RESEARCH ARTICLE
T ansc ip ome changes in esponse o
empe a u e in he ish pa hogen
Pho obac e ium damselae subsp. damselae:
Clues o unde s and he eme gence o
disease ou b eaks a inc eased seawa e
empe a u es
Xose
´M. Ma anza, Ca los R. Oso ioID*
Depa amen o de Mic obioloxı
´a e Pa asi oloxı
´a, Ins i u o de Acuicul u a, Uni e sidade de San iago de
Compos ela, San iago de Compos ela, Spain
*[email p o ec ed]
Abs ac
The ma ine bac e ium Pho obac e ium damselae subsp. damselae (Pdd) is a gene alis and
acul a i e pa hogen ha causes disease in a wide ange o ma ine animals including ish
species o impo ance in aquacul u e. Disease ou b eaks in ish a ms ha e been co ela ed
wi h an inc eased wa e empe a u e du ing summe mon hs. In his s udy, we ha e used
RNA sequencing o analyze he ansc ip ome o Pdd RM-71 cul u ed a wo di e en em-
pe a u es, which simula ed empe a u e condi ions expe ienced du ing ee swimming li e-
s yle a mid la i udes in win e mon hs (15˚C) and du ing ou b eaks in aquacul u e in wa m
summe mon hs (25˚C). The enhanced bac e ial g ow h o Pdd obse ed a 25˚C in compa -
ison o 15˚C sugges s ha an ele a ed seawa e empe a u e con ibu es o he build-up o a
su icien bac e ial popula ion o cause disease. In compa ison o g ow h a 15˚C, g ow h a
25˚C esul ed in he up egula ion o genes in ol ed in DNA syn hesis, nu ien up ake, che-
mo axis, lagella mo ili y, sec e ion sys ems and an imic obial esis ance. Plasmid-encoded
i ulence ac o s, which include a pu a i e adhesin/in asin OmpU, a ans e in ecep o
and a se um esis ance p o ein, we e also up egula ed. T ansc ip ion ac o RpoS, genes
in ol ed in cold shock esponse, modula ion o cell en elope and amino acid me abolism,
as well as genes o ye unknown unc ion we e down egula ed a 25˚C. No ably, he gene
encoding damselysin cy o oxin (Dly) was among he mos highly ansc ibed genes a he
wo assayed empe a u es, a le els compa able o he mos highly exp essed housekeep-
ing genes. This s udy con ibu es o ou unde s anding o he egula o y ne wo ks and biol-
ogy o a gene alis ma ine bac e ial pa hogen, and p o ides e idence ha empe a u e
egula es mul iple physiological and i ulence- ela ed unc ions in Pdd.
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0210118 Decembe 31, 2018 1 / 23
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OPEN ACCESS
Ci a ion: Ma anza XM, Oso io CR (2018)
T ansc ip ome changes in esponse o empe a u e
in he ish pa hogen Pho obac e ium damselae
subsp. damselae: Clues o unde s and he
eme gence o disease ou b eaks a inc eased
seawa e empe a u es. PLoS ONE 13(12):
e0210118. h ps://doi.o g/10.1371/jou nal.
pone.0210118
Edi o : Diogo Ne es P oenc¸a, Uni e sidade de
Coimb a, PORTUGAL
Recei ed: Sep embe 18, 2018
Accep ed: Decembe 17, 2018
Published: Decembe 31, 2018
Copy igh : ©2018 Ma anza, Oso io. This is an
open access a icle dis ibu ed unde he e ms o
he C ea i e Commons A ibu ion License, which
pe mi s un es ic ed use, dis ibu ion, and
ep oduc ion in any medium, p o ided he o iginal
au ho and sou ce a e c edi ed.
Da a A ailabili y S a emen : All ele an da a a e
wi hin he manusc ip and i s Suppo ing
In o ma ion iles.
Funding: This wo k has been suppo ed by g an
AGL2016-79738-R (AEI/FEDER, EU) om he S a e
Agency o Resea ch (AEI) o Spain, and co- unded
by he FEDER P og amme om he Eu opean
Union, o CRO. The suppo o Xun a de Galicia
(Spain) wi h g an GRC-2014/007 o CRO and
In oduc ion
The Vib ionaceae amily includes aqua ic bac e ia ound in ocean en i onmen s and empe a-
u e and salini y a e he main abio ic ac o s ha shape hei abundance and dis ibu ion [1,
2]. Rising ocean empe a u es con ibu e o he inc ease in p e alence and se e i y o a wide
ange o Vib io- ela ed diseases in ma ine o ganisms and also in humans [3,4]. Vib io and
Pho obac e ium in ec ions ha e a ma ked seasonal dis ibu ion, and mos cases occu du ing
he wa me summe mon hs [5].
Pho obac e ium damselae subsp. damselae (he ea e Pdd), a ma ine bac e ium o he
Vib ionaceae amily, is a pa hogen o a wide ange o ma ine animals, including ish, molluscs,
c us aceans and ce aceans. I is also an eme ging pa hogen in ma ine aquacul u e sys ems ha
causes wound in ec ions and sep icaemia in economically impo an ish species [6–8]. In
addi ion, i is an oppo unis ic human pa hogen which causes se e e wound in ec ions ha
can ha e a a al ou come [9].
No ably, disease ou b eaks in aquacul u e caused by Pdd ha e been associa ed wi h an
inc eased seawa e empe a u e du ing summe mon hs. This has been he case o ou b eaks
in a ms ea ing u bo (Scoph halmus maximus) [10], ainbow ou (Onco hynchus mykiss)
[11,12], seabass (Dicen a chus lab ax) [13–16], gil head seab eam (Spa us au a a) [14,15,
17], cobia (Rachycen on canadum) [18] and sil e pom e (Pampus a gen eus) [19]. The e is
inc easing e idence ha ou b eaks o Pdd in ish a ms a e caused by gene ically he e oge-
neous popula ions exis ing in he en i onmen [7,20]. I is belie ed ha unde ad an ageous
en i onmen al condi ions hese popula ions cause disease by aking ad an age o s essed ish
hos s.
The majo epo ed i ulence ac o s o Pdd a e cy o oxins wi h hemoly ic ac i i y [8].
Highly i ulen s ains ha bo he i ulence plasmid pPHDD1 ha ca y he cy o oxin genes
dly and hlyA
pl
[21]. dly encodes damselysin oxin (Dly), a phospholipase-D ac i e agains
sphingomyelin [22], and hlyA
pl
encodes he po e- o ming oxin phobalysin P (PhlyP) [23].
In addi ion, he po e- o ming oxin phobalysin C (PhlyC), encoded by he hlyA
ch
gene
loca ed on ch omosome I, and he phospholipase PlpV also con ibu e o i ulence in
ish and o cell oxici y [24,25]. These ou cy o oxins a e sec e ed ia a ype II sec e ion sys-
em [26]. The wo-componen egula o y sys em Rs AB posi i ely egula es ansc ip ion o
he hemolysin genes dly,hlyA
pl
and hlyA
ch
, and i s inac i a ion se e ely impai s i ulence
[27].
Despi e he inc easing e idence sugges ing ha ou b eaks caused by Pdd in ish a ms a e
igge ed by ises in seawa e empe a u e du ing summe mon hs, he ole o empe a u e in
he physiology and gene egula ion o his pa hogen has no been s udied so a . The aim o
his s udy was o in es iga e he ansc ip ome o Pdd a wo di e en empe a u es, 15˚C and
25˚C, using an RNA-seq app oach. We iden i ied a numbe o genes up egula ed a 25˚C— he
empe a u e a which mos ou b eaks occu in aquacul u e a ms— ha likely con ibu e o an
inc eased bac e ial g ow h a e and o an enhanced abili y o colonize and su i e in ish hos s.
Addi ionally, we ound genes wi h highe exp ession a 15˚C which migh aid Pdd o adap o
li e in colde wa e s du ing win e mon hs. The global ansc ip ome da a also shed ligh on
he ela i e exp ession alues o genes encoding i ulence ac o s compa ed o housekeeping
genes and demons a ed ha he damselysin oxin gene is one o he mos highly exp essed
genes in he cell. Finally, his s udy has b ough o he o e on many p e iously o e looked
gene ic ne wo ks and gene clus e s o his pa hogen. O e all, he in o ma ion gene a ed in his
s udy is expec ed o p o ide no el app oaches o he p e en ion and con ol o ib iosis
caused by Pdd in ma ine ish aquacul u e.
RNA sequencing o Pho obac e ium damselae subsp. damselae
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0210118 Decembe 31, 2018 2 / 23
XMM is also acknowledged. The unde s had no
ole in s udy design, da a collec ion and analysis,
decision o publish, o p epa a ion o he
manusc ip . Funde s websi es: h p://www.ciencia.
gob.es/,h p://www.edu.xun a.gal/po al/.
Compe ing in e es s: The au ho s ha e decla ed
ha no compe ing in e es s exis .
Ma e ials and me hods
G ow h analysis
Cells we e ou inely g own a 15 o 25˚C on yp ic soy aga (TSA) o in yp ic soy b o h
(TSB) supplemen ed wi h NaCl up o 1% (TSA-1 and TSB-1, espec i ely). Fo g ow h cu es,
h ee eplica es o each empe a u e o he assay (15 and 25˚C) we e g own in TSB-1 un il
ob aining exponen ially g owing p ecul u es (OD
600
: 0.3). Then, 1:100 dilu ions o each p e-
cul u e we e g own in 100 μl o TSB-1 in 96 well pla es and he op ical densi y (OD
600
) was
measu ed du ing 48h using he spec opho ome e Epoch2 mic opla e eade (Bio ek). Th ee
eplica es we e measu ed o each empe a u e condi ion.
RNA-seq
Expe imen al design, RNA ex ac ion and pu i ica ion. As o RNA-seq app oach, 3
biological eplica es we e pe o med o each condi ion. 15˚C was chosen as he con ol condi-
ion and is close o he empe a u e ha his bac e ium inds du ing ee swimming li es yle in
mid la i udes, whils 25˚C ep esen s he highe empe a u e condi ion ha usually p ecedes
aquacul u e ou b eaks. Fo each empe a u e, h ee independen p ecul u es we e s a ed and
g own un il an OD
600
: 0.3. Then, 1:100 dilu ions o each p ecul u e we e g own in 10 ml o
TSB-1 in 100 ml lasks un il hey eached a sha p OD
600
o 0.55. Cells we e immedia ely ea ed
wi h RNAp o ec Bac e ia Reagen (Qiagen) o s abiliza ion o RNA ollowing manu ac u e ’s
ins uc ions. Pelle ed cells we e hen ca e ully esuspended in TE bu e (30mM T is�Cl, 1mM
EDTA, pH 8.0) con aining 15mg/ml lysozyme (Sigma Ald ich) and he app op ia e olume o
P o einase K (Qiagen). RNA ex ac ion was subsequen ly ca ied ou using RNeasy Mini Ki
(Qiagen) ollowing manu ac u e s’ ins uc ions. An ex a DNase I ea men was ca ied ou
using he on-column ki RNase- ee DNase (Qiagen) o elimina e genomic DNA con amina-
ion. RNA was elu ed using nuclease- ee wa e . The quali y and he quan i y o he o al RNA
was de e mined using a Bioanalyze 2100 (RNA 6000 Nano chip assay) and a Qubi 3.0
(Quan -I dsRNA BR Assay).
Lib a ies p epa a ion and sequencing. To al RNA was RNA-deple ed using he Ribo-
Ze o RNA Remo al Ki (G am Nega i e Bac e ia) (Illumina) and cDNA lib a ies we e
ob ained using he T uSeq RNA ki ollowing Illumina´s ecommenda ions. B ie ly, RNA-
deple ed RNA was chemically agmen ed p io o e e se ansc ip ion and cDNA gene a ion.
The cDNA agmen s hen wen h ough an end epai p ocess, he addi ion o a single ‘A’
base o he 3’ end and hen liga ion o he adap e s. Finally, he p oduc s we e pu i ied and
en iched by PCR o c ea e he indexed inal double s anded cDNA lib a y. The pool o lib a -
ies was sequenced on an Illumina HiSeq 2500 sequence .
Mapping and quan i ica ion o ansc ip s. The quali y con ol o he aw da a ( aw
eads) was pe o med using he Fas QC [h p://www.bioin o ma ics.bab aham.ac.uk/p ojec s/
as qc/] p og am. The aw pai -end eads we e i s mapped agains he e e ence genome o
he Pho obac e ium damselae subsp. damselae ype s ain CIP102761 (GenBank Acc. No.
NZ_ADBS00000000.1). Reads ha did no map o he e e ence genome (co esponding o
genes o RM-71 s ain no p esen in CIP102761) we e subsequen ly mapped o he d a
genome sequence o s ain RM-71 (GenBank Acc. No. NZ_LYBT00000000.1). The wo p o-
cesses we e comple ed using he Bow ie2 [28] 2.2.6 algo i hm. Se e al quali y con ol s eps
we e pe o med. Reads displaying a e y low quali y we e emo ed by using Sam ools [29] and
Pica d Tools so wa e [30]. Fu he mo e, one o he key ac o s ha can condi ion he
sequencing p ocess is he GC con en o samples which was checked as no mal (dis ibu ion
be ween 40–60%) in ou expe imen . Likewise, dis ibu ion o duplica es was e alua ed o
RNA sequencing o Pho obac e ium damselae subsp. damselae
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0210118 Decembe 31, 2018 3 / 23
con i m he no mal small p opo ion. The p ocess o gene ic quan i ica ion was ca ied ou by
he HTSeq [31] so wa e (0.6.1 e sion).
Compa ison be ween samples. Conco dance be ween samples o he same condi ion
( eplica es o each o he wo assayed empe a u es) was ca ied ou by a s udy o co ela ion
and dis ance conside ing he whole ansc ip ome no malized by he size o he lib a y. This
p ocess was made using he s a is ics p og am R. Di e en ial exp ession analysis was assessed
using DESeq2 [32] me hod (1.18.1 e sion). The analysis o Di e en ially Exp essed Genes
(DEG) was done by using s a is ical packages designed by Py hon and R, using he DESseq2
[32] algo i hm applying a di e en ial nega i e binomial dis ibu ion o he s a is ics signi i-
cance. Compa ison be ween he wo di e en condi ions (25˚C s. 15˚C) was se as ixed e ec
in DEseq2. A Py hon sc ip de eloped a Sis emas Geno
´micos (Valencia, Spain) was employed
o gene a e a da a ma ix o each g oup condi ion wi h he coun s ob ained om HTSeq
coun o each sample (each o he h ee eplica es a each o he wo empe a u es). We con-
side ed di e en ially exp essed genes hose wi h Fold Change (FC) alue lowe han -1.5 o
highe han 1.5 and a P alue adjus ed by False Disco e y Ra e (FDR) [33]�0.05. FPKM (F ag-
men s pe kilobase pe million agmen s mapped) alues calcula ed wi h Cu links 2.2.1 [34]
we e used o ep esen he exp ession o each indi idual gene. FPKM is used o no maliza ion
o he da a since i indica es he numbe o lec u es o a gi en gene pe kilobase (independen ly
o he leng h o he gene), and pe million eads (independen ly o he size o he lib a y).
Resul s and discussion
G ow h dynamics o Pdd a 15 and 25˚C
Pdd RM-71 was he s ain selec ed o he p esen s udy. I was isola ed du ing a disease ou b eak
in a u bo (Scoph halmus maximus) a m in Galicia (NW Spain), when he wa e empe a u e
inc eased suddenly om 18˚C o 22–24˚C in he summe o 1988 [10]. I is a highly i ulen ,
s ongly hemoly ic and cy o oxic s ain and con ains he pPHDD1 i ulence plasmid [21]. G ow h
o RM-71 was analyzed in 48-h con inuous cul u es a 15 and 25˚C in TSB-1 medium (S1 Table).
The wo assayed empe a u es simula ed an a p io i non- isky condi ion (15˚C) and wa m wa e
episodes ha igge aquacul u e ou b eaks (25˚C). The beginning o he exponen ial phase was
delayed a 15˚C compa ed o g ow h a 25˚C and he e was a g ea di e ence be ween op ical den-
si y alues a e 15 h o cul i a ion a 15˚C (OD
600
: 0.129) and 25˚C (OD
600
: 0.527) (Fig 1). These
obse a ions sugges ha 25˚C is close o he op imal g ow h empe a u e o Pdd han 15˚C.
This subs an ial inc ease in bac e ial p oli e a ion a 25˚C wi h espec o 15˚C du ing he
i s 15 h o cul i a ion migh con ibu e o he apid p og ession o Pdd ou b eaks when he
sea wa e empe a u e inc eases. P oli e a ion o species o he Vib ionaceae amily is a ou ed
by wa m (>15˚C) sea wa e s [35], and ecen s udies ha e demons a ed ha ollowing an
inc ease in wa e empe a u e, Vib ios can go om ba ely de ec able o being he p edominan
bac e ia in a e y sho ime [36,37]. Pdd isola ion was i s epo ed om ulce s in damsel ish
(Ch omis punc ipinnis) du ing he summe and all seasons in sou he n Cali o nia and was
shown o ollow a seasonal pa e n o in ec i i y. I was p oposed ha ele a ed wa e empe a-
u es migh allow he build-up o su icien bac e ial popula ions o cause disease in damsel-
ish, hence he seasonal in ec i i y o Pdd [38]. P e ious s udies ha e p o ided sound e idence
ha seawa e ansmi s he disease caused by Pdd and ha he sp ead o his bac e ium la gely
depends on wa e empe a u e [39]. Skin is sugges ed o be a po en ial ou e o pene a ion o
his pa hogen, which is able o speci ically adhe e o ish mucus [39]. Hence, e en i ish a e
colonized by a small numbe o bac e ia, as p oli e a ion enhanced by wa m empe a u e will
acili a e he in ec ing bac e ial popula ion o e ade hos immune esponses by a a ie y o
mechanisms [40]. High numbe s o bac e ial cells a he in ec ion si e migh cause exhaus ion
RNA sequencing o Pho obac e ium damselae subsp. damselae
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0210118 Decembe 31, 2018 4 / 23
o complemen componen s as well as o phagocy es [41] leading o sys emic in ec ion and ish
dea h. O e all, he esul s o he g ow h dynamics analysis a 15 and 25˚C con ibu e o unde -
s and why inc eased wa e empe a u es p ecede mos ou b eaks caused by his pa hogen.
RNA sequencing esul s
S ain RM-71 was g own a 15 and 25˚C, and cDNA p epa ed om mRNA isola ed om cul-
u es a he wo di e en empe a u es was subjec ed o Illumina sequencing. A ound 55 o 71
million eads we e gene a ed o each biological eplica e (S2 Table). G ow h a 15˚C was
de ined as he con ol condi ion. The compa a i e analyses o he ansc ip omes a 15 and
25˚C esul ed in a o al o 1195 di e en ially exp essed genes (DEGs): 641 genes wi h lowe
exp ession a 25˚C (FC lowe han -1.5) and 554 genes wi h highe exp ession a 25˚C (FC
highe han 1.5) (Fig 2,S3 and S4 Tables).
Simila o o he membe s o he Vib ionaceae amily, Pdd RM-71 con ains wo ch omo-
somes. In addi ion, his s ain ha bo s he i ulence plasmid pPHDD1 [21]. Using he com-
ple e sequences o ch omosome I and II o he ype s ain CIP102761, and he comple e
pPHDD1 plasmid sequence o s ain RM-71 (GenBank Acc. No. NC_014653) as e e ences,
we dis ibu ed he DEGs in o each eplicon. No ably, we obse ed an imbalance in he numbe
o DEGs be ween he wo ch omosomes (Fig 3). In ch omosome I simila numbe s o DEGs
a e up egula ed and down egula ed. Howe e , ch omosome II con ains 204 down egula ed
genes and only 77 up egula ed genes a 25˚C. The ch omosome I o Vib ionaceae species con-
ains mos o he essen ial genes, whe eas ch omosome II has a mo e lexible gene con en and
is esponsible o adap a ion o en i onmen al changes [42,43]. In e es ingly, among he 31
DEGs in he i ulence plasmid pPHDD1, 24 co esponded o genes whose exp ession is up e-
gula ed a 25˚C, an obse a ion o special in e es since his plasmid cons i u es a hallma k o
highly i ulen isola es [8]. Indeed, he p esen s udy has un eiled po en ial i ulence ac o s
Fig 1. The in luence o empe a u e cul i a ion in g ow h dynamics o Pdd.G ow h o RM-71 s ain was assayed a 15˚C and 25˚C in TSB-1
o 48 h. Ve ical e o ba s ep esen s anda d de ia ion o biological iplica es.
h ps://doi.o g/10.1371/jou nal.pone.0210118.g001
RNA sequencing o Pho obac e ium damselae subsp. damselae
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encoded by pPHDD1 plasmid among he g oup o genes up egula ed a 25˚C (see below), hus
highligh ing he impo ance o his i ulence plasmid in he pa hobiology o Pdd.
Genes in ol ed in g ow h and i ulence a e up egula ed a 25˚C
G ow h a 25˚C esul ed in he up egula ion o 533 genes ha mapped o he genome o he
ype s ain (S3 Table) and o 21 addi ional genes unique o s ain RM-71 (S4 Table). A lis o
he 50 op DEGs up egula ed a 25˚C plus addi ional selec ed genes is shown in Table 1.
Fig 2. Smea plo o di e en ially exp essed genes (DEGs) in Pdd RM-71 exposed o wo empe a u es, 15˚C and 25˚C. The smea plo shows he
ela ionship be ween he log FC and mean o no malized coun s. G ey poin s ep esen genes wi h non-signi ican changes in exp ession, whe eas ed poin s
ep esen genes ha a e signi ican ly di e en ially exp essed.
h ps://doi.o g/10.1371/jou nal.pone.0210118.g002
RNA sequencing o Pho obac e ium damselae subsp. damselae
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Nu ien acquisi ion and me abolism. Genes encoding memb ane p o eins, nu ien
anspo e s and po ins we e up egula ed a 25˚C (Table 1). The nucleoside pe mease NupC
(VDA_001789) was he mos up egula ed gene a 25˚C. A V.chole ae nupC dele ion mu an
was impai ed o nucleoside acquisi ion leading o diminished i ness in nu ien -limi ed en i-
onmen s [44]. G ow h a 25˚C up egula ed wo ibonucleo ide educ ases belonging o class
Ia and II, one o hem, VDA_001894, is coenzyme B12-dependen . In acco dance, he i amin
B12 ecep o B uB was also up egula ed (Table 1). Up egula ion o he u acyl phospho ibosyl-
ans e ase VDA_001405, an enzyme necessa y o he syn hesis o p ecu so s o all py imi-
dine nucleo ides, was also obse ed. Among up egula ed amino acid biosyn hesis enzymes
and amino ans e ases was he glu amine syn he ase ype I, which has a cen al ole in amino
acid biosyn hesis. The pu a i e ypsin supe amily p o ein encoded by A0J47_09785, which
has a possible unc ion in pep ide deg ada ion, as well as a numbe o ibosomal and ansla-
ion- ela ed p o eins we e up egula ed sugges ing ha g ow h a 25˚C enhances p o ein syn-
hesis and eno a ion.
Pdd deg ades ex acellula lipids, which may se e as ca bon and ene gy sou ces [10,11,
25]. An ope on encoding an ex acellula lipase (VDA_001610) and a a y acid anspo e
Fig 3. G aphical ep esen a ion o DEGs dis ibu ion among he wo Pdd ch omosomes and pPHDD1 i ulence plasmid. Numbe s deno e DEGs up egula ed a
25˚C ( ed), down egula ed a 25˚C (blue), and genes no di e en ially egula ed (g ey).
h ps://doi.o g/10.1371/jou nal.pone.0210118.g003
RNA sequencing o Pho obac e ium damselae subsp. damselae
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Table 1. Lis o selec ed DEGs wi h enhanced exp ession a 25˚C including he 50 op up egula ed genes a 25˚C. Enhanced exp ession a 25˚C is deno ed by posi i e
FC alues. Genes wi h VDA codes co espond o he anno a ion in he CIP102761 genome, and genes wi h A0J47 codes co espond o he anno a ion in he RM-71
genome.
Gene ID P oduc /Func ion Fold Change p- alue Loca ion
Nu ien acquisi ion/me abolism
VDA_001789 Nucleoside pe mease NupC 5.1 2.2182E-122 Ch I
VDA_001833 GPR1/FUN34/yaaH pu a i e ace a e anspo e 3.6 5.07132E-17 Ch I
VDA_002532 Po in 5.1 1.08929E-77 Ch I
VDA_003254 Po in 3.0 3.53973E-49 Ch I
VDA_001005 Po in 2.5 3.2873E-69 Ch II
VDA_003133 Glu amine syn he ase ype I 4.7 2.8174E-108 Ch I
VDA_000463 L-aspa aginase 3.3 8.16042E-85 Ch II
VDA_003226 Glucosamine uc ose-6-phospha e amino ans e ase 3.5 3.71313E-35 Ch I
VDA_001560 Aspa a e/ y osine/a oma ic amino ans e ase 2.8 1.19637E-65 Ch I
A0J47_09785 Pu a i e ypsin supe amily p o ein 4.3 4.6629E-132 Ch I
VDA_002568 Long-chain a y acid anspo p o ein 2.5 4.75483E-39 Ch I
VDA_000298 A ginine deca boxylase ca abolic 2.6 7.62289E-83 Ch II
VDA_002194 Manganese-dependen ino ganic py ophospha ase 2.5 2.24757E-83 Ch I
VDA_003183 Oligopep idase A 2.7 2.38104E-73 Ch I
VDA_003148 Vi amin up ake anspo e 2.6 6.42707E-28 Ch I
DNA syn hesis and epai
VDA_002372 Ribonucleo ide educ ase o class Ia (ae obic) alpha subuni 3.5 1.03533E-70 Ch I
VDA_001894 Ribonucleo ide educ ase o class II (coenzyme B12-dependen ) 3.1 6.75604E-90 Ch I
VDA_003108 Ou e memb ane i amin B12 ecep o B uB 3.0 9.66178E-20 Ch I
VDA_001405 U acil phospho ibosyl ans e ase 3.0 1.51133E-50 Ch I
T ansla ion
VDA_003390 LSU ibosomal p o ein L31p 2.7 1.98928E-09 Ch I
VDA_003099 T ansla ion elonga ion ac o Tu 2.6 3.07515E-36 Ch I
VDA_002952 SSU ibosomal p o ein S21p 2.6 1.9713E-24 Ch I
Vi ulence and an imic obial esis ance
VDA_000110 Se um esis ance p o ein Vep07-like 2.0 5.76819E-33 pPHDD1
VDA_000111 T ans e in ecep o Vep20-like 3.0 5.67092E-76 pPHDD1
VDA_000113 OmpU 2.5 4.19858E-66 pPHDD1
VDA_000794 TonB-dependen side opho e ecep o 2.8 5.7247E-111 Ch II
VDA_000157 TolC 2.8 2.115E-103 pPHDD1
VDA_000158 Ac A/MacA-like memb ane usion p o ein 3.2 7.9453E-112 pPHDD1
A0J47_18110 Unknown p o ein ela ed o T6SS 2.8 1.0749E-24 pPHDD1
A0J47_18115 RNase oxin N ox44 2.7 9.41221E-55 pPHDD1
A0J47_18120 P oline-alanine-alanine-a ginine (PAAR) domain p o ein 2.9 1.3511E-60 pPHDD1
Mo ili y and chemo axis
VDA_003029 Flagella p o ein Mo X 2.6 9.54565E-60 Ch I
VDA_002607 Flagellin p o ein FlaB 3.6 1.0743E-118 Ch I
VDA_002671 Flagella mo o o a ion p o ein Mo A 2.8 5.26275E-79 Ch I
VDA_002604 Flagella biosyn hesis p o ein FliS 2.7 6.88724E-93 Ch I
VDA_003044 Me hyl-accep ing chemo axis p o ein 3.1 7.48432E-68 Ch I
VDA_001198 Me hyl-accep ing chemo axis p o ein 2.7 1.10855E-85 Ch I
S ess esponse and de ence mechanisms
VDA_003059 Chape onin complex G oEL-G oES 3.1 2.1437E-116 Ch I
VDA_003060 Chape onin complex G oEL-G oES 3.5 1.517E-130 Ch I
(Con inued)
RNA sequencing o Pho obac e ium damselae subsp. damselae
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FadL (VDA_001611) we e 2- old up egula ed a 25˚C, and so was he long-chain a y acid
anspo e VDA_002568 (Table 1), sugges ing ha exogenous lipid deg ada ion and up ake o
a y acids migh cons i u e an ad an age o Pdd as eplica ion a 25˚C.
I on acquisi ion plays a ole in Pdd i ulence o ish [45–47]. Ou analysis un eiled up e-
gula ion a 25˚C o a pPHDD1 plasmid-bo ne gene ha encodes a pu a i e ans e in binding
p o ein (VDA_000111) (see below), and o VDA_000794 encoding a TonB-dependen side o-
pho e ecep o . The ligand(s) anspo ed h ough VDA_000794 a e unknown, bu a ecen
s udy demons a ed ha exp ession o his gene is enhanced in Pdd unde i on-limi a ion con-
di ions [48].
Mo ili y and chemo axis. Mo ili y and issue coloniza ion cons i u e impo an ac o s in
Pdd pa hogenici y [39,49]. Fou lagellum- ela ed genes we e ound among he 50 mos up e-
gula ed genes (Table 1). Addi ional up egula ed genes included lagella hook p o ein FlgE
(VDA_002616), lagella mo o o a ion p o ein Mo B (VDA_002670), lagella hook-associ-
a ed p o ein FlgK (VDA_002609) and lagella basal-body od modi ica ion p o ein FlgD
(VDA_002617) (S3 Table). P e ious s udies ha e epo ed ha Pdd can in ec new hos s
h ough seawa e and ha inc eased wa e empe a u e boos ed in ec ion by his ou e [49].
The up egula ion o mo ili y- ela ed genes a 25˚C suppo s hese p e ious obse a ions and
su ely calls o u he s udies along hese lines.
Chemo axis is ini ia ed by memb ane chemo ecep o s dubbed me hyl-accep ing chemo-
axis p o eins, which bind ligands and ansduce a signal cascade ha modula es lagellum
ac i i y. No ably, wo chemo axis- ela ed genes we e ound among he 50 mos up egula ed
genes a 25˚C and co espond o he me hyl-accep ing chemo axis p o eins VDA_003044 and
VDA_001198 (Table 1). A ecen s udy showed ha mu an s in chemo axis genes a e no only
impai ed in swimming mo ili y in Pdd bu also exhibi diminished p oduc ion o he majo
i ulence ac o PhlyP and impai ed adhesion o euka yo ic cells [50]. Vib io ische i and V.
Table 1. (Con inued)
Gene ID P oduc /Func ion Fold Change p- alue Loca ion
VDA_002771 DnaK chape onin 3.4 7.7384E-130 Ch I
VDA_001553 Pep idyl-p olyl cis- ans isome ase PpiD 2.5 3.30237E-65 Ch I
VDA_002523 H pG chape onin 2.8 3.0531E-107 Ch I
VDA_001325 ClpB chape onin 2.5 3.25923E-77 Ch I
VDA_003124 Ribosome associa ed hea shock p o ein 3.0 1.98913E-71 Ch I
VDA_003529 Ou e memb ane s ess senso p o ease DegQ 3.3 1.13294E-15 Ch I
VDA_003386 ATP-dependen p o ease HslV 3.1 4.0286E-132 Ch I
VDA_001154 Pe oxidase 4.9 6.6194E-111 Ch II
VDA_000806 Pe oxidase 3.5 5.64336E-61 Ch II
VDA_000771 I on-sul u clus e assembly p o ein Su B 2.6 1.95494E-63 Ch II
T ansc ip ional egula ion and signalling
VDA_003227 DeoR amily ansc ip ional egula o 3.3 1.4446E-35 Ch I
VDA_001088 XRE amily egula o 2.6 7.86116E-25 Ch II
VDA_002825 Cyclic-di-GMP phosphodies e ase A 3.7 6.3346E-128 Ch I
Cell wall/memb ane/en elope biogenesis
VDA_003228 N-ace ylglucosamine-1-phospha e u idyl ans e ase GlmU 2.7 4.3747E-86 Ch I
Hypo he ical p o eins o unknown unc ion
VDA_003431 Hypo he ical p o ein 3.4 3.96176E-86 Ch I
VDA_000943 Hypo he ical p o ein 2.6 3.53416E-24 Ch II
VDA_000598 Hypo he ical p o ein 2.5 8.47329E-75 Ch II
h ps://doi.o g/10.1371/jou nal.pone.0210118. 001
RNA sequencing o Pho obac e ium damselae subsp. damselae
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Vep07 and Vep20 showed ansc ip abundance le els la gely in e io o Dly cy o oxin, again
ein o cing he dominance o Dly as he op-exp essed i ulence ac o in Pdd. This obse a-
ion is in ag eemen wi h ea ly s udies which desc ibed highly i ulen Pdd s ains a e p oduc-
e s o “la ge amoun s o a cy oly ic oxin in i o” [93] and ein o ces he majo ole o
Table 4. Lis o mos exp essed genes a 25˚C and hei co esponding FPKM alues. FPKM alues shown co e-
spond o mean alues o he h ee biological eplica es.
Locus ag P o ein FPKM
VDA_003450 LSU ibosomal p o ein L24p (L26e) 16486
VDA_003447 LSU ibosomal p o ein L29p (L35e) 15622
VDA_003093 LSU ibosomal p o ein L7/L12 (L23e) 15513
VDA_003446 LSU ibosomal p o ein L16p (L10e) 14870
VDA_003461 SSU ibosomal p o ein S13p (S18e) 13609
VDA_003444 LSU ibosomal p o ein L22p (L17e) 13577
VDA_003449 LSU ibosomal p o ein L14p (L23e) 13557
VDA_003456 SSU ibosomal p o ein S5p (S2e) 13188
VDA_003244 LSU ibosomal p o ein L34p 12838
VDA_003463 SSU ibosomal p o ein S4p (S9e) 12813
h ps://doi.o g/10.1371/jou nal.pone.0210118. 004
Fig 5. Damselysin oxin gene dly is one o he mos highly exp essed genes in Pdd.FPKM (F agmen s Pe Kilobase o ansc ip pe Million mapped eads)
alues a he wo assayed empe a u es (S6 Table) we e ob ained o a selec ion o i ulence and egula o y genes, sec e ion sys em genes and housekeeping genes
including he op-exp essed gene encoding ibosomal p o ein L24p, and compa ed using a loga i hmic scale. Ve ical e o ba s ep esen s anda d de ia ion o
biological iplica es.
h ps://doi.o g/10.1371/jou nal.pone.0210118.g005
RNA sequencing o Pho obac e ium damselae subsp. damselae
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pPHDD1 as a i ulence plasmid ha con ibu ed o he e olu ion o highly hemoly ic and
highly i ulen lineages o Pdd.
Conclusions
Regula ion o exp ession o i ulence ac o s in esponse o empe a u e has been widely s ud-
ied in pa hogenic bac e ia in ec ing homeo he ms [94–96]. Meanwhile, much less is known
abou he empe a u e-dependen egula ion o i ulence ac o s in ish bac e ial pa hogens
[59,97]. The aim o he cu en s udy was o in es iga e which genes a e di e en ially egu-
la ed in Pdd in a hea s ess condi ion (25˚C), ela i e o a colde condi ion (15˚C), conside ing
ha he majo i y o ish a m ou b eaks occu du ing wa m summe mon hs a empe a u es
close o 25˚C. Fig 6 ea u es a diag am o di e en ially exp essed unc ions ha a e up egu-
la ed ( igh panel) o down egula ed (le panel) a 25˚C.
G ow h a 25˚C esul ed in he up egula ion o mo ili y- and chemo axis- ela ed unc ions,
as well as nu ien up ake and u iliza ion genes. No ably, po en ial i ulence ac o s encoded
Fig 6. Diag amma ic summa y o genes up egula ed ( igh side) and down egula ed (le side) a 25˚, ela i e o 15˚C, in Pdd.G ow h a 25˚C up egula ed
mo ili y and chemo axis- ela ed unc ions, as well as nu ien up ake and u iliza ion genes. Po en ial i ulence ac o s encoded wi hin pPHDD1 plasmid we e also
up egula ed a 25˚C. Genes wi h lowe exp ession a 25˚C include a numbe o gene ope ons o ye unknown unc ion, unc ions ela ed o cell en elope, poS, and
speci ic amino acid biosyn hesis ou es, among o he s. This diag am has been cons uc ed based on ansc ip omic da a, and u he s udies a e needed o es ablish he
p oposed model.
h ps://doi.o g/10.1371/jou nal.pone.0210118.g006
RNA sequencing o Pho obac e ium damselae subsp. damselae
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wi hin pPHDD1 ela ed o i on acquisi ion ( ans e in ecep o ), adhesion (OmpU), se um
esis ance (Vep07-like) and de ence agains compe i o s, we e also up egula ed a 25˚C (Fig 6).
O e all, his s udy un eils a la ge se o p e iously o e looked gene ic ne wo ks in Pdd and
poin s a a numbe o cell unc ions ela ed o i ulence and acclima iza ion o changes in he
en i onmen .
No ably, damselysin oxin was one o he mos highly exp essed genes in he cell a he wo
assayed empe a u es, wi h exp ession le els compa able o he mos exp essed genes encoding
ibosomal p o eins. This inding highligh s he impo ance o his phospholipase D o he bac-
e ium and sugges s ha his oxin migh ul il o he biological oles in addi ion o i ulence.
To he bes o ou knowledge, his s udy ep esen s he i s ansc ip ome-based analysis o
Pdd, and i has allowed us o iden i y a la ge se o gene unc ions ha su ely will cons i u e a
ounda ion o u u e s udies. Cu en ly we a e in he p ocess o in es iga ing he ole o he
op-di e en ially exp essed genes iden i ied in his s udy in he pa hobiology o Pdd.
Suppo ing in o ma ion
S1 Table. OD
600
da a o each biological eplica e a 15˚C and 25˚C used o gene a e
g ow h cu es depic ed in Fig 1.
(XLSX)
S2 Table. Reads mapping o each biological eplica e.
(XLSX)
S3 Table. Lis o Di e en ially Exp essed Genes (DEGs) a 25˚C s 15˚C mapped o ype
s ain genome (CIP102761).
(XLSX)
S4 Table. Lis o Di e en ially Exp essed Genes (DEGs) a 25˚C s 15˚C mapped o RM-71
genome.
(XLSX)
S5 Table. Lis o Di e en ially Exp essed Genes (DEGs) wi hin i ulence plasmid
pPHDD1.
(XLSX)
S6 Table. FPKM alues o each biological eplica e a 15˚C and 25˚C.
(XLSX)
Acknowledgmen s
XM hanks Xun a de Galicia, Spain, o a p edoc o al ellowship. We would like o hank Gen-
e ie e Ga iss a Ka olinska Ins i u e , Sweden, o c i ical eading o he manusc ip and o
use ul commen s.
Au ho Con ibu ions
Concep ualiza ion: Xose
´M. Ma anza, Ca los R. Oso io.
Da a cu a ion: Xose
´M. Ma anza, Ca los R. Oso io.
Fo mal analysis: Xose
´M. Ma anza, Ca los R. Oso io.
Funding acquisi ion: Ca los R. Oso io.
In es iga ion: Xose
´M. Ma anza, Ca los R. Oso io.
RNA sequencing o Pho obac e ium damselae subsp. damselae
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0210118 Decembe 31, 2018 18 / 23
Me hodology: Xose
´M. Ma anza, Ca los R. Oso io.
P ojec adminis a ion: Ca los R. Oso io.
Resou ces: Ca los R. Oso io.
So wa e: Xose
´M. Ma anza.
Supe ision: Ca los R. Oso io.
Valida ion: Xose
´M. Ma anza, Ca los R. Oso io.
Visualiza ion: Ca los R. Oso io.
W i ing – o iginal d a : Xose
´M. Ma anza, Ca los R. Oso io.
W i ing – e iew & edi ing: Ca los R. Oso io.
Re e ences
1. Thompson JR, Randa MA, Ma celino LA, Tomi a-Mi chell A, Lim E, Polz MF. Di e si y and dynamics o
a no h a lan ic coas al Vib io communi y. Appl En i on Mic obiol. 2004; 70(7): 4103–4110. h ps://doi.
o g/10.1128/AEM.70.7.4103-4110.2004 PMID: 15240289
2. Takemu a AF, Chien DM, Polz MF. Associa ions and dynamics o Vib ionaceae in he en i onmen ,
om he genus o he popula ion le el. F on . Mic obiol. 2014; 5: 38. h ps://doi.o g/10.3389/ micb.2014.
00038 PMID: 24575082
3. Le Roux F, Wegne KM, Bake -Aus in C, Vezzulli L, Oso io CR, Ama o C, e al. The eme gence o Vib-
io pa hogens in Eu ope: ecology, e olu ion, and pa hogenesis (Pa is, 11-12 h Ma ch 2015). F on
Mic obiol. 2015; 6: 830. h ps://doi.o g/10.3389/ micb.2015.00830 PMID: 26322036
4. Bake -Aus in C, T inanes J, Gonzalez-Escalona N, Ma inez-U aza J. Non-Chole a Vib ios: The Mic o-
bial Ba ome e o Clima e Change. T ends Mic obiol. 2017; 25: 76–84. h ps://doi.o g/10.1016/j. im.
2016.09.008 PMID: 27843109
5. Bake -Aus in C, Oli e JD, Alam M, Ali A, Waldo MK, Qad i F, e al. Vib io spp. in ec ions. Na Re Dis
P ime s. 2018; 4(1): 8. h ps://doi.o g/10.1038/s41572-018-0005-8 PMID: 30002421
6. Labella A, Be bel C, Manchado M, Cas o D, Bo ego JJ. Pho obac e ium damselae subsp. damselae,
an Eme ging Pa hogen A ec ing New Cul u ed Ma ine Fish Species in Sou he n Spain. Recen Ad
Fish a ms. 2011; 136–152.
7. Te ce i MS, Ogu H, Oso io CR. Pho obac e ium damselae subsp. damselae, an eme ging ish pa ho-
gen in he Black Sea: e idence o a mul iclonal o igin. Appl En i on Mic obiol. 2016; 82: 3736–3745.
h ps://doi.o g/10.1128/AEM.00781-16 PMID: 27084008
8. Oso io CR, Vences A, Ma anza XM, Te ce i MS. Pho obac e ium damselae subsp. damselae, a gene -
alis pa hogen wi h unique i ulence ac o s and high gene ic di e si y. J Bac e iol. 2018; 15: e00002–
18.
9. Hundenbo n J, Thu ig S, Komme ell M, Haag H, Nol e O. Se e e wound in ec ion wi h Pho obac e ium
damselae ssp. damselae and Vib io ha eyi, ollowing a lace a ion inju y in ma ine en i onmen : a case
epo and e iew o he li e a u e. Case Rep Med. 2013;610632.
10. Fouz B, La sen JL, Nielsen B, Ba ja JL, To anzo AE. Cha ac e iza ion o Vib io damsela s ains isola ed
om u bo Scoph halmus maximus in Spain. Dis Aqua O g. 1992; 12: 155–166.
11. Pede sen K, Dalsgaa d I, La sen JL. Vib io damsela associa ed wi h diseased ish in Denma k. Appl
En i on Mic obiol. 1997; 63: 3711–3715. PMID: 9293023
12. Pede sen K, Skall HF, Lassen-Nielsen AM, Bje um L, Olesen NJ. Pho obac e ium damselae subsp.
damselae, an eme ging pa hogen in Danish ainbow ou , Onco hynchus mykiss (Walbaum), ma icul-
u e. J Fish Dis. 2009; 32: 465–472. h ps://doi.o g/10.1111/j.1365-2761.2009.01041.x PMID:
19364386
13. Uzun E, Ogu H. The isola ion equency o bac e ial pa hogens om sea bass (Dicen a chus lab ax) in
he Sou heas e n Black Sea. Aquacul u e. 2015; 437: 30–37.
14. Essam HM, Abdell azeq GS, Tayel SI, To ky HA, Fadel AH. Pa hogenesis o Pho obac e ium damselae
subspecies in ec ions in sea bass and sea b eam. Mic ob Pa hog. 2016; 99: 41–50. h ps://doi.o g/10.
1016/j.micpa h.2016.08.003 PMID: 27497892
15. Eissa IAM, De wa HI, Ismail M, El-Lamie M, Dessouki AA, Elshesh awy H, Bayoumy EM. Molecula
and pheno ypic cha ac e iza ion o Pho obac e ium damselae among some ma ine ishes in Lake
RNA sequencing o Pho obac e ium damselae subsp. damselae
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0210118 Decembe 31, 2018 19 / 23
Temsah. Mic ob Pa hog. 2018; 114: 315–322. h ps://doi.o g/10.1016/j.micpa h.2017.12.006 PMID:
29225092
16. Mahmoud SA, El-Bouhy ZM, Hassanin ME, Fadel AH. Vib io alginoly icus and Pho obac e ium damse-
lae subsp. damselae: p e alence, his opa hology and ea men in sea bass Dicen a chus lab ax. J
Pha m Chem Biol Sci. 2017; 5(4): 354–364.
17. Ve a P, Na as JI, Fouz B. Fi s isola ion o Vib io damsela om seab eam (Spa us au a a). Bull Eu Ass
Fish Pa hol. 1991; 11: 112–113.
18. Sha ma SRK, P adeep MA, Sadu N, Dube PN, Vijayan KK. Fi s epo o isola ion and cha ac e iza ion
o Pho obac e ium damselae subsp. damselae om cage- a med cobia (Rachycen on canadum). J
Fish Dis. 2017; 40: 953–958. h ps://doi.o g/10.1111/j d.12557 PMID: 27696450
19. Tao Z, Shen C, Zhou SM, Yang N, Wang GL, Wang YJ, e al. An ou b eak o Pho obac e ium damselae
subsp. damselae in ec ion in cul u ed sil e pom e Pampus a gen eus in Eas e n China. Aquacul u e.
2018; 492: 201–205.
20. Te ce i MS, Vences A, Ma anza XM, Dalsgaa d I, Pede sen K, Oso io CR. Molecula epidemiology o
Pho obac e ium damselae subsp. damselae ou b eaks in ma ine ainbow ou a ms e eals ex ensi e
ho izon al gene ans e and high gene ic di e si y. F on Mic obiol. 2018; h ps://doi.o g/10.3389/ micb.
2018.02155 PMID: 30283411
21. Ri as AJ, Balado M, Lemos ML, Oso io CR. The Pho obac e ium damselae subsp. damselae hemoly-
sins damselysin and HlyA a e encoded wi hin a new i ulence plasmid. In ec Immun. 2011; 79: 4617–
4627. h ps://doi.o g/10.1128/IAI.05436-11 PMID: 21875966
22. K ege AS, Be nheime AW, E kin LA, Daniel LW. Phospholipase D ac i i y o Vib io damsela cy olysin
and i s in e ac ion wi h sheep e y h ocy es. In ec Immun. 1987; 55: 3209–3212. PMID: 2890587
23. Ri as AJ, on Ho en G, Neuki ch C, Meyenbu g M, Qin Q, Fuse S, e al. Phobalysin, a small be a-
po e- o ming oxin o Pho obac e ium damselae subsp. damselae. In ec Immun. 2015; 83: 4335–4348.
h ps://doi.o g/10.1128/IAI.00277-15 PMID: 26303391
24. Ri as AJ, Balado M, Lemos ML, Oso io CR. Syne gis ic and addi i e e ec s o ch omosomal and plas-
mid-encoded hemolysins con ibu e o hemolysis and i ulence in Pho obac e ium damselae subsp.
damselae. In ec Immun. 2013; 81: 3287–3299. h ps://doi.o g/10.1128/IAI.00155-13 PMID: 23798530
25. Vences A, Ri as AJ, Lemos ML, Husmann M, Oso io CR. Ch omosome-encoded hemolysin, phospho-
lipase, and collagenase in plasmidless isola es o Pho obac e ium damselae subsp. damselae con ib-
u e o i ulence o ish. Appl En i on Mic obiol. 2017;83.
26. Ri as AJ, Vences A, Husmann M, Lemos ML, Oso io CR. Pho obac e ium damselae subsp. damselae
majo i ulence ac o s Dly, plasmid-encoded HlyA, and ch omosome-encoded HlyA a e sec e ed ia
he ype II sec e ion sys em. In ec Immun. 2015; 83: 1246–1256. h ps://doi.o g/10.1128/IAI.02608-14
PMID: 25583529
27. Te ce i MS, Ri as AJ, Al a ez L, Noia M, Ca a F, Oso io CR. s B Regula es exp ession o he Pho o-
bac e ium damselae subsp. damselae majo i ulence ac o s damselysin, phobalysin P and phobalysin
C. F on Mic obiol. 2017; 8: 582. h ps://doi.o g/10.3389/ micb.2017.00582 PMID: 28443076
28. Langmead B, Salzbe g S. Fas gapped- ead alignmen wi h Bow ie 2. Na u e Me hods. 2012; 9: 357–
359. h ps://doi.o g/10.1038/nme h.1923 PMID: 22388286
29. Li H, Handsake B, Wysoke A, Fennell T, Ruan J, Home N, e al. The sequence alignmen /map o ma
and SAM ools. Bioin o ma ics. 2009; 25: 2078–209. h ps://doi.o g/10.1093/bioin o ma ics/b p352
PMID: 19505943
30. Ins i u e B oad. (Accessed: 2018/02/21; e sion 2.17.8). “Pica d Tools.” B oad Ins i u e, Gi Hub eposi-
o y. h p://b oadins i u e.gi hub.io/pica d/.
31. Ande s S, Pyl PT, Hube W. HTSeq—a Phy on amewo k o wo k wi h high- h oughpu sequencing
da a. Bioin o ma ics. 2015; 31: 166–169. h ps://doi.o g/10.1093/bioin o ma ics/b u638 PMID:
25260700
32. Lo e MI, Hube W, Ande s S. Mode a ed es ima ion o old change and dispe sion o RNA-seq da a
wi h DESeq2. Genome Biol. 2014; 15: 550. h ps://doi.o g/10.1186/s13059-014-0550-8 PMID:
25516281
33. Benjamini Y, Hochbe g Y. Con olling he alse disco e y a e: a p ac ical and powe ul app oach o mul-
iple es ing. J Roy S a Soc B. 1995; 57: 289–300.
34. T apnell C, Williams BA, Pe ea G, Mo aza i A, Kwan G, an Ba en MJ, e al. T ansc ip assembly and
quan i ica ion by RNA-Seq e eals unanno a ed ansc ip s and iso o m swi ching du ing cell di e en ia-
ion. Na Bio echnol. 2010; 28: 511–515. h ps://doi.o g/10.1038/nb .1621 PMID: 20436464
35. Bake -Aus in C, S ockley L, Rangdale R, Ma inez-U aza J. En i onmen al occu ence and clinical
impac o Vib io ulni icus and Vib io pa ahaemoly icus: a Eu opean pe spec i e. En i on Mic obiol
Rep. 2010; 2: 7–18. h ps://doi.o g/10.1111/j.1758-2229.2009.00096.x PMID: 23765993
RNA sequencing o Pho obac e ium damselae subsp. damselae
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0210118 Decembe 31, 2018 20 / 23
36. Gilbe JA, S eele JA, Capo aso JG, S einb u¨ck L, Reede J, Tempe on B, e al. De ining seasonal
ma ine mic obial communi y dynamics. ISME J. 2012; 6(2): 298–308. h ps://doi.o g/10.1038/ismej.
2011.107 PMID: 21850055
37. Ma in-Pla e o AM, Clea y B, Kau man K, P eheim SP, McGillicuddy DJ, Alm EJ, e al. High esolu ion
ime se ies e eals cohesi e bu sho -li ed communi ies in coas al plank on. Na Commun. 2018; 9(1):
266. h ps://doi.o g/10.1038/s41467-017-02571-4 PMID: 29348571
38. Lo e M, Teebken-Fishe D, Hose JE, Fa me JJ 3 d, Hickman FW, Fanning GR. Vib io damsela, a
ma ine bac e ium, causes skin ulce s on he damsel ish Ch omis punc ipinnis. Science. 1981; 214:
1139–1140. h ps://doi.o g/10.1126/science.214.4525.1139 PMID: 17755898
39. Fouz B, To anzo AE, Milan M, Ama o C. E idence ha wa e ansmi s he disease caused by he ish
pa hogen Pho obac e ium damselae subsp. damselae. J Appl Mic obiol. 2000; 88: 531–535. PMID:
10747234
40. Finlay BB, McFadden G. An i-immunology: e asion o he hos immune sys em by bac e ial and i al
pa hogens. Cell. 2006; 124: 767–782. h ps://doi.o g/10.1016/j.cell.2006.01.034 PMID: 16497587
41. Lew DP, Despon JP, Pe in LH, Aguado MT, Lambe PH, Wald ogel FA. Demons a ion o a local
exhaus ion o complemen componen s and o an enzyma ic deg ada ion o immunoglobulins in pleu al
empyema: a possible ac o a ou ing he pe sis ence o local bac e ial in ec ions. Clin Exp Immunol.
1980; 42: 506–514. PMID: 6908546
42. Okada K, Iida T, Ki a-Tsukamo o K, Honda T. Vib ios commonly possess wo ch omosomes. J Bac e -
iol. 2005; 187: 752–757. h ps://doi.o g/10.1128/JB.187.2.752-757.2005 PMID: 15629946
43. D yselius R, Ku okawa K, Iida T. Vib ionaceae, a e sa ile bac e ial amily wi h e olu iona ily conse ed
a iabili y. Res Mic obiol. 2007; 158: 479–486. h ps://doi.o g/10.1016/j. esmic.2007.04.007 PMID:
17590316
44. Gumpenbe ge T, Vo kapic D, Zingl FG, P essle K, Lackne S, Sepe A, Reidl J, Schild S. Nucleoside
up ake in Vib io chole ae and i s ole in he ansi ion i ness om hos o en i onmen . Mol Mic obiol.
2016; 99: 470–483 h ps://doi.o g/10.1111/mmi.13143 PMID: 26202476
45. Fouz B, To anzo AE, Biosca EG, Mazoy R, Ama o C. Role o i on in he pa hogenici y o Vib io damsela
o ish and mammals. FEMS Mic obiol Le . 1994; 121: 181–188. h ps://doi.o g/10.1111/j.1574-6968.
1994. b07097.x PMID: 7926669
46. Fouz B, Biosca EG, Ama o C. High a ini y i on-up ake sys ems in Vib io damsela: ole in he acquisi ion
o i on om ans e in. J Appl Mic obiol. 1997; 82: 157–167. PMID: 12452589
47. Rio SJ, Oso io CR, Lemos ML. Heme up ake genes in human and ish isola es o Pho obac e ium dam-
selae: exis ence o hu A pseudogenes. A ch Mic obiol. 2005; 183: 347–358. h ps://doi.o g/10.1007/
s00203-005-0779-4 PMID: 15918073
48. Puen es B, Balado M, Be mudez-C espo J, Oso io CR, Lemos ML. A p o eomic analysis o he i on
esponse o Pho obac e ium damselae subsp. damselae e eals me abolic adap a ions o i on le els
changes and no el po en ial i ulence ac o s. Ve Mic obiol. 2017; 201: 257–264. h ps://doi.o g/10.
1016/j. e mic.2017.01.040 PMID: 28284618
49. Fouz B, To anzo AE, Ma co-Noales E, Ama o C. Su i al o ish- i ulen s ains o Pho obac e ium
damselae subsp. damselae in seawa e unde s a a ion condi ions. FEMS Mic obiol Le . 1998; 168:
181–186. h ps://doi.o g/10.1111/j.1574-6968.1998. b13271.x PMID: 9835027
50. Von Ho en G, Neuki ch C, Meyenbu g M, Schmid S, Vences A, Oso io CR, e al. Cy o oxin- and che-
mo axis-genes coope a e o p omo e adhesion o Pho obac e ium damselae subsp. damselae. F on
Mic obiol. 2018; h ps://doi.o g/10.3389/ micb.2018.02996
51. O’Toole R, Mil on DL, Wol -Wa z H. Chemo ac ic mo ili y is equi ed o in asion o he hos by he ish
pa hogen Vib io anguilla um. Mol Mic obiol. 1996; 19: 625–637. PMID: 8830252
52. DeLoney-Ma ino CR, Wol e AJ, Visick KL. Chemoa ac ion o Vib io ische i o se ine, nucleosides,
and N-ace ylneu aminic acid, a componen o squid ligh -o gan mucus. Appl En i on Mic obiol. 2003;
69: 7527–7530. h ps://doi.o g/10.1128/AEM.69.12.7527-7530.2003 PMID: 14660408
53. Ou en FW, Djaman O, S o z G. A su ope on equi emen o Fe-S clus e assembly du ing i on s a a-
ion in Esche ichia coli. Mol Mic obiol. 2004; 52: 861–872. h ps://doi.o g/10.1111/j.1365-2958.2004.
04025.x PMID: 15101990
54. Haghjoo E, Galan JE. Iden i ica ion o a ansc ip ional egula o ha con ols in acellula gene exp es-
sion in Salmonella yphi. Mol Mic obiol. 2007; 64: 1549–1561. h ps://doi.o g/10.1111/j.1365-2958.
2007.05754.x PMID: 17555437
55. Mo is CR, G assel CL, Redman JC, Sahl JW, Ba y EM, Rasko DA. Cha ac e iza ion o in acellula
g ow h egula o icgR by u ilizing ansc ip omics o iden i y media o s o pa hogenesis in Shigella lex-
ne i. In ec Immun. 2013; 81: 3068–3076. h ps://doi.o g/10.1128/IAI.00537-13 PMID: 23753632
RNA sequencing o Pho obac e ium damselae subsp. damselae
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0210118 Decembe 31, 2018 21 / 23
56. Ayala JC, Wang H, Sil a AJ, Beni ez JA. Rep ession by H-NS o genes equi ed o he biosyn hesis o
he Vib io chole ae bio ilm ma ix is modula ed by he second messenge cyclic diguanylic acid. Mol
Mic obiol. 2015; 97: 630–645. h ps://doi.o g/10.1111/mmi.13058 PMID: 25982817
57. Pu sley BR, Maiden MM, Hsieh M-L, Fe nandez NL, Se e in GB, Wa e s CM. Cyclic di-GMP Regula es
T oY in Vib io chole ae o con ol mo ili y by bo h ansc ip ional and pos ansc ip ional mechanisms. J
Bac e iol. 2018;200.
58. Fe nandez NL, S i as a a D, Ngouajio AL, Wa e s CM. Cyclic di-GMP Posi i ely Regula es DNA
Repai in Vib io chole ae. J Bac e iol. 2018;200.
59. Mendez J, Cascales D, Ga cı
´a-To ico AI, Guija o JA. Tempe a u e-dependen gene exp ession in
Ye sinia ucke y: acking speci ic genes by bioluminescence du ing in i o coloniza ion. F on Mic o-
biol. 2018; 9: 1098. h ps://doi.o g/10.3389/ micb.2018.01098 PMID: 29887855
60. Hinchli e P, Symmons MF, Hughes C, Ko onakis V. S uc u e and ope a ion o bac e ial ipa i e
pumps. Annu Re Mic obiol. 2013; 67: 221–242. h ps://doi.o g/10.1146/annu e -mic o-092412-
155718 PMID: 23808339
61. Roig F, Ama o C. Plasmid di e si y in Vib io ulni icus bio ypes. Mic obiology. 2009; 155: 489–497.
h ps://doi.o g/10.1099/mic.0.023424-0 PMID: 19202097
62. Ama o C, Sanjua
´n E, Fouz B, Pajuelo D, Lee CT, Ho LI, e al. The ish pa hogen Vib io ulni icus Bio-
ype 2: epidemiology, phylogeny, and i ulence ac o s in ol ed in wa m-wa e ib iosis. Mic obiol
Spec . 2015; 3.
63. Dupe huy M, Schmi P, Ga zon E, Ca o A, Rosa RD, Le Roux F, e al. Use o OmpU po ins o a ach-
men and in asion o C assos ea gigas immune cells by he oys e pa hogen Vib io splendidus. P oc
Na l Acad Sci U S A. 2011; 108: 2993–2998. h ps://doi.o g/10.1073/pnas.1015326108 PMID:
21282662
64. Goo SY, Lee HJ, Kim WH, Han KL, Pa k DK, Lee HJ, e al. Iden i ica ion o OmpU o Vib io ulni icus as
a ib onec in-binding p o ein and i s ole in bac e ial pa hogenesis. In ec Immun. 2006; 74: 5586–5594.
h ps://doi.o g/10.1128/IAI.00171-06 PMID: 16988233
65. Nyholm SV, S ewa JJ, Ruby EG, McFall-Ngai MJ. Recogni ion be ween symbio ic Vib io ische i and
he haemocy es o Eup ymna scolopes. En i on Mic obiol. 2009; 11: 483–493. h ps://doi.o g/10.1111/
j.1462-2920.2008.01788.x PMID: 19196278
66. Pajuelo D, Lee C-T, Roig FJ, Ho L-I, Ama o C. No el hos -speci ic i on acquisi ion sys em in he zoo-
no ic pa hogen Vib io ulni icus. En i on Mic obiol. 2015; 17: 2076–2089. h ps://doi.o g/10.1111/1462-
2920.12782 PMID: 25630302
67. Shneide MM, Bu h SA, Ho BT, Basle M, Mekalanos JJ, Leiman PG. PAAR- epea p o eins sha pen
and di e si y he ype VI sec e ion sys em spike. Na u e. 2013; 500: 350–353. h ps://doi.o g/10.1038/
na u e12453 PMID: 23925114
68. Zhang D, de Souza RF, Anan ha aman V, Iye LM, A a ind L. Polymo phic oxin sys ems: Comp ehen-
si e cha ac e iza ion o a icking modes, p ocessing, mechanisms o ac ion, immuni y and ecology
using compa a i e genomics. Biol Di ec . 2012; 7: 18. h ps://doi.o g/10.1186/1745-6150-7-18 PMID:
22731697
69. Ma ı
´nez-U aza J, T iñanes J, Aban o M, Lozano-Leo
´n A, Llo o-Taboada J, Ga cia-Campello M, e al.
Epidemic dynamics o Vib io pa ahaemoly icus illness in a ho spo o disease eme gence, Galicia,
Spain. Eme g In ec Dis. 2018; 24: 852–859. h ps://doi.o g/10.3201/eid2405.171700 PMID: 29664388
70. Pensinge DA, Boldon KM, Chen GY, Vincen WJB, She man K, Xiong M, e al. The Lis e ia monocy o-
genes PASTA kinase P kA and i s subs a e Y cK a e equi ed o cell wall homeos asis, me abolism,
and i ulence. PLoS Pa hog. 2016; 12: e1006001. h ps://doi.o g/10.1371/jou nal.ppa .1006001 PMID:
27806131
71. Chen YH, Lu CW, Shyu YT, Lin SS. Re ealing he saline adap a ion s a egies o he halophilic bac e-
ium Halomonas beimenensis h ough high- h oughpu Omics and ansposon mu agenesis
app oaches. Sci Rep. 2017; 7: 13037. h ps://doi.o g/10.1038/s41598-017-13450-9 PMID: 29026163
72. Liu X, Luo Y, Li Z, Wei G. Func ional analysis o P kA—a pu a i e se ine p o ein kinase om Meso hizo-
bium alhagi CCNWXJ12-2—in s ess esis ance. BMC Mic obiol. 2016; 16: 1–8. h ps://doi.o g/10.
1186/s12866-015-0617-z
73. Beall B, Mo an CPJ. Cloning and cha ac e iza ion o spoVR, a gene om Bacillus sub ilis in ol ed in
spo e co ex o ma ion. J Bac e iol. 1994; 176: 2003–2012. PMID: 8144469
74. G oiche KH, Fi ek BA, Fujimo o DF, Bayles KW. The S aphylococcus au eus l gAB ope on modula es
mu ein hyd olase ac i i y and penicillin ole ance. J Bac e iol. 2000; 182: 1794–1801. PMID: 10714982
75. Yao J, Rock CO. Phospha idic acid syn hesis in bac e ia. Biochim Biophys Ac a. 2013; 1831: 495–502.
h ps://doi.o g/10.1016/j.bbalip.2012.08.018 PMID: 22981714
RNA sequencing o Pho obac e ium damselae subsp. damselae
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0210118 Decembe 31, 2018 22 / 23
76. Mansilla MC, Cybulski LE, Albanesi D, de Mendoza D. Con ol o memb ane lipid luidi y by molecula
he mosenso s. J Bac e iol. 2004; 186: 6681–6688. h ps://doi.o g/10.1128/JB.186.20.6681-6688.2004
PMID: 15466018
77. Sinensky M. Homeo iscous adap a ion—A homeos a ic p ocess ha egula es he iscosi y o mem-
b ane lipids in Esche ichia coli. P oc Na l Acad Sci U S A. 1974; 71: 522–525. PMID: 4360948
78. B aiban M, Gilo P, Con en J. The ATP binding casse e (ABC) anspo sys ems o Mycobac e ium
ube culosis. FEMS Mic obiol Re . 2000; 24: 449–467. h ps://doi.o g/10.1111/j.1574-6976.2000.
b00550.x PMID: 10978546
79. Liu W, Huang L, Su Y, Qin Y, Zhao L, Yan Q. Con ibu ions o he oligopep ide pe meases in mul is ep
o Vib io alginoly icus pa hogenesis. Mic obiology Open. 2017; 6(5): e00511.
80. Rubio LM, Ludden PW. Biosyn hesis o he i on-molybdenum co ac o o ni ogenase. Annu Re Mic o-
biol. 2008; 62: 93–111. h ps://doi.o g/10.1146/annu e .mic o.62.081307.162737 PMID: 18429691
81. Tibbles BJ, Rawlings DE. Cha ac e iza ion o ni ogen- ixing bac e ia om a empe a e sal ma sh
lagoon, including isola es ha p oduce e hane om ace ylene. Mic ob Ecol. 1994; 27: 65–80. h ps://
doi.o g/10.1007/BF00170115 PMID: 24190169
82. Balado M, Puen es B, Coucei o L, Fuen es-Mon e e de JC, Rod iguez J, Oso io CR, e al. Sec e ed Ci -
a e Se es as I on Ca ie o he Ma ine Pa hogen Pho obac e ium damselae subsp damselae. F on
Cell In ec Mic obiol. 2017; 7: 361. h ps://doi.o g/10.3389/ cimb.2017.00361 PMID: 28848719
83. Ma Y, Wang Q, Gao X, Zhang Y. Biosyn hesis and up ake o glycine be aine as cold-s ess esponse o
low empe a u e in ish pa hogen Vib io anguilla um. J Mic obiol. 2017; 55: 44–55. h ps://doi.o g/10.
1007/s12275-017-6370-2 PMID: 28035596
84. Townsley L, Sison Mangus MP, Mehic S, Yildiz FH. Response o Vib io chole ae o low- empe a u e
shi s: CspV egula ion o Type VI sec e ion, bio ilm o ma ion, and associa ion wi h zooplank on. Appl
En i on Mic obiol. 2016; 82: 4441–4452. h ps://doi.o g/10.1128/AEM.00807-16 PMID: 27208110
85. Yang L, Zhou D, Liu X, Han H, Zhan L, Guo Z, Zhang L, Qin C, Wong HC, Yang R. Cold-induced gene
exp ession p o iles o Vib io pa ahaemoly icus: a ime-cou se analysis. FEMS Mic obiol Le . 2009; 291:
50–58. h ps://doi.o g/10.1111/j.1574-6968.2008.01434.x PMID: 19054071
86. Webe H, Polen T, Heu eling J, Wendisch VF, Hengge R. Genome-wide analysis o he gene al s ess
esponse ne wo k in Esche ichia coli: sigmaS-dependen genes, p omo e s, and sigma ac o selec i -
i y. J Bac e iol. 2005; 187: 1591–1603. h ps://doi.o g/10.1128/JB.187.5.1591-1603.2005 PMID:
15716429
87. Chang S-C, Lee C-Y. OpaR and RpoS a e posi i e egula o s o a i ulence ac o P A in Vib io pa a-
haemoly icus. Mic obiology. 2018; 164: 221–231. h ps://doi.o g/10.1099/mic.0.000591 PMID:
29256853
88. Wu m P, Tu z S, Mu sam B, Vo kapic D, Heyne B, G abne C, Kleewein K, Halscheid A, Schild S, Reidl
J. S ingen ac o and p o eolysis con ol o sigma ac o RpoS exp ession in Vib io chole ae. In J Med
Mic obiol. 2017; 307: 154–165. h ps://doi.o g/10.1016/j.ijmm.2017.01.006 PMID: 28228329
89. Zhao B, Hou y WA. Acid s ess esponse in en e opa hogenic gammap o eobac e ia: an ap i ude o
su i al. Biochem. Cell Biol. 2010; 88: 301–314. h ps://doi.o g/10.1139/o09-182 PMID: 20453931
90. Me ell DS, Camilli A. Regula ion o Vib io chole ae genes equi ed o acid ole ance by a membe o
he “ToxR-like” amily o ansc ip ional egula o s. J Bac e iol. 2000; 182: 5342–5350. PMID: 10986235
91. B o N, Weissbach H. Pep ide me hionine sul oxide educ ase: biochemis y and physiological ole. Bio-
polyme s. 2000; 55: 288–296. h ps://doi.o g/10.1002/1097-0282(2000)55:4<288::AID-BIP1002>3.0.
CO;2-M PMID: 11169920
92. B eme H, Dennis PP. Modula ion o chemical composi ion and o he pa ame e s o he cell by g ow h
a e. In: Neidha d F, Cu iss R, Ing aham JL, Lin ECC, Low KB, Magasanik B, e al. edi o s. Esche ichia
coli and Salmonella: Cellula and Molecula Biology. Washing on, DC: Ame ican Socie y o Mic obiol-
ogy P ess; 1996. pp. 1553–1569.
93. K ege AS. Cy oly ic ac i i y and i ulence o Vib io damsela. In ec Immun. 1984; 44: 326–331. PMID:
6715036
94. Konkel ME, Tilly K. Tempe a u e- egula ed exp ession o bac e ial i ulence genes. Mic obes In ec .
2000; 2: 157–166. PMID: 10742688
95. Johansson J, Cossa P. RNA-media ed con ol o i ulence gene exp ession in bac e ial pa hogens.
T ends Mic obiol. 2003; 11: 280–285. PMID: 12823945
96. S einmann R, De sch P. The mosensing o adjus bac e ial i ulence in a luc ua ing en i onmen .
Fu u e Mic obiol. 2013; 8: 85–105. h ps://doi.o g/10.2217/ mb.12.129 PMID: 23252495
97. Guija o JA, Cascales D, Ga cı
´a-To ico AI, Ga cı
´a-Domı
´nguez M, Me
´ndez J. Tempe a u e-dependen
exp ession o i ulence genes in ish-pa hogenic bac e ia. F on Mic obiol. 2015; 6: 700. h ps://doi.o g/
10.3389/ micb.2015.00700 PMID: 26217329
RNA sequencing o Pho obac e ium damselae subsp. damselae
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0210118 Decembe 31, 2018 23 / 23
