i ied axa
belonging o di e se phylogene ic lineages ha inhabi hype saline
en i onmen s ac oss he globe ha e been examined h ough he u ilisa ion o
cu ing-edge me agenomics and o he cul u e-independen me hodologies. In
ac , i was epo ed ha among halophiles, he MDM includes 42 “Candida us”
phyla including “Zixibac e io a”, “Pa cubac e io a”, “Asga da chaeo a”,
“Ba hya chaeo a” and membe s o he Nanobdella i kingdom. Albei hese
s udies yield subs an ial in o ma ion, he ul ima e objec i e should be o
cul i a e hese no el species in a labo a o y se ing (21). The success o
p e ious s udies wi h Spi ibac e (32), Haloquad a um (33) o Salinibac e
(34) p o ides a posi i e ou look o he u u e isola ion o new halophilic axa,
including membe s classi ied as “Candida us” who ha e no ye been isola ed.
Me agenomic da a allow esea ch eams o de elop ailo ed s a egies o isola e
membe s o uncha ac e ised dominan popula ions, wi h a ying deg ees o
success. “Cul u omics" me hods can be use ul, as demons a ed in he isola ion
o he a chaeon Halo u ilus (35). O he s we e se endipi ous disco e ies, such
as he ascina ing Ac ina chaeum halophilum, which g ew du ing a s udy o
isola e halophiles o Ac inomyce es class om a Chinese sal lake (36). Isola ion
in pu e cul u e may no always be possible when di e en halophile species a e
no in e dependen due o mu ualism. An example o his is he me abolic c oss-
eeding disco e ed be ween a Halo ub um and a Ma inococcus s ain om he
Cua o Cienegas Basin (Mexico) g owing oge he a 250 g/L sal (37).
In his con ex , i is no ewo hy ha , despi e he g owing global end owa ds
an o e - eliance on molecula app oaches o s udy mic obial communi ies,
e o s con inue o be made o conduc cul u e-based s udies a ge ing no el
halophilic axa (38). Cu en p oka yo ic axonomy is based on phylogene ics
and ollows wha is known as polyphasic cha ac e isa ion. Phylogeny uses
e olu iona y associa ions o conse ed gene sequences, mainly 16S RNA gene
in he case o p oka yo es, o de e mine he phylogene ic ela ionships o an
isola e. Polyphasic cha ac e isa ion combines genomic, chemo axonomic,
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physiological and cul u al cha ac e is ics. Polyphasic cha ac e isa ion is
ecognised as he s anda d me hod o he comp ehensi e classi ica ion o new
axa based on pheno ypic, geno ypic and chemo axonomic cha ac e s (39). As
a esul , he numbe o axa e ec i ely ecognised, o example in he class
Halobac e ia, has inc eased signi ican ly in ecen yea s, by a ound 50%
be ween 2017 and 2023 (21).
Bio echnological ele ance o halophilic and halo ole an mic oo ganisms
The use o ex emophilic mic oo ganisms as cellula ac o ies o he
p oduc ion o biomolecules has become o pa icula in e es o he disco e y
o new biomolecules wi h mul iple applica ions, om biomedical o
bio echnological (40). Halophiles a e ex emophiles ha ha e adap ed o he
high osmo ic p essu es o hei en i onmen . They a e also conside ed
polyex emophiles because hey ha e adap ed no only o high salini y bu also
o o he en i onmen al ex emes such as ex emely high o low pH o
empe a u e (4). The gene ic adap a ion o halophilic and halo ole an
mic oo ganisms o ex eme condi ions ha e led o he p oduc ion o a a ie y
o compounds o g ea in e es o indus ial applica ions. Some o he mos
impo an a e lis ed in Table 1.
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Table 1. Some cu en and po en ial bio echnological uses o compounds p oduced by halophilic and halo ole an mic oo ganisms.
P oduc
Applica ion
Examples o ep esen a i e
p oduce s
Bac e io hodopsin
Pho ochemical (op ical memo ies) and pho oelec ic (mo ion biosenso s, X- ay
senso s and pho o ol aic cells) applica ions. Comme cialised by a Ge man
company (MIB, Munich Inno a i e Bioma e ials).
Halobac e ium halobium
Halobac e ium salina um
Ca o enoid pigmen s
Bac e io ube in
An ioxidan and ood colou ing agen .
Halo ub um sp.
β-Ca o ene
Addi i e in cosme ics and ood p oduc s.
Dunaliella sp.
Compa ible solu es
Ec oine
Addi i e in de ma ological mois u ize s.
Halomonas sp.
Ch omohalobac e is aelensis
Hyd oxyec oine
P o ec ion o p o eins agains mis olding deg ada ion and eezing.
Halomonas elonga a
Ma inococcus M52
Be aine
In biomedicine, as a po en ial ea men o adipose in il a ion o he li e a he
onse o ci hosis and an icoagulan .
Halo hodopi a halochlo is
Thioalkalo ib io e su us
Halophilic enzymes
Amylases
In he ood indus y, in baking p ocesses, b ewing and ui juices, whe e hey a e
added o deg ade he s a ch.
Halomonas sp.
Halobac e ium salina um
Halo e ax medi e anei
P o eases
Addi i es in pha maceu icals and laund y de e gen s.
Bacillus sp.
Halobacillus sp.
Halobac e ium halobium
Xylanases and cellulases
Biobleaching and o accele a e he p ocess o hyd olysis in plan s o he ex ac ion
o ui juices.
Halo e ax sul u i on is
S ep omonospo a sp.
Con inued
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P oduc
Applica ion
Examples o ep esen a i e
p oduce s
Polyhyd oxyalkanoa es (PHAs)
Polyhyd oxybu y a e
Biodeg adable plas ics and medical ma e ials.
Halomonas boli iensis
Halo e ax medi e anei
Biosu ac an s and bioemulsi ie s
Rhamnolipids
Remo al o c ude oil, ea men o soil and was ewa e , an i i al, an i ungal, and
an ibac e ial agen s and cosme ics. Ma ke ed by a numbe o US companies:
GlycoSu , AGAE Technologies LLC and Logos Technologies.
Pseudomonas ae uginosa
Pseudomonas pu ida
Ma inobac e sp.
Su ac in
Hea y me als emo al.
Bacillus sub ilis
T ehalose lipids
Bio emedia ion o cold ma ine en i onmen s.
Ec o hio hodospi a sp.
Exopolysaccha ides
Ac i e hyd oca bon emulsi ie s.
Halomonas sp.
Halo e ax olcanii
Halobac e ium salina um
Emulsan
Emulsion o ming and s abilising p ope ies especially in ela ion o hyd oca bon
biodeg ada ion.
Acine obac e ene ianus
An imic obial compounds
Ac inomycin C2 and py ole (1,2-A
(py azine-1,4‑dione, hexahyd o-3-
(2me hylp opyl)-)
An imic obial ac i i y agains Esche ichia coli, S hapylococcus au eus and
Pseudomonas ae uginosa.
Noca diopsis sp.
Py olo[1,2-a]py azine-1,4-
dione,hexahyd o
An imic obial ac i i y agains mul id ug esis an S. au eus.
Bacillus equilensis
Chaxamycins A–D
An imic obial ac i i y agains S. au eus.
S ep omyces sp.
De ailed in o ma ion in e e ences (4,40–47).
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Among hem, new an imic obial compounds, as well as biosu ac an and
bioemulsi ie compounds, a e in high demand due o he need o add ess oday's
p essing issues such as an imic obial esis ance and en i onmen al
con amina ion, which h ea en bo h human and en i onmen al heal h. In his
con ex , bioac i e p oduc s om mic obes in ex eme en i onmen s, including
halophile and halo ole an mic oo ganisms, ha e demons a ed a g ea capaci y
o p oduce aluable compounds. Thus, he upwa d end o scien i ic esea ch
in he ield o halophiles and halo ole an bioac i i y is e iden om he
inc ease in publica ions on he subjec yea on yea (Figu e 5).
Figu e 5. Numbe o publica ions ound in he ScienceDi ec da abase a e sea ching o s udies
on an imic obials (blue) (a o al o 819) and su ace-ac i e compounds (g een) (a o al o 388)
ela ed o halophilic and halo ole an mic oo ganisms be ween 2000 and 2025 (accessed Ma ch
2025).
The con inuing ise o an ibio ic esis ance among many ypes o bac e ia is one
o he mos se ious heal h p oblems we ace oday. Acco ding o he da a
epo ed by he Lance , nea ly 5 million people died om d ug- esis an
0
20
40
60
80
100
120
Numbe o publica ions
Publica ion yea
Sea ch que y: (biosu ac an OR bioemulsi ie ) AND (halophil OR halo ole an )
Sea ch que y: ("an i-in ec i e agen " OR an imic obial) AND (halophil OR
halo ole an )
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bac e ial in ec ions in 2019 and he c isis is expec ed o wo sen in he nea
u u e (48). Finding new an imic obial he apies is he e o e one o he
undamen al goals o science. In his sense, an agonis ic in e ac ions and
an imic obial compounds o halophilic bac e ia and a chaea ha e been s udied.
I has been shown ha ex eme condi ions can inc ease compe i ion and
p omo e he e olu ion o biosyn hesis o an imic obial compounds wi h a
na ow spec um o ac i i y bu high s abili y and po ency (49). Among
halophilic and halo ole an bac e ia, Ac inomyce o a a e a p edominan phylum
o an imic obial p oducing s ains. Some examples o an imic obial compounds
p oduced by hem a e chaxamycins A–D (S ep omyces sp. om Sala de
A acama, Chile) (Figu e 6A), an h acimycin (S ep omyces sp. om San a
Ba ba a, Cali o nia) and abyssomicins B–D (Ve ucosispo a sp. om Sea o
Japan) (50). In addi ion o Ac inomyce es class membe s, o he halophilic and
halo ole an gene a, including Bacillus, Kocu ia, Vib io and Halomonas, ha e
been shown o p oduce molecules such as indole de i a i es, alkaloids,
ypenoids and pep ides ha a e bioac i e agains ce ain pa hogens.
In e es ingly, he e icacy o hese halophilic biomolecules agains
d ug- esis an pa hogens such as me hicillin- esis an S aphylococcus au eus
(MRSA), luconazole- esis an Candida albicans, no loxacin and
cip o loxacin- esis an "Klebsiella quasi a iicola" and penicillin- esis an
S ep ococcus pneumoniae has al eady been demons a ed (44).
Recen ad ances sugges ha halogena ed an imic obials may enhanced
ac i i y agains d ug- esis an bac e ia. Indeed, s udies ha e shown ha he
inco po a ion o halogena ed compounds in o pep ides enhances an imic obial
ac i i y agains mul i- esis an pa hogens, wi h b omina ed analogues showing
32- old inc eased ac i i y agains MRSA and 16-64- old inc eased ac i i y
agains Pseudomonas ae uginosa and Esche ichia coli (51,52). In his con ex ,
species o he halophilic genus Pseudoal e omonas, o en isola ed om
sal e ns, ha e been desc ibed as p oduce s o halogena ed seconda y
me aboli es (Figu e 6B). Mos o hem ha e s ong an ibac e ial and an i ouling
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ac i i y (53,54). In ac , genome mining app oach o seconda y me aboli e
disco e y in Pseudoal e omonas genomes (42 publicly a ailable) e ealed ha
he pigmen ed s ains had an a e age densi y o 10 biosyn he ic gene clus e s
(BGCs) pe genome, sugges ing an un apped sou ce o po en ially in e es ing
me aboli es in his genus (55).
Figu e 6. Examples o he chemical s uc u es o some an imic obial compounds p oduced by
A, S ep omyces and B, Pseudoal e omonas, bo h halophilic and halo ole an bac e ia.
On he o he hand, su ace-ac i e biomolecules a e ano he in e es ing g oup o
compounds syn hesised by a ious mic oo ganisms as seconda y me aboli es.
These biomolecules a e essen ial o he bioa ailabili y o subs a es and hus
o he su i al o he mic oo ganisms. Su ace-ac i e biomolecules ha e a
hyd ophilic (a suga o pep ide) and a hyd ophobic ( a y acid chain) domain in
he same molecule and a e p oduced ex acellula ly o as pa o he cell
memb ane.
In gene al, su ace-ac i e biomolecules can be di ided in o wo ypes acco ding
o hei molecula weigh (Figu e 7). On he one hand, low molecula weigh
compounds, called biosu ac an s, which educe he su ace and/o in e acial
ension be ween wo immiscible phases o liquid, liquid and solid o liquid and
gas. On he o he hand, high molecula weigh compounds, e med
bioemulsi ie s, o en e e ed o as exopolysaccha ides (EPS), which allow he
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o ma ion o oil-in-wa e o wa e -in-oil emulsions (43,56). Con e sely, based
on hei molecula s uc u e, su ace-ac i e biomolecules a e u he classi ied
as, lipop o eins, lipopep ides, glycolipids, a y acids and polyme s (Figu e 7)
(57).
Figu e 7. Classi ica ion o mic obial su ace-ac i e compounds, g ouped in o biosu ac an s and
bioemulsi ie s. Pic u e om Sánchez (58).
Su ace-ac i e biomolecules a e a sui able al e na i e o syn he ic su ac an s,
as many o hem a e syn hesised mainly om pe oleum. They ha e been
widely used in nume ous indus ial sec o s, including hygiene,
pha maceu icals, medicine, cosme ics and ag ochemicals (59). A wide ange o
su ace ac i i ies including emulsi ying, dispe sing, solubilising, we ing and
oaming ha e been demons a ed in su ace-ac i e biomolecules (60). As a
esul , biosu ac an s and bioemulsi ie s ha e demons a ed po en ial
applica ion in a ious ields (Figu e 8) (57,61).
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Wa e samples om each si e we e collec ed di ec ly in s e ile labelled glass
bo les. A Niskin bo le (Aqua ic BioTechnology, El Pue o de San a Ma ía,
Spain) was used o collec wa e om he San a Eng acia and El Cau i o
sp ings, bo h a 2 m dep h. The g oundwa e om he piezome e S8 (a a dep h
o 60 m) was collec ed using a manual baile sys em (Eijkelkamp, Giesbeek,
The Ne he lands). A all sampling si es, wa e empe a u e, pH and elec ical
conduc i i y we e measu ed wi h a Combo es e (Hanna Ins umen s, Eiba ,
Spain) and salini y wi h a densi y hyd ome e (Ha wig Ins umen s, The
Ne he lands), all pa ame e s measu ed in si u. All o he samples we e hen
anspo ed o he labo a o y o u he p ocessing and analysis.
O he 12 sampling si es, eigh ( he six sp ings, he s eam and he piezome e )
a e pa o a hyd ogeological moni o ing ne wo k o su ace and g oundwa e
s a ed in 2017, so da a on he ionic composi ion o hese wa e samples we e
a ailable (Sec ion 3, APPENDIX I, Table S1). This ionic composi ion is
pe iodically analysed by ion ch oma og aphy a he Ad anced Resea ch
Facili ies (SGIke ) o he Uni e si y o he Basque Coun y UPV/EHU.
Assessmen o mic obial di e si y and communi y dis ibu ion using
amplicon sequencing echnique
Mos ecen s udies assessing he di e si y and composi ion o he mic obial
communi y in hype saline en i onmen s ha e used cul u e-independen
me hods, as hese p o ide a much mo e accu a e ep esen a ion o he mic obial
composi ion, as many o hem a e challenging o cul i a e and he e o e hei
p esence is unknown (21). The use o Nex Gene a ion Sequencing (NGS)
echnology and echniques such as amplicon sequencing (sequencing o
ampli ied genes o in e es ) has allowed he analysis o mic obial communi ies
and hei po en ial ecological unc ion by examining DNA ex ac ed om wa e
samples con aining mixed assemblages o o ganisms. The ela i ely low cos
o amplicon sequencing compa ed o o he echnologies (such as sho gun
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sequencing, which in ol es sequencing agmen s o all he gene ic ma e ial
p esen ) led o i s widesp ead use (68).
Wa e samples collec ed om he Añana Sal Valley we e p ocessed (en iched
and sequenced) a Biogene ics, S.L. (Ála a, Spain). B ie ly, 5 L o wa e we e
il e ed unde asep ic condi ions using a cus om-buil acuum/comp ession
pumping de ice (Labbox, P emia de Dal , Spain), ending wi h a 0.22 μm il e .
P oka yo ic DNA was ex ac ed using he Genomic Mini AX Bac e ia Ki
(A&A Bio echnology, Gdansk, Poland) and euka yo ic DNA was ex ac ed
using he Genomic Mini AX Yeas Ki (A&A Bio echnology, Gdansk, Poland).
The DNA ob ained was quan i ied using he Quan iFluo dsDNA Sys em
(P omega, Uni ed S a es). The V3-V4 hype a iable egion o he 16S RNA
gene was ampli ied o simul aneous de ec ion o bac e ia and a chaea. On he
o he hand, he nuclea ibosomal in e nal ansc ibed space 1 (ITS1) egion
was ampli ied o he molecula iden i ica ion o euka yo es. Lib a ies we e
p epa ed using a Nex e a DNA Lib a y P ep Ki (Illumina, Uni ed S a es) and
sequencing was pe o med on an Illumina MiSeq pla o m, gene a ing pai ed-
end sequences in FASTQ o ma . The nucleo ide sequence da a om he
p oka yo ic and ungal s udies a e a ailable in he DDBJ/EMBL/GenBank
da abases unde accession numbe s PRJNA1115049 and PRJNA749727
espec i ely.
The amplicon sequencing da a se s we e analysed using he Quan i a i e
Insigh s In o Mic obial Ecology (QIIME2) bioin o ma ics pipeline ( e sion
2021.4) (69). This pipeline coo dina es he inpu s and ou pu s o di e en
bioin o ma ics ools o acili a e he analysis o samples ha equi e many
di e en analy ical s eps. The implemen a ion o he denoising ool, DADA2,
allows quali y con ol o sequencing by emo ing sequencing e o s and
gene a ing Amplicon Sequence Va ian s (ASVs) ha e ain all he obse ed
biological a ia ion. This da a was used o axonomic composi ion s udies,
di e en ial abundance analysis and di e si y analyses. Taxonomic assignmen
o ASVs based on simila i y o sequences in 16S RNA gene da abase SILVA
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da abase .138 (70), allowed he cons uc ion o he p oka yo ic axonomic
communi y. Simila ly, ASVs de i ed om ITS1 egion sequences we e
compa ed o he UNITE e e ence da abase (71), which allowed iden i ica ion
o ungal communi y.
Di e si y analysis was assessed a di e en scales, om wi hin-sample (alpha)
o be ween-sample (be a), using he q2‐di e si y plugin in QIIME2 p og am.
Alpha di e si y me ics include obse ed ASVs, Chao1, Pielou's e enness,
Shannon's and Simpson's di e si y indexes. Di e si y es ima o like obse ed
ASVs and Chao1 index, es ablish he mic obial communi y ichness.
Quan i a i e communi y ichness o di e si y was assessed by Shannon's and
Simpson's indexes and communi y equali y o e enness by Pielou's e enness.
On he o he hand, be a di e si y me ics include B ay‐Cu is dissimila i y,
which iden i ies dissimila i ies in ASV composi ion be ween he samples.
The unc ional po en ial o he p oka yo ic communi y was p edic ed using
16S RNA gene abundance da a ia Phylogene ic In es iga ion o
Communi ies by Recons uc ion o Unobse ed S a es (PICRUS 2). Simila ly,
he FUNGuild da abase was used o assign ecological guilds o all ungal ASVs.
Figu e 11 shows he gene al wo k low employed o amplicon sequencing o
he wa e samples.
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Figu e 11. Scheme o he wo k low pe o med o he amplicon sequencing analysis o he
wa e samples.
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Isola ion and iden i ica ion o p oka yo ic s ains
Wa e samples collec ed a he di e en si es men ioned abo e we e also used
o he isola ion o halophilic and halo ole an bac e ia. To ca y ou his
app oach, wa e samples we e subjec ed o a se ies o il a ion s ages
(Wha man® G ade 113V and Millipo e 5 µm and 0.22 µm il e s), ollowed by
cul i a ion on ma ine aga (MA; Condalab, Spain) and MA supplemen ed wi h
23% NaCl (Sigma-Ald ich, Spain), wi h duplica e cul u es o each sample. The
media we e hen incuba ed a 25 °C. Colony o ma ion was moni o ed
pe iodically o up o 2 mon hs. Subsequen sub-cul u ing o he isola es was
ca ied ou on he same medium un il a pu e cul u e was ob ained. Pu e cul u es
we e main ained a -80 °C in ma ine b o h (MB; Condalab, Spain) in 25% ( / )
glyce ol. The diame e , edge, pigmen a ion, ex u e, heigh , su ace, ha dness
and emulsi iabili y o he colonies we e s udied (72). A he same ime, G am
s aining and o ange-ac idine s aining we e pe o med o de e mine he
mic oscopic cha ac e is ics o he colony cells acco ding o he p o ocol
p oposed by Vázquez e al. (73).
To iden i y he isola es, ch omosome DNA was ex ac ed using he P epMan®
Ul aReac i e (Applied Biosys ems, Uni ed S a es) acco ding o he
manu ac u e 's ins uc ions. In case o ailu e o his echnique, ex ac ion was
pe o med by he use o glass beads and boiling. The concen a ion o he DNA
was measu ed using a NanoD op® 2000 spec opho ome e (The mo
Scien i ic, Uni ed S a es). The 16S RNA gene was ampli ied by PCR using
speci ic p ime s: 27F (5′-GAGTTTGATCMTGGCTCAG-3′)
and 1492R (5′-GGTTACCTTGTTACGACTT-3')
o bac e ia (74) and 21F (5′-TCCGGTTGATCCYGCCGG-3′)
and 1492R
(5′-GGTTACCTTGTTACGACTT-3') o a chaea (75). In cases whe e no
ampli ica ion was ob ained, uni e sal p ime s ITS1
(5'-TCCGTAGGTGAACCTGCGG-3') and ITS4
(5'-TCCTCCGCTTATTGATATGC-3') we e ied o ungal iden i ica ion
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(76). Ampli ica ion was pe o med on a T100 he mal cycle (Bio-Rad, Uni ed
S a es) using he app op ia e ampli ica ion p og amme.
Visualisa ion o he ampli ied agmen was pe o med by 1% aga ose gel
elec opho esis (Condalab, Spain) in 1X TAE bu e (The mo Scien i ic,
Uni ed S a es) a 100 V o 50 min. GelRed 0.5X (Bio ium, Uni ed S a es) was
used o isualise nucleic acids and he molecula weigh ma ke Hype Ladde
I (Bioline, Spain). Ampli ied agmen s we e isualised using a ChemiDoc
XRS+ Sys em Ul a iole (UV) ansillumina o (Bio-Rad, Uni ed S a es). The
esul ing amplicon was pu i ied using he NucleoSpin Gel and PCR Clean-Up
Ki (Mache ey-Nagel, Ge many).
Pu i ied PCR p oduc was sequenced by he Sange me hod (S ab Vida,
Po ugal) and he esul ing sequences we e analysed using Ch omas 2.6.6
so wa e and CLUSTAL W webse e e sion 2.0.12 (77). The EzBioCloud
websi e (www.ezbiocloud.ne /iden i y) (78) was used o iden i y closely ela ed
species and hei 16S RNA gene sequence simila i y. Fo isola es o which i
was no possible o ob ain a comple e o su icien ly long 16S RNA gene
sequence, a agmen simila i y sea ch was ca ied ou using he BLAST web
se e (79) o hei app oxima e iden i ica ion. A 16S RNA gene-based
phylogene ic ee was cons uc ed using he Neighbou -joining me hod in
MEGA X so wa e (80) o es ablish he phylogene ic ela ionships o all he
isola ed s ains.
Polyphasic cha ac e isa ion o po en ial no el axa
Taxonomy p o ides he in o ma ion necessa y o iden i y and cha ac e ise a
species in i s ecological, clinical and indus ial niche. The polyphasic
axonomic app oach in ol es a combina ion o geno ypic, pheno ypic and
chemo axonomic da a o he nomencla u e and sys ema ics o new axa (39).
Figu e 12 summa ised he polyphasic s a egy used in his s udy o he
axonomic cha ac e isa ion o po en ial new isola es.
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Geno ypic me hods p o ide in o ma ion de i ed p ima ily om nucleic acids,
DNA o RNA, o he isola ed o ganism. 16S RNA gene sequencing is a apid
and accu a e iden i ica ion me hod o bac e ial and a chaeal isola es. In his
s udy, isola es wi h 16S RNA gene sequence simila i ies be ween he s ain
unde s udy and he closes ela ed ype species ≤ 98.7% ( he h eshold
p oposed by Chun e al. (81) o dis inguishing wo species) and ≤ 95.0% ( he
h eshold p oposed by Hö d e al. (82) o dis inguishing wo gene a) we e
conside ed po en ial new axa. The 16S RNA gene sequences o closely ela ed
species we e e ie ed om he GenBank da abase o pe o m a compa a i e
phylogene ic analysis based on ha gene. The sequences we e aligned by
Clus alW 2.0.12. Phylogene ic ees we e cons uc ed using Neighbou -joining,
Minimum-e olu ion and Maximum-likelihood me hods in MEGA X so wa e.
E olu iona y dis ances o Neighbou -joining analysis we e calcula ed using
Kimu a's wo-pa ame e model algo i hm wi h boo s ap alues based on 1,000
eplica es. The phylogene ic ee shows he posi ion o he s ain s udied
ela i e o i s nea es neighbou s by compa ison wi h o he sequences in he
da abase, om which u he geno ypic, chemo axonomic and pheno ypic
analyses a e designed (39).
The DNA o whole genome sequencing (WGS) was ob ained by using he
Nucleo Spin Tissue DNA ex ac ion ki (Mache ey-Nagel, Ge many) acco ding
o he manu ac u e 's p o ocol. De no o assembly o con igs om aw pai ed-
end eads ob ained using he Illumina Miseq pla o m was pe o med using
SPAdes ( 3.13.0) (83) o Vel e (1.1.04) (84). Gene anno a ion was pe o med
using he NCBI (Na ional Cen e o Bio echnology In o ma ion) P oka yo ic
Genome Anno a ion pipeline and he Rapid Anno a ion wi h Subsys ems
Technology (RAST) pipeline (85).
A basic geno ypic me hod o bac e ial classi ica ion is he measu emen o he
mol% o guanosine and cy osine bases. Wi hin a well-de ined species and
genus, he a ia ion in G+C % con en should no exceed 3% and 10%,
espec i ely (39). In addi ion, he O e all Genomic Rela edness Index (OGRI)
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e lec s he simila i y be ween wo genomic sequences. The e a e many
algo i hms o calcula ing OGRI alues, bu he mos widely used algo i hm o
axonomic s udies is A e age Nucleo ide Iden i y (ANI), which was compu ed
in his s udy using he O hoANI Calcula o ool a ailable a EzBiocloud (86).
In addi ion, A e age Amino Acid Iden i y (AAI) alues we e calcula ed using
he Kos as Lab AAI calcula o (h p:// en e- omics. ce. ga ech. edu/ aai/) (87)
and Digi al DNA-DNA hyb idiza ion (dDDH) alues we e compu ed using he
Genome- o-Genome Dis ance Calcula o (h p://ggdc. dsmz. de/ ggdc. php)
(88). The AAI cu -o o 95% co ela es well wi h 95% o ANI and 70% o
dDDH when compa ing simila mic obial species, making hem eliable ools
o mic obial axonomy o p oka yo es (39). In addi ion, whole genome
sequence-based ee was in e ed using Fas ME 2.1.6.1 (89) om he GBDP
dis ances calcula ed om he genome sequences using he Type S ain
Genome Se e (h ps:// ygs.dsmz.de) (90).
In o de o assess genus a ilia ions, amino acid-le el compa isons we e
pe o med using he AAI and he Pe cen age O Conse ed P o eins (POCP)
o e e y pai wise combina ion o sequences. The POCP alues we e calcula ed
using a Py hon sc ip wi h he o mula [(C1 + C2)/(T1 + T2)] x 100%, whe e
C1 and C2 ep esen he conse ed numbe o p o eins in he wo genomes
being compa ed and T1 and T2 ep esen he o al numbe o p o eins in he wo
genomes being compa ed (91).
Pheno ypic es s, including chemo axonomic echniques, a e used o desc ibe
axa, species and subspecies up o genus and amily le el. The mo phology o
he cells, hei size and he p esence o lagella we e obse ed by ansmission
elec on mic oscopy (1400 Plus, Jeol). The mo ili y o he cells was checked by
he hanging d op me hod and on semi-solid aga con aining MB and 0.7%
bac e iological aga (Scha lau, Spain). Colony mo phology, size and colou
we e obse ed on MA a e 3 days a 25 °C. The cellula pigmen s om he
cul u es g own on MB we e analysed by means o High-Pe o mance Liquid
Ch oma og aphy (HPLC). G ow h a di e en empe a u es (4, 10, 15, 20, 25,
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30, 37 and 42 °C), pH ange (pH 4.5-10.5 in inc emen s o 0.5 pH uni s) and
NaCl concen a ion (0, 0.5, 1.0, 2.0, 3.0, 5.0, 10.0, 15.0, 20.0 and 23.0 g/L)
we e de e mined. Anae obic g ow h was es ed on MA using GENbox anae
(bioMé ieux, F ance) in an anae obic chambe a 25 °C o 15 days.
Oxidase ac i i y was assessed using Bac iden oxidase s ips (Me ck, Uni ed
S a es) and ca alase ac i i y using ID colou ca alase (bioMé ieux, F ance).
Hyd oly ic capaci y was de e mined on MA pla es supplemen ed wi h 1% (w/ )
skim milk, 1% (w/ ) Tween 20 and 1% (w/ ) Tween 80. API ZYM and API
20NE s ips (bioMé ieux, F ance) we e used o de e mine o he physiological
and biochemical cha ac e is ics acco ding o he manu ac u e 's ins uc ions.
An ibio ic suscep ibili y was de e mined on MA pla es.
Whole cell a y acid composi ion was de e mined a he Spanish Type Cul u e
Collec ion (CECT) acco ding o he p o ocol ecommended by he MIDI
Mic obial Iden i ica ion Sys em (92). She lock MIDI e sion 6.1 was used o
iden i ica ion and TSBA6 lib a y o analysis o cellula a y acid con en .
Analyses o pola lipids and espi a o y quinones we e pe o med by he
iden i ica ion se ice o he Leibniz Ins i u e DSMZ (Deu sche Sammlung on
Mik oo ganismen und Zellkul u en GmbH, B aunschweig, Ge many).
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Figu e 12. Schema ic wo k low o desc ibing no el bac e ial axa using a polyphasic axonomic
app oach. PCR, Polyme ase chain eac ion; WGS, Whole genome sequencing; OGRI, O e all
Genomic Rela edness Index; ANI, A e age Nucleo ide Iden i y; AAI, A e age Amino Acid
Iden i y; dDDH, Digi al DNA-DNA hyb idiza ion.
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Cha ac e isa ion o bioac i e compounds p oduced by he ASV78 isola e
Among all he isola es es ed he isola e ASV78 was selec ed o u he
analysis as i showed posi i e esul s o bo h he su ace-ac i e ac i i y and he
an ibac e ial ac i i y. To op imise he p oduc ion o he compounds o in e es ,
MB medium supplemen ed wi h wo ca bon sou ces (1% w/ glyce ol o
glucose) was es ed a wo empe a u es (25 o 30 °C) and wo condi ions (125
pm o s a ic) o a pe iod o 5 days. The p esence o he ac i i ies o in e es
was de e mined by he well di usion assay (METHODOLOGY, Figu e 14) and
he Pa a ilm M, oil sp eading and emulsi ica ion assays (METHODOLOGY,
Figu e 15). The mos a ou able condi ions o p oduc ion condi ions we e
es ablished and used o he subsequen ex ac ion and cha ac e isa ion
analyses, summa ised in Figu e 16.
Th ee di e en ex ac ions we e pe o med o sepa a ely ob ain c ude
an ibac e ial ex ac , c ude biosu ac an ex ac and c ude bioemulsi ie
ex ac . The eason why wo di e en me hods a e used o ex ac su ac an
compounds is due o he di e en chemical na u e o biosu ac an s (low
molecula weigh molecules) and bioemulsi ie s (high molecula weigh
molecules).
The c ude an ibac e ial ex ac was ob ained using he CFS om 1 L cul u e
and ex ac ed wice wi h an equal olumes o e hyl ace a e by igo ous shaking
in a sepa a ing unnel. Fo he c ude biosu ac an ex ac , he CFS om 1 L
cul u e was i s acidi ied o pH 2.0 wi h 1 M HCl and hen ex ac ed wice wi h
equal olumes o e hyl ace a e. In bo h cases, he sol en o he o ganic phase
was hen e apo a ed unde acuum using a Ro a apo R100 (Buchi,
Swi ze land). Finally, o ob ain he c ude bioemulsi ie ex ac , he CFS was
mixed wi h cold e hanol (1:1) and incuba ed o e nigh a -20 ºC. The
bioemulsi ie was hen pelle ed by cen i uga ion a 9,000 pm o 15 min a 4
°C and dissol ed in dis illed wa e (1% w/ ) be o e lyophilisa ion. The h ee
c ude ex ac s we e weighed and s o ed a - 20 ºC un il u he analysis.
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Each c ude ex ac was hen es ed o he p esence o he co esponding
ac i i ies as desc ibed in he METHODOLOGY subsec ion in he Figu e 14
and Figu e 15. Addi ionally, he c ude an ibac e ial ex ac was sepa a ed by
Thin Laye Ch oma og aphy (TLC) (Me ck, Ge many) and a con ac
bioau og aphy analysis was used o de e mine which o he bands sepa a ed by
TLC had an ibac e ial ac i i y (98). Fo his pu pose, he TLC pla e was placed
on MH aga inocula ed wi h a pa hogen s ain a a concen a ion o
0.5 McFa land and i was le o 30 minu es o allow he compounds o di use
om he silica pla e o he aga medium. A e incuba ion a 37 °C o 24 hou s,
he appea ance o a g ow h inhibi ion zone on he aga indica ed which band
was ac i e agains he es ed mic obe.
De eplica ion o he ac i e c ude ex ac s was pe o med using liquid
ch oma og aphy high esolu ion mass spec ome y (LC–HRMS) in an Agilen
1200RR HPLC sys em coupled wi h a B uke maXis Q-TOF mass analyze ,
unde analy ical condi ions p e iously desc ibed (99–101). De eplica ion is
de ined as he analy ical me hod used o apidly iden i y al eady known na u al
p oduc s in samples om na u al sou ces (102).
In o de o pu i y he compounds o in e es p esen in he c ude an ibac e ial
ex ac , a i s ac iona ion was pe o med on an au oma ic lash
ch oma og aphy sys em (CombiFlash R , Teledyne Isco) using a linea g adien
om 5% o 100% ace oni ile in wa e (in 35 min) wi h a inal s ep o 100%
ace oni ile ( o 10 min), collec ing 47 ac ions. The ac ions we e
concen a ed o d yness in a cen i ugal e apo a o . F ac ions 27, 28 and 29 on
he one hand and ac ions 37, 38 and 39 on he o he hand we e pooled as hey
con ained he wo compounds o in e es . These ac ions we e u he pu i ied
sepa a ely by semi-p epa a i e e e sed-phase HPLC (XB idge® P ep C18, 10
× 150 mm, 5 μm, 3.8 mL/min, UV de ec ion a 210 and 280 nm, 1.8
mL/ ac ion). A linea g adien o wa e -ace oni ile om 50% o 100%
ace oni ile o e 30 min was es ablished.
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Nuclea Magne ic Resonance (NMR) spec a we e eco ded in CDCl3 on a
B uke A ance III spec ome e (500 and 125 MHz o 1H and 13C NMR,
espec i ely) equipped wi h a 1.7 mm TCI Mic oC yoP obe.
The an ibac e ial ac i i y o he wo pu i ied compounds was es ed using he
b o h mic odilu ion me hod in a 96-well pla e o ma agains S aphylococcus
au eus ATCC 29213, Pseudomonas ae uginosa ATCC 27853, Acine obac e
baumannii ATCC 19606, En e ococcus aecalis ATCC 29212 and
ancomycin- esis an En e ococcus aecium anA 15167 (clinical isola e)
(103,104). Vancomycin, cip o loxacin and az eonam we e used as e e ence
an ibio ics (posi i e con ols). A concen a ion o 80 μg/mL o he pu e
compounds was used o he ini ial assay. To e alua e any syne gis ic ac i i y
an ini ial concen a ion o 40 μg/mL was used o each o he pu e compounds.
Blank and g ow h con ols we e included as in e nal pla e con ols.
To es he g ow h o he pa hogens ubidi y (abso bance [Abs] a 600 nm) was
measu ed a baseline (T0) and a e 24 hou s o incuba ion (T ) a 37 °C, using
an EnVision™ Mic opla e Reade (Pe kinElme , Uni ed S a es). The Minimum
Inhibi o y Concen a ion (MIC) is de ined by he Eu opean Commi ee on
An imic obial Suscep ibili y Tes ing (EUCAST) as he lowes concen a ion o
an imic obial agen ha inhibi s isible g ow h o a mic oo ganism, exp essed
in mg/L o μg/mL (105). Th ee independen biological expe imen s (n = 3) we e
pe o med o de e mine he MIC alues. The Geneda a Sc eene so wa e,
e sion 18.0.4-S anda d (Geneda a, Swi ze land) was used o p ocess and
analyze he da a.
On he o he hand, in a ed (IR) spec oscopy was used o analyse he bioac i e
compounds o in e es in he c ude ex ac s o biosu ac an s and
bioemulsi ie s. The elucida ion o unc ional g oups was ob ained om IR da a
spec a using a JASCO FT/IR-4100 spec ome e equipped wi h a PIKE
MIRacle single e lec ion ATR accesso y (JASCO Co p., Japan). All
measu emen s we e made a oom empe a u e.
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Figu e 16. O e iew o he ex ac ion and cha ac e isa ion p ocess o he bioac i e compounds
p esen in he ex ac s o his s udy. TLC, Thin Laye Ch oma og aphy; HPLC, High-Pe o mance
Liquid Ch oma og aphy; UV, Ul a iole spec a; MS, Mass spec a; NMR, Nuclea Magne ic
Resonance; IR, In a ed spec oscopy. C ea ed in h ps://BioRende .com.
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Finally, o iden i y any po en ial biosyn he ic clus e s in he ASV78 genome,
WGS was pe o med on he Illumina Nex Seq pla o m a he UPV/EHU
Ad anced Resea ch Facili ies (SGIke ), combined wi h long- ead sequencing
on he MinION sequence (Ox o d Nanopo e Technologies) a he Uni e si y
Hospi al o Ála a. A e il e ing o sequence quali y, he aw Illumina eads
and MinION long eads we e assembled de no o using he Unicycle pipeline
(Galaxy e sion 0.5.0+galaxy1). Gene anno a ion was pe o med using he
Rapid Anno a ion wi h Subsys ems Technology (RAST) pipeline (85) and
P okka 1.14.5 (106). Biosyn he ic gene clus e (BGC) analysis was pe o med
using an iSMASH 7.0 (107).
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SUMMARY OF THE RESULTS AND DISCUSSION
The i s pa o his sec ion consis s o a summa y o he esul s on he
mic obial di e si y and communi y composi ion in he Añana Sal Valley,
which a e p esen ed in APPENDIX I and APPENDIX II. The second pa
summa ises he esul s ob ained wi h ega d o he cha ac e isa ion o new axa
isola ed om he San a Eng acia sp ing, which a e included in APPENDIX III
and APPENDIX IV. Finally, he esul s ob ained wi h ega d o he collec ion
o p oka yo ic isola es and he sc eening o halophilic and halo ole an bac e ia
o hei abili y o p oduce an imic obial and/o su ace-ac i e compounds, wi h
u he cha ac e isa ion o he compounds o in e es p oduced by he s ain
ASV78, a e p esen ed in he Sec ion 4, APPENDIX V.
Mic obial di e si y and communi y composi ion in he Añana Sal Valley
The s udy o he mic obial communi y by Illumina-based 16S RNA gene and
ITS1 egion sequencing in hype saline en i onmen s e eals he possibili ies
and limi s o li e in he mos ex eme condi ions (108), no ing ha i is based
on he pu a i e associa ion o an amplicon wi h a axon, de ined as an Amplicon
Sequence Va ian (ASV). These s udies, oge he wi h he analysis o
physicochemical pa ame e s o wa e samples, could be used o iden i y wa e
physicochemical a iables ha p edic communi y s uc u e.
In he Añana Sal Valley, physicochemical moni o ing o he main
wa e cou ses ha eed he alley has e ealed he p esence o sal y and b ackish
wa e s o di e en o igins. This can be a ibu ed o he exis ence o mul iple
pa hways o unsa u a ed wa e o in il a e he sal deposi s in he subsoil,
whe e sal dissolu ion akes place (109). This p ocess gi es ise o sp ings wi h
a ying salini ies, caused by he sal iness o unde g ound wa e . Consequen ly,
i can be assumed ha he di e en wa e lows in he Añana diapi ic s uc u e
a e esponsible o he di e ences in he sp ing wa e 's hyd ochemical
cha ac e is ics.
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The dissolu ion o e apo i e (hali e) hund eds o me e s deep in he diapi
s uc u e is cha ac e ised by a high and s able ionic composi ion such as Cl−,
Na+ and K+, and ela i ely high empe a u e (16-20 °C), obse ed in sal y wa e
sp ings (Table 3). On he o he hand, he dissolu ion o gypsum and anhyd i e
and o NO3
− ions, a e he esul o he mixing o deep lows wi h shallowe
lows, cha ac e izing he b ackish wa e s, wi h compa a i ely highe p esence
o SO4
2−, Ca2+ and Mg2+ ions (Table 3). The wa e om he S8 piezome e has
a highe SO4
2−con en han he o he b ackish wa e s (Table 3), due o i s
pa icula posi ion in he low scheme (Figu e 17). While b ackish wa e is close
o neu al pH (7.2 o 7.4), sal y wa e is sligh ly acidic (6.2 o 6.6) (Table 3).
The low a iabili y o he sal acies is a peculia i y o his alley and indica es
a s able ionic en i onmen , con a y o wha has been desc ibed in o he diapi ic
en i onmen s (e.g., Zechs ein 2, Ge many) (109).
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Table 3. Physicochemical pa ame e s o he wa e a he di e en sampling si es analysed.
Sampling
si es
Physicochemical pa ame e s
Measu ed in si u
Measu ed by ionic ch oma og aphy* (mg/L)
Salini y
(g/L)
T
(°C)
pH
Cl-
𝐒𝐎𝟒
𝟐−
𝐍𝐎𝟑
−
Na+
Ca2+
Mg2+
K+
Sal y
[1]
200
16
6.6
153,813
4,697
nd
106,513
1,860
286
519
[2]
200
17
6.6
ND
ND
ND
ND
ND
ND
ND
[4]
220
17
6.6
142,721
4,475
nd
100,710
1,933
283
476
[6]
205
16
7.5
168,361
5,493
nd
118,100
1,823
300
567
[8]
210
16
6.2
148,983
4,778
nd
99,312
1,751
266
464
[9]
200
20
6.5
152,187
4,669
nd
102,345
1,845
281
580
[10]
220
29
7.6
ND
ND
ND
ND
ND
ND
ND
[11]
210
27
7.5
ND
ND
ND
ND
ND
ND
ND
[12]
200
18
7.8
ND
ND
ND
ND
ND
ND
ND
B ackish
[3]
20
13
7.4
5,515
921
17
3,853
391
65
19
[5]
10
13
7.3
5,460
939
17
3,812
396
62
19
[7]
4
13
7.2
3,060
2,374
6
2,380
673
193
21
Sampling si es: [1], San a Eng acia sp ing; [2], San a Eng acia channel; [3], San Juan s eam; [4], El Cau i o
sp ing; [5], El Pico Dulce sp ing; [6], El Pico sp ing; [7], S8 piezome e ; [8], Hon ana sp ing; [9], Fuen e iba
sp ing; [10], Pond III; [11], Pond II and [12], Pond I.
ND, no de e mined; nd, no de ec ed; *, hese da a a e he medium o he da a ob ained om he moni o ing
ne wo k du ing 2017-1022. Conduc i i y was measu ed in si u a [1], [6], [10], [11] and [12] sampling si ea
and was sa u a ed in all cases.
Figu e 17. The Añana Sal Valley map showing he 12 sampling loca ions included in he
ep esen a ion o he di e en wa e lows in he alley.
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Illumina sequencing was used o analyse p oka yo ic communi ies a 11
sampling si es and euka yo ic communi ies a se en sampling si es. The
mic obial communi y analysis e ealed a o al o 583,454 p oka yo ic
sequences and 704,874 ungal sequences, co esponding o 1,801 and 2,204
di e en ASVs, espec i ely. The main ac o in luencing he mic obial
dis ibu ion in his alley was ound o be salini y, as shown by he be a
di e si y indices. In ac , signi ican di e ences we e ound be ween sampling
si es (ANOSIM es , p alue < 0.05) o bo h p oka yo ic and ungal di e si y
when sampling si es we e g ouped acco ding o salini y (sal y and b ackish).
Howe e , no s a is ically signi ican di e ences we e obse ed when
compa ing samples acco ding o hei sampling loca ion (sp ing, pond, e c.).
This was also epo ed ela ed wi h he p oka yo es in he sal e n o Ma ghe i a
di Sa oia (I aly) (110) o ungi in Rhode Island´s sal e ns (Uni ed S a es) (111).
Alpha di e si y me ics e ealed ha p oka yo ic and ungal communi y
ichness (Obse ed ASVs and Chao1), quan i a i e communi y ichness o
di e si y (Shannon and Simpson indexes) and communi y equali y o e enness
(Pielou’s e enness) a ied widely among he samples (Table 4). The lowes
p oka yo ic and ungal ichness and di e si y was ound in wo sal y sp ings,
he Pico sp ing [6] and he San a Eng acia sp ing [1], espec i ely (Table 4).
On he con a y, he b ackish sp ing El Pico Dulce [5] is cha ac e ised by he
highes p oka yo ic ichness and di e si y, as well as by he high di e si y o
he ungi, al hough in he case o ungi, his can be equalled o exceeded in sal y
wa e s (Table 4). This phenomenon could be pa ly explained by he highe
salini y s ess encoun e ed by mic oo ganisms li ing in high salini y
en i onmen s, which may ha e limi ed di e si y due o he ene ge ically cos ly
li es yle (112). Howe e , o he aspec s, such as he a ailabili y o nu ien s and
oxygen, which we e no de e mined in his s udy, mus be conside ed among
he main limi ing ac o s o mic obial di e si y in wa e s o igina ing om e y
deep lows (113), which is he case o he sal y wa e sp ing in his alley.
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The dominance o some p oka yo ic axa in he San Juan s eam [3] (membe s
o he genus A cobac e , acco ding o subsequen analysis) and o ungal axa
in he San a Eng acia sp ing [1] (membe s o he genus Saccha omyces,
acco ding o subsequen analysis), could be obse ed (Table 4). This could be
ela ed o he abili y o ce ain mic oo ganisms in ce ain habi a s o
signi ican ly inc ease hei popula ion and become dominan . In he case o
A cobac e dominance, wo possible scena ios could be specula ed. The i s is
ela ed o i s abili y o adhe e and o m bio ilms (114), since in his loca ion, a
s eam, he e could be p olonged con ac wi h ege a ion and ocks. The second
is ela ed o he abili y o membe s o he genus A cobac e o oxidise sulphide
and educe ni a e. This sugges s ha sulphide may ac as an elec on dono in
deni i ica ion and inhibi ni ous oxide educ ion, making his niche speci ic
and educing species ichness (115).
Table 4. Alpha di e si y indexes calcula ed o he loca ions o he sal e n analysed in he s udy.
Sampling
si es
Obse ed
ASVs
Chao1
Simpson’s
index
Shannon’s
index
Pielou’s
e enness
P oka yo es
Sal y
[1]
104
104
0.95
5.07
0.76
[2]
196
197
0.96
5.56
0.73
[4]
169
169
0.96
5.50
0.74
[6]
69
69
0.83
3.68
0.60
[9]
294
301
0.93
5.48
0.67
[10]
147
147
0.96
5.59
0.78
[11]
74
74
0.86
3.88
0.62
[12]
120
120
0.89
4.49
0.65
B ackish
[3]
155
158
0.83
3.50
0.48
[5]
541
541
1.00
8.76
0.97
[7]
284
284
0.99
7.17
0.88
Fungi
Sal y
[1]
71
116
3.59
0.77
0.58
[6]
354
633
6.27
0.95
0.74
[10]
263
337
6.07
0.94
0.75
[11]
396
699
6.47
0.94
0.75
[12]
264
452
5.44
0.93
0.67
B ackis
h
[5]
262
430
6.15
0.96
0.76
[7]
93
93
4.89
0.91
0.74
Sampling si es: [1], San a Eng acia sp ing; [2], San a Eng acia channel; [3], San Juan s eam; [4], El
Cau i o sp ing; [5], El Pico Dulce sp ing; [6], El Pico sp ing; [7], S8 piezome e ; [9], Fuen e iba sp ing;
[10], Pond III; [11], Pond II and [12], Pond I.
ASVs, Amplicon Sequence Va ian s; Chao1, Con idence in e al o ichness es ima o .
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In conside a ion o he axonomic-in e ed me abolic p edic ion de i ed om
16S RNA gene amplicon sequencing, i was de e mined ha he mic obiome
engaged in chemohe e o ophy (also e e ed o as ae obic) and e men a ion
was he mos p e alen and ex ensi e along he alley. I is known ha mos
halophilic p oka yo es a e ae obic chemoo gano ophs capable o decomposing
o ganic compounds up o NaCl sa u a ion (5). Howe e , oxygen is poo ly
soluble in b ines, allowing anae obic halophilic he e o ophs o h i e (5) and
ac i ely coexis in he same niche. Acco ding o Wang and Bao (118), he main
bac e ial unc ion is chemohe e o ophy, a p ocess which also p edomina es
among a chaea; con e sely, e men a ion is widesp ead among he bac e ial
domain. On he o he hand, ITS1 egion sequencing-based p edic ion o ungal
ecological unc ions e ealed he ubiqui y o sap o ophs h oughou he sal y
wa e s, eaching up o 95.5% in he San a Eng acia sp ing, p obably associa ed
o he la ge p esence o he genus Saccha omyces, a well-known sap o oph. In
con as , b ackish wa e s p esen ed a high abundance o p oka yo ic and ungal
mic obio a in ol ed in animal/plan pa asi es o symbion s.
Finally, he high pe cen age o p oka yo ic and ungal ASVs assigned o
Unclassi ied axa, oge he wi h he la ge numbe o axa g ouped as
Uncul i a ed (12.6%) in he San a Eng acia sp ing, was s iking in his s udy.
This could imply ha hey co espond o sequences o axa ha ha e no ye
been iden i ied. A he same ime, his migh mean ha dominan o abundan
axa could be o e aken by new o uniden i ied axa, as no ed by O en (129).
This p emise highligh s he need o ca y ou cul u e-based s udies in his
hype saline en i onmen , as hey could p o ide ele an scien i ic knowledge.
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Sec ion 1 - SUMMARY OF THE RESULTS AND DISCUSSION
63
Isola ion and iden i ica ion o p oka yo ic isola es
Cul u e-based app oaches, ca ied ou o he i s ime in he Añana Sal
Valley, iden i ied a o al o 150 halophilic and halo ole an isola es wi h
di e en colony and cell mo phologies. All isola es we e bac e ia, excep o
wo ungi and wo a chaea. 89,3% o he s ains (134/150) we e isola ed in MA
medium and 10,7% (16/150) in MA con aining 23% NaCl (Sec ion 4,
APPENDIX V, Table S1). The Pseudomonado a phylum p edomina ed wi h
106 isola es (70.7%), in ag eemen wi h wha has been desc ibed in he Rambla
Salada, whe e he Pseudomonado a phylum ep esen s 72.5% o he o al
isola es isola ed he e (130). Mo eo e , he ou comes a e deemed o be logical
in iew o he cul u e media employed in he p esen s udy, as hese media a e
designed o he cul i a ion o he e o ophic ma ine bac e ia (131). The es o
he isola es we e di ided in o he phylum Ac inomyce o a (21 isola es, 14.0%),
Bacillo a (12 isola es, 8.0%), Bac e oido a (se en isola es, 4.7%),
Me hanobac e io a ( wo isola es, 1.3%) and Basidiomyco a ( wo isola es,
1.3%).
A he genus le el, he isola es we e dis ibu ed ac oss 48 di e en gene a. In
addi ion, a o al o 95 di e en species we e iden i ied. The genus Halomonas
was he genus wi h he highes numbe o isola es (36 isola es; 24.0%), as
epo ed in he s udy o Rambla Salada (130), ollowed by Pseudoal e omonas
(15 isola es; 10.0%). In pa icula , Halomonas sabkhae, Halomonas aeanensis
and Pseudoal e omonas neus onica we e he mos equen ly isola ed species,
7 isola es each. The phylogene ic ela ionship be ween he isola es is shown in
Sec ion 4, APPENDIX V, Figu e S1. Con e sely, 24.7% o he iden i ied
isola es (37/150) exhibi ed alues o less han 98.7% in he 16S RNA gene
sequence simila i y o axa p e iously desc ibed. These alues a e conside ed
below he h eshold p oposed by Chun e al. o designa ing such a axon o a
known species (81). Consequen ly, i can be deduced ha 37 o he isola es o
he cul u e collec ion ob ained in his s udy may ep esen new axa.
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Polyphasic cha ac e isa ion o new axa isola ed om San a Eng acia
sp ing
A polyphasic s udy was conduc ed o cha ac e ise he SALINAS58 (also named
ASV58, in his s udy) and ASV31 s ains isola ed om he San a Eng acia
sp ing, which is he main sou ce o b ine o he sal e n. The polyphasic s udy
consis ed on geno ypic, pheno ypic and chemo axonomic cha ac e isa ion o
he isola ed s ains.
Cha ac e isa ion o s ain SALINAS58
Phylogene ic analysis based on he 16S RNA gene sequence indica ed ha
s ain SALINAS58 belonged o he genus Al e e y h obac e . The 16S RNA
gene sequence o he po en ially no el isola e was deposi ed in GenBank unde
accession numbe MN918268. Ne e heless, in o de o enhance he con idence
o he esul s, he 16S RNA gene sequence ob ained om he WGS o he
selec ed s ain was u ilised o subsequen phylogene ic analysis, due o i s
g ea e leng h. WGS da a o s ain SALINAS58 was deposi ed a
DDBJ/ENA/GenBank unde he accession numbe s JAAFZT000000000.
The 16S RNA gene sequence (1,436 bp) analysis o s ain SALINAS58
showed he highes simila i y o Al e e y h obac e ma ensis MSW-14T
(96.6%), ollowed by Al e e y h obac e aquaemix ae JSSK-8T (96.5%),
Pon ixan hobac e lu eolus SW-109T (96.5%) and Al e e y h obac e
a lan icus 26DY36T (96.4%). Taking in o accoun he eclassi ica ion o he
amily E y h obac e aceae p oposed by Xu e al. (132), a he ime o w i ing
Al e e y h obac e ma ensis, Al e e y h obac e aquaemix ae and
Al e e y h obac e a lan icus a e homo ypic synonyms, alidly published
unde he In e na ional Code o Nomencla u e o P oka yo es (ICNP), bu hei
co ec names a e Pelage y h obac e ma ensis, Pon ixan hobac e
aquaemix ae and C oceibac e ium a lan icum, espec i ely. The sequence
simila i y o he 16S RNA gene be ween he s ain SALINAS58 and o he
ela ed species was lowe han 98.7%, he h eshold alue p oposed by Chun e
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65
al. (81) o dis inguishing wo species. Neighbou -joining phylogene ic
analysis based on he 16S RNA gene sequence showed ha s ain SALINAS58
o med a dis inc b anch wi hin he genus Al e e y h obac e , despi e no being
suppo ed by a high boo s ap alue (Figu e 21).
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Figu e 21. Neighbou -joining phylogene ic ee om 16S RNA gene sequences showing he
ela ionships be ween s ain SALINAS58, he ype s ains o Al e e y h obac e species and
ep esen a i es o se e al o he ela ed axa. Only boo s ap alues ≥ 50% (exp essed as
pe cen age o 1,000 eplica es) a e shown a di e gence poin s. Filled ci cles indica e ha he
co esponding nodes we e also ound in bo h he minimum-e olu ion and maximum-likelihood
ees (Sec ion 3, APPENDIX III, Fig. S2 and Fig. S3, espec i ely). Pa acoccus paci icus F14T
(GenBank accession numbe KF924610) was used as an ou g oup. Ba , 0.020 subs i u ions pe
nucleo ide posi ion.
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The genome size o s ain SALINAS58 was 2.8 Mbp and was assembled in o
56 con igs, wi h a N50 alue o 1.9 Mbp. The DNA G+C con en o s ain
SALINAS58 was 61.4%, wi hin he ange o he genus Al e e y h obac e
(54.5–69.0%) (133). The use o OGRI alues o he cons uc ion o
phylogenomic ees mus necessa ily be aken in o accoun o he e ec i e
de e mina ion o he axonomic posi ion o a s ain (39). Among OGRIs, he
ANI alues ela i e o s ain SALINAS58 anged om 71.5 o 73.1% compa ed
o P. aquaemix ae JSSK-8T and P. ma ensis MSW-14T, espec i ely. The
dDDH alues ( esul s ob ained om he ecommended o mula 2) anged om
18.3 o 18.9% wi h P. aquaemix ae JSSK-8T and P. lu eolus SW-109T,
espec i ely. AAI alues anged om 66.6 o 68.1% compa ed o
A. epoxidi o ans JCS350T and P. ma ensis MSW-14T, espec i ely. All o
hese alues we e below he h esholds speci ied o bac e ial species
iden i ica ion (39,81). The whole genome phylogene ic ee con i med ha
s ain SALINAS58 o ms a sepa a e b anch be ween ela i e ype species
(Sec ion 3, APPENDIX III, Fig. S4). In addi ion, genome anno a ion p edic ed
genes ela ed o ehalose biosyn hesis, N-ce ylneu amina e syn hase and he
mul id ug e lux pump componen M F in s ain SALINAS58, which we e no
ound in ela ed axa (Sec ion 3, APPENDIX III, Table S1).
Pheno ypic and chemo axonomic cha ac e isa ion analyses we e also ca ied
ou o dis inguish he SALINAS58 s ain om he ecognised species. Fo his
pu pose, all hese analyses we e ca ied ou simul aneously wi h he pu chased
ype s ains P. ma ensis MSW-14T and P. aquaemix ae SSK-8T. The main
pheno ypic cha ac e is ics ha dis inguished s ains SALINAS58 om i s
closes ela i es a e summa ised in Table 5.
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Table 5. Di e en ial pheno ypic cha ac e is ics o s ain SALINAS58 and phylogene ically ela ed ype s ains. S ains: 1, SALINAS58 ( his s udy); 2, P. ma ensis
MSW-14T ( his s udy); 3, P. aquaemix ae SSK-8T ( his s udy); 4, P. lu eolus SW-109T (134,135); 5, Al e e y h obac e epoxidi o ans JCS350T (134,135). +,
posi i e; −, nega i e; w, weakly posi i e; NA, no da a a ailable; ND, no de e mined. None o he s ains we e able o g ow in anae obic condi ions o u ilize ace a e.
In API ZYM s ips, all he s ains we e posi i e o es e ase (C4) and es e ase lipase (C8) ac i i ies and nega i e o β-glucu onidase, N-ace yl-β-glucosaminidase,
α-mannosidase and α- ucosidase ac i i ies.
Cha ac e is ic
1
2
3
4
5
Colony colou
O ange
Beige
Yellow
Yellow
Yellow
Cell shape
Rod
Rod
Rod
Rod
O oid– od
Oxidase ac i i y
-
+
w
+
+
Ni a e educ ion
-
-
+
-
-
G ow h ange (op imum):
NaCl (%, w/ )
0-5 (1)
0-10 (2)
0-5 (1)
0.5-9 (2)
0.5-9 (2)
Tempe a u e (°C)
15-30 (30)
4-42 (30)
15-37 (30)
4-36 (30)
20-40 (35)
pH
5.5-9 (6-6.5)
7-10 (7)
7-7.5 (7)
5.5-8 (7-8)
6-8.5 (6.5)
Hyd olisis o :
Casein
+
-
-
+
+
S a ch
-
-
-
w
-
Tween 80
+
-
-
+
+
Tween 20
+
+
-
+
NA
Gela in
-
-
+
-
-
U iliza ion o :
Inulin
+
-
ND
NA
NA
Succina e
-
-
+
-
-
D-Xylose
-
+
-
+
-
Melibiose
+
+
-
-
NA
So bi ol
+
+
-
-
NA
D-F uc ose
+
+
-
+
+
Con inued
Sec ion 1 - SUMMARY OF THE RESULTS AND DISCUSSION
68
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Cha ac e is ic
1
2
3
4
5
Inosi ol
+
+
-
-
NA
D-Galac ose
+
+
-
+
+
Glyce ol
-
+
-
NA
NA
Assimila ion o :
Mala e
-
w
-
-
-
Manni ol
-
-
-
-
+
N-Ace ylglucosamine
-
-
-
+
+
Mal ose
-
-
-
+
+
Adipa e
-
-
-
NA
+
Enzyme ac i i y:
Lipase (C 14)
w
-
-
+
-
Valine a ylamidase
w
+
+
-
+
Cys ine a ylamidase
-
w
w
w
+
T ypsin
w
+
+
w
+
α-Chymo ypsin
+
+
-
+
-
Acid phospha ase
w
w
-
-
+
Naph hol-AS-BI-phospho ohyd olase
+
w
w
+
-
α-Glukosidase
w
-
-
w
-
β-Glucosidase
w
+
+
+
+
β-Galac osidase
-
-
-
+
+
Pigmen (nm)
465
307, 340, 381, 415*a
449, 476, 477*b
340, 417, 449, 447
340, 381, 417, 541, 477
*Da a om: a, Seo and Lee (136) and b, Pa k e al. (135).
Sec ion 1 - SUMMARY OF THE RESULTS AND DISCUSSION
69
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A chemo axonomic app oach showed ha he a y acid p o ile o s ain
SALINAS58 was simila o ha o ela ed species, al hough di e ences in he
p opo ions o some a y acids we e obse ed. The p esence o C14:0, C18:0,
C18:0 2OH and C16:0 iso 3OH only in s ain SALINAS58 and he absence o
C12:0 2OH, dis inguish his s ain om he o he wo s ains analysed in his
s udy. The p edominan espi a o y quinone iden i ied in s ain SALINAS58
was ubiquinone Q-10, which is cha ac e is ic o he genus Al e e y h obac e
(132). In addi ion, he majo pola lipids de ec ed in s ain SALINAS58 we e
diphospha idylglyce ol, phospha idyle hanolamine, phospha idilglyce ol, ou
uniden i ied glycolipids and one uniden i ied phospholipid (Sec ion 3,
APPENDIX III, Fig. S6).
O e all, he analyses e ealed di e ences in genomic, pheno ypic and
chemo axonomic cha ac e is ics, indica ing ha s ain SALINAS58 was a new
species, o which he name Al e e y h obac e mu iae sp. no . was p oposed,
being he ype s ain SALINAS58T.
A he ime o w i ing, Al e e y h obac e mu iae is a homo ypic synonym,
alidly published unde he ICNP, conside ing Al e ipon ixan hobac e mu iae
o be he co ec name since he las eclassi ica ion in 2021 by Kim e al. (137).
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Desc ip ion o Al e ipon ixan hobac e mu iae sp. no .
Table 6. Desc ip ion o Al e ipon ixan hobac e mu iae s ain SALINAS58T.
E ymology
mu iae (mu’ i.ae. L. gen. em.
n. mu iae, o b ine, subs ance
om which he s ain was
isola ed).
Type s ain
SALINAS58T
(=CECT 30029T=LMG
31726T).
Taxonomy
Phylum Pseudomonado a,
class Alphap o eobac e ia,
o de Sphingomonadales,
amily E y h obac e aceae
and genus
Al e ipon ixan hobac e .
Cells
G am-nega i e, ae obic, non-
mo ile, ods 0.40.9 μm wide
and 1.83.0 μm long and non
lagella .
Colonies
O ange, pigmen ed,
anslucen , smoo h and hey
o m a con ex ele a ion a e 3
days a 25 °C, wi h 1 mm o
diame e .
Pigmen s
Ca o enoid pigmen s, i does
no con ain
bac e iochlo ophyll a.
G ow h
A pH 5.5-9 (op imum, pH
6-6.5), be ween 15 and 30 °C
(op imum, 30 °C) and in he
p esence o 0-5% o NaCl
(op imum 1%).
Enzyma ic
ac i i ies
Ca alase-posi i e and oxidase-nega i e. I hyd olyses casein, Tween
20 and Tween 80, bu no s a ch. I can use inulin, melibiose, so bi ol,
D- uc ose, inosi ol and D-galac ose as ca bon and ene gy sou ces, bu
no succina e, D-xylose, ace a e o glyce ol. In API 20NE es s, i is
only posi i e o aesculin hyd olysis. In API ZYM es s, i shows
s ong alkaline phospha ase ac i i y; in e media e es e ase (C4),
es e ase lipase (C8), leucine a ylamidase, α-chymo ypsin and
naph hol-AS-BI-phosphohyd olase ac i i y; and weak lipase (C14),
aline a ylamidase, ypsin, acid phospha ase, β-glucosidase ac i i y.
Con inued
Figu e 23. Al e ipon ixan hobac e
mu iae s ain SALINAS58T g ow h on MA.
Colony mo phology and colou a e
shown.
Figu e 22. T ansmission elec on
mic og aph o a cell o SALINAS58T.
Ba 500.0 nm.
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Cha ac e is ics
1
2
3
4
5
Enzyma ic ac i i ies:
Es e ase (C 4)
+
w
w
NA
+
Es e ase lipase (C 8)
+
w
-
NA
+
Valine a ylamidase
+
-
w
NA
-
Acid phospha ase
w
w
+
NA
+
Naph hol-AS-BI-phosphohyd olase
w
w
+
NA
w
Cys ine a ylamidase
w
-
-
NA
-
α-glucosidase
+
+
-
NA
-
β-glucosidase
-
w
-
NA
-
Pigmen s
Sphe oidenone,
sphe oidene and
BChl a
No de ec ed*a
Sphe oidenone and
BChl a*b
ND
ND
*Da a om: a, So okin e al. (143); b, Ramap asad e al. (144). BChl a, bac e iochlo ophyll a.
Sec ion 1 - SUMMARY OF THE RESULTS AND DISCUSSION
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A chemo axonomic app oach showed ha he main a y acid, C18:0 cyclo ω8c,
p esen in s ain ASV31 was no p esen in T. paci ica DSM 10166T o R. algae
LMG 29228T. In addi ion, ASV31 was he only s ain wi hou hyd oxy a y
acids and he only one wi h unsa u a ed a y acids. Mo eo e , summed ea u e
8 (C18:1 ω7c and/o C18:1 ω6c) was he majo a y acid ound in T. paci ica DSM
10166T (83.08%) and R. algae LMG 29228T (70.12%), while in ASV31 i was
de ec ed a a lowe pe cen age (37.34%). Ubiquinone Q-10 was iden i ied in
s ain ASV31, which is ypical o mos gene a o he Pa acoccaceae amily
(138). In addi ion, he pola lipid p o ile o s ain ASV31 comp ised
phospha idylglyce ol, aminolipid, wo uniden i ied glycolipids, wo
uniden i ied phospholipids and wo uniden i ied lipids (Sec ion 3, APPENDIX
IV, Figu e S4). I was obse ed ha he phospha idyle hanolamine, which is
one o he mos impo an pola lipid in T. dalianensis and he membe s o
Rhodo ulum (138,145), did no ma ch wi h he p o ile o s ain ASV31.
O e all, he analyses e ealed di e ences in genomic, pheno ypic and
chemo axonomic cha ac e is ics, indica ing ha s ain ASV31 was he i s
species o a new genus wi hin he amily Pa acoccaceae, o which he name
Anianabac e salinae gen. no . sp. no . was p oposed, being he ype s ain
ASV31T.
A he ime o w i ing, Anianabac e salinae is alidly published in acco dance
wi h he ICNP bu he designa ion is add essed o Anianibac e salina um due
o o he spelling o name o inaccu a e spelling (146).
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Desc ip ion o Anianibac e salina um gen. no . sp. no .
Table 8. Desc ip ion o Anianibac e salina um s ain ASV31T.
E ymology
(A.ni.a.ni.bac’ e . N.L. masc.
n. bac e , a od; N.L. masc. n.
Anianibac e , a od om he
Añana sola sal e n) and
(sa.li.na’ um. L. gen. pl. n.
salina um, o sal wo ks).
Type s ain
ASV31T (=CECT 30309T =
LMG 32242T)
Taxonomy
Phylum Pseudomonado a,
class Alphap o eobac e ia,
o de Rhodobac e ales and
amily Pa acoccaceae.
Cells
Non-mo ile G am-nega i e
ods ha occu singly o in
agg ega es wi h a mucous
polysaccha ide capsule a ound
hem. PHA g anules a e
p esen inside he cells.
Colonies
Beige- o-pink colou ed. The
od cells a e 2.4-2.5 µm in
leng h and 0.4-0.5 µm wide.
Pigmen s
Sphe oidenone, sphe oidene
and bac e iochlo ophyll a a e
p esen .
G ow h
The species g ows a pH
6.5-9.5 (op imum 7-9.5) and a
empe a u es be ween 18-
37 ºC (op imum 30 ºC). I has
a high NaCl ole ance o 0-
23% (w/ ) (op imum 35%).
Enzyma ic
ac i i ies
Ca alase and oxidase posi i e and u ease nega i e. I hyd olyses aesculin and
Tween 20. Ni a e educ ion occu s in his species. In API ZYM es s, i
shows s ong alkaline phospha ase, es e ase (C 4), es e ase lipase (C 8),
leucine a ylamidase, aline a ylamidase and α-glucosidase ac i i y; and
weak cys eine a ylamidase, acid phospha ase and naph hol-AS-BI-
phosphohyd olase ac i i y.
Con inued
Figu e 23. T ansmission elec on
mic og aph o a cell o ASV31T.
Ba 500.0 nm.
Figu e 26. Anianibac e salina um s ain
ASV31T g ow h on MA. Colony
mo phology and colou a e shown.
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An ibio ic
suscep ibili y
Resis an o polymyxin B (300 IU), ancomycin (30 µg) and
s ep omycin (10 µg).
Chemo axonomy
The unique ubiquinone is Q-10 and he majo a y acids a e C18:0 cyclo
ω8c and summed ea u e 8 (C18:1 ω7c and/o C18:1 ω6c). The majo
pola lipids a e phospha idylglyce ol, aminolipid, an uniden i ied
glycolipid, an uniden i ied phospholipid and an uniden i ied lipid.
WGS
The genome size is 3.6 Mbp and he DNA G+C con en o he ype
s ain is 65.7 mol%.
CECT, Spanish Type Cul u e Collec ion; LMG, Labo a o ium oo Mic obiologie Gen
(Belgium); WGS, Whole genome sequencing.
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Sc eening o an imic obial and/o su ace-ac i e compound p oduce s
Wi h he excep ion o wo isola es ha p o ed un eco e able, he emaining
148 isola es we e subjec ed o a sc eening o an imic obial p oduc ion
po en ial. This was based on hei abili y o inhibi he g ow h o pa hogen
s ains using he de e ed an agonism assay. Twen y isola es showed clea
ac i i y agains a leas one o he pa hogen s ains (Table 9). These
an imic obial p oducing isola es we e hen subjec ed o he well di usion assay
whe e nine o hem we e ound o be posi i e o an imic obial p oduc ion
(Table 9). Eigh o hese halo ilic o halo ole an isola es p oduced compounds
ha inhibi ed he g ow h o bo h G am-posi i e and G am-nega i e bac e ial
s ains, while an i ungal ac i i y was only obse ed o isola e ASV55 in he
well di usion assay (Table 9). Illus a ions o some o he esul s ob ained in
bo h an agonism and well di usion assays a e shown in Figu e 27A and Figu e
27B, espec i ely.
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Sec ion 1 - SUMMARY OF THE RESULTS AND DISCUSSION
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Figu e 27. Example o he de ec ion o an imic obial compounds p oduced by some halophilic
and halo ole an isola es. A, pa hogen g ow h inhibi ion halo in he an agonism assay
p oduced by isola es ASV12, ASV10, ASV14, ASV25, ASV89 and ASV106 and B, pa hogen
g ow h inhibi ion halo in he di usion assay p oduced by isola es ASV12, ASV13, ASV55,
ASV128, ASV106, ASV129 and ASV136.
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In addi ion, he di usion assay sugges ed ha he an imic obial compounds
we e p oduced by he hi d day o incuba ion and ha deg ada ion was e iden
a e he en h day (Table 9). On he o he hand, isola es o which ac i i y was
only obse ed in he delayed an agonism assay may imply ha he p oduc ion
o he an imic obial compound was based on nu ien compe i ion wi h he
pa hogen s ain, as desc ibed in o he s udies (147).
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Table 9. Isola es showing an imic obial ac i i y agains a leas one o he pa hogenic s ains used. The diame e (mm) o he inhibi ion zone in he delayed
an agonism assay (DAA) and he well di usion assay (WDA) is gi en.
Pa hogen
s ains
P. ae uginosa
ATCC 27853T
E. coli
ATCC25922T
B. sub illis
CECT 356T
S. au eus
ATCC 29213T
E. aecalis
ATCC 29212T
C. albicans
ATCC 90029T
Mean o he inhibi ion halo (mm)
DAA
WDA
DAA
WDA
DAA
WDA
DAA
WDA
DAA
WDA
DAA
WDA
day
day
day
day
day
day
Isola e ID
3
10
17
3
10
17
3
10
17
3
10
17
3
10
17
3
10
17
ASV1
8
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV3
11
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV7
20
-
-
-
-
-
-
-
12
-
-
-
25
-
-
-
-
-
-
-
-
-
-
-
ASV10
10
-
-
-
15
-
-
-
-
-
-
-
12
-
-
-
-
-
-
-
-
-
-
-
ASV11
10
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV12
27
-
-
-
27
14
12
-
25
-
-
-
30
-
-
-
30
-
-
-
-
-
-
-
ASV13
-
-
-
-
9
15
11
-
-
-
-
-
-
-
-
-
31
-
-
-
-
-
-
-
ASV14
12
-
15
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV25
12
-
12
-
15
-
-
-
16
-
-
-
10
-
-
-
13
-
-
-
-
-
-
-
ASV55
-
15
13
-
-
16
15
10
13
20
16
13
-
12
18
13
-
7
6
-
-
15
13
-
ASV78
12
-
-
-
-
-
-
-
20
-
-
-
17
-
-
-
-
-
-
-
-
-
-
-
ASV89
9
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV106
-
-
-
-
-
-
-
-
21
18
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV117
9
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV118
9
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV120
-
-
-
-
-
-
-
-
6
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV121
-
-
-
-
-
-
-
-
6
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV128
-
-
-
-
-
-
-
-
20
15
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV129
-
-
-
-
-
-
-
-
19
17
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV136
6
-
-
-
-
-
-
-
14
9
-
-
10
-
-
-
-
-
-
-
-
-
-
-
-, nega i e
Sec ion 1 - SUMMARY OF THE RESULTS AND DISCUSSION
85
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The genus Pseudoal e omonas (isola es ASV1, ASV3, ASV10, ASV11,
ASV12, ASV13, ASV14, ASV25, ASV78, ASV89, ASV117, ASV118 and
ASV136) was he main con ibu o o an ibac e ial ac i i y in his s udy. I is
no ewo hy ha he majo i y o he isola es, wi h he excep ion o ASV13,
inhibi ed he g ow h o P. ae uginosa. In addi ion o he possibili y o an
inhibi o y e ec due o nu ien compe i ion, ano he hypo hesis o his
occu ence is ela ed o he abili y o Pseudoal e omonas species o p oduce
an ibio ilm agen s such as al e ocin, he eby supp essing he g ow h o P.
ae uginosa, a ecognised bio ilm- o ming bac e ium (148). The emaining o
he isola es wi h an imic obial capaci y belonged o he gene a Salini ib io
(ASV7), Pseudomonas (ASV55), Kocu ia (ASV106 and ASV128),
Halomonas (ASV120 and ASV121) and S ep omyces (ASV129). These
gene a ha e p e iously been documen ed o possess an imic obial ac i i y
(149–152), wi h he excep ion o he genus Salini ib io. Despi e some epo s
o an i ungal (153) and insec icidal (154) ac i i y o membe s o he genus
Salini ib io, hei an ibac e ial capaci y, as seen in s ain ASV7 in his s udy,
has no been desc ibed.
Con e sely, all 148 isola es we e also sc eened o biosu ac an p oduc ion
po en ial, based on hei abili y o educe su ace ension using oil sp eading
and Pa a ilm-M es s, and o bioemulsi ie po en ial, based on hei abili y o
emulsi y oil and hyd oca bon compounds by EI24. Illus a ions o some o he
esul s a e shown in Figu e 28, while he de ailed esul s a e summa ised in
Sec ion 3, APPENDIX III, Table S2. The mean esul s ob ained o each isola e
in he Pa a ilm-M es and he oil sp eading es a e shown in Figu e 29 and
Figu e 30, espec i ely, g ouped by genus. Isola es we e designa ed as posi i e
o biosu ac an p oduc ion i he d op diame e in he Pa a ilm-M es and he
clea ing zone hey p oduced in he oil sp eading es we e g ea e han he
nega i e con ol (BPM) (≥ 3.4 mm and ≥ 7.0 mm, espec i ely). Simila cu -o
alues ha e been used in o he s udies o es posi i e o biosu ac an
p oduc ion (95,155).
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Figu e 248. Examples o some o he halophilic and halo ole an s ains ha p oduce
su ace-ac i e compounds in A, Pa a ilm-M es ; B, oil sp eading es ; C, emulsi ica ion es ,
compa ed wi h he nega i e con ol.
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Sec ion 1 - SUMMARY OF THE RESULTS AND DISCUSSION
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indica ed ha he ASV78 s ain was able o syn hesize biomolecules o
di e en ac i i ies simul aneously and unde he same condi ions and ha hey
could be ex ac ed by he di e en echniques used.
Figu e 33. TLC-based bioau hog aphy o he an imic obial c ude ex ac o ASV78 exhibi ing
an ibac e ial ac i i y agains S. au eus ATCC 29213.
To iden i y he componen s o he an imic obial ex ac , LC-HRMS
de eplica ion was pe o med. The de eplica ion p ocess yielded h ee peaks,
which we e iden i ied as lumich ome, pen ab omopseudilin and b omophene.
Lumich ome was de eplica ed as a ma ch agains he Fundación MEDINA
lib a y by ma ching he e en ion ime, UV spec um and molecula o mula o
he analysed peak wi h a s anda d s o ed in he spec al da abase (Sec ion 4,
APPENDIX V, Figu e S5). Lumich ome is a known deg ada ion p oduc o
ibo la in, by pho odeg ada ion in neu al o acidic solu ions and by enzymes
in bac e ia, wi h plan g ow h p omo ing ac i i y (163).
On he o he hand, pen ab omopseudilin was de eplica ed by in e p e ing he
molecula o mula o a UV peak ha was e y clea ly de ined. The MS
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spec um (Sec ion 3, APPENDIX III, Figu e S5) showed a cha ac e is ic
iso opic pa e n wi h i e b omine a oms, hus sugges ing C10H4B 5NO as he
molecula o mula, wi h pen ab omopseudilin being he unique ma ch in he
Dic iona y o Na u al P oduc s. The compound was pu i ied om a bioac i e
ex ac using e e sed phase ch oma og aphy and p epa a i e/semi-p epa a i e
HPLC. A R 22 min, a o al o 0.9 mg o pu e compound was ob ained. Then,
(-)-MS spec um and 1H-NMR analysis (Figu e 34) con i med i s iden i y.
Finally, he peak iden i ied as b omophene did no gi e an in e p e able
(+)-HRMS spec um. Consequen ly, pu i ica ion o he compound was
unde aken om he bioac i e ex ac by e e sed phase ch oma og aphy,
ollowed by p epa a i e/semi-p epa a i e HPLC, he eby yielding a ac ion
ha was deemed sui able o NMR analysis. Nega i e mode LC-HRMS
p oduced a (-)-MS spec um ha was in e p e ed, based on i s iso opic
dis ibu ion, o co espond o a molecula o mula o C12H6B 4O2 wi h eigh
coincidences in he Dic iona y o Na u al P oduc s. A R 20 min, a o al o
0.5 mg o pu e compound was ob ained. Then, (-)-MS spec um and 1H-NMR
analysis (Figu e 35) con i med he p esence o b omophene by compa ison
wi h li e a u e da a (164).
As desc ibed in his s udy, highly b omina ed me aboli es om
Pseudoal e omonas ha e also been documen ed. Speci ically,
pen ab omopseudilin was p e iously isola ed om P. phenolica and
P. lu eo iolacea and b omophene om P. phenolica (165). Howe e , his s udy
desc ibes he abili y o simul aneously p oduce bo h compounds by he s ain
ASV78, o which he phylogene ically closes species is Pseudoal e omonas
neus onica (16S RNA gene sequence simila i y o 98.57%).
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Figu e 34. (-) MS spec um ( op) and 1H-NMR spec a wi h he s uc u e (bo om) o
pen ab omopseudilin p oduced by s ain ASV78.
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Figu e 35. (-) MS spec um ( op) and 1H-NMR spec a wi h he s uc u e (bo om) o
b omophene p oduced by s ain ASV78.
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Despi e he epo ed an ibac e ial ac i i y o bo h pen ab omopseudilin and
b omophene agains me hicillin- esis an S. au eus, li le is known abou hei
b oad spec um ac i i y (165,166). Fu he mo e, he simul aneous p oduc ion
o bo h compounds may no be common. To he bes o ou knowledge, his has
only been documen ed in one s ain, Pseudoal e omonas sp. CMMED 290
(165). Despi e he same me abolic pa hway o syn hesising bo h compounds
(167). In his espec , i would be wo hwhile o in es iga e he po en ial
syne gis ic e ec s o hese compounds, as his has no been demons a ed o
da e. Such an in es iga ion could p o e bene icial o hei p ac ical
applica ion.
In his ega d, he assay hold wi h he pu e compounds showed ha bo h
compounds exhibi ed an imic obial ac i i y agains G am-posi i e bac e ia,
wi h pen ab omopseudilin addi ionally exhibi ing ac i i y agains
G am-nega i e bac e ia (Table 10). In pa icula , pen ab omopseudilin
exhibi ed MIC alues anging om 0.02 o 0.04 µg/mL agains S. au eus ATCC
29213 and MIC alues anging om 10 o 20 µg/mL agains P. ae uginosa
ATCC 27853 (Table 10, op). Con e sely, b omophene showed MIC alues
anging om 2.5 o 5 µg/mL agains S. au eus ATCC 29213 (Table 10, op).
Table 10. An ibac e ial ac i i y o pu e compounds and con ols. MIC alues a op and
an ibac e ial ac i i y (MIC alues ob ained om one eplica e) a bo om.
Compounds
MIC (µg/mL)
S.au eus
ATCC 29213
P.ae uginosa
ATCC 27853
Pen ab omopseudilin
0.02-0.04
10-20
B omophene
2.5-5
> 80
Syne gy (1:1)
0.04-0.08
20-40
Vancomycin
0.5-1
ND
Cip o loxacin
ND
0.25-0.5
Con inued
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Compounds
Ac i i y (µg/mL)
A.baumannii
ATCC 19606
E. aecalis
ATCC 29212
E. aecium
anA 15167
Pen ab omopseudilin
10-20
10-20
1.25-2.5
B omophene
> 80
40-80
20-40
Syne gy (1:1)
20-40
20-40
2.5-5
Vancomycin
ND
4-8
> 32
Az eonam
80-160
ND
ND
ND; no de e mined
MIC alues agains E. aecalis ATCC 29212, ancomycin- esis an E. aecium
anA 15167 and A. baumannii ATCC 19606 we e de e mined using a single
eplica e assay. This was due o he pauci y o samples and may equi e u he
alida ion in u u e s udies. Pen ab omopseudilin inhibi ed g ow h o
E. aecalis ATCC 29212 a 10-20 µg/mL, ancomycin- esis an E. aecium
anA 15167 a 1.25-2.5 µg/mL and A. baumannii ATCC 19606 a 10-20 µg/mL
(Table 10, bu om). On he o he hand, i was obse ed ha b omophene
inhibi ed he g ow h o E. aecalis ATCC 29212 a 40-80 µg/mL and he g ow h
o ancomycin- esis an E. aecium anA 15167 a 20-40 µg/mL (Table 10,
bu om). Fo none o he pa hogens es ed, syne gis ic ac i i y be ween he wo
compounds was obse ed.
Pen ab omopseudilin's an ibac e ial ac i i y agains clinically ele an species
including A. baumannii, E. aecium and S. au eus should be conside ed. The
low concen a ion o pen ab omopseudilin (MIC alues anging om 0.02 o
0.04 µg/mL) a which i is e ec i e agains S. au eus ATCC 29213 is s iking,
especially when compa ed o he ancomycin concen a ions (MIC alues o
0.5 o 1 µg/mL in his s udy, and 0.52 o 0.59 µg/mL acco ding o Lepe e al.
(168). I would he e o e be e y in e es ing o e alua e he spec um o
pen ab omopseudilin agains a wide ange o pa hogens and o u he
in es iga e i s mechanism o ac ion, which may be di e en om ha o
ancomycin, as e idenced by i s ac i i y agains ancomycin- esis an
E. aecium and G am-nega i e bac e ia. Indeed, mul i- a ge inhibi o s
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Sec ion 1 - SUMMARY OF THE RESULTS AND DISCUSSION
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ep esen he nex on ie in he wo ld o an ibio ics, o e ing signi ican
ad an ages o e he de elopmen o d ug esis ance (169).
Wi h ega d o he biosu ac an and bioemulsi ie componen s o he bioac i e
ex ac s, IR spec oscopy was used o iden i y he chemical na u e o hese
componen s. On he one hand, he IR spec um o he biosu ac an ex ac
e ealed he p esence o alipha ic hyd oca bon chains along wi h a
polysaccha ide moie y, he eby sugges ing a glycolipid na u e o he
biosu ac an (Figu e 36A). The p esence o abso p ion bands a 3,311 cm−1
indica ed he p esence o -OH s e ching o he hyd oxy g oups. The p esence
o bands a 2,919 and 2,847 cm−1 co esponded o he C-H s e ching mode o
an alipha ic chain. The peak a 1,652 cm−1 could be co ela ed wi h he p esence
o C=O g oups. A peak a 1,408 cm−1 indica ed ha p esence o C-H bending
ib a ions o CH3 and CH2 ha may be a ibu ed o polysaccha ides. The peaks
be ween 1,317-1,013 cm−1 indica ed he p esence o C-O s e ching
co esponding o he suga moie y. The IR spec um o he biosu ac an ex ac
p oduced by s ain ASV78 was simila o ha o o he glycolipid biosu ac an s
epo ed in p e ious s udies (170–172).
On he o he hand, he IR spec um o he bioemulsi ie ex ac is shown in
Figu e 36B. B oad abso p ion bands indica ing he p esence o ca boxylic
g oups we e obse ed (3,369 cm-1, hyd oxy s e ching; 1,630 cm-1, ca boxyl
C=O s e ching; 1,114 cm-1, ca boxyl -C-O s e ching). In addi ion, s e ching
ib a ions a he wa eleng h o 1,556 cm-1 we e obse ed o he amide g oup.
They a e associa ed wi h C-N and N-H g oups, indica ing he p esence o
acyla ed amino suga s and p o eins/pep ides in he bioemulsi ie s uc u e,
o ming glycop o eins. The emulsi ying ac i i y o a glycop o ein exopolyme
p oduced by Pseudoal e omonas spp. has al eady been desc ibed (173,174).
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Figu e 36. IR spec a o A, c ude biosu ac an ex ac and B, c ude bioemulsi ie ex ac ob ained om he e men a ion b o h o s ain ASV78.
Sec ion 1 - SUMMARY OF THE RESULTS AND DISCUSSION
101
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Sec ion 1 - SUMMARY OF THE RESULTS AND DISCUSSION
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I is well known ha su ace-ac i e compounds a e in ol ed in he
"pseudosolubilisa ion" s a egy, which aims o imp o e bioa ailabili y and
acili a e access o hyd ophobic compounds. In addi ion, he genus
Pseudoal e omonas is ypical among hyd oca bon-deg ading mic oo ganisms,
so i is no su p ising ha a signi ican p opo ion o biosu ac an o
bioemulsi ie p oduce s a e ound in his genus (60). Consequen ly, u he
cha ac e isa ion and in es iga ion o he po en ial applicabili y o he ac i e
compounds p oduced by s ain ASV78 could be p omising.
Finally, he WGS o s ain ASV78, ob ained using a combina ion o MinION
and Illumina app oaches, yielded 19 con igs ( he da a is accessible a he NCBI
unde he BioP ojec accession numbe PRJNA1218939). The genome had a
G+C DNA con en o 41.5% and 5,215,181 bp. The assembled con igs we e
analysed using P okka and a o al o 4,674 genes we e de ec ed. O hese, 4,548
we e iden i ied as p o ein coding genes (CDSs) and hei unc ions we e
assigned. A unc ion was assigned o only 55.5% o he CDSs, while he es
we e anno a ed as hypo he ical p o ein CDSs. This esul highligh s he
necessi y o ca y ou u he esea ch in o de o in es iga e he unc ions o
hese genes.
The analysis o he genome using an iSMASH e ealed a o al o se en egions
ha a e hough o encode biosyn he ic gene clus e s (BGCs) (Table 11). The
o al leng h o he p edic ed BGCs is app oxima ely 250 Kb, ep esen ing
~4.8% o he ASV78 genome. The numbe o p edic ed BGCs in ela ion o he
size o he genome is in he a e age ange ound in p oka yo es (175). I is
in e es ing o no e ha only one o he p edic ed BGCs showed 100% simila i y
o he al eady desc ibed BGC o pen ab omopseudilin (167). In his ega d,
Aga wal e al. emphasised ha he bmp biosyn he ic gene clus e is esponsible
o he p oduc ion o a limi ed numbe o polyb omina ed diphenyl e he
(PBDE) compounds, including b omophene and pen ab omopseudilin. (167).
In his sense, ou esul s a e consis en wi h he con i med p esence o bo h
compounds in he ex ac ia LC–HRMS. Howe e , a phylogeny o he genus
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e ealed ha nume ous clades do no con ain hese genes (176). In ac , he
de ec ion o he bioac i e compounds was mos ly es ic ed o he clades
con aining he P. lu eo iolacea and P. phenolica ype s ains (176), and no o
P. neus onica as was he case o s ain ASV78. Two o he BGSs showed 75%
and 45% simila i y wi h he des e ioxamine E BGC (177) and APE V BGC
(175), espec i ely. Des e ioxamine E and i s analogues a e side opho es
widely p oduced by S ep omyces and ela ed bac e ia (178). Howe e , in he
Pseudoal e omonas gene a whe e des e ioxamine is ound (such as P.
spongiae, P. u henica, P. pi a ica and P. unica a), i is des e ioxamine B
(179). Finally, a yl polyenes a e yellow pigmen s, widesp ead among G am-
nega i e pa hogens, such as, APE Ec om u opa hogenic E. coli (180). They
a e s uc u ally and unc ionally ela ed o ca o enoids and con e p o ec ion
agains pho o-oxida i e damage and lipid pe oxida ion (181). The yellowish
colony colo was obse ed when ASV78 was cul u ed on aga pla es and is
p obably ela ed o i s genomic abili y o syn hesize a yl polyenes. The es o
he BGCs showed low (8% wi h N-my is oyl-D-aspa agine) o no simila i y o
known BGCs, sugges ing he po en ial o encode no el molecules. Among he
uniden i ied BGCs could be biosu ac an s p oduced by s ain ASV78, since in
o he s udies non- ibosomal pep ide syn he ases (NRPSs) ha e been iden i ied
as su ac ins o engycin (182), which a e ecognised biosu ac an s.
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109. Zhang B, K ause M, Mu i M. The Fo ma ion and S uc u e E olu ion o
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112. O en A. The modynamic limi s o mic obial li e a high sal
concen a ions. En i onmen al Mic obiology. 2011;13(8):1908-23.
h ps://doi.o g/10.1111/j.1462-2920.2010.02365.x.
113. Ven osa A, de la Haba RR, Sánchez-Po o C, Papke RT. Mic obial
di e si y o hype saline en i onmen s: a me agenomic app oach. Cu en
Opinion in Mic obiology. 2015;25:80-7.
h ps://doi.o g/10.1016/j.mib.2015.05.002.
114. Doll PW, Doll K, Winkel A, Thelen R, Ah ens R, S iesch M, e al.
In luence o he A ailable Su ace A ea and Cell Elas ici y on Bac e ial
Adhesion Fo ces on Highly O de ed Silicon Nanopilla s. ACS Omega.
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115. Rami ez LYA, Angell IL, Nilsen T, Rudi K. Delayed Shi in Mic obio a
Composi ion in a Ma ine Mic ocosm Pollu ion Expe imen . Cu
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116. Simachew A, Lanzén A, Gessesse A, Ø eås L. P oka yo ic Communi y
Di e si y Along an Inc easing Sal G adien in a Soda Ash Concen a ion
Pond. Mic ob Ecol. 2016;71(2):326-38. h ps://doi.o g/10.1007/s00248-
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117. Liu D, Liu G, Chen L, Wang J, Zhang L. Soil pH de e mines ungal
di e si y along an ele a ion g adien in Sou hwes e n China. Sci China
Li e Sci. 2018;61(6):718-26. h ps://doi.o g/10.1007/s11427-017-9200-
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118. Wang Y, Bao G. Di e si y o p oka yo ic mic oo ganisms in alkaline
saline soil o he Qa han Sal Lake a ea in he Qinghai–Tibe Pla eau.
Scien i ic Repo s. 2022;12(1):3365. h ps://doi.o g/10.1038/s41598-
022-07311-3.
119. Zhao S, Liu J, Bane jee S, Whi e JF, Zhou N, Zhao Z, e al. No by
Salini y Alone: How En i onmen al Fac o s Shape Fungal Communi ies
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in Saline Soils. Soil Science Soc o Ame J. 2019;83(5):1387-98.
h ps://doi.o g/10.2136/sssaj2019.03.0082.
120. Luo Y, Wei X, Yang S, Gao YH, Luo ZH. Fungal di e si y in deep-sea
sedimen s om he Magellan seamoun s as e ealed by a me aba coding
app oach a ge ing he ITS2 egions. Mycology. 2020;11(3):214-29.
h ps://doi.o g/10.1080/21501203.2020.1799878.
121. Tang X, Yu L, Xu W, Zhang X, Xu X, Wang Q, e al. Fungal di e si y o
deep-sea sedimen s in Mid-Oceanic Ridge a ea o he Eas Paci ic and he
Sou h Indian Oceans. Bo anica Ma ina. 2020;63(2):183-96.
h ps://doi.o g/10.1515/bo -2018-0112.
122. Heo YM, Lee H, Kim K, Kwon SL, Pa k MY, Kang JE, e al. Fungal
Di e si y in In e idal Mud la s and Abandoned Sola Sal e ns as a Sou ce
o Biological Resou ces. Ma D ugs. 2019;17(11):601.
h ps://doi.o g/10.3390/md17110601.
123. Za illa B, Ma ínez-Espinosa RM, Alonso MA, Bone e MJ. Biodi e si y
o A chaea and lo al o wo inland sal e n ecosys ems in he Al o
Vinalopó Valley, Spain. Saline Sys ems. 2010;6(1):10.
h ps://doi.o g/10.1186/1746-1448-6-10.
124. Kalwasińska A, Deja-Siko a E, Bu kowska-Bu A, Szabó A, Fel öldi T,
Kosobucki P, e al. Changes in bac e ial and a chaeal communi ies du ing
he concen a ion o b ine a he g adua ion owe s in Ciechocinek spa
(Poland). Ex emophiles. 2018;22(2):233-46.
h ps://doi.o g/10.1007/s00792-017-0992-5.
125. Zhu D, Han R, Long Q, Gao X, Xing J, Shen G, e al. An e alua ion o
he co e bac e ial communi ies associa ed wi h hype saline en i onmen s
in he Qaidam Basin, China. A ch Mic obiol. 2020;202(8):2093-103.
h ps://doi.o g/10.1007/s00203-020-01927-7.
126. Alsamma H, Delne i D. An upda e on he di e si y, ecology and
biogeog aphy o he Saccha omyces genus. FEMS Yeas Res.
2020;20(3): oaa013. h ps://doi.o g/10.1093/ emsy / oaa013.
127. Blombe g A. Me abolic su p ises in Saccha omyces ce e isiae du ing
adap a ion o saline condi ions: ques ions, some answe s and a model.
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Analisi kimioo axonomikoek e aku si zuen ASV31 anduian nagusia zen
gan z-azidoa, C18:0 cyclo ω8c, ez zegoela T. paci ica DSM 10166T e a R. algae
LMG 29228T anduie an. Gaine a, ASV31 hid oxi gan z-azido ik gabeko andui
baka a zen, e a gan z-azido asegabeak zi uen baka a. A e gehiago, summed
ea u e 8 (C18:1 ω7c edo a C18:1 ω6c) T. paci ica DSM 10166T (% 83,08) e a R.
algae LMG 29228T (% 70,12) anduien gan z-azido nagusia izan zen, aldiz
ASV31 anduian po zen aje baxuagoan (% 37,34) de ek a u zen. Ubikinona
Q-10 ASV31 anduian iden i ika u zen, Pa acoccaceae amiliako gene o
gehiene an ohikoa den a nas kinona izanik (138). Gaine a, ASV31 anduia en
lipido pola en pe ilak honako hauek zi uen: os a idilglize ola, aminolipidoa,
iden i ika u gabeko bi glikolipido, iden i ika u gabeko bi os olipido e a
iden i ika u gabeko bi lipido (3. a ala, IV. ERANSKINA, S4 i udia).
Fos a idile anolamina ez de ek a zeak, T. dalianensis-en e a Rhodo ulum
gene oko kideen lipido pola ga an zi suene a iko ba izanik (138,145),
ASV31 anduia desbe din zen zuen.
O o ha , analisiek ezauga i genomiko, eno ipiko e a kimio axonomikoen
desbe din asunak age ian u zi zi uz en, e a ho ek adie az en zuen ASV31
anduia gene o be i ba en lehen espeziea zela Pa acoccaceae amilia en
ba uan, e a ho ega ik Anianabac e salinae gen. no . sp. no . izena p oposa u
zen, ASV31T andui ipoa izanik.
Idaz eko unean, Anianabac e salinae ICNP en a abe a balio osoz a gi a a ua
dago baina izendapena Anianibac e salina um-e a zuzen zen da, bes e izen
o og a ia edo o og a ia zehaz ugabe ba zuk di ela e a (146).
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Desk ibapena: Anianibac e salina um gen. no . sp. no .
8. aula. Anianibac e salina um ASV31T anduia en desk ibapena.
E imologia
(A.ni.a.ni.bac’ e . N.L. masc.
n. bac e , a od; N.L. masc. n.
Anianibac e , Añanako
ga zagako bazilo ba ) e a
(sa.li.na’ um. L. gen. pl. n.
salina um, ga zagakoa).
Andui ipoa
ASV31T (=CECT 30309T =
LMG 32242T)
Taxonomia
Pseudomonado a iluma,
Alphap o eobac e ia klasea,
Rhodobac e ales o dena e a
Pa acoccaceae amilia.
Zelulak
Ez-mugiko ak, G am-
nega iboa di en baziloak,
banaka edo ingu uan
polisaka ido mukosozko
kapsula ba du en ag ega ue an
age zen di enak. PHA
piko ak bil zen di uz e zelulen
ba uan.
Koloniak
Beix-a osa kolo ekoak.
Zelulek 2,4-2,5 µm-ko luze a
e a 0,4-0,5 µm-ko zabale a
du e.
Pigmen uak
Es e oidenona, es e oidenoa
e a bak e ioklo o ila a age zen
di a.
Hazkun za
pH 6,5-9,5 (op imoa 7-9,5) e a
18-37 ºC (op imoa 30 ºC)
a ean haz en da. NaCl
ole an zia handia du, % 0-23
(p/b) (op imoa % 35).
Ak ibi a e
en zima ikoa
Ka alasa e a oxidasa posi iboa e a u easa nega iboa. Esculina e a Tween 20
hid oliza zen di u. Ni a oen e edukzioa bu u zen du. API ZYM p obe an
os a asa alkalino, es e asa (C 4), es e asa lipasa (C 8), leuzina a ilamidasa,
balina a ilamidasa e a α-glukosidasa ak ibi a e handia e akus en di u; e a
ak ibi a e ahulak zis eina a ilasa, os a asa azidoa e a na ola-AS-BI-
os ohid olasa en zako.
Ja ai zen du
25. i udia. ASV31T anduia en zelula ba en
ansmisioko mik oskopia elek onikoa
e abiliz egindako mik og a ia. 500,0 nm
ba a.
26. i udia. Anianibac e salina um ASV31T
anduia MA hazkun za medioan. Kolonien
mo ologia e a kolo ea e akus en di a.
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An ibio ikoekiko
suszep ibili a ea
B polimixina (300 IU), bankomizina (30 µg) e a es ep omizina ekiko
(10 µg) e esis en ea.
Kimio axonomia
Ubikinona baka a Q-10 da e a gan z-azido nagusiak C18:0 cyclo ω8c
e a summed ea u e 8 (C18:1 ω7c edo a C18:1 ω6c). Lipido pola
nagusiak os a idilglize ola, aminolipida, iden i ika u gabeko
glikolipido ba , iden i ika u gabeko os olipido ba e a iden i ika u
gabeko lipido ba di a.
WGS
Genoma en amaina 3.6 Mbp- akoa da e a andui ipoa en DNA en
G+C edukia % 65.7 mol-ekoa da.
CECT, Espainiako mik oo ganismo ipo-en bilduma; LMG, Gan eko Mik obiologiako
Labo a egia (Belgika); WGS, Genoma osoa en sekuen ziazioa.
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Konposa u an imik obianoen edo a en sioak iboen ekoizleak di en
isola uen baheke a
Be esku aezinak sue a u zi en bi isola u en salbuespena ekin, gainon zeko
148 isola uei an imik obianoen ekoizpen ahalmena en baheke a egin zi zaien.
Hau, an agonismo en segua e abiliz, andui pa ogenoen hazkun za inhibi zeko
zu en gai asunean oina i zen da. Hogei isola uk ja due a a gia e aku si zu en
gu xienez pa ogeno anduie ako ba en au ka (4. aula). Ondo en,
an imik obianoak ekoiz en zi uz en 20 isola u ho iekin di usio en segua bu u u
zen, e a ho ie a ik bede a zik an imik obianoak ekoiz eko gai asuna azaldu
zu en (4. aula). Isola u halo ilo edo halo ole an e ho ie a ik zo zik bak e io
andui G am-posi iboen zein G am-nega iboen hazkun za inhibi zen zu en
konposa uak ekoiz u zi uz en; aldiz, ASV55 isola uak di usio en seguan
e aku si zuen soilik ak ibi a e an i ungikoa (4. aula). An agonismo zein di usio
en segue an lo u ako emai z ba zuen ilus azioak 27. i udian age zen di a.
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27. i udia. Isola u halo ilo e a halo ole an e ba zuek ekoz u ako konposa u an imik obianoen
de ekzioa en adibidea. A, an agonismo en seguan pa ogenoen hazkun za en inhibizio haloa,
ASV12, ASV10, ASV14, ASV25, ASV89 e a ASV106 isola uek so ua e a B, di usio en seguan
pa ogenoen hazkun za en inhibizio haloa, ASV12, ASV13, ASV55, ASV128, ASV106, ASV129 e a
ASV136 isola uek so ua.
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Gaine a, di usio en seguak i adoki zuen konposa u an imik obianoak
inkubazioa en hi uga en egunean ekoiz uak zeudela e a deg adazioa naba ia
zela hama ga en eguna en ondo en (4. aula). Bes alde, soilik an agonismo
en seguan ak ibi a ea e aku si zu en isola uek, konposa u an imik obianoa en
ekoizpena pa ogenoa ekiko man enugaien lehian oina i u zela suposa
dezake e, bes e ike ke a ba zue an desk iba u den bezala (147).
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9. aula. Az e u ako andui pa ogenoe ako ba en au ka, gu xienez, ak ibi a e an imik obianoa e aku si zu en isola uak. An agonismo en seguan (DAA) e a di usio
en seguan (WDA) jaso ako inhibizio haloa en diame oak (mm) ema en di a.
Andui
pa ogenoak
P. ae uginosa
ATCC 27853T
E. coli
ATCC25922T
B. sub illis
CECT 356T
S. au eus
ATCC 29213T
E. aecalis
ATCC 29212T
C. albicans
ATCC 90029T
Inhibizio haloa en ba ezbes ekoa (mm)
DAA
WDA
DAA
WDA
DAA
WDA
DAA
WDA
DAA
WDA
DAA
WDA
eguna
eguna
eguna
eguna
eguna
eguna
Isola uak
3
10
17
3
10
17
3
10
17
3
10
17
3
10
17
3
10
17
ASV1
8
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV3
11
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV7
20
-
-
-
-
-
-
-
12
-
-
-
25
-
-
-
-
-
-
-
-
-
-
-
ASV10
10
-
-
-
15
-
-
-
-
-
-
-
12
-
-
-
-
-
-
-
-
-
-
-
ASV11
10
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV12
27
-
-
-
27
14
12
-
25
-
-
-
30
-
-
-
30
-
-
-
-
-
-
-
ASV13
-
-
-
-
9
15
11
-
-
-
-
-
-
-
-
-
31
-
-
-
-
-
-
-
ASV14
12
-
15
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV25
12
-
12
-
15
-
-
-
16
-
-
-
10
-
-
-
13
-
-
-
-
-
-
-
ASV55
-
15
13
-
-
16
15
10
13
20
16
13
-
12
18
13
-
7
6
-
-
15
13
-
ASV78
12
-
-
-
-
-
-
-
20
-
-
-
17
-
-
-
-
-
-
-
-
-
-
-
ASV89
9
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV106
-
-
-
-
-
-
-
-
21
18
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV117
9
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV118
9
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV120
-
-
-
-
-
-
-
-
6
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV121
-
-
-
-
-
-
-
-
6
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV128
-
-
-
-
-
-
-
-
20
15
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV129
-
-
-
-
-
-
-
-
19
17
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ASV136
6
-
-
-
-
-
-
-
14
9
-
-
10
-
-
-
-
-
-
-
-
-
-
-
-, nega ibo
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Pseudoal e omonas gene oa (ASV1, ASV3, ASV10, ASV11, ASV12, ASV13,
ASV14, ASV25, ASV78, ASV89, ASV117, ASV118 e a ASV136 isola uak)
ak ibi a e an imik obianoa e aku si zu en isola uen gene o nagusia izan zen
ike ke a hone an. Aipaga ia da haue ako isola u gehienek, ASV13a salbu,
P. ae uginosa- en hazkun za inhibizio gai asuna age u zu ela. Man enugaien
konpe en ziak e agindako e ek u inhibi zaile ba iza eko auke az gain, bes e
hipo esi ba e e badago: Pseudoal e omonas espezieek gai asun handia du e
al e ozina bezalako pelikulen au kako agen eak ekoiz eko e a modu ho e an,
P. ae uginosa- en (bio ilmak so zen di uen bak e io ezaguna) inhibizioa
e agi eko (148). Gai asun an imik obianoa e aku si zu en gaine ako isola uak
Salini ib io (ASV7), Pseudomonas (ASV55), Kocu ia (ASV106 e a ASV128),
Halomonas (ASV120 e a ASV121) e a S ep omyces (ASV129) gene oe an
bil zen zi en. Gene o haue ako kide ba zuk ak ibi a e an ibak e ianoa du ela
jaso a dago (149–152), Salini ib io gene oko kideak izan ezik. Haue an,
ak ibi a e an i ungikoa (153) e a in sek izidaz (154) gain, ez da desk iba u
bak e ioen au kako ak ibi a e ik, ike ke a hone an ASV7 anduian ikusi den
bezala.
Ai zi ik, 148 isola u en biosu ak an e ekoizpen ahalmena e e az e u zen,
olioa en dispe sio es a e a Pa a ilm-M es a e abiliz gainazaleko en sioa
mu iz eko du en gai asuna de e mina uz. Bai a bioemul si ika zaile ekoizpen
ahalmena e e, emul si ikazio es a en bidez olio zein hid oka bu oak
e aginko asunez emul siona zeko du en ahalmena ebalua uz. 28. i udian,
lo u ako emai z ba zuen a gazkiak age i di a; xeha asun gehiago, be iz,
4. a ala, V. ERANKINA, S2 aulan au ki u dai ezke. Pa a ilm-M e a olioa en
dispe sio es e an isola u bakoi za en za lo u ako ba ez bes eko emai zak
age i di a, 29. i udian e a 30. i udian hu enez hu en, gene oa en a abe a
mul zoka u a. Biosu ak an eak ekoiz eko gai asuna zu ela kon side a u zen,
baldin e a Pa a ilm-M es ean an a en diame oa e a olioa en dispe sio es ean
halo ga bia en diam oa kon ol nega iboa enak (BPM) baino handiagoak
bazi en (≥ 3,4 mm e a ≥ 7,0 mm, hu enez hu en). Bes e ike ke a ba zue an
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an zeko mozke a pun uak e abili di a biosu ak an een ekoizpena
de e mina zeko (95,155).
28. i udia. Konposa u en sioak iboak ekoiz en di uz en zenbai andui halo ilo en e a
halo ole an e en adibideak A, Pa a ilm-M es ean; B, olioa en dispe sio es ean e a
C, emul si ikazio es ean, kasu guz ie an kon ol nega iboa ekin alde a u a.
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29. i udia. Biosu ak an eak ekoiz eko isola uen baheke a en emai zak Pa a ilm-M es ean.
Isola uak gene oa en a abe a aldeka u ako pun uen bidez i udika u a daude, an a en
ba ezbes eko diame oa e aku siz. Kon ol nega iboa en (BPM, go iz) e a SDS kon ol
posi iboa en (% 1, p/b) (u dinez) emai zak e e age i di a.
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Bes alde, pen ab omopseudilin konposa ua en iden i ikazioa oso a gia izan zen
be e UV espek oa e a o mula molekula a in e p e a uz. Izan e e, MS
espek oak (4. a ala, V. ERANSKINA, S6 i udia) bos b omo a omo di uen
pa oi iso opiko be eizga i ba e aku si zuen, modu ho e an o mula
molekula a baka a, C10H4B 5NO, i adokiz, zeinak pen ab omopseudilin
konposa ua ekin soilik koin zidi zen zuen P oduk u Na u alen Hiz egia en
a abehe a. Jakineko ak ibi a ea medio, konposa u hau an imik obiano es ak u
go dine ik pu i ika u zen alde an zizko asean HPLC semi-p epa a i e
eknika en bidez. R 22 minu u an, 0,9 mg konposa u pu u lo u zi en. Ondo en,
(-) MS espek oak e a 1H-NMR analisiak (34. i udia) kon i ma u zuen o mula
e a pu u asuna.
Azkenik, b omophene bezala iden i ika u ako pikoak ez zuen (+) MS espek o
in e p e aga i ik eman. Ondo ioz, an imik obiano es ak u go dine ik abia u a,
konposa ua en pu i ikazioa i ekin zi zaion alde an zizko asean HPLC semi-
p epa a i e eknika en bidez, e a, ho ela, RMN analisi ako egoki za jo zen
akzio ba lo u zen. F akzio hone an egindako (-) MS espek oan age u ako
banake a iso opikoan oina i u a, P oduk u Na u alen Hiz egian zo zi
koin ziden zia zu uen C12H6B 4O2 o mula molekula a ekin ba ze o ela
ondo ioz a u zen. R 20 minu u an, 0,5 mg konposa u pu u lo u zi en.
Ondo engo (-) MS espek oak e a 1H-NMR analisiak (35. i udia) b omophene
konposa ua en p esen zia baiez a u zu en, li e a u a en da uekin alde a uz
(164).
Ike ke a hone an desk iba u den bezala, Pseudoal e omonas ge e oan
me aboli o b oma uen ekoizpena dokumen a ua izan da. Zehazki,
pen ab omopseudilin, P. phenolica e a P. lu eo iolacea espeziee a ik isola ua
izan da e a b omophene aldiz P. phenolica espezie ik (165). Deiga ia dena da,
ike ke a hone an ASV78 anduiak bi konposa uak aldi be ean ekoiz eko
gai asuna azaldu duela, e a ASV78 andui ik ilogene ikoki ge uen dagoen
espeziea Pseudoal e omonas neus onica dela (% 98,57ko 16S RNA gene-
sekuen zia en an zeko asuna ekin).
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34. i udia. ASV78 anduiak ekoiz u ako pen ab omopseudilin konposa ua en (-) MS espek oa
(goian) e a 1H-NMR espek oa be e egi u a ekin (behean).
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35. i udia. ASV78 anduiak ekoiz u ako b omophene konposa ua en (-) MS espek oa (goian) e a
1H-NMR espek oa be e egi u a ekin (behean).
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Me izilina ekiko e esis en ea den S. au eus anduia en au kako ak ibi a ea
pen ab omopseudilin zein b omophene konposa ue an desk iba u bada e e, ez
da asko ik ezagu zen hauen ak ibi a e espek oa en ingu uan (165,166).
Gaine a, bi konposa uak aldi be ean ekoiz ea ez da ohikoena; izan e e,
o aingoz, Pseudoal e omonas sp. CMMED 290 andui baka ean baino ez da
dokumen a u (165), nahiz e a bide me abolikoa pa eka ua izan bi konposa uak
ekoiz eko (167). Ho i ho ela izanda, balo a zekoa da konposa u ho iek izan
dezake en e ek u sine gikoa ike zea, o ain a e ez bai a halako ik oga u.
Ike ke a ho en emai zak onu aga ia izan dai ezke konposa u hauek p ak ikan
aplika zeko.
Tes uingu u hone an, konposa u pu uekin egindako en seguek e aku si zuen bi
konposa uek bak e io G am-posi iboen au kako ak ibi a e an imik obianoa
e akus en zu ela; pen ab omopseudilin-ak gaine a bak e io G am-nega iboen
au kako ak ibi a ea e e e akus en zuela ik (10. aula). Zehazki,
pen ab omopseudilin-ak 0,02 e a 0,04 µg/mL bi a eko MIC balioak age u
zi uen S. au eus ATCC 29213 anduia en au ka e a 10 e a 20 µg/mL bi a eko
MIC balioak P. ae uginosa ATCC 27853 anduia en au ka (10. aula, goiko
aldean). Aldiz, b omophene-k 2,5 e a 5 µg/mL bi a eko MIC balioak e aku si
zi uen S. au eus ATCC 29213 anduia en au ka (10. aula, goiko aldean).
10. aula. Konposa u pu uen e a kon olen ak ibi a e an ibak e ianoa. MIC balioak goiko aldean
e a bak e ioen au kako ak ibi a ea (e eplika ba e ik lo u ako MIC balioak) behekaldean.
Konposa uak
MIC (µg/mL)
S. au eus
ATCC 29213
P. ae uginosa
ATCC 27853
Pen ab omopseudilin
0.02-0.04
10-20
B omophene
2.5-5
> 80
Sine gia (1:1)
0.04-0.08
20-40
Bankomizina
0.5-1
ND
Zip o loxazinoa
ND
0.25-0.5
Ja ai zen du
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Konposa uak
Ak ibi a ea (µg/mL)
A.baumannii
ATCC 19606
E. aecalis
ATCC 29212
E. aecium
anA 15167
Pen ab omopseudilin
10-20
10-20
1.25-2.5
B omophene
> 80
40-80
20-40
Sine gia (1:1)
20-40
20-40
2.5-5
Bankomizina
ND
4-8
> 32
Az eonama
80-160
ND
ND
ND; zehaz u gabe.
MIC balioak E. aecalis ATCC 29212, bankomizina ekiko e esis en ea den
E. aecium anA 15167 e a A. baumannii ATCC 19606 andui pa ogenoen au ka
de e mina u zi en baina soilik e eplika baka a e abiliz. Ho i konposa u pu uen
kan i a e eskasiaga ik ge a u zen, e a ho az, bali eke e o kizuneko ike ke e an
balioz a ze handiagoa beha iza ea. Pen ab omopseudilin konposa uak
E. aecalis ATCC 29212 anduia en hazkun za inhibi u zuen 10-20 µg/mL- an,
bankomizina ekiko e esis en ea den E. aecium anA 15167 anduia ena
1,25-2,5 µg/mL- an e a A. baumannii ATCC 19606 anduia ena 10-20 µg/mL-
an (10. aula, behekaldean). Bes alde, ikusi zen b omophene-k E. aecalis
ATCC 29212 anduia en hazkun za 40-80 µg/mL- an inhibi zen zuela e a
bankomizina ekiko e esis en ea zen E. aecium anA 15167 anduia ena
20-40 µg/mL- an (5. aula, behekaldean). Az e u ako pa ogeno baka ba en za
e e ez zen ikusi bi konposa uen a eko ak ibi a e sine giko ik.
Pen ab omopseudilin konposa uak e aku si zuen ak ibi a e an ibak e ianoa
kon uan ha zekoa da klinikoki ga an zi suak di en espezieen au ka, bes eak
bes e, A. baumannii, E. aecium e a S. au eus. Deiga ia da
pen ab omopseudilin-a en kon zen azio baxua (0,02 e a 0,04 μg/mL bi a eko
MIC balioak) zeine an S. au eus ATCC 29213 anduia en au ka e aginko a
den, ba ez e e bankomizina en kon zen azioekin alde a uz (0,5 e a 1 μg/mL
bi a eko MIC balioak ike ke a hone an, e a 0,52 e a 0,59 μg/mL bi a ekoak
Lepe e al.-en ike ke an (168)). Be az, oso in e esga ia izango li za eke
pen ab omopseudilin-a en ak ibi a e espek oa ebalua zea pa ogeno so a
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zabalago ba en au ka, e a ha en ekin za mekanismoa ike zen ja ai zea, zeina
bankomizina ena ekiko desbe dina izan dai ekeen, bankomizina ekiko
e esis en ea den E. aecium e a bak e io G am-nega iboen au kako ja due ak
age ian uz en duen bezala. Izan e e, diana ani zeko inhibi zaileek an ibio ikoen
munduko hu engo belaunaldia age zen du e, an imik obianoekiko
e esis en zia en ga apena ekiko aban aila naba menak eskainiz (169).
Biosu ak an e e a bioemul si ika zaile es ak u go dinei dagokienez, IR e abili
zen osagai ho ien izae a kimikoa iden i ika zeko. Alde ba e ik, biosu ak an e
es ak ua en IR espek oak e aku si zuen hid oka bu o ali a ikoen ka eak
zeudela polisaka ido akzio ba ekin ba e a, ho ek biosu ak an ea en izae a
glikolipidikoa i adokiz (36A i udia). Izan e e, 3.311 cm−1- an xu gapen banda
zabala ego eak hid oxi aldeen -OH luzake ak zeudela adie az en zuen. Gaine a,
2.919 e a 2.847 cm–1-ko banden p esen zia ka e ali a ikoen C-H luzake a
modua i zegokion. 1.652 cm−1-ko banda C=O aldeen p esen zia ekin
elaziona u a egon li eke e a 1.408 cm–1-ko bandak polisaka idoei ego z
zekizkiekeen CH3 e a CH2-a en C-H aldeei. Azkenik, 1.317-1,013 cm−1 a eko
bandek, azuk ea en honda a i dagokion C-O luzake a en p esen zia adie az en
zu en. O o ha , ASV78 anduiak ekoiz u ako biosu ak an ea en es ak u
go dina en IR espek oa bes e ike ke e a ba zue an (170–172) adie azi ako
biosu ak an e glikolipidikoen an zekoa izan zen.
Bes alde, bioemul si ika zaile es ak u go dina en IR espek oa 36B i udian
e akus en da. Talde ka boxilikoen p esen zia adie az en du en xu gapen banda
zabalak ikusi zi en (3.369 cm-1, hid oxi-luzake a; 1.630 cm-1, ka boxilo C=O
luzake a; 1.114 cm-1, ka boxilo -C-O luzake a). Gaine a, amida aldea en za
1.556 cm-1-ko uhin luze an luza ze bib azioak ikusi zi en. Hauek C-N e a N-H
aldeekin lo zen di a, bioemul si ika zailea en egi u an aminoazuk e e a
p o eina/pep ido azila uak daudela adie aziz, glikop o einak e a uz.
Au kikun za hau ba da o bes e Pseudoal e omonas spp. isola u ba zue an
desk iba u den glikop o eina izae adun exopolime oekin, zein zuek gaine a
ja due a emul siona zailea e aku si du en (173,174).
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36. i udia. ASV78 anduia en ha zidu a salda ik lo u ako A, biosu ak an e es ak u go dina en e a B, bioemul si ika zaile es ak u go dina en IR espek oak.
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Ezaguna da konposa u en sioak iboek "pseudosolubilizazio" es a egian pa e
ha zen du ela; es a egia ho en helbu ua da bioe abilga i asuna hobe zea e a
konposa u hid o oboen esku aga i asuna e az ea. Gaine a,
Pseudoal e omonas gene oa ohikoa da hid oka bu oak deg ada zen di uz en
mik oo ganismoen a ean, e a, be az, ez da ha i zekoa gene o ho e an
biosu ak an e edo bioemul si ika zaile ekoizleak di en isola uen p opo zio
esangu a su ba au ki zea (60). Ondo ioz, ASV78 anduiak ekoiz u ako
konposa u en sioak iboen aplikaga i asun po en ziala en ka ak e izazio e a
ike ke a gehiago e o kizun handikoak izan li ezke.
Azkenik, ASV78 anduia en genoma az e u zen MinION e a Illumina ekniken
konbinazioa e abiliz. Genomak 5.215.181 bp zi uen e a 19 con ig-e an
mihiz a u zen (NCBIn esku aga i PRJNA1218939 a xiki ze zenbakia ekin),
be e DNA G+C edukia % 41,5-ekoa izanik. Con ig-ak P okka e abiliz az e u
zi en e a guz i a 4.674 gene de ek a u zi en. Ho ie a ik 4.548 p o einak
kode zen di uz en gene (CDS) gisa iden i ika u zi en, zein zuen a ean soilik
% 55,5a i eslei u zi zaien un zio ba , gaine akoak p o eina hipo e iko gisa
sailka u bai zi en. Emai za ho ek age ian uz en du beha ezkoa dela ike ke a
gehiago egi ea gene ho ien un zioak zehaz eko.
ASV78 anduia en genoma an iSMASH bidez az e u zen e a gene-mul zo
biosin e ikoak (BGC) kode zen zi uz en zazpi eskualde au ki u zi en (11. aula).
De ek a u ako BGCen luze a osoa 250 Kb ingu ukoa izan zen, ASV78 en
genoma en ~ % 4,8koa alegia. Genoma en amaina ekiko au esandako BGCen
kopu ua p oka io oe an au ki u ako ba ez bes eko a ean dago (175).
I aga i ako BGCen a ean baka ba ek e aku si zuen % 100eko an zeko asuna
pen ab omopseudilin konposa ua en sin sia ekin e laziona u a (167). Ho en
ha i a, Aga wal e al.-ek azpima a u zuen bpm gene biosin e ikoen klus e
ho i, di enil e e polib oma u (PBDE) konposa uen kopu u muga u ba en
ekoizpenaz a du a zen dela, hauen a ean, pen ab omopseudilin e a
b omophene konposa uen sin esiaz (167). Lan honek ho i kon i ma zen du,
izan e e LC-HRMS eknika en bidez es ak uan bi konposa uen ekoizpena
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baiez a u zen. Dena den, Pseudoal e omonas gene oa en ilogeniak age ian u zi
zuen klado uga ik ez di uz ela gene ho iek (176). Izan e e, konposa u
bioak iboen de ekzioa P. lu eo iolacea e a P. phenolica espeziee ako anduiak
zi uz en kladoe a a muga u zen gehienba (176), e a ez P. neus onica- a ASV78
anduia en kasuan ge a zen zen bezala.
Bes e bi BGC E des e ioxamina en (177) e a APE V - en (175) BGCekin
an zeko asuna e aku si zu en. E des e ioxamina e a be e analogoak
S ep omyces e a e laziona u ako bak e ioek maiz ekoiz en di uz en
side o o oak di a (178). Hala e e, des e ioxamina de ek a u den
Pseudoal e omonas gene oe an (P. spongiae, P. u henica, P. pi a ica e a P.
unica a kasu), B des e ioxamina izan da (179). Azkenik, a il polienoak G am-
nega ibo pa ogenoen a ean heda u ako pigmen u ho iak di a, hala nola E. coli
u opa ogenikoa en APE Ec (180). Es uk u alki e a un zionalki
ka o enoideekin e laziona u a daude e a kal e o ooxida iboen e a lipidoen
pe oxidazioa en au kako babesa ema en du e (181). Li ekeena da ASV78
anduia en koloniek aga plake an kul iba ze akoan e akus en zu en kolo e
ho ixka a il polienoak sin e iza zeko duen gai asun genomikoa ekin
e laziona u a ego ea. Gaine ako BGC an zeko asun baxua e aku si zu en
(% 8 N-my is oyl-D-aspa agine- ekin) edo ez zu en an zeko asunik e aku si
BGC ezagunekin, hauei molekula be iak kode zeko ahalmena i adokiz. Zen zu
hone an, iden i ika u gabeko BGCen a ean ASV78 anduiak so u ako
biosu ak an eak egon li ezke, izan e e bes e ike ke a ba zue an e ibosomikoak
ez di en pep ido sin e asak (NRPS) iden i ika u di a, su ac in edo engycin
bezalako biosu ak an e ai o uekin e laziona u a (182).
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11. aula. An iSMASH e abili a ASV78 anduia en genoman de ek a u ako us ezko gene-mul zo
biosin e ikoak (BGC).
E emua
Mo a
Hasie a
(bp)
Bukae a
(bp)
Ezagu zen den
alde ik an zekoena
(MIBiG E e .)
An zeko asuna
E emua 1
PBDE
865.555
889.181
Pen ab omopseudilin
(BGC0000890)
100%
E emua 2
NRPS,
Type I PKS
1.224.295
1.296.460
E emua 3
RiPP-like
1.498.707
1.509.561
N-my is oyl-D-
aspa agine
(BGC0000972)
8%
E emua 4
NRPS
2.574.952
2.643.824
E emua 5
NI-
side o o oa
3.032.695
3.063.100
Des e ioxamine E
(BGC0001572)
75%
E emua 6
A il
polienoak
478.747
522.306
APE V
(BGC0000837)
45%
E emua 7
RiPP-like
804.360
816.540
PBDE, di enil e e polib oma uak; NRPS, e ibosomikoak ez di en pep ido sin e asak; PKS,
polyke ide sin asak; RiPP, e ibosomikoki sin e iza u ako e a i zulpen os ean alda u ako
pep ido p oduk ua; NI-side o o oa, NRPS-side o o oa independen ea; APE V , Alii ib io
ische i ES114 anduian de ek a u ako a il polienoa en gene-mul zo biosin e ikoa.
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Sec ion 3. a ala - APPENDIX I. ERANSKINA
245
APPENDIX I. ERANSKINA
P oka yo ic Di e si y and Communi y
Dis ibu ion in he Complex
Hyd ogeological Sys em o he Añana
Con inen al Sal e n
Re e ence: Azpiazu-Muniozgu en M, Ga cía-Ma ínez M, Zabale a A,
An iguedad I, Ga aiza J, Lao den L, Ma inez-Malaxe xeba ia, I, and
Ma inez-Balles e os, I. P oka yo ic Di e si y and Communi y Dis ibu ion in
he Complex Hyd ogeological Sys em o he Añana Con inen al Sal e n.
Mic ob Ecol. 2024; 87(1):171.
Mic obial Ecology (2025); 87(1):171
DOI: 10.1007/s00248-025-02488-2
Impac ac o 2023: 3.3
Rela i e posi ion: 70/161, Q2 Mic obiology
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Vol.:(0123456789)
Mic obial Ecology (2024) 87:171
h ps://doi.o g/10.1007/s00248-025-02488-2
RESEARCH
P oka yo ic Di e si y andCommuni y Dis ibu ion in heComplex
Hyd ogeological Sys em o  heAñana Con inen al Sal e n
MaiaAzpiazu‑Muniozgu en1· Mine aGa cía‑Ma ínez1· AneZabale a2· IñakiAn iguedad2· Ja ie Ga aiza 1,3·
Lo enaLao den1,3· I a iMa inez‑Malaxe xeba ia1,3· Ila giMa inez‑Balles e os1,3
Recei ed: 10 Oc obe 2024 / Accep ed: 2 Janua y 2025
© The Au ho (s) 2025
Abs ac
The Añana Sal Valley (no he n Spain) is a con inen al sal e n consis ing o a se ies o na u al sp ings ha ha e been used
o sal p oduc ion o a leas 7000yea s. This habi a has been ela i ely unde s udied; he e o e, p oka yo ic di e si y was
in es iga ed h ough Illumina-based 16S RNA gene sequencing o de e mine i he wa e s wi hin he alley exhibi dis inc-
i e mic obiological cha ac e is ics. Two main ypes o wa e we e ound in he alley: sal y (app oxima ely 200g/L salini y)
om he diapi ic s uc u e and b ackish (≤ 20g/L salini y) om shallow s eams. The be a di e si y indices showed ha
salini y was he p ima y ac o in luencing he p oka yo ic dis ibu ion. Howe e , a niche-speci ic in luence was obse ed
be ween wa e s o he same o igin, wi h signi ican di e ences in he ela i e abundance o he ASVs. The mic obiome o
he sal e n e ealed ha he a chaeal domain was mainly es ic ed o sal y wa e s, while he bac e ial domain was ubiqui-
ous h oughou he sal e n, wi h a no able p e alence in b ackish wa e s. The main bac e ial and a chaeal phyla iden i ied
we e Pseudomonado a and Halobac e o a, espec i ely. The genus Halo ub um was abundan and widesp ead in sal y
wa e s, while Pseudomonas was a signi ican pa o he p oka yo e communi y, mainly in b ackish wa e s. The ela i e
abundance o he gene a Haloplanus and Salinibac e inc eased in he sal ponds used o sal p oduc ion. The axa in ol ed
in chemohe e o ophy and e men a ion we e widesp ead, sha ing he same niche. O e all, he loca ion o his sal e n on a
diapi ic s uc u e a o s he occu ence o wa e s wi h di e en o igins ha a ec he p oka yo ic dis ibu ion beyond he
niche loca ion in he alley.
Keywo ds Sal diape · Con inen al sal e n· 16S RNA gene sequencing· P oka yo ic di e si y
In oduc ion
Hype saline en i onmen s ep esen highly biop oduc i e
habi a s wi h a la ge mic obial di e si y adap ed o hese
condi ions [1]. To be e unde s and hese en i onmen s, i
is essen ial o cha ac e ize he mic obial communi ies due
o hei con ibu ion o nu ien cycling and ecosys em unc-
ioning [2]. The e o e, he s udy o hype saline en i onmen s
such as sola sal e ns [3], sal mines [4], and sal lakes [5]
has ecei ed conside able in e es . Many di e en ypes
o halophilic and halo ole an mic oo ganisms including
a chaea o he o de Halobac e iales and bac e ial species
o he o de Halanae obiales and Halomonadaceae, Desul-
ohalobiaceae, and Salinibac e iaceae amilies, among o h-
e s [6], a e ound in hese en i onmen s. While halophiles
a e less common wi hin he Euca ya domain, he g een alga
Dunaliella is ypically p esen in ae obic en i onmen s wi h
high sal con en [5]. As mic obial communi ies a e i al
o ecosys em unc ions and a e in luenced by he physical
and geological cha ac e is ics o he si e, pa allel s udies in
hese o he a eas a e also necessa y o gain a comp ehensi e
unde s anding o hese ecosys ems [1, 7].
*Ila gi Ma inez-Balles e os
[email p o ec ed]
1 Mik oIke Resea ch G oup, Immunology, Mic obiology
andPa asi ology Depa men , Facul y o Pha macy,
Uni e si y o  heBasque Coun y UPV/EHU, Paseo de La
Uni e sidad 7, 01006Vi o ia-Gas eiz, Spain
2 Hyd o-En i onmen P ocesses Resea ch G oup. Geology
Depa men , Facul y o Science andTechnology, Uni e si y
o  heBasque Coun y UPV/EHU, Ba io Sa iena S/N,
48940Leioa, Spain
3 Bioa aba, Mic obiology, In ec ious Diseases, An imic obial
Agen s, andGene The apy, 01006Vi o ia-Gas eiz, Spain
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Among he di e en ypes o sal e ns ha exis , con i-
nen al ones a e less well known, and no many a e s ill in
use oday. The Añana Sal Valley (Ála a, Basque Coun y,
Spain) is placed in one o he bes -p ese ed con inen al
sal e ns in Eu ope. Sal p oduc ion is ac i e in he alley
since a leas 7000yea s, and i has been ecognized by Food
and Ag icul u e O ganiza ion (FAO) as being Eu ope’s i s
Globally Impo an Ag icul u al He i age Sys em (GIAHS).
The unde g ound e apo i ic ocks, comp ising sal , gypsum,
clay and o he s, we e o med du ing he ini ial s ages o he
agmen a ion o Pangaea supe con inen (app oxima ely
200 million yea s ago). These ocks now o m a geologi-
cally complex diapi ic s uc u e whe e he sal s a e ac i ely
ising [8]. The geological complexi y o he a ea leads o
hyd ogeological complexi y, wi h e y slow deep lows mix-
ing wi h shallowe lows in some a eas o he alley. This
esul s in he eme gence o sp ings wi h a ying salini y
le els, dependen on he ou e aken by he wa e h ough he
subsoil. This gi es ise o he p esence o sp ings wi h sal y
(≈ 200g/L salini y) o b ackish (≤ 20g/L salini y) wa e ha
a e e y close o each o he .
Despi e he singula i y o his en i onmen , and al hough
s udies ha e been ca ied ou in his sal e n on a cheology,
hyd ogeology, o he sal i sel , he e is limi ed mic obiologi-
cal da a a ailable. The e is only one publica ion on mic obial
and i al changes om g oundwa e o su ace wa e [9] and
one on ungal communi y di e si y [10], so u he esea ch
is needed o ully unde s and his ecosys em. The e o e,
in his con ex , his wo k p esen an ex ended s udy along
his ancien sal e n in which he p oka yo ic di e si y was
s udied by Illumina-based 16S RNA gene sequencing o
de e mine whe he he wa e s wi hin he alley ha e dis inc-
i e mic obiological cha ac e is ics. Taking in o accoun he
physico-chemical pa ame e s o he main wa e s supplying
he alley (six sp ings, a s eam ha c oss he sal e n, and
g oundwa e ) and wa e in ol ed in sal p oduc ion (wa e
om a b ine dis ibu ion channel and h ee b ine es ing
ponds), p oka yo ic communi y s uc u e and composi ion
was s udied. The in eg a ion o all da a is no only impo an
o expanding scien i ic unde s anding bu also o acili a -
ing he eco e y and enhancemen o his ac i e sal e n.
Ma e ial andMe hods
Sampling Si e Fea u es andSampling P ocedu e
Añana Sal Valley (42°47′59.82″ N, 2°59′3.23″ W; al i ude
570–598m) is in wes e n Ála a, Basque Coun y, Spain.
Fo his s udy, 12 sampling si es (Fig.1a) we e selec ed
based on hei accessibili y and minimal human in e en-
ion. The San a Eng acia sp ing is si ua ed a he highes
poin o he alley, and i is he main b ine supplie o he
sal e n. Immedia ely, i s wa e is channeled (called in his
s udy San a Eng acia channel) and dis ibu ed h ough-
ou he alley o he empo a y accumula ion o es ing
ponds (i.e., Pond I, Pond II, and Pond III) om which he
b ine is dis ibu ed o he c ys alliza ion pans a he end o
he sal p oduc ion sys em. San a Eng acia sp ing is e y
close o he b ackish wa e sou ce o he San Juan s eam,
which uns all along he sal e n, bu i is no pa o he
sal p oduc ion sys em. A li le u he no h a e he o he
sp ings, some sal y (El Cau i o, El Pico, Fuen e iba, and
Hon ana) and some b ackish (El Pico Dulce, which is only
wo me e s om El Pico). To he eas in he alley, he e is
a piezome e (called S8) om which b ackish g oundwa e
can be sampled a a dep h o 60m. Mos o he sp ings
a e in di ec con ac wi h he na u al su ounding en i on-
men , excep o he San a Eng acia sp ing and he El Cau-
i o sp ing, which ha e a wooden s uc u e a ound hem
(Fig.1a). Mo e in o ma ion abou he complex geological
and hyd ogeological con ex o he alley is p esen ed in
he Supplemen a y Ma e ial.
Gi en he impo ance o sal p oduc ion in he eco-
nomic and social de elopmen o he a ea and i s ele-
ance as a na u al, cul u al and he i age si e, he e is since
2017 a su ace- and g ound-wa e moni o ing ne wo k
h oughou he alley. O he 12 sampling si es, eigh ( he
six sp ings, he s eam, and he piezome e ) a e pa o
his moni o ing ne wo k; he e o e, he ionic composi-
ion o he wa e samples was a ailable. This composi-
ion is pe iodically analyzed by ion ch oma og aphy a
he SGIke Ad anced Resea ch Facili ies o he Uni e -
si y o he Basque Coun y UPV/EHU. In a i s phase o
his s udy (sp ing 2018), wa e samples om h ee sp ings
(San a Eng acia sp ing, El Pico sp ing, and El Pico Dulce
sp ing), g oundwa e om S8 piezome e , and wa e om
he ponds (pond I, II, and III) we e collec ed. In a sec-
ond phase o he s udy (sp ing 2021), he sampling was
ex ended o he es o he sp ings (Fuen e iba sp ing,
El Cau i o sp ing, and Hon ana sp ing), he San Juan
s eam, and he b ine dis ibu ion channel (San a Eng a-
cia channel).
Wa e samples we e collec ed di ec ly using s e ile glass
bo les. Wa e om he San a Eng acia and El Cau i o
sp ings was collec ed using a Niskin bo le (Aqua ic Bio-
Technology, El Pue o de San a Ma ía, Spain) bo h a 2m
dep h. G oundwa e om he S8 piezome e (60m dep h)
was collec ed using a manual baile sys em (Eijkelkamp,
Giesbeek, The Ne he lands). A all sampling si es, wa e
empe a u e, pH, and elec ical conduc i i y we e meas-
u ed wi h a Combo es e (Hanna Ins umen s, Eiba ,
Spain) and salini y wi h a densi y hyd ome e (Ha wig
Ins umen s, Ne he lands), all pa ame e s measu ed insi u.
The samples we e hen anspo ed o he labo a o y in
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p ope ly labeled con aine s a 4°C and wi hin 1h o u -
he p ocessing. P oka yo ic Communi y andDi e si y S udy by16S
RNA Gene Deep Sequencing
DNA Ex ac ion andSequencing
Fig. 1 The Añana Sal Valley; a map showing he 12 sampling loca ions and hei pic u es (A-L) and b he ep esen a ion o he di e en wa e
lows in he alley
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M.Azpiazu-Muniozgu en e al. 171 Page 4 o 15
Wa e samples we e en iched by il e ing 5 L o wa e unde
asep ic condi ions h ough a cus om-made de ice d i en
by acuum/comp ession pumps (Labbox, P emia de Dal ,
Spain) and ending up in a 0.22-µm po e size il e . DNA was
ex ac ed using he Genomic Mini AX Bac e ia Ki (A&A
Bio echnology, Gdansk, Poland) and quan i ied using he
Quan iFluo dsDNA Sys em (P omega, Madison, WI, USA).
The V3-V4 hype a iable egion o he 16S RNA gene was
ampli ied [11], and he amplicons we e sequenced using he
pai ed-end me hod on a MiSeq Illumina pla o m a Bioge-
ne ics (Ála a, Spain). Lib a ies we e p epa ed om isola ed
DNA using he Nex e a DNA Lib a y p ep ki (Ilumina, San
Diego, CA, USA). Sequencing da a om he s udy a e a ail-
able unde he accession numbe PRJNA1115049.
P oka yo e Di e si y andDis ibu ion
Sequence analysis was pe o med using he packages and
pipelines o he Quan i a i e Insigh s In o Mic obial Ecol-
ogy (QIIME2) p og am ( e sion 2021.4) [12]. Joining o
pai ed-end eads, sequence quali y con ol, and ea u e able
cons uc ion we e pe o med by denoising wi h DADA2
plugin (q2-dada2) wi h s anda d pa ame e s [13]. Taxonomic
assignmen o DADA2-gene a ed amplicon sequence a i-
an s (ASVs) was pe o med using he Bayesian axonomy
classi ie classi y-sklea n wi h he q2- ea u e-classi ie add-
on [14] wi h axonomy.py using he SILVA da abase .138
[15], clus e ed a 99% sequence simila i y.
Alpha di e si y me ics (obse ed ASVs, Fai h’s Phyloge-
ne ic Di e si y, Pielou’s e enness, Shannon’s and Simpson’s
di e si y indexes) and be a di e si y me ic (B ay‐Cu is
dissimila i y) we e es ima ed using q2‐di e si y a e sam-
ples we e a e ied o he smalles numbe o non-chime ic
sequences o all samples es ed. S a is ical signi icance o he
alpha di e si y alues be ween sampling si es was assessed
using he K uskal–Wallis H es . To assess he ela ionship
o he wa e p ope ies wi h α-di e si y indices, Pea son’s
coe icien es was ca ied ou . Fo compa ing he mic o-
bial communi y s uc u e among samples, B ay–Cu is dis-
simila i y coe icien was assessed by analysis o simila i ies
(ANOSIM) wi h 999 pe mu a ions. In all analyses, i was
assumed a p < 0.05 o s a is ically signi ican di e ences.
An analysis o composi ion o mic obiome (ANCOM)
es was applied o assess di e en ially abundance gene a
using he q2-composi ion QIIME2 plugin [16]. A hea map
was pe o med by ggplo 2 R package o isualize di e -
ences a genus-le el composi ion be ween samples. Asso-
cia ion be ween samples we e es ablished by he B ay–Cu -
is dissimila i y. Venn analysis was pe o med by Venn
diag am so wa e (a ailable online a : h p:// bioin o ma
ics. psb. ugen . be/ web o ols/ Venn/, accessed on 13 Oc obe
2023) o de e mine common and unique p oka yo ic axa
on samples. A Canonical Co espondence Analysis (CCA)
was pe o med o co ela e en i onmen al a iables wi h
p oka yo ic axa and samples. To make he CCA, PAST 4
so wa e package [17] was used and a Mon e Ca lo es wi h
999 un es ic ed pe mu a ions was ca ied.
Func ional P edic ion o P oka yo ic Communi ies
The unc ional po en ial o he mic obial communi y was
p edic ed using 16S RNA gene abundance da a ia Phy-
logene ic In es iga ion o Communi ies by Recons uc ion
o Unobse ed S a es (PICRUS 2) [18]. The ecommended
maximum NSTI cu -o poin o wo was implemen ed
by de aul in PICRUS 2, which excluded 1.3% o ASVs.
P edic ed pa hways we e ca ego ized acco ding o KEGG
(Kyo o Encyclopedia o Genes and Genomes) o hology
g oups and compa ed be ween samples. In addi ion, he
unc ional anno a ion o p oka yo ic axa ia Func ional
Anno a ion o P oka yo ic Taxa (FAPROTAX) was ca ied
ou (h p:// www. ehbio. com/ Image GP/ index. php/ Home/
Index/ FAPRO TAX. h ml) [19].
Resul s
Physico‑chemical Cha ac e is ics o Wa e s
The physico-chemical analysis o he wa e samples e ealed
ha hey could be classi ied in o wo dis inc ca ego ies:
sal y and b ackish (Table1). The able p esen s wo se s
o da a on which he classi ica ion o he ype o wa e is
based: he i s se comp ises da a ob ained insi u a all sam-
pling si es, while he second se comp ises ion concen a-
ion da a o si es belonging o he moni o ing ne wo k. The
alues displayed o he la e da ase ep esen he mean
alue ob ained om measu emen s conduc ed be ween 2017
and 2022. De ailed in o ma ion is gi en in Supplemen a y
TableS1. Moni o ing showed ha he e is e y li le change
in ion concen a ion o e he yea s, hus adequa ely e lec -
ing he s able cha ac e o he physico-chemical cha ac e is-
ics o he wa e s o he alley, which a e conside ed s able
o e he yea s. They also main ain hei low a es (a o al
o abou 3 L/s o he sal y and 5 L/s o he b ackish) ai ly
cons an o e ime.
Bo h sal y and b ackish wa e show a clea sodium chlo-
ide acies, a di ec esul o he dissolu ion o hali e in he
diapi ic s uc u e. Howe e , i is impo an o no e he high
p esence o sul a e and calcium, due o he dissolu ion o
gypsum-anhyd i e, which is compa a i ely highe in he
b ackish wa e s (Table1). The wa e aken om he S8
piezome e sampling si e has a highe sul a e con en han
he o he b ackish wa e (Table1), due o i s pa icula posi-
ion in he low scheme (Fig.1b). I is also wo h no ing he
high concen a ion o magnesium and po assium in he sal y
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Table 1 Physico-chemical pa ame e s o he wa e a he di e en sampling si es analyzed
NA no analyzed, nd no de ec ed
*These da a a e ob ained om he moni o ing ne wo k in he alley ha measu es hese pa ame e s pe iodically. The da a shown a e he media om he da a ob ained du ing 2017–2022
Sampling si es Physico-chemical pa ame e s
Measu ed insi u Measu ed by ionic ch oma og aphy*
Salini y (g/L) Conduc i i y T (°C) pH Cl− (mg/L)
SO2−
4
(mg/L)
NO−
3
(mg/L) Na+ (mg/L) Ca2+ (mg/L) Mg2+ (mg/L) K+ (mg/L)
Sal y San a Eng acia sp ing 200 Sa u a ed 16 6.6 153,813 4697 nd 106,513 1860 286 519
San a Eng acia channel 200 NA 17 6.6 NA NA NA NA NA NA NA
El Pico sp ing 205 Sa u a ed 16 7.5 168,361 5493 nd 118,100 1823 300 567
El Cau i o sp ing 220 NA 17 6.6 142,721 4475 nd 100,710 1933 283 476
Hon ana sp ing 210 NA 16 6.2 148,983 4778 nd 99,312 1751 266 464
Fuen e iba sp ing 200 NA 20 6.5 152,187 4669 nd 102,345 1845 281 580
Pond I 200 Sa u a ed 18 7.8 NA NA NA NA NA NA NA
Pond II 210 Sa u a ed 27 7.5 NA NA NA NA NA NA NA
Pond III 220 Sa u a ed 29 7.6 NA NA NA NA NA NA NA
B ackish San Juan s eam 20 NA 13 7.4 5515 921 17 3853 391 65 19
El Pico Dulce sp ing 10 NA 13 7.3 5460 939 17 3812 396 62 19
S8 piezome e 4 NA 13 7.2 3060 2374 6 2380 673 193 21
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wa e s (Table1). In addi ion, as expec ed, ni a es a e only
p esen in he shallowe b ackish wa e s (Table1).
The wa e s o he i e sal y sp ings o igina e om e y
slow deep lows (hund eds o me e s) and ha e a e y high
salini y (220–240g/L) and ela i ely high empe a u e
(16–20°C). The wo b ackish wa e s sp ings ha e a much
lowe salini y (15–30g/L) and co espond o he mix u e o
deep wa e s wi h shallowe cu en s. The pH o he b ackish
wa e is close o neu al (7.2 o 7.4) while he pH o he sal y
wa e is sligh ly lowe (6.2 o 6.6).
P oka yo ic Di e si y andCommuni y Composi ion
A o al o 583,454 sequences co esponding o 1801 di -
e en ASVs we e ob ained om 11 sampling si es. The
da a om he Hon ana sp ing had o be disca ded due o
he low numbe o sequences ob ained. Ra e ac ion cu es
we e compu ed by a e ying each sample o he minimum
equency o sequences, which was 14,641 eads (Fig.S1).
Di e si y was calcula ed a e no maliza ion o he samples.
P oka yo ic Di e si y
Alpha di e si y analysis e ealed ha p oka yo ic com-
muni y ichness (Obse ed ASVs and Chao1), quan i a i e
communi y ichness o di e si y (Shannon and Simpson
indexes), and communi y equali y o e enness (Pielou’s
e enness) a ied widely among he samples (Table2). In
pa icula , he highes p oka yo ic ichness, di e si y, and
e enness we e obse ed in he El Pico Dulce sp ing, while
he lowes ichness was ound in he El Pico sp ing. In con-
as , he lowes di e si y and e enness we e ound in he
San Juan s eam, wi h alues simila o hose o he El Pico
sp ing.
Pea son’s coe icien es showed a s a is ically sig-
ni ican nega i e co ela ion be ween salini y and ASVs,
and also be ween salini y and Chao1 di e si y indexes (p
alue < 0.05).
Be a di e si y iden i ies dissimila i ies be ween he sam-
pling si es. Dissimila i y in ASV composi ion was ep e-
sen ed by he P incipal Coo dina e Analysis (PCoA) plo .
The i s h ee p incipal componen s explained 57.61%
(PC1 + PC2 + PC3) o he o al a ia ion (Fig.2). Fo
he analysis o mul i a ia e homogenei y among g oups,
he analysis o simila i ies (ANOSIM) es showed sig-
ni ican di e ences in he p oka yo ic di e si y be ween
g oups based on hei salini y (sal y and b ackish wa e )
(p- alue < 0.05). Howe e , no s a is ically signi ican di e -
ences we e obse ed when compa ing samples acco ding o
hei sampling si e (sp ing, pond, s eam, o g oundwa e ).
Assessmen o  heP oka yo ic Communi y Composi ion
andI s Dis ibu ion
The 1801 di e en ASVs we e subsequen ly assigned o
di e en axonomic le els. Fi y-nine o he ASVs (3.3%)
could no be assigned o any known phylum. The axo-
nomic assignmen o each ASV is shown in Supplemen-
a y TableS2. The axonomic assignmen showed ha he
a chaeal domain was essen ially es ic ed o sal y wa e s,
whe eas bac e ia we e p esen a all sampling si es, wi h he
majo i y in b ackish wa e (99.4 o 99.8%) (Fig.3a). The
bac e ial and a chaeal domains we e dis ibu ed in 31 phyla,
57 classes, 120 o de s, 182 amilies, and 258 gene a. The
dis ibu ion o gene a whose ela i e abundance was g ea e
han 3% in a leas one o he analyzed si es is shown in
Fig.3b. I can be obse ed ha he 60.7% o he ASVs we e
classi ied a genus le el (6.3% o hem as uncul u ed o gan-
isms), while 39.3% emained Unclassi ied.
The majo bac e ial and a chaeal phyla we e Pseudomon-
ado a and Halobac e o a, espec i ely. A he genus le el,
Table 2 Alpha di e si y indexes
calcula ed o he loca ions o
he sal e n analyzed in he s udy
ASVs amplicon sequence a ian s, Chao1 con idence in e al o ichness es ima o
Sampling si e Obse ed
ASVs
Chao1 Simpson’s index Shannon’s index Pielou’s
e enness
San a Eng acia sp ing 104 104 0.95 5.07 0.76
San a Eng acia channel 196 197 0.96 5.56 0.73
El Pico sp ing 69 69 0.83 3.68 0.60
El Cau i o sp ing 169 169 0.96 5.50 0.74
Fuen e iba sp ing 294 301 0.93 5.48 0.67
Pond I 120 120 0.89 4.49 0.65
Pond II 74 74 0.86 3.88 0.62
Pond III 147 147 0.96 5.59 0.78
San Juan s eam 155 158 0.83 3.50 0.48
El Pico Dulce sp ing 541 541 1.00 8.76 0.97
S8 piezome e 284 284 0.99 7.17 0.88
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he mos ep esen a i e bac e ial gene a ( ela i e abun-
dance > 1%) we e Pseudomonas, Undibac e ium, Salini-
bac e , Ma inomonas, and Bac e oides. Simila ly, he mos
abundan a chaeal gene a ( ela i e abundance > 1%) we e
Halo ub um, Halono ius, Haloplanus, Halomic oa cula,
and Na onomonas. Each o hese gene a a ied g ea ly in
abundance a di e en sampling si es (Fig.3b).
Al hough some gene a we e ound in mo e han one loca-
ion, di e en composi ional p o iles can be de ined a he
genus le el. Clus e ing using B ay–Cu is dissimila i y coe -
icien based on genus abundance con i med ha b ackish
wa e s showed g ea e simila i y o one ano he , as did sal y
wa e s (Fig.4).
ANCOM analysis iden i ied he gene a Na onomonas
and Halo ub um as signi ican ly mo e abundan gene a in
he sal y wa e samples. Fu he mo e, when he analysis was
pe o med acco ding o he wa e salini y o he samples,
only 25 ou o 258 gene a we e sha ed (Fig.5a). Howe e ,
64 and 169 gene a we e unique o sal y and b ackish wa e ,
espec i ely.
The dis ibu ion o gene a be ween sal y sp ings e ealed
12 gene a sha ed among hese samples (Fig.5b); includ-
ing Haloma ina, Halono ius, Owenweeksia, Halohas a,
Na onomonas, Flexis ipes, Salinibac e , Halo ubellus,
Haloplanus, Halo ub um, Halobaculum, and Halomic o-
a cula. Halo ub um was he mos abundan (8.6 o 61.2%)
among hem, excep in he San a Eng acia sp ing, whe e
Halohas a was he mos abundan one. In he Fuen e iba
sp ing, 22 gene a we e iden i ied no p esen in he o he
saline sp ings. Howe e , he gene a ound he e we e in he
mino i y (Acine obac e , 0.8%; Ma inobac e , 0.4%; En e-
ococcus, 0.2%).
The in es iga ion o he possibili y o g oundwa e con-
ac be ween he ElPico and he ElPico Dulce sp ings,
which a e only wo me e s apa bu ha e di e en physico-
chemical pa ame e s (Fig.1 and Table1), e ealed a di e -
en axonomic composi ion. O he 132 gene a de ec ed, he
majo i y (n = 111) we e only p esen a he ElPico Dulce
sp ing and only wo (Halomonas and Cellulosimic obium)
we e sha ed (Fig.5c). Mo eo e , he ela i e abundance o
bo h gene a based on ASV axonomic assignmen was e y
low in bo h sampling si es, 0.05% and 0.71% and 0.19% and
0.54%, espec i ely.
Fig. 2 UniF ac dis ance-based Jackkni e clus e ing based on he ASVs da a, wi h he i s h ee p incipal coo dina es (PCs) shown: unweigh ed
UniF ac wi h PC1 (26.90%), PC2 (19.21%), and PC3 (11.50%)
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The supplemen a y igu es and ables in APPENDIX I can be downloaded by
scanning his QR code.
I. ERANSKINeko i udi e a aula osaga iak QR kode hau eskanea u a deska ga
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APPENDIX II. ERANSKINA
Fungal Di e si y and Composi ion o
he Con inen al Sola Sal e n in Añana
Sal Valley (Spain)
Re e ence: Azpiazu-Muniozgu en M, Pe ez A, Remen e ia A, Ma inez-
Malaxe xeba ia I, Alonso R, Lao den L, Gamboa J, Bikandi J, Ga aiza J, and
Ma inez-Balles e os I. Fungal di e si y and composi ion o he con inen al
sola sal e n in Añana Sal Valley (Spain). Jou nal o Fungi. 2021; 7(12):1074.
Jou nal o Fungi (Basel) (2021); 7(12):1074
DOI: 10.3390/jo 7121074
Impac ac o 2021: 5.724
Rela i e posi ion: 40/137, Q2 Mic obiology
263
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Fungi
Jou nal o
A icle
Fungal Di e si y and Composi ion o he Con inen al Sola
Sal e n in Añana Sal Valley (Spain)
Maia Azpiazu-Muniozgu en 1, Alba Pe ez 1, Ai o Remen e ia 2, I a i Ma inez-Malaxe xeba ia 1,
Rod igo Alonso 1, Lo ena Lao den 1, Ja ie Gamboa 3, Joseba Bikandi 1, Ja ie Ga aiza 1
and Ila gi Ma inez-Balles e os 1,*


Ci a ion: Azpiazu-Muniozgu en, M.;
Pe ez, A.; Remen e ia, A.;
Ma inez-Malaxe xeba ia, I.; Alonso,
R.; Lao den, L.; Gamboa, J.; Bikandi,
J.; Ga aiza , J.; Ma inez-Balles e os, I.
Fungal Di e si y and Composi ion o
he Con inen al Sola Sal e n in
Añana Sal Valley (Spain). J. Fungi
2021,7, 1074. h ps://doi.o g/
10.3390/jo 7121074
Academic Edi o s:
Nalin Wijayawa dene and
Na awu Boonyuen
Recei ed: 26 Oc obe 2021
Accep ed: 12 Decembe 2021
Published: 14 Decembe 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
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4.0/).
1
Mik oIke Resea ch G oup, Depa men o Immunology, Mic obiology and Pa asi ology, Facul y o Pha macy,
Uni e si y o he Basque Coun y UPV/EHU, Paseo de la Uni e sidad 7, 01006 Vi o ia-Gas eiz, Spain;
[email p o ec ed] (M.A.-M.); [email p o ec ed] (A.P.); [email p o ec ed] (I.M.-M.);
[email p o ec ed] (R.A.); lo [email p o ec ed] (L.L.); [email p o ec ed] (J.B.);
ja ie [email p o ec ed] (J.G.)
2
Depa men o Immunology, Mic obiology and Pa asi ology, Facul y o Science and Technology, Uni e si y o
he Basque Coun y UPV/EHU, Ba io Sa iena s/n, 48940 Leioa, Spain; ai o [email p o ec ed]
3Biogene ics, Po al de Zu bano 3, 6-B, 01013 Vi o ia-Gas eiz, Spain; [email p o ec ed]
*Co espondence: ila [email p o ec ed]; Tel.: +34-945-013904
Abs ac :
The Añana Sal Valley in Spain is an ac i e con inen al sola sal e n o med 220 million
yea s ago. To da e, no ungal genomic s udies o con inen al sal e ns ha e been published, al hough
DNA me aba coding has ecen ly expanded esea che s’ abili y o s udy mic obial communi y
s uc u es. Acco dingly, he aim o his p esen s udy was o e alua e ungal di e si y using he
in e nal ansc ibed space (ITS) me aba coding a di e en loca ions along he sal e n (sp ings,
ponds, and g oundwa e ) o desc ibe he ungal communi y o his saline en i onmen . A o al o
380 ungal gene a we e de ec ed. The ubiqui y o Saccha omyces was obse ed in he sal e n, al hough
o he halo ole an and halophilic ungi like Wallemia,Cladospo ium, and T imma os oma we e also
de ec ed. Mos o he ungi obse ed in he sal e n we e sap o ophs. The ungal dis ibu ion
appea ed o be in luenced by su ounding condi ions, such as he plan and soil con ac , ce eal ields,
and ineya ds o his ag icul u al egion.
Keywo ds: ITS; me aba coding; ungi; biodi e si y; con inen al sal e n
1. In oduc ion
Saline and hype saline en i onmen s a e widely dis ibu ed a ound he wo ld. The
s udy o hese habi a s is becoming mo e common, no only as a means o inc ease knowl-
edge ega ding he s uc u e o hei mic obial communi ies bu also o gain insigh s in o
he adap a ions o hese o ganisms o hese unique loca ions. Among hype saline en i on-
men s, sola sal e ns a e o pa icula in e es . Di e en ypes o sal e ns exis ha may
be classi ied as li o al sal e ns and con inen al o inland sal e ns. Con inen al sal e ns a e
hose ha use b ine ob ained om sp ing sou ces, o he p oduc ion o sal ia e apo a ion
and sola ac i i y [1].
The Añana Sal Valley (30 km sou hwes o Vi o ia-Gas eiz, Ála a, No he n Spain;
42.80 N 2.98 W) is an ac i e con inen al sola sal e n o med abou 220 million yea s ago
ollowing he e apo a ion o wa e om he g ea ocean (Te hys Ocean) ha co e ed mos o
he Ea h. This p ocess led o he deposi o ex ensi e laye s o e apo i es (sal s, anhyd i e,
and gypsum), which, emana ing om highly plas ic s a ig aphic le els and subjec ed
o g ea p essu e, ose h ough he sedimen a y laye s o he Ea h’s c us , c ossing and
de o ming hese laye s and esul ing in he o ma ion o a diapi . Rainwa e c osses he
uppe s a um o he diapi ock, and hen laye s o sal appea on he su ace decades
la e in he o m o hype saline o b ackish sp ings [
2
]. Depending on he pa h he il a ed
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wa e akes h ough he ocks, i encoun e s a eas o highe o lowe sal con en . Fo his
eason, sp ings ha a e nex o each o he con ain wa e wi h di e en le els o salini y.
The hype saline sp ings loca ed in he Añana Sal Valley a e wa e sou ces ha supply
b ine a he su ace le el in a na u al and con inuous way wi hou any need o d illing o
o he use o pumps. A chaeological emains show ha hese wa e sou ces ha e been
used by locals o 7000 yea s, o he ex ac ion o sal [
3
,
4
]. Consequen ly, he physical,
chemical, and an h opogenic cha ac e is ics o he Añana Sal Valley en i onmen make i
an excep ional se ing o s udy and esea ch. Wha is mo e, hese sal la s con ain deposi s
wi h impo an paleo-en i onmen al and paleo-clima ic in o ma ion, in addi ion o he
biodi e si y o hei ex eme en i onmen s [3,4].
Fo many yea s, saline and hype saline en i onmen s we e hough o be popula ed
almos exclusi ely by p oka yo ic mic oo ganisms. Recen ly, i has been shown ha
euka yo ic mic oo ganisms a e p esen oo [
5
–
7
]. S udies ega ding p oka yo ic di e si y
and dis ibu ion ha e been conduc ed a di e en sal e ns using bo h cul u e-dependen
and cul u e-independen me hods [
8
–
12
]. Fewe s udies ha e been conduc ed on he
examina ion o ungal communi ies [
13
–
15
]. In he las ew yea s, DNA me aba coding has
become an e ec i e ool o he iden i ica ion o yeas and o he ungal species. Di e en
s udies ha e been conduc ed on soil, ma ine, and saline ecosys ems; ineya ds and wine-
p oduc ion sys ems; and in glacie s [
16
–
23
], among o he ecosys ems. Al hough a s udy
on p oka yo ic and i us di e si y in he Añana Sal Valley has been published [
24
], no
ungal me aba coding su ey in his ecosys em has been published no o o he con inen al
o inland sal e ns wo ldwide. The aim o his p esen s udy, he e o e, was o e alua e
ungal di e si y in he ex eme ecosys em o he Añana Sal Valley using me aba coding o
desc ibe he composi ion o he ungi p esen in his loca ion.
2. Ma e ials and Me hods
2.1. Sampling Si es and P oceedings
Wa e samples om di e se si es a ound he Añana Sal Valley we e collec ed o
pe o m he ungal di e si y s udy (Figu e 1). Indi idual wa e samples om each o he
se en si es we e collec ed du ing he su ey and s udied o ungal di e si y. Two o hese
si es, San a Eng acia (SE) Sp ing and Pico Sp ing, we e sp ings con aining high-salini y
wa e . SE Sp ing is he main b ine supplie o sal p oduc ion in he sal e n, wi h an
a e age low o 3 L/s and 200 g o sal /L. Pico Sp ing p o ides a lowe b ine low o
he sys em. The sal p oduc ion b ine is dis ibu ed by an in e connec ed wooden canal
sys em, channeling he b ine o a se ies o dis ibu ion ponds and c ys allizing pans. Th ee
o hese ponds (Pond I, II, and III), con aining high-salini y wa e and loca ed along he
sal -p oduc ion sys em, we e also selec ed o his s udy. In he alley, o he sp ings wi h
a lowe -salini y wa e con en a ise na u ally. F om among hese, b ackish wa e om
he Pico Dulce Sp ing was analyzed. Finally, b ackish g oundwa e om an aqui e a
60 m dep h, accessible h ough a piezome e called S8, was also analyzed. Wa e samples
we e collec ed in Oc obe 2017 (SE, Pico Sp ing, and ponds) and in June 2018 (S8 and Pico
Dulce Sp ing).
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Figu e 1.
Loca ions along he Añana Sal Valley s udied o ungal di e si y and composi ion
de e mina ion (pho og aph ob ained om Viso geoEuskadi a h ps://www.geo.euskadi.eus/s69-
biso ea/es/x72aGeoeuskadiWAR/index.jsp, accessed on 26 July 2021). The exac coo dina es o each
loca ion a e as ollows: Pond I, 42.8014 N 2.9860 W; Pond II, 42.8009 N 2.9852 W; Pond III, 42.8006 N
2.9851 W; Pico Dulce, 42.7989 N 2.9858 W; Pico, 42.7986 N 2.9856 W; San a Eng acia, 42.7965 N 2.9863
W; S8 piezome e , 42.7970 N 2.9838 W.
Wa e samples we e di ec ly ob ained using s e ile glass bo les o a Niskin bo le
(Aqua ic BioTechnology, El Pue o de San a Ma ía, Spain). G oundwa e om he S8
piezome e was aken a a 60 m dep h using a manual baile sys em (Eijkelkamp, Giesbeek,
The Nede lands). Physicochemical pa ame e s, such as wa e empe a u e, pH, NaCl con-
cen a ion, and conduc i i y ( ela ed o sal con en ), we e de e mined in si u (Combo es e ,
Hanna Ins umen s, Eiba , Spain). Wa e samples we e aken di ec ly o he labo a o y
o p ocessing.
2.2. DNA Ex ac ion and Sequencing
Samples we e en iched by il e ing app oxima ely 5 L o wa e in asep ic condi ions
h ough a ailo ed de ice, d i en by acuum/comp ession pumps, which concen a ed he
samples ia a se ies o dec easing low- a e polyca bona e memb anes (Labbox, P emia
de Dal , Spain), ending in a 0.2
µ
m po e size il e . DNA ex ac ion and quan i ica ion o
he samples we e conduc ed acco ding o he manu ac u e ’s ins uc ions ia he Genomic
Mini AX Yeas Ki (A&A Bio echnology, Gdansk, Poland) and he Quan iFluo dsDNA
Sys em (P omega, Madison, WI, USA), espec i ely. The ungal in e nal ansc ibed space
1 (ITS1) egion was ampli ied, as sugges ed in he ITS Me agenomics P o ocol o Illumina
by modi ied ITS1-F and ITS2 p ime s se [
25
]. The lib a y was p epa ed by a Nex e a
DNA Lib a y p ep ki (Ilumina, San Diego, CA, USA) acco ding o ha p o ocol. High-
h oughpu sequencing (HTS) was pe o med on an Illumina MiSeq pla o m, which
gene a es pai ed-end sequences in FASTQ o ma . The nucleo ide sequence da a om he
s udy a e a ailable in he DDBJ/EMBL/GenBank da abases unde he accession numbe
PRJNA749727.
2.3. Sequencing Da a and S a is ical Analysis
Mos o he analyses was ca ied ou using QIIME2 e sion 2021.2 so wa e ools
and pipelines [
26
]. Joining o pai ed-end eads, sequence quali y con ol, and ea u e
able cons uc ion we e pe o med by denoising wi h DADA2 plugin (q2-dada2) [
27
].
Du ing his s ep, sequence denoising, de eplica ion, and chime a il e ing we e unde aken.
Sequences we e immed a a 200 bp leng h. Amplicon sequence a ian s (ASVs) [
28
]
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gene a ed by DADA2 we e assigned axonomically using he q2- ea u e-classi ie [
29
]
classi y-sklea n naï e Bayes axonomy classi ie wi h he UNITE 8.3 dynamic da abase [
30
]
a 97% simila i y le el. Manually sea ching o uniden i ied ASVs was pe o med by BLAST
(h ps://blas .ncbi.nlm.nih.go /Blas .cgi, accessed on 6 Oc obe 2021).
Alpha and be a di e si y me ics we e es ima ed using q2-di e si y plunging a e a -
e ac ion o he samples o 2270 sequences pe sample. Alpha di e si y es ima o s, such as
obse ed ASVs and Chao1 index, we e de e mined o es ablish he species ichness a each
poin . To s udy he di e si y o he species in he samples,
α
es ima o s, such as Shannon
and Simpson indexes, we e used: he highe he Shannon index, he g ea e he di e si y,
and he close he Simpson index is o 0, he lowe he di e si y. Pielou’s e enness, which
quan i ies how equal a communi y is, was also de e mined ( he close o 0, he highe he
dominance o a species). S a is ical signi icance o he alpha di e si y alues be ween sam-
pling si es was assessed using he K uskal–Wallis H es . A
p alue < 0.05
was conside ed
s a is ically signi ican . To es ablish he co ela ion o di e si y indexes wi h physicochemi-
cal cha ac e is ics, Pea son’s coe icien es was pe o med (p alue < 0.05 was conside ed
s a is ically signi ican ). Dissimila i y be ween sample g oups was also examined by he
B ay–Cu is coe icien and di e ences among g oups we e assessed by ANOSIM wi h
999 pe mu a ions (p< 0.05). A hea map was pe o med by ggplo 2 R package o isualize
di e ences a he genus le el composi ion among samples. Associa ions be ween samples
we e es ablished by he B ay–Cu is dissimila i y. Venn analysis was pe o med by Venn di-
ag am so wa e (a ailable online a : h p://bioin o ma ics.psb.ugen .be/web ools/Venn/,
accessed on 21 July 2021) o de e mine common and unique ungal axa on samples.
The FUNGuild da abase was used o assign ecological guilds wi hin h ee ophic
modes (i.e., pa ho oph, symbio oph, and sap o oph) o all ASVs [
31
]. Only ungal ASVs
wi h p obable and highly p obable con idence ma ches we e conside ed o u he analysis.
3. Resul s
3.1. Wa e Physicochemical Da a
The physicochemical cha ac e is ics o wa e samples measu ed in si u a e shown in
Table S1. Two di e en deg ees o salini y we e obse ed, which we e used o es ablish
g oups o u he analysis: SE Sp ing, Pico Sp ing, and he h ee b ine ponds we e
classi ied as sal y wa e , and Pico Dulce Sp ing and S8 piezome e we e classi ied as
b ackish wa e due o hei lowe sal concen a ion.
3.2. Sequence Analysis and ASV De e mina ion
The ungal di e si y and communi y s uc u e in he wa e samples aken om
ep esen a i e si es in he Añana Sal Valley we e in es iga ed by HTS (Figu e 1). A e
denoising and quali y con ol o sequenced DNA, a mean o 132,520 eads pe sample was
ob ained. A o al o 704,874 sequences we e ob ained co esponding o 2204 di e en ASVs
de e mined by DADA2. These ASVs we e subsequen ly assigned a di e en axonomy
le els o ungal iden i ica ion. Among he ASVs, 853 ailed o be iden i ied a any known
phylum (38.7% o he o al).
3.3. Fungal Di e si y in he Sal e n
Ra e ac ion cu es we e compu ed by a e ying each sample o he minimum numbe
o sequences (2270 eads). Ra e ac ion cu es we e no pa allel o all he samples analyzed
bu s a ed o la en (Figu e S1). The alpha di e si y was calcula ed a e no maliza ion o
he samples. Table 1shows he axa ichness and di e si y alues ob ained by he di e en
alpha di e si y es ima o s. SE Sp ing had he lowes di e si y. The e, he smalles numbe
o ASVs (71 ASVs) was de ec ed and he Shannon index was 3.59 ( anging om 4.89–6.47
o he es o he loca ions). The o he six loca ions demons a ed a simila di e si y, excep
o S8, which was less di e se. While 93 ASVs and a Shannon index o 4.89 we e ob ained a
S8, he obse ed ASVs om he o he i e loca ions anged om 262–396 and he Shannon
index anged om 5.44–6.47. Pielou’s e enness o SE was 0.58 ( he closes alue o 0 in he
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s udy), which indica ed he p esence o a dominan axon (p esumably Saccha omyces). No
s a is ically signi ican di e ences we e obse ed in he alpha di e si y indexes be ween
sample si es.
Table 1. Alpha di e si y indexes calcula ed o he loca ions o he sal e n analyzed in he s udy.
Sample Si e Obse ed
ASVs Chao1 Shannon Simpson Pielou’s
E enness
San a
Eng acia
Sp ing
71 116 3.59 0.77 0.58
Pico Sp ing 354 633 6.27 0.95 0.74
Pond I 264 452 5.44 0.93 0.67
Pond II 396 699 6.47 0.94 0.75
Pond III 263 337 6.07 0.94 0.75
Pico Dulce
Sp ing 262 430 6.15 0.96 0.76
S8 93 93 4.89 0.91 0.74
ASVs, amplicon sequence a ian s.
The di e si y and pH showed a s a is ically signi ican co ela ion based on Pea son’s
coe icien es (Table 2), indica ing ha en i onmen al di e si y dec eases as he pH o he
wa e dec eases oo. No co ela ion was obse ed in ega d o he NaCl concen a ion o
empe a u e.
Table 2.
Co ela ion be ween wa e physicochemical pa ame e s and ungal di e si y. The analysis
was ca ied ou using Pea son’s coe icien es .
Pa ame e Pea son’s Coe icien Tes (pValues)
Shannon Index Simpson Index
NaCl 0.896 0.732
Tempe a u e 0.314 0.262
pH 0.029 * 0.015 *
* s a is ically signi ican co ela ion based on p- alue (p< 0.05).
The ANOSIM es based on B ay–Cu is dissimila i y iden i ied di e ences in he un-
gal di e si y among g oups based on hei salini y (sal y and b ackish wa e ) (
p- alue 0.046
).
No di e ences we e obse ed ega ding di e en wa e ypes (sp ing, pond, o g oundwa e ).
3.4. Fungal Taxonomic Assignmen and Communi y Composi ion
The axonomic assignmen o ASVs showed ha Basidiomyco a and Ascomyco a we e
he phyla p esen in he sal e n (0.18–63.6% and 23–81.1%, espec i ely) (Figu e 2). No
o he ungal phylum was de ec ed in he samples. As e e enced abo e, 38.7% o he ASVs
we e classi ied only as ungi. The manual iden i ica ion o uniden i ied ASVs by BLAST
sea ch did no imp o e hese esul s, so a low axonomic assignmen o he species le el
was ob ained in he s udy. The axonomic assignmen o each ASV can be ound in
Table S2
.
A o al o 380 ungal gene a we e de ec ed in he su ey (Table S3). In Figu e 2, gene a
whose ela i e abundance was mo e han 3% in a leas one o he loca ions analyzed a he
sal e n a e shown. Rega ding SE Sp ing, 30 ASVs we e axonomically assigned o genus
le el; 184 wi h espec o Pico Sp ing; 147, 196, and 110 gene a wi h espec o Pond I, II,
and III, espec i ely; 81 a Pico Dulce Sp ing; and 28 gene a a S8.
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APPENDIX II. ERANSKINA
The supplemen a y igu es and ables in APPENDIX II can be downloaded by
scanning his QR code.
II. ERANSKINeko i udi e a aula osaga iak QR kode hau eskanea u a
deska ga dai ezke.
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Sec ion 3. a ala - APPENDIX III. ERANSKINA
APPENDIX III. ERANSKINA
Al e e y h obac e mu iae sp. no .,
isola ed om hype saline Añana Sal
Valley sp ing wa e , a con inen al
halassohaline- ype sola sal e n
Re e ence: Azpiazu-Muniozgu en M, Ma inez-Balles e os I, Gamboa J,
Seoane S, Alonso R, Lao den L, Ga aiza J, and Bikandi J. Al e e y h obac e
mu iae sp. no ., isola ed om hype saline Añana Sal Valley sp ing wa e , a
con inen al halassohaline- ype sola sal e n. In e na ional Jou nal o
Sys ema ic and E olu iona y Mic obiology. 2021; 71(3):004734.
INTERNATIONAL JOURNAL OF SYSTEMATIC AND
EVOLUTIONARY MICROBIOLOGY (2021); 71(3)
DOI: 10.1099/ijsem.0.004734
Impac ac o 2021: 2.689
Rela i e posi ion: 106/137, Q4 Mic obiology
281
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1
Al e e y h obac e mu iae sp. no ., isola ed om hype saline
Añana Sal Valley sp ing wa e , a con inen al halassohaline- ype
sola sal e n
MaiaAzpiazu- Muniozgu en1, Ila giMa inez- Balles e os1,*, Ja ie Gamboa2, Se gioSeoane3, Rod igoAlonso1,
Lo enaLao den1, Ja ie Ga aiza 1 and JosebaBikandi1
TAXONOMIC DESCRIPTION
Azpiazu-Muniozgu en e al., In . J. Sys . E ol. Mic obiol. 2021;71:004734
DOI 10.1099/ijsem.0.004734
Au ho a ilia ions: 1Mik oIke Resea ch G oup. Immunology, Mic obiology and Pa asi ology Depa men , Fa macy Facul y, Uni e si y o he Basque
Coun y UPV/EHU, Vi o ia- Gas eiz, Ála a, Spain; 2Biogene ics, Vi o ia- Gas eiz, Ála a, Spain; 3Plan Biology and Ecology Depa men , Facul y o Science
and Technology, Uni e si y o he Basque Coun y UPV/EHU, Leioa, Spain.
*Co espondence: Ila gi Ma inez- Balles e os, ila gi. ma inez@ ehu.eus
Keywo ds: Al e e y h obac e mu iae; sal e n; halophile; poliphasic axonomy.
Abb e ia ions: AAI, a e age aminoacid iden i y; ANI, a e age nucleo ide iden i y; dDDH, digi al DDH; DDH, DNA–DNA hyb idiza ion; MA, ma ine aga ;
MB, ma ine b o h; OGRI, o e all genome ela ed index.
The Genbank accession numbe o he 16S RNA gene sequence o s ain SALINAS58T is MN918268. The Whole Genome Sho gun sequence was
deposi ed a DDBJ/ENA/GenBank unde he accession JAAFZT000000000.
Six supplemen a y igu es and one supplemen a y able a e a ailable wi h he online e sion o his a icle.
004734 © 2021 The Au ho s
Abs ac
A no el sal - ole an alpha- p o eobac e ium, designa ed SALINAS58T, was isola ed om San a Eng acia hype saline sp ing
wa e in he Añana Sal Valley, Ála a, Spain. The isola e was G am- nega i e, ae obic, non- mo ile, ca alase- posi i e, oxidase-
nega i e, od- shaped and o med o ange colonies on ma ine aga . Op imal g ow h was obse ed a pH 6.0–6.5, a 30 °C and in
he p esence o 1% (w/ ) NaCl. The main cellula a y acids (>20%) we e summed ea u e 8 (C18 : 1 ω7c and/o C18 : 1 ω6c) and
summed ea u e 3 (C16 : 1 ω7c and/o C16 : 1 ω6c). The majo espi a o y quinone was ubiquinone Q-10 and he majo pola lipids
de ec ed we e diphospha idylglyce ol, phospha idyle hanolamine, phospha idilglyce ol, ou uniden i ied glycolipids and one
uniden i ied phospholipid. S ain SALINAS58T had he highes 16S RNA gene sequence simila i y o Al e e y h obac e ma en-
sis MSW-14T (96.6%), Al e e y h obac e aquaemix ae JSSK-8T (96.5%) and Pon ixan hobac e lu eolus SW-109T (96.5%) ollowed
by Al e e y h obac e a lan icus 26DY36T (96.4%). Resul s o he phylogene ic analysis, based on 16S RNA gene sequences,
and phylogene ic app oaches based on whole genome nucleo ide di e ences, showed ha s ain SALINAS58T could be dis-
inguished om ecognized species o he genus Al e e y h obac e . The genomic DNA G+C con en was 61.4 mol%. Digi al
DNA–DNA hyb idiza ion, a e age nucleo ide iden i y and a e age aminoacid iden i y alues be ween he genome o s ain SALI-
NAS58T and A. ma ensis MSW-14T we e 18.4, 73.1 and 68.1%, espec i ely. Based on da a om his polyphasic cha ac e iza ion,
s ain SALINAS58T (=CECT 30029T=LMG 31726T) is conside ed o be classi ied as ep esen ing a no el species in he genus
Al e e y h obac e , o which he name Al e e y h obac e mu iae sp. no . is p oposed.
Phylogene ic analyses based on 16S RNA gene sequences
ha e shown ha he genus Al e e y h obac e alls wi hin he
amily E y h obac e aceae [1]. The ecen genomic- based
axonomic classi ica ion o he amily E y h obac e aceae
eclassi ies all axons in o 16 gene a, including 11 no el ones
[2]. The genus Al e e y h obac e was p oposed in 2007 by
Kwon e al. h ough he desc ip ion o a single species, Al e -
e y h obac e epoxidi o ans ( ype species) [1, 3, 4]. The genus
Al e e y h obac e comp ises, a he ime o w i ing, 44 alidly
published species, including synonyms (h ps:// lpsn.dsmz.
de/). The cha ac e is ic ea u es o his genus include G am-
nega i e, ae obic g ow h, non- mo ile, gene ally od- shaped
cells, oxidase- and ca alase- posi i e and Q-10 as dominan
espi a o y quinone wi h he absence o bac e iochlo ophyll
a (BChl a) [2, 3, 5]. Membe s o he genus equi e NaCl o
g ow h and hey can be cha ac e ized by yellow o o ange- ed
colony colou s on aga pla es. The empe a u e ange o op i-
mal g ow h is 15–35 °C [2–4]. The dominan a y acid (>10%)
is C18 : 1 ω7c and he majo pola lipids a e phospha idyle ha-
nolamine, phospha idylglyce ol and sphingoglycolipid. The
DNA G+C con en is 52.0–61.8 mol% [2].
The habi a ange o he genus Al e e y h obac e includes
mainly aqua ic en i onmen s, equen ly seawa e
[6, 7]. Howe e , he e a e also isola ion epo s om di e se
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Azpiazu-Muniozgu en e al., In . J. Sys . E ol. Mic obiol. 2021;71:004734
en i onmen s such as cold- seep sedimen [1], deep- sea sedi-
men [8], lagoon sedimen s [9], mang o e sedimen [10],
es ua y en i onmen wa e [11, 12], he junc ion be ween
he ocean and a eshwa e sp ing [13], sal ma sh plan [14],
sea u chin [15], ho sp ing [16], ai [5] and dese soil [17].
Al e e y h obac e has also been eco e ed om polycyclic
a oma ic hyd oca bons and c ude oil deg ading conso ia
en iched om ma ine sedimen s [4].
In his wo k, we desc ibe s ain SALINAS58T isola ed om
hype saline sp ing wa e collec ed in he Añana Sal Valley,
Spain. The pheno ipic analyses, he compa a i e 16S RNA
gene sequence analysis and he genomic analysis indica ed
ha s ain SALINAS58
T
is a ilia ed phylogene ically o he
genus Al e e y h obac e . The e o e, acco ding o he esul s,
he species Al e e y h obac e mu iae sp. no . is p oposed.
ISOLATION AND ECOLOGY
S ain SALINAS58T was isola ed om a hype saline wa e
sample collec ed om he San a Eng acia na u al sp ing in
he Añana Sal Valley (Spain; Fig. S1, a ailable in he online
e sion o his a icle), an ac i e con inen al halassohaline-
ype sola sal e n decla ed as a Globally Impo an Ag icul u al
He i age Sys em by he Food and Ag icul u e O ganiza ion
o he Uni ed Na ions (FAO). The hype salini y o he wa e
sou ces in his alley is explained by he phenomenon known
as diapi , whose o igin lies in ma ine wa e om he T iassic
pe iod. The San a Eng acia sp ing is loca ed a 598 m abo e
sea le el and he app oxima e geog aphic coo dina es a e
42.79665° N 2.98602° W. Wa e samples we e collec ed in May
2017. Wa e empe a u e was 16.0 °C, pH 6.6 and salini y
200 g l−1, and he conduc i i y was sa u a ed. The s ain
was isola ed by mul iple il a ion s eps (Wha man G ade
113V, and Millipo e 5 and 0.22 µm il e s), hen 100 µl o he
il e ed ou wa e was cul i a ed a 25 °C on ma ine aga (MA;
Conda). A e 20 days o incuba ion, a isible o ange- colou ed
colony was ans e ed and subcul u ed on MA medium un il
a pu e cul u e was ob ained and cul i a ed ou inely a 25
°C on MA. S ain SALINAS58T was main ained a −80 °C in
ma ine b o h (MB; Conda) and 25% ( / ) glyce ol.
16S RNA GENE PHYLOGENY
A e incuba ion a 25 °C o 3 days, genomic DNA was
ex ac ed om s ain SALINAS58T using P epMan Ul a
DNA ex ac ion eagen (Applied Biosys ems), acco ding
o he manu ac u e ’s ins uc ions. The 16S RNA gene was
ampli ied by PCR wi h he p ime s 27F (5′- AGAGTTT-
GATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTT-
GTTACGACTT-3′) [18]. The amplicons we e pu i ied and
Sange sequencing was pe o med (S ab Vida). Sequence
da a we e aligned and analysed by Ch omas 2.6.6 so -
wa e (Technelysium). The 16S RNA gene sequence (1296
bp) was deposi ed a Genbank unde accession numbe
MN918268. Due o he sho 16S RNA gene sequence
ob ained, he 1436 bp 16S RNA sequence om he genome
sequencing was used o u he phylogene ic analysis
(accession numbe JAAFZT000000000; NODE 27). Iden i-
ica ion o closely ela ed species was ca ied ou using he
EzBioCloud websi e (www.ezbiocloud.ne /iden i y) [19]. The
16S RNA gene sequence indica ed ha s ain SALINAS58T
belongs o he genus Al e e y h obac e . The 16S RNA gene
sequence analysis o s ain SALINAS58T showed he highes
simila i y o Al e e y h obac e ma ensis MSW-14T (96.6%),
ollowed by Al e e y h obac e aquaemix ae JSSK-8
T
(96.5%),
Pon ixan hobac e lu eolus SW-109T (96.5%) and Al e e y h-
obac e a lan icus 26DY36T (96.4%). The 16S RNA gene
sequence simila i ies among s ain SALINAS58
T
and o he
ela ed species we e lowe han 98.7%, he h eshold p oposed
by Chun e al. [20] o di e en ia ing wo species.
The 16S RNA gene sequences o ep esen a i es o he genus
Al e e y h obac e we e e ie ed om he GenBank da abase
and we e aligned by Clus alW 2.0.12 (Kyo o Uni e si y Bioin-
o ma ics Cen e). Phylogene ic ees we e econs uc ed
by mega X so wa e [21] using he neighbou - joining [22],
minimum- e olu ion [23] and maximum- likelihood [24]
me hods. E olu iona y dis ances o he neighbou - joining
analysis we e calcula ed using he algo i hm o Kimu a’s wo-
pa ame e model wi h boo s ap alues based on 1000 eplica-
ions [25]. The neighbou - joining phylogene ic analysis based
on 16S RNA gene sequence, showed ha s ain SALINAS58
T
o med a dis inc b anch wi hin he genus Al e e y h obac e ,
albei no suppo ed by a high boo s ap alue (Fig.1). Simila
esul s we e obse ed in bo h he minimum- e olu ion and he
maximum- likelihood ees (Figs S2 and S3).
GENOME FEATURES
Whole genome sequencing o s ain SALINAS58T was
pe o med using he Illumina Miseq pla o m a SGIke
Gene al Se ices o he Uni e si y o he Basque Coun y
UPV/EHU (Leioa, Spain). The DNA o genome sequencing
was ob ained by using he Nucleo Spin Tissue DNA ex ac ion
ki (Mache ey- Nagel) acco ding o manu ac u e ’s p o ocol.
De no o assembly om aw pai ed- end eads o con igs was
pe o med using Vel e 1.1.04 so wa e [26]. The au hen-
ici y was con i med by he p esence o he 16S RNA gene
sequence ob ained by PCR on he assembled genome, and
he con amina ion o he genome sequence was checked by
GenomeQC pla o m (h ps:// genomeqc. maizegdb.o g/) [27].
Genome sequences we e anno a ed by he NCBI P oka yo ic
Genome Anno a ion Pipeline and by he Rapid Anno a ion
wi h Subsys ems Technology (RAST) [28]. Whole genome
sequencing da a we e deposi ed a DDBJ/ENA/GenBank
unde he accession numbe JAAFZT000000000. The genome
size o s ain SALINAS58T was 2.8 Mbp and was assembled
in o 56 con igs, wi h an N50 alue o 1.9 Mbp. The DNA G+C
con en o s ain SALINAS58T was 61.4%, wi hin hose o he
genus Al e e y h obac e (54.5–69.0%) [5, 17].
The combina ion o 16S RNA sequence simila i y and he
o e all genome ela ed index (OGRI) can be used sys ema i-
cally o iden i y and ecognize bac e ial s ains a he le el o
species. Among he OGRIs, he a e age nucleo ide iden i y
(ANI) alue has been mos widely used o species delinea ion.
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3
Azpiazu-Muniozgu en e al., In . J. Sys . E ol. Mic obiol. 2021;71:004734
Al e e y h obac e lu ipelagi GH1-16T (LT797153)
88 Al e e y h obac e ae ophilus E y1T (MG183676)
Al e e y h obac e hizo icinus AY-3RT (MH611372)
Al e e y h obac e xinjiangensis S3-63T (HM028673)
Al e e y h obac e soli MN-1T (KT906300)
73 91
90
Al e e y h obac e ae ius 100921-2T
(KU311004)
Al e e y h obac e igui WW3T (KP997219)
Al e e y h obac e dese i THG-S3T (KY287245)
Al e e y h obac e ul us S-54T (KY117470)
Al e e y h obac e amyloly icus NS1T
(KX601069)
87 Al e e y h obac e oi sensis KMM 6042T (AY676115)
86 Tsuneonella dong anensis JM27T (GU166344)
80 Al e e y h obac e la us MS1-4T (KX099616)
57 Al e e y h obac e mang o i C9-11T (MF034045)
Al e e y h obac e oceanensis Y2T (KF924606)
Al e e y h obac e xiamenensis LY02T (KC520828)
Al e e y h obac e epoxidi o ans JCS350T (DQ304436)
Al e e y h obac e insulae BPTF-M16T (MH206217)
Al e e y h obac e ishigakiensis NITE-AP48T (AB363004)
89
Pelage y h obac e ma inus H32T (EU726272)
62 Al e e y h obac e ma ensis MSW-14T (FM177586)
Al e e y h obac e mu iae SALINAS58T (JAAFZT000000000)
87 Al e e y h obac e aquaemix ae JSSK-8T (KY614064)
Al e e y h obac e aes iaquae HDW-31T (KJ658262)
Al e e y h obac e gangjinensis KJ7T (JF751048)
96
55 82
Al e e y h obac e aquiagge is KEM-3T (KX812543)
Al e e y h obac e con luen is KEM-4T (KX129915)
99 Al e e y h obac e sediminis CAU 1172T (KP779619)
Pon ixan hobac e lu eolus SW-109T (AY739662)
66 Al e e y h obac e spongiae HN-Y73T (MG437235)
Al e ic oceibac e ium endophy icum BR-75T (KY310591)
84 Al e ic oceibac e ium indicum MSSRF26T (DQ399262)
83 Al e e y h obac e a lan icus 26DY36T (KC018454)
Al e e y h obac e xixiisoli S36T (KJ150597)
89
Al e e y h obac e salegens XY-R17T (KT886062)
Al e au an iacibac e buc ensis M0322T (KJ599648)
88
Al e au an iacibac e aquimix icola SSKS-13T (MK194299)
83
51 Al e e y h obac e lau a is YIM 75003T (KX808673)
100 Al e e y h obac e palmi a is YIM 75004T (KX808674)
54 Al e e y h obac e halimionae CPA-5T (KY310593)
Al e au an iacibac e aes ua ii KYW147T (FJ997597)
Pa au an iacibac e namhicola KYW48T (FJ935793)
Pa apon ixan hobac e au an iacus O30T (KF924607)
99 Al e ipon ixan hobac e ma i imus HME9302T (KF385494)
E y h obac e spongiae HN-E23T (MG655147)
97 E y h obac e a lan icuss 21-N3T (KP994305)
E y h obac e longus JCM 6170T (D12699)
91 E y h obac e ci eus RE35F/1T (AF118020)
Pa acoccus paci icus F14
T
(KF924610)
0.020
Fig. 1. Neighbou - joining phylogene ic ee based on 16S RNA gene sequences, showing he ela ionships be ween s ain SALINAS58T,
he ype s ains o Al e e y h obac e species and ep esen a i es o some o he ela ed axa. Only boo s ap alues ≥50% (exp essed
as pe cen ages o 1000 eplica ions) a e shown a b anching poin s. Filled ci cles indica e ha he co esponding nodes we e also
eco e ed in bo h he minimum- e olu ion and maximum- likelihood ees. Pa acoccus paci icus F14T (GenBank accession numbe
KF924610) was used as he ou g oup. Ba , 0.02 subs i u ions pe nucleo ide posi ion.
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APPENDIX III. ERANSKINA
The supplemen a y igu es and ables in APPENDIX III can be downloaded by
scanning his QR code.
III. ERANSKINeko i udi e a aula osaga iak QR kode hau eskanea u a
deska ga dai ezke.
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Sec ion 3. a ala - APPENDIX VI. ERANSKINA
APPENDIX IV. ERANSKINA
Anianabac e salinae gen. no ., sp. no .
ASV31T, a Facul a i e Alkaliphilic and
Ex emely Halo ole an Bac e ium
Isola ed om B ine o a Millennial
Con inen al Sal e n
Re e ence: Azpiazu-Muniozgu en M, Ga cía M, Lao den Muñoz L, Ma ínez
Malax-Eche a ía I, Seoane Pa a S, Bikandi Bikandi J, Ga aiza J, and
Ma ínez-Balles e os I. Anianabac e salinae gen. no ., sp. no . ASV31T, a
Facul a i e Alkaliphilic and Ex emely Halo ole an Bac e ium Isola ed om
B ine o a Millennial Con inen al Sal e n. 2022; 14(11):1009.
Di e si y (2022); 14(11)
DOI: 10.3390/d14111009
Impac ac o 2022: 2.4
Rela i e posi ion: 26/65, Q2 Biodi e si y conse a ion
293
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Ci a ion: Azpiazu-Muniozgu en, M.;
Ga cía, M.; Lao den, L.;
Ma inez-Malaxe xeba ia, I.; Seoane,
S.; Bikandi, J.; Ga aiza , J.;
Ma ínez-Balles e os, I. Anianabac e
salinae gen. no ., sp. no . ASV31T, a
Facul a i e Alkaliphilic and
Ex emely Halo ole an Bac e ium
Isola ed om B ine o a Millennial
Con inen al Sal e n. Di e si y 2022,
14, 1009. h ps://doi.o g/10.3390/
d14111009
Academic Edi o s: Michael Wink and
S ua Donachie
Recei ed: 25 Sep embe 2022
Accep ed: 18 No embe 2022
Published: 21 No embe 2022
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2022 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
di e si y
A icle
Anianabac e salinae gen. no ., sp. no . ASV31T, a Facul a i e
Alkaliphilic and Ex emely Halo ole an Bac e ium Isola ed
om B ine o a Millennial Con inen al Sal e n
Maia Azpiazu-Muniozgu en 1, Mine a Ga cía1, Lo ena Lao den 1,2 , I a i Ma inez-Malaxe xeba ia 1,2 ,
Se gio Seoane 3, Joseba Bikandi 1, Ja ie Ga aiza 1,2 and Ila gi Ma ínez-Balles e os 1,2,*
1
Mik oIke Resea ch G oup, Depa men o Immunology, Mic obiology and Pa asi ology, Facul y o Pha macy,
Uni e si y o he Basque Coun y UPV/EHU, Paseo de la Uni e sidad 7, 01006 Vi o ia-Gas eiz, Ála a, Spain
2Bioa aba, Mic obiology, In ec ious Disease, An imic obial Agen s and Gene The apy,
01006 Vi o ia-Gas eiz, Ála a, Spain
3
Plan Biology and Ecology Depa men , Facul y o Science and Technology, Uni e si y o he Basque Coun y
UPV/EHU, Ba io Sa iena s/n, 48940 Leioa, Bizkaia, Spain
*Co espondence: ila [email p o ec ed]
Abs ac :
Du ing a p oka yo ic di e si y s udy in Añana Sal Valley, a new Rhodobac e aceae membe ,
designa ed ASV31
T
, was isola ed om San a Eng acia sp ing wa e . I was ex emely halo ole an ,
ole a ing up o 23% NaCl, and acul a i ely alkaliphilic, g owing a pH 6.5–9.5 (op imum a 7.0–9.5).
The isola e was a G am-nega i e, od-shaped, ae obic and non-mo ile bac e ium ha o med beige-
o-pink colonies on ma ine aga . Acco ding o a 16S RNA gene-based phylogene ic analysis, s ain
ASV31
T
o ms a dis inc b anch o he amily Rhodobac e aceae, wi h Thiocla a paci ica DSM 10166
T
being i s closes ype s ain (95.3%). This was con i med wi h a phylogenomic ee and he alues o
ANI (73.9%), dDDH (19.3%), AAI (63.5%) and POCP (56.0%), which we e below he genus/species
le el bounda y. Addi ionally, an abili y o deg ade a oma ic compounds and biosyn hesise seconda y
me aboli es was sugges ed by he genome o s ain ASV31
T
. Dis inguishing a y acid p o iles and po-
la lipid con en we e also obse ed. The genome size was 3.6 Mbp, wi h a DNA G+C con en o 65.7%.
Based on he da a ob ained, i was conside ed ha s ain ASV31
T
(
=CECT 30309T= LMG 32242T
) ep-
esen s a new species o a new genus in he amily Rhodobac e aceae, o which he name Anianabac e
salinae gen. no ., sp. no . is p oposed.
Keywo ds:
con inen al sal e n; ex emely halo ole an bac e ia; alkaliphilic; Anianabac e salinae;
bio echnological applica ions
1. In oduc ion
Sola sal e ns a e ex eme habi a s in which halophilic mic oo ganisms wi h di e en
adap a ions o such en i onmen s a e ound. Fo his eason, he s udy o mic obial
communi ies o hese en i onmen s is impo an , no only o inc ease knowledge a ound
hei di e si y, bu also because he me aboli es p oduced by membe s o hese communi ies
could be in e es ing o di e en bio echnological applica ions. An ac i e con inen al sola
sal e n, loca ed in Añana Sal Valley (Ála a, No he n Spain, 42.82 N 2.98 W), was ecen ly
decla ed a Globally Impo an Ag icul u al He i age Sys em by he Food and Ag icul u e
O ganiza ion o he Uni ed Na ions (FAO). I s o igin lies in he ma ine wa e o he T iassic
pe iod (be ween 251 and 208 million yea s ago), when a diapi was o med by he deposi
o e apo i e laye s ha ose h ough he Ea h’s su ace, esul ing in he hype salini y o
he wa e ha is now ound in his alley [
1
]. Du ing a s udy o he mic obial di e si y
o his sal e n, a beige- o pink-pigmen ed bac e ial s ain om he San a Eng acia sp ing
( he main b ine suppo o he sal p oduc ion in he sal e n) was isola ed. The isola e was
designa ed ASV31
T
, which ep esen ed a po en ial no el species o a no el genus in he
Rhodobac e aceae amily.
Di e si y 2022,14, 1009. h ps://doi.o g/10.3390/d14111009 h ps://www.mdpi.com/jou nal/di e si y
Sec ion 3. a ala - APPENDIX VI. ERANSKINA
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Di e si y 2022,14, 1009 2 o 17
The amily Rhodobac e aceae, belonging o he o de Rhodobac e ales, class Alphap o-
eobac e ia, was i s p oposed in 2005 by Ga i y e al. [
2
] and was amended by Hö d e al.
in 2020 [
3
]. This amily is a la ge pheno ypically, me abolically and ecologically di e se
axonomic g oup. Mos membe s o he amily o igina ed om aqua ic en i onmen s
and many o hem equi e sodium ions o combined sal s o g ow h [
4
]. A he ime o
w i ing, he amily Rhodobac e aceae consis s o 227 alidly published gene a, including
synonyms (h ps://lpsn.dsmz.de/ amily/ hodobac e aceae, accessed on 20 Janua y 2022)
which a e di ided in o six di e en phylogene ic subg oups: S appia,Ama icoccus,Pa a-
coccus,Rhodobac e ,Rhodo ulum and Roseobac e [
5
]. The la e has been p oposed o be
spli om he Rhodobac e aceae amily o o m a new one, he Roseobac e aceae amily [
6
].
In gene al, membe s o he amily Rhodobac e aceae a e G am-nega i e and mul iply by
bina y ission o budding, ollowing monopola g ow h. While hey a e mainly ae obic
pho o- and chemohe e o ophs, pu ple non-sulphu bac e ia ha pe o m pho osyn hesis
in anae obic en i onmen s a e also ound. They a e cha ac e ised chemo axonomically
by he p esence o ubiquinone-10 (Q-10) and C18:1
ω
7c is he p edominan cellula a y
acid. They a e conside ed o be he majo g oup o ma ine he e o ophic bac e ia and ha e
di e en oles such as con ibu ing o sulphu , ni ogen and ca bon cycles, decomposing
a ious compounds and gene a ing seconda y me aboli es [7,8].
Membe s o he Rhodobac e aceae amily could be u ilised o indus ial applica ions.
Exopolysaccha ide (EPS)-p oducing species, such as Palle onia ma ismino is [
7
], can be
ound in his amily. Mic obial EPSs can be used in he ood indus y as iscosi y agen s,
s abilise s, emulsi ie s, gelling agen s and wa e -binding agen s. Mo eo e , in he medical
indus y, mic obial EPSs a e eme ging as p omising ma e ials o d ug- elease sys ems,
due o hei abili y o e ain la ge amoun s o wa e and emain insoluble, as well as d ug-
a ge ing ca ie s, based on hei pa icula binding and pene a ing ea u es o cellula
ecep o s [
9
]. O he gene a, such as Me hyla cula, a e able o syn hesise ec oine as a majo
compa ible solu e [
5
]. Ec oine is comme cially a ailable as a p o ec an o p o eins, DNA
and mammalian cells [
10
]. O he s, such as Palle onia,Salipige and T anquillimonas, can syn-
hesise polyhyd oxyalkanoa es (PHAs), o , mo e commonly, polyhyd oxybu y a e (PHB),
as a ca bon ese e ma e ial [
5
]. PHAs a e a amily o biodeg adable and biocompa ible
polyes e s accumula ed by many mic oo ganisms and a e de eloped o indus ial usage
(e.g., as bioplas ics, bio uels and ine chemicals) o o medicinal use [
10
]. Fu he mo e,
ex emophilic ca bohyd a e-ac i e enzymes (CAZymes) ha e ecen ly ga ne ed a lo o
a en ion due o hei ad an ages [
11
]. In his con ex , a genomic anno a ion allows o he
iden i ica ion o a ious me abolic pa hways ei he di ec ly in ol ed o no in he syn hesis
o hese me aboli es o indus ial in e es .
Using a polyphasic app oach, he aim o his s udy was o cha ac e ise he s ain
ASV31
T
(isola ed om he b ine o a sal e n) and o de e mine i s axonomic posi ion
wi hin he amily Rhodobac e aceae.
2. Ma e ials and Me hods
2.1. S ain Isola ion and Main enance
S ain ASV31
T
was isola ed om he San a Eng acia na u al sp ing in he con inen al
sal e n a Añana Sal Valley, loca ed 598 m abo e sea le el ( he app oxima e geog aphic
coo dina es a e 42.79 N 2.98 W). Wa e samples we e collec ed in June 2016. The wa e
empe a u e was 16.0
◦
C, pH 6.6 and sal con en 20%. In o de o isola e halophilic and
halo ole an bac e ia, he wa e samples we e i s p ocessed h ough mul iple il a ion
s eps (Wha man
®
G ade 113 V, Maids one, UK, and Millipo e 5
µ
m and 0.22
µ
m il e s
(Bu ling on, MA, USA)). Subsequen ly, 100
µ
L o il e ed wa e was cul i a ed on ma ine
aga (MA; Conda) and incuba ed a 25
◦
C. A e 7 days o incuba ion, a isible beige- o-pink
colony was obse ed and subcul u ed on MA, un il pu e cul u e was ob ained. S ain
ASV31Twas s o ed a −80 ◦C in ma ine b o h (MB; Conda) and 25% ( / ) glyce ol.
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2.2. 16S RNA Gene-Based Iden i ica ion and Phylogeny
S ain ASV31
T
ch omosomal DNA was ex ac ed om h ee-day colonies on MA using
P epMan Ul a Reac i e (Applied Biosys ems, Wal ham, MA, USA) ollowing he manu ac-
u e ’s speci ica ions. The DNA concen a ion was measu ed using a NanoD op 2000 spec-
opho ome e (The mo Scien i ic, Wal ham, MA, USA). 16S RNA gene sequencing was
used o s ain iden i ica ion. The gene was ampli ied using he uni e sal bac e ial p ime s
27F (5
0
-AGAGTTTGATCMTGGCTCAG-3
0
) and 1492R (
50-GGTTACCTTGTTACGACTT-30
) [
12
],
and was, subsequen ly, pu i ied by a NucleoSpin Gel and PCR clean-up ki (Mache ey-
Nagel, Dü en, Ge many). Pu i ied PCR p oduc s we e sequenced using he Sange me hod
(S ab Vida, Capa ica, Po ugal) and he sequencing da a we e analysed wi h Ch omas 2.6.6
so wa e (Technelysium, Sou h B isbane, Aus alia). Iden i ica ion o closely ela ed species
and he measu emen o hei 16S RNA sequence simila i y we e ca ied ou using he
EzBioCloud da abase (www.ezbiocloud.ne /iden i y, accessed on 15 June 2021) [13].
The 16S RNA gene sequences o ep esen a i es o he amily Rhodobac e aceae we e
e ie ed om he GenBank da abase and a phylogene ic ee was cons uc ed wi h MEGA
X so wa e [14] using he neighbou -joining [15], minimum-e olu ion [16] and maximum-
likelihood [
17
] me hods. E olu iona y dis ance ma ices we e calcula ed using he al-
go i hm o Kimu a’s wo-pa ame e model wi h boo s ap alues based on 1000 eplica-
ions [17].
2.3. Pheno ype and Chemo axonomy
Type s ains Thiocla a paci ica DSM 10166
T
and Rhodo ulum algae LMG 29228
T
we e
es ed alongside ASV31
T
o compa ison. The pheno ypic cha ac e is ics o cells and
colonies we e de e mined a e h ee days o g ow h a 30
◦
C on MA. Cell mo phology,
size and p esence o lagella we e de e mined wi h ansmission elec on mic oscopy (1400
Plus, JEOL, Tokyo, Japan) a he UPV/EHU Ad anced Resea ch Facili ies (SGIke , Leioa,
Spain). G am s aining was pe o med using he me hod desc ibed by Smi h e al. [
18
].
Cellula pigmen s we e analysed by HPLC. Pigmen ex ac ion was pe o med wi h 90%
ace one ollowing he me hod desc ibed by Zapa a e al. [
19
], wi h he modi ica ions
explained by Seoane e al. [
20
]. The abso bance ch oma og am was ex ac ed a 440 nm.
PHA p oduc ion was es ed using Sudan black B s aining acco ding o he p ocedu es
o Smi h e al. [
21
] and San hanam e al. [
22
], inocula ing 10 mL o MB supplemen ed
wi h glucose (2% w/ ) and yeas ex ac (2 g/L) as he ca bon and ni ogen sou ces,
espec i ely, and incuba ing his o 48 h a 28
◦
C and 150 pm. Cell mo ili y was measu ed
using he hanging d op me hod in semisolid aga including MB and 0.7% bac e iological
aga (Condalab, Mad id, Spain). Anae obic g ow h was es ed a 30
◦
C o 15 days on
MA in an anae obic chambe wi h GENbox anae (bioMé ieux, Ma cy-l
'
É oile, F ance).
G ow h a di e en empe a u es (4, 8, 15, 20, 25, 30, 37 and 42
◦
C) was de e mined on MA
a e 3 o 15 days o incuba ion. Sal ole ance and equi emen o g ow h we e es ed
in MB supplemen ed wi h NaCl a inal concen a ions o 0, 0.5, 1.0, 2.0, 3.0, 5.0, 10.0,
15.0, 20.0 and 23.0 g/L. These media we e p epa ed acco ding o he MB o mula, bu
wi hou NaCl. The pH ange suppo ing g ow h was de e mined in MB a 30
◦
C, wi h he
pH adjus ed (pH 4.5–10.5 in inc emen s o 0.5 pH uni s) wi h di e en bu e s: sodium
ace a e/ace ic acid ( o pH 4.5–6.0) and NaHCO
3
/Na
2
CO
3
( o pH 6.5–10.5). Ene ge ic
me abolism was cha ac e ised on a modi ied MA medium o pho o- (anae obic, ligh
2400 lx) and chemo- (ae obic, da k)o ganohe e o ophy (wi h py u a e (0.03%, w/ ) as
he ca bon sou ce/elec on dono ), pho oli hoau o ophy (anae obic, ligh (2400 lx), wi h
Na
2
S
2
O
3
.5H
2
O (1 mM) as he elec on dono and NaHCO
3
(0.1%, w/ ) as he ca bon
sou ce), chemoli hoau o ophy (da k, ae obic, wi h Na
2
S
2
O
3
.5H
2
O (1 mM) as he elec on
dono and NaHCO
3
(0.1%, w/ ) as he ca bon sou ce) and e men a i e g ow h (da k,
anae obic, wi h py u a e/glucose (0.3%, w/ ) as he e men able subs a es).
Oxidase ac i i y was es ed wi h Bac iden Oxidase s ips (Me ck, Rahway, NJ, USA)
and ca alase ac i i y wi h ID colou ca alase (bioMé ieux). Hyd olysis capaci y was de e -
mined on MA pla es supplemen ed wi h 1% (w/ ) skimmed milk, 1% (w/ ) Tween 20 and
Sec ion 3. a ala - APPENDIX VI. ERANSKINA
REGISTRO TELEMÁTICO
Sa e en E egis o
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01/07/2025 13:49
EHU2025E029511

Di e si y 2022,14, 1009 4 o 17
1% (w/ ) Tween 80. O he physiological and biochemical ea u es we e examined using
API ZYM and API 20NE s ips (bioMé ieux) acco ding o he manu ac u e ’s ins uc ions,
wi h cells suspended in a saline solu ion. Suscep ibili y o an ibio ics was es ed on MA
pla es incuba ed a 30
◦
C o h ee days using discs con aining he ollowing: cip o loxacin
(10
µ
g), e y h omycin (15
µ
g), o loxacin (5
µ
g), penicillin G (5 IU), cephazolin (30
µ
g),
i ampicin (2
µ
g), kanamycin (5
µ
g), gen amicin (10
µ
g), polymyxin B (300 IU), ancomycin
(30 µg) and s ep omycin (10 µg).
Whole-cell a y acid composi ion was de e mined ollowing he p o ocol ecom-
mended by he MIDI Mic obial Iden i ica ion Sys em [
23
] a he Spanish Type Cul u e
Collec ion (CECT). Iden i ica ion was pe o med wi h he She lock MIDI e sion 6.1 and
cellula a y acid con en was analysed using he TSBA6 lib a y [
24
]. Cells we e g own
o 72 h a 30
◦
C on MA o ob ain hei biomass. Analyses o pola lipids and espi a o y
quinones we e ca ied ou using he Iden i ica ion Se ice a he Leibniz-Ins i u DSMZ
(Deu sche Sammlung on Mik oo ganismen und Zellkul u en GmbH, B aunschweig, Ge -
many) ollowing he p o ocol based on Bligh and Dye [25] and Tindall e al. [26].
2.4. Genome Sequencing and Analysis
Whole-genome sequencing o he ASV31
T
isola e was pe o med using he Illumina
Miseq pla o m a he UPV/EHU Ad anced Resea ch Facili ies (SGIke ). DNA ex ac ion
was conduc ed using he NucleoSpin Tissue DNA ex ac ion ki (Mache ey-Nagel) acco d-
ing o he manu ac u e ’s p o ocol. Assembly om aw pai ed-end eads o con igs was
pe o med using he SPAdes assemble 3.13.0 a ailable in PATRIC 3.6.9 so wa e [
27
].
Au hen ici y was con i med wi h he p esence o he 16S RNA gene sequence ob ained
using PCR on he assembled genome, and he genome’s quali y was con i med using
CheckM 1.0.18 [28].
Genome-based compa isons o s ain ASV31
T
wi h i s closes species we e pe o med
wi h a e age nucleo ide iden i y (ANI) alues compu ed using he O hoANI Calcula o
ool a ailable a EzBiocloud (www.ezbiocloud.ne / ools/ani, accessed on
20 Janua y 2022
) [
29
]
and digi al DNA–DNA hyb idisa ion (dDDH) alues compu ed using he genome- o-
genome dis ance calcula o (h p://ggdc.dsmz.de/ggdc.php, accessed on
20 Janua y 2022
) [
30
].
In o de o assess genus a ilia ions, amino acid-le el compa isons we e pe o med using
he a e age amino acid iden i y (AAI) and pe cen age o conse ed p o eins (POCP) o e -
e y pai wise combina ion o genomes. The AAI alues we e calcula ed using he Kos as Lab
AAI calcula o (h p://en e-omics.ce.ga ech.edu/aai/, accessed on
20 Janua y 2022
) [
31
].
The POCP alues we e calcula ed using a Py hon sc ip wi h he o mula
[(C1 + C2)/(T1 + T2)] ×100%
, whe e C1 and C2 ep esen he conse ed numbe o p o-
eins in he wo genomes being compa ed, and T1 and T2 ep esen he o al numbe o
p o eins in he wo genomes being compa ed [32].
A whole-genome-based axonomic analysis was pe o med on he Type S ain Genome
Se e (h ps:// ygs.dsmz.de, accessed on 18 Feb ua y 2022) [
30
] and a genome-based
phylogene ic ee was in e ed wi h Fas ME 2.1.6.1 [
33
] om he GBDP dis ances calcula ed
om genome sequences. B anch leng hs we e scaled in e ms o GBDP dis ance o mula d5.
In o de o p o ide u he in o ma ion abou he axonomic posi ion o s ain ASV31
T
, he
amino acid sequences o RpoC, 2-oxoglu a a e dehyd ogenase and acyl-CoA syn he ase
p o eins we e ob ained om he genome. These sequences we e analysed wi h BLAST
in o de o ind he mos simila axa. Phylogene ic ees based on hese p o eins we e
cons uc ed wi h MEGA X using he maximum-likelihood me hod. Amino acid sequences
we e e ie ed om he GenBank da abase.
Gene anno a ion was pe o med using he NCBI P oka yo ic Genome Anno a ion
Pipeline and he Rapid Anno a ion wi h Subsys ems Technology (RAST) pipeline [
34
]. The
de ec ion o gene clus e s ela ed o seconda y me aboli e p oduc ion was pe o med using
he an iSMASH 6.0.1 webse e (h ps://an ismash.seconda yme aboli es.o g, accessed on
11 May 2022) [
35
]. The dbCAN me a se e (h ps://bcb.unl.edu/dbCAN2/, accessed on
Sec ion 3. a ala - APPENDIX VI. ERANSKINA
REGISTRO TELEMÁTICO
Sa e en E egis o
O oko a / Regis o Gene al
de En adas
01/07/2025 13:49
EHU2025E029511
Di e si y 2022,14, 1009 5 o 17
25 May 2022) [
36
] was used o iden i y he coding sequences (CDSs) encoding ca bohyd a e-
ac i e enzymes (CAZymes) using an HMMER anno a ion.
3. Resul s and Discussion
A polyphasic s udy was ca ied ou in o de o cha ac e ise s ain ASV31
T
, which was
isola ed om a hype saline wa e sp ing in an ac i e con inen al sal e n. This sal e n was
o med as a esul o he deposi o e apo i es millions o yea s ago du ing he desicca ion
o he g ea ocean ha co e ed almos he en i e Ea h.
3.1. 16S RNA Gene-Based Iden i ica ion and Phylogeny
Phylogene ic analysis showed ha s ain ASV31
T
belonged o he Bac e ia domain,
P o eobac e ia phylum, Alphap o eobac e ia class, Rhodobac e ales o de and Rhodobac e aceae
amily. Due o he sho 16S RNA gene sequence (1337 bp) ob ained by PCR (sequence
deposi ed a GenBank unde accession numbe MW691205), he 1468 bp 16S RNA se-
quence om genome sequencing was used o u he phylogene ic analysis (accession
numbe JAHQZS010000014.1). A compa ison o 16S RNA sequences de e mined ha
Thiocla a GU322906_s YC6923 was he mos closely ela ed s ain (95.4% simila i y be ween
sequences), ollowed by Thiocla a paci ica DSM 10166
T
(95.3%) and Thiocla a sediminum
TAW-CT134
T
(95.2%) ype s ains. O he ela ed axa we e T opicimonas AM176881_s SZB22
(95.1%), Haema obac e massiliensis CCUG 47968
T
(95%) and Rhodo ulum ad ia icum DSM
2781
T
(94.9%). Hö d e al. [
3
] p oposed ha when he 16S RNA gene sequence simila i y
is below 95%, he axa ep esen di e en gene a. The simila i y o he 16S RNA sequences
o Thiocla a species and s ain ASV31
T
was jus a his limi , which was no su icien
o suppo he placemen o s ain ASV31
T
in o he Thiocla a genus. Fu he mo e, he
phylogene ic ee, cons uc ed wi h he neighbou -joining me hod based on he 16S RNA
gene sequence, showed ha s ain ASV31
T
was no de ined by any genus in he amily
Rhodobac e aceae, as i clus e ed in a di e en ia ed b anch be ween he Rhodobac e and
Roseobac e g oups (Figu e 1). In ac , s ain ASV31
T
was in an independen b anch ha did
no show a de ined a ilia ion ei he o Thiocla a spp. (Rhodobac e g oup) o T opicimonas
spp. (Roseobac e g oup). Simila esul s we e obse ed in bo h he maximum-likelihood
and he minimum-e olu ion ees (Figu es S1 and S2). These esul s sugges ed ha s ain
ASV31Tmay ep esen a no el genus o he Rhodobac e aceae amily.
3.2. Pheno ypic and Chemo axonomic Cha ac e isa ion
S ain ASV31
T
is a G am-nega i e bacillus 2.4–2.5
µ
m in leng h and 0.4–0.5
µ
m in
wid h, wi h a mucous polysaccha ide capsule (slime) all a ound (Figu e 2A). This ea u e
was also seen gene ically in he me abolic econs uc ion ( esul s shown below). Unlike
Thiocla a membe s, which a e desc ibed as a ely mo ile by means o a pola lagellum [
37
],
and Rhodo ulum membe s, which also exhibi mo ili y by a pola lagellum [
5
], no lagella
we e obse ed in ASV31
T
using ansmission elec on mic oscopy. Mo ili y was nei he
obse ed using he hanging d op me hod no in semisolid aga . The p esence o PHA as
in acellula g anules was con i med h ough Sudan black B s aining (Figu e 2B). This ca-
paci y has also been epo ed in Rhodobac e capsula us,Palle onia ma ismino is,Salipige spp.,
T anquillimonas spp., Lab enzia spp. and Rhodo ulum sul idophilum in he Rhodobac e aceae
amily, wi h PHB being he main PHA class [5].
Sec ion 3. a ala - APPENDIX VI. ERANSKINA
REGISTRO TELEMÁTICO
Sa e en E egis o
O oko a / Regis o Gene al
de En adas
01/07/2025 13:49
EHU2025E029511
Di e si y 2022,14, 1009 6 o 17
Di e si y 2022, 14, x FOR PEER REVIEW 6 o 17
Figu e 1. Neighbou -joining phylogene ic ee based on 16S RNA gene sequences ha shows he
ela ionship be ween s ain ASV31T and he ype s ains o Rhodobac e , Roseobac e and Rhodo ulum
g oups o he amily Rhodobac e aceae. The black ci cles indica e he clades ha we e also conse ed
in bo h he maximum-likelihood and minimum-e olu iona y phylogene ic ees. Boo s ap alues
(exp essed as pe cen ages o 1000 eplica ions) g ea e han 50% in he clade nodes a e shown. Esch-
e ichia coli NCCB 54008T was used as ou g oup. Scale: 0.02 subs i u ions pe nucleo ide posi ion.
3.2. Pheno ypic and Chemo axonomic Cha ac e isa ion
S ain ASV31T is a G am-nega i e bacillus 2.4–2.5 μm in leng h and 0.4–0.5 μm in
wid h, wi h a mucous polysaccha ide capsule (slime) all a ound (Figu e 2A). This ea u e
was also seen gene ically in he me abolic econs uc ion ( esul s shown below). Unlike
Thiocla a membe s, which a e desc ibed as a ely mo ile by means o a pola lagellum
[37], and Rhodo ulum membe s, which also exhibi mo ili y by a pola lagellum [5], no
lagella we e obse ed in ASV31T using ansmission elec on mic oscopy. Mo ili y was
nei he obse ed using he hanging d op me hod no in semisolid aga . The p esence o
PHA as in acellula g anules was con i med h ough Sudan black B s aining (Figu e 2B).
This capaci y has also been epo ed in Rhodobac e capsula us, Palle onia ma ismino is,
Salipige spp., T anquillimonas spp., Lab enzia spp. and Rhodo ulum sul idophilum in he
Rhodobac e aceae amily, wi h PHB being he main PHA class [5].
Figu e 1.
Neighbou -joining phylogene ic ee based on 16S RNA gene sequences ha shows he
ela ionship be ween s ain ASV31
T
and he ype s ains o Rhodobac e ,Roseobac e and Rhodo ulum
g oups o he amily Rhodobac e aceae. The black ci cles indica e he clades ha we e also conse ed
in bo h he maximum-likelihood and minimum-e olu iona y phylogene ic ees. Boo s ap alues
(exp essed as pe cen ages o 1000 eplica ions) g ea e han 50% in he clade nodes a e shown.
Esche ichia coli NCCB 54008
T
was used as ou g oup. Scale: 0.02 subs i u ions pe nucleo ide posi ion.
Di e si y 2022, 14, x FOR PEER REVIEW 7 o 17
(a)
(b)
Figu e 2. Anianabac e salinae ASV31
T
cell cha ac e is ics obse ed using mic oscopy: (a) T ansmis-
sion elec on mic og aph o a cell. A polysaccha ide mucous capsule was obse ed a ound he cell
and da ke spo s in he cy oplasm. Ba 500.0 nm. (b) Sudan black B s aining o PHA g anules ob-
se ed as black in acellula g anules unde 100× oil imme sion objec i e.
Colonies o s ain ASV31
T
we e less han 1 mm in diame e and beige- o-pink col-
ou ed, wi h a ci cula shape, con ex ele a ion, anspa en smoo h edge and hey glis-
ened on he MA medium (Figu e S3). The colony mo phology was di e en om he
Thiocla a paci ica DSM 10166
T
and Rhodo ulum algae LMG 29228
T
s ains ha we e s udied
alongside ASV31
T
in his s udy. ASV31
T
was no capable o g owing unde anae obic con-
di ions. S ain ASV31
T
g ew in a empe a u e ange o 18–37 °C, wi h an op imum em-
pe a u e o 30 °C, he same as i s ela i es. The pH ange o g ow h was pH 6.5–9.5, wi h
an op imum o pH 7.0–9.5, indica ing i was sligh ly alcaliphilic compa ed wi h i s close
ela i es. The NaCl ole ance o g ow h was 0.0–23.0%, wi h an op imum o 3.0–5.0%. In
con as wi h i s ela i es, s ain ASV31
T
could g ow a highe sal concen a ions and did
no equi e NaCl o g ow like mos Thiocla a and Rhodo ulum membe s [5]. Membe s o
Rhodobac e aceae a e mainly ae obic pho o- and chemohe e o ophs [5]. In his sense, s ain
ASV31
T
is an ae obic chemoo ganohe e o oph. This capaci y was also obse ed in Thio-
cla a paci ica DSM 10166
T
and Rhodo ulum algae LMG 29228
T
. Chemoli oau o ophic, pho-
oli hoau o ophic and pho oo ganohe e o ophic g ow h could no be demons a ed in
ASV31
T
. Mo eo e , Thiocla a membe s a e conside ed acul a i e sulphu chemo-
li ho ophs, a e included in he Rhodobac e g oup and a e able o g ow using hiosul a e
oxida ion and ino ganic ca bon ixa ion h ough he Cal in cycle [5]. This cha ac e is ic
was obse ed in his s udy. Pho o ganohe e o ophy was also obse ed in Rhodo ulum
algae LMG 29228
T
(as desc ibed by Ramap asad e al. [38]). Fe men a i e g ow h was no
seen in any o he s ains analysed, as desc ibed in [5].
Sphe oidenone was he main pigmen in ASV31
T
, while sphe oidene and bac e io-
chlo ophyll a (BChl a) we e also de ec ed in he s ain. I is known ha Rhodo ulum mem-
be s can also syn hesise BChl a and ca o enoids o he sphe oidene se ies (sphe oidene,
sphe oidenone, deme hylsphe oidene, hyd oxysphe oidene and neu ospo ene, among
o he s) in di e en p opo ions. Howe e , Thiocla a membe s do no syn he ise BChl a o
ca o enoids [5,39].
Oxidase ac i i y was posi i e in ASV31
T
and i s ela i es, while ca alase was p esen
in ASV31
T
and Rhodo ulum algae LMG 29228
T
. U ease was only p esen in Rhodo ulum
algae LMG 29228
T
. A capaci y o Tween 20 hyd olysis was only seen in s ain ASV31
T
and
none o he s ains had a casein o Tween 80 hyd olysis abili y. In API 20NE es s, ASV31
T
was posi i e o aesculin hyd olysis and ni a e educ ion, bu nega i e o indole p oduc-
ion, glucose e men a ion, gela ine hyd olysis, a ginine dihyd olase, u ease and β-galac-
osidase, and he assimila ion o glucose, a abinose, mannose, manni ol, N-ace ylglucosa-
mine, mal ose, glucona e, cap a e, adipa e, mala e, ci a e and phenylace a e. The API
Figu e 2.
Anianabac e salinae ASV31
T
cell cha ac e is ics obse ed using mic oscopy: (
a
) T ansmission
elec on mic og aph o a cell. A polysaccha ide mucous capsule was obse ed a ound he cell and
da ke spo s in he cy oplasm. Ba 500.0 nm. (
b
) Sudan black B s aining o PHA g anules obse ed as
black in acellula g anules unde 100×oil imme sion objec i e.
Sec ion 3. a ala - APPENDIX VI. ERANSKINA
REGISTRO TELEMÁTICO
Sa e en E egis o
O oko a / Regis o Gene al
de En adas
01/07/2025 13:49
EHU2025E029511
Di e si y 2022,14, 1009 7 o 17
Colonies o s ain ASV31
T
we e less han 1 mm in diame e and beige- o-pink colou ed,
wi h a ci cula shape, con ex ele a ion, anspa en smoo h edge and hey glis ened on
he MA medium (Figu e S3). The colony mo phology was di e en om he Thiocla a
paci ica DSM 10166
T
and Rhodo ulum algae LMG 29228
T
s ains ha we e s udied alongside
ASV31
T
in his s udy. ASV31
T
was no capable o g owing unde anae obic condi ions.
S ain ASV31
T
g ew in a empe a u e ange o 18–37
◦
C, wi h an op imum empe a u e o
30
◦
C, he same as i s ela i es. The pH ange o g ow h was pH 6.5–9.5, wi h an op imum
o pH 7.0–9.5, indica ing i was sligh ly alcaliphilic compa ed wi h i s close ela i es. The
NaCl ole ance o g ow h was 0.0–23.0%, wi h an op imum o 3.0–5.0%. In con as wi h i s
ela i es, s ain ASV31
T
could g ow a highe sal concen a ions and did no equi e NaCl
o g ow like mos Thiocla a and Rhodo ulum membe s [
5
]. Membe s o Rhodobac e aceae
a e mainly ae obic pho o- and chemohe e o ophs [
5
]. In his sense, s ain ASV31
T
is an
ae obic chemoo ganohe e o oph. This capaci y was also obse ed in Thiocla a paci ica DSM
10166
T
and Rhodo ulum algae LMG 29228
T
. Chemoli oau o ophic, pho oli hoau o ophic
and pho oo ganohe e o ophic g ow h could no be demons a ed in ASV31
T
. Mo eo e ,
Thiocla a membe s a e conside ed acul a i e sulphu chemoli ho ophs, a e included in
he Rhodobac e g oup and a e able o g ow using hiosul a e oxida ion and ino ganic
ca bon ixa ion h ough he Cal in cycle [
5
]. This cha ac e is ic was obse ed in his s udy.
Pho o ganohe e o ophy was also obse ed in Rhodo ulum algae LMG 29228
T
(as desc ibed
by Ramap asad e al. [
38
]). Fe men a i e g ow h was no seen in any o he s ains analysed,
as desc ibed in [5].
Sphe oidenone was he main pigmen in ASV31
T
, while sphe oidene and bac e i-
ochlo ophyll a (BChl a) we e also de ec ed in he s ain. I is known ha Rhodo ulum
membe s can also syn hesise BChl a and ca o enoids o he sphe oidene se ies (sphe oidene,
sphe oidenone, deme hylsphe oidene, hyd oxysphe oidene and neu ospo ene, among
o he s) in di e en p opo ions. Howe e , Thiocla a membe s do no syn he ise BChl a o
ca o enoids [5,39].
Oxidase ac i i y was posi i e in ASV31
T
and i s ela i es, while ca alase was p esen
in ASV31
T
and Rhodo ulum algae LMG 29228
T
. U ease was only p esen in Rhodo ulum algae
LMG 29228
T
. A capaci y o Tween 20 hyd olysis was only seen in s ain ASV31
T
and none
o he s ains had a casein o Tween 80 hyd olysis abili y. In API 20NE es s, ASV31
T
was
posi i e o aesculin hyd olysis and ni a e educ ion, bu nega i e o indole p oduc ion,
glucose e men a ion, gela ine hyd olysis, a ginine dihyd olase, u ease and
β
-galac osidase,
and he assimila ion o glucose, a abinose, mannose, manni ol, N-ace ylglucosamine,
mal ose, glucona e, cap a e, adipa e, mala e, ci a e and phenylace a e. The API ZYM es s
showed alkaline phospha ase, es e ase (C 4), es e ase lipase (C 8), leucine a ylamidase,
aline a ylamidase and
α
-glucosidase ac i i y, weak ac i i y o cys eine a ylamidase, acid
phospha ase and naph hol-AS-BI- phosphohyd olase, and nega i e ac i i y o lipase (C 14),
α
-chymo ypsin, ypsin,
α
- and
β
-galac osidases,
β
-glucu onidase,
β
-glucosidase, N-
ace yl-
β
-glucosaminidase,
α
-mannosidase and
α
- ucosidase. S ain ASV31
T
was esis an
o polymyxin B (300 IU), ancomycin (30
µ
g) and s ep omycin (10
µ
g). The mos ele an
pheno ypic cha ac e is ics ha dis inguished s ain ASV31
T
om i s closes ela i es a e
summa ised in Table 1.
Sec ion 3. a ala - APPENDIX VI. ERANSKINA
REGISTRO TELEMÁTICO
Sa e en E egis o
O oko a / Regis o Gene al
de En adas
01/07/2025 13:49
EHU2025E029511
Di e si y 2022,14, 1009 14 o 17
Supplemen a y Ma e ials:
The ollowing suppo ing in o ma ion can be downloaded a h ps:
//www.mdpi.com/a icle/10.3390/d14111009/s1: Figu e S1: Maximum-likelihood phylogene ic
ee based on 16S RNA gene sequences showing he ela ionships be ween Anianabac e salinae
ASV31
T
and o he s ains o Rhodobac e aceae amily. Figu e S2: Minimum e olu ion phylogene ic
ee based on 16S RNA gene sequences showing he ela ionships be ween Anianabac e salinae
ASV31
T
and o he species o Rhodobac e aceae amily. Figu e S3: Anianabac e salinae ASV31
T
g ow h
on ma ine aga medium. Figu e S4: Thin-laye ch oma og am o pola lipids o Anianabac e salinae
ASV31
T
. Figu e S5: Whole-genome-based axonomic ee, showing he posi ion o he s ain ASV31
T
and ela ed s ains o Rhodobac e aceae amily. Figu e S6: RpoC-based maximum-likelihood ee:
A, pe o med wi h he mos simila amino acidic sequences; B, pe o med wi h sequences om
ep esen a i es o he Rhodobac e aceae amily. T ee was gene a ed using MEGA X. Only boo s ap
alues (exp essed as pe cen ages o 1000 eplica ions) g ea e han 50% a e shown a b anching poin s.
The scale ba indica es he numbe o subs i u ions pe si e. Figu e S7: Acyl-CoA syn he ase-based
maximum-likelihood ee: A, pe o med wi h he mos simila amino acidic sequences; B, pe o med
wi h sequences om ep esen a i es o he Rhodobac e aceae amily. T ee was gene a ed using MEGA
X. Only boo s ap alues (exp essed as pe cen ages o 1000 eplica ions) g ea e han 50% a e shown
a b anching poin s. The scale ba indica es he numbe o subs i u ions pe si e. Figu e S8: 2-
oxoglu a a e dehyd ogenase-based maximum likelihood ee; A, pe o med wi h he mos simila
amino acidic sequences, B, pe o med wi h sequences om ep esen a i es o he Rhodobac e aceae
amily. T ee was gene a ed using MEGA X. Only boo s ap alues (exp essed as pe cen ages o
1000 eplica ions) g ea e han 50% a e shown a b anching poin s. The scale ba indica es he numbe
o subs i u ions pe si e. Table S1: AAI and POCP ma ix om pai wise whole-genome compa ison.
Table S2: P esence and absence o subsys ems in s ain ASV31
T
compa ed o i s closes ela i es.
Table S3: CAZyme ypes p esen in ASV31Ts ain.
Au ho Con ibu ions:
Concep ualisa ion, I.M.-B., J.G. and J.B.; me hodology, I.M.-B. and J.G.; o -
mal analysis, M.A.-M., M.G., L.L., I.M.-M., S.S. and I.M.-B.; esou ces, J.B.; da a cu a ion,
M.A.-M.
,
M.G., L.L., I.M.-M., S.S. and I.M.-B.; w i ing—o iginal d a p epa a ion, M.A.-M. and I.M.-B.;
w i ing— e iew
and edi ing, all au ho s; unding acquisi ion, J.G. and I.M.-B. All au ho s ha e
ead and ag eed o he published e sion o he manusc ip .
Funding:
This esea ch was unded by he Uni e si y o he Basque Coun y UPV/EHU (g an
numbe US19/01) and he Añana Sal Valley Founda ion (speci ic ag eemen be ween he Añana Sal
Valley Founda ion and he Uni e si y o he Basque Coun y UPV/EHU).
Ins i u ional Re iew Boa d S a emen : No applicable.
Acknowledgmen s:
The au ho s a e g a e ul o he echnical and human suppo p o ided by he
UPV/EHU Ad anced Resea ch Facili ies (SGIke ).
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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de En adas
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APPENDIX IV. ERANSKINA
The supplemen a y igu es and ables in APPENDIX IV can be downloaded by
scanning his QR code.
VI. ERANSKINeko i udi e a aula osaga iak QR kode hau eskanea u a
deska ga dai ezke.
Sec ion 3. a ala - APPENDIX VI. ERANSKINA
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Sec ion 4. a ala – Wo k unde e iew /
Be ikuspenean dagoen lana
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APPENDIX V. ERANSKINA
Isola ion and sc eening o bioac i e
compounds p oduced by halophiles and
cha ac e izing compounds p oduced by
Pseudoal e omonas spp. ASV78
Unde e iew: Azpiazu-Muniozgu en M, Valgañón-Pé ez E, Ga cía‑Ma ínez
M, Rod iguez-Paniagua A, Poppy Cla k H, Jus icia C, Ma ín J, de la C uz
Mo eno M, Reyes F, Lao den L, Ma inez-Malaxe xeba ia I, Ma inez-
Balles e os I.
En i onmen al Mic obiology Repo s
Impac ac o 2023: 3.6
Rela i e posi ion: 66/161, Q2 Mic obiology
315
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Isola ion and sc eening o bioac i e compounds p oduced by
halophiles and cha ac e iza ion o compounds p oduced by
Pseudoal e omonas spp. ASV78
Maia Azpiazu-Muniozgu ena,e, Elena Valgañón-Pé eza, Mine a
Ga cía‑Ma íneza, Alba Rod iguez-Paniaguab, H. Poppy Cla kc, Ca los Jus iciad,
Jesús Ma índ, Me cedes de la C uz Mo enod, Fe nando Reyesd, Lo ena
Lao dena,e, I a i Ma inez-Malaxe xeba iaa,e, Ila gi Ma inez-Balles e osa,e*
aMik oIke Resea ch G oup, Immunology, Mic obiology and Pa asi ology Depa men , Facul y
o Pha macy, Uni e si y o he Basque Coun y UPV/EHU, Paseo de la Uni e sidad 7, 01006,
Vi o ia-Gas eiz, Spain. [email p o ec ed]; e [email protected];
mine g[email p o ec ed]; lo ena.lao [email protected]; [email p o ec ed];
ila gi.ma [email protected]
bDepa men o O ganic Chemis y I, Facul y o Pha macy and Lasca ay Resea ch Cen e ,
Uni e si y o he Basque Coun y UPV/EHU, Paseo de la Uni e sidad 7, 01006, Vi o ia-Gas eiz,
Spain. [email p o ec ed]
cMa ine Biodisco e y Cen e, Depa men o Chemis y, Uni e si y o Abe deen, Abe deen AB24
3UE, UK. [email p o ec ed]
dFundación MEDINA, Cen o de Excelencia en In es igación de Medicamen os Inno ado es en
Andalucía, A da. del Conocimien o 34, 18016 A milla G anada, Spain.
ca los.jus [email protected]; jesus.ma [email protected];
me cedes.[email p o ec ed]; [email p o ec ed]
eBioa aba, Mic obiology, In ec ious Diseases, An imic obial Agen s, and Gene The apy, 01006,
Vi o ia-Gas eiz, Spain. [email protected]; [email p o ec ed];
i a i.ma [email protected]; [email p o ec ed]
*Co esponding au ho : ila [email p o ec ed] (I. Ma inez-Balles e os). Mik oIke Resea ch
G oup, Immunology, Mic obiology and Pa asi ology Depa men , Facul y o Pha macy,
Uni e si y o he Basque Coun y UPV/EHU, Paseo de la Uni e sidad 7, 01006, Vi o ia-Gas eiz,
Spain. Tel.: +34 945013288; ORCID: 0000-0002-8867-1487
Sec ion 4. a ala - APPENDIX V. ERANSKINA
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2.5. Cha ac e iza ion o isola e ASV78
Among all he isola es es ed, he isola e ASV78 was selec ed o u he
analysis as i exhibi ed posi i e esul s o bo h, su ace-ac i e and
an imic obial ac i i ies. To op imize he p oduc ion o he compounds o
in e es , MB medium supplemen ed wi h wo ca bon sou ces (1% w/ glyce ol
o glucose) was es ed a wo empe a u es (25 o 30 °C) and wo condi ions
(125 pm o s a ic) o a pe iod o 5 days. Cell cul u es we e hen cen i uged
a 3,100 g o 15 minu es a 4 °C. The p esence o ac i i ies o in e es was
hen de e mined in acco dance wi h he p o ocols ou lined in sec ions 2.3 and
2.4. The mos a o able p oduc ion condi ions we e es ablished o all he
subsequen analyses.
2.5.1. Ob aining he c ude ex ac s
In o de o ex ac he an imic obial compounds, he CFS was ex ac ed wice
wi h an equal olume o e hyl ace a e, by means o igo ous shaking in a
sepa a ing unnel. Fo he biosu ac an ex ac ion, he CFS was acidi ied o
pH 2.0 wi h 1 M HCl p io o ex ac ion wice wi h an equal olume o e hyl
ace a e. The sol en o he o ganic phase in each case was hen e apo a ed
unde acuum using a Ro a apo R100 (Buchi, Swi ze land) o ob ain he c ude
ex ac s. Finally, o ex ac he bioemulsi ie , he CFS was mixed wi h cold
e hanol (1:1) and incuba ed o e nigh a - 20 ºC. The bioemulsi ie was hen
pelle ed by cen i uga ion a 9,000 pm o 15 minu es a 4 °C and dissol ed in
dis illed wa e (1% w/ ) be o e lyophiliza ion. The h ee c ude ex ac s we e
weighed and s o ed a - 20 ºC un il u he analysis. Each c ude ex ac was
es ed o he p esence o co esponding ac i i ies as desc ibed in he
Supplemen a y Ma e ial.
Sec ion 4. a ala - APPENDIX V. ERANSKINA
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