micb-10-02104 Sep embe 10, 2019 Time: 15:59 # 1
ORIGINAL RESEARCH
published: 11 Sep embe 2019
doi: 10.3389/ micb.2019.02104
Edi ed by:
And é An unes,
Edge Hill Uni e si y, Uni ed Kingdom
Re iewed by:
Ma in W. Hahn,
Uni e si y o Innsb uck, Aus ia
Tomoo Sawabe,
Hokkaido Uni e si y, Japan
*Co espondence:
An onio Ven osa
[email p o ec ed]
Special y sec ion:
This a icle was submi ed o
Ex eme Mic obiology,
a sec ion o he jou nal
F on ie s in Mic obiology
Recei ed: 09 May 2019
Accep ed: 27 Augus 2019
Published: 11 Sep embe 2019
Ci a ion:
de la Haba RR,
López-He moso C, Sánchez-Po o C,
Kons an inidis KT and Ven osa A
(2019) Compa a i e Genomics
and Phylogenomic Analysis o he
Genus Salini ib io.
F on . Mic obiol. 10:2104.
doi: 10.3389/ micb.2019.02104
Compa a i e Genomics and
Phylogenomic Analysis o he Genus
Salini ib io
Ra ael R. de la Haba1, Cla a López-He moso1, C is ina Sánchez-Po o1,
Kons an inos T. Kons an inidis2and An onio Ven osa1*
1Depa men o Mic obiology and Pa asi ology, Facul y o Pha macy, Uni e si y o Se ille, Se ille, Spain, 2School o Ci il
and En i onmen al Enginee ing, Geo gia Ins i u e o Technology, A lan a, GA, Uni ed S a es
In he genomic e a phylogene ic ela ionship among p oka yo es can be in e ed om
he co e o hologous genes (OGs) o p o eins in o de o elucida e hei e olu iona y
his o y and cu en axonomy should bene i s o ha . The genus Salini ib io belongs o
he amily Vib ionaceae and cu en ly includes only i e halophilic species, in spi e he
ac ha new s ains a e e y equen ly isola ed om hype saline en i onmen s. Species
belonging o his genus ha e unde gone se e al eclassi ica ions and, mo eo e , he e
a e many s ains o Salini ib io wi h a ailable genomes which ha e no been a ilia ed
o he exis ing species o ha e been w ongly designa ed. The e o e, a phylogene ic
s udy using he a ailable genomic in o ma ion is necessa y o cla i y he ela ionships
o exis ing s ains wi hin his genus and o e iew hei axonomic a ilia ion. Fo ha
pu pose, we ha e also sequenced he i s comple e genome o a Salini ib io species,
Salini ib io kushne i AL184T, which was employed as a e e ence o o de he con igs
o he d a genomes o he ype s ains o he cu en species o his genus, as well as
o pe o m a compa a i e analysis wi h all he o he a ailable Salini ib io sp. genomes.
The genome o S. kushne i AL184Twas assembled in wo ci cula ch omosomes (wi h
sizes o 2.84 Mb and 0.60 Mb, espec i ely), as ypically occu s in membe s o he
amily Vib ionaceae, wi h nine comple e ibosomal ope ons, which migh explain he
as g owing a e o salini ib ios cul u ed unde labo a o y condi ions. Syn eny analysis
among he ype s ains o he genus e ealed a high le el o genomic conse a ion
in bo h ch omosomes, which allow us o hypo hesize a slow specia ion p ocess o
homogeniza ion e en s aking place in his g oup o mic oo ganisms o be es ed
expe imen ally in he u u e. Phylogenomic and o hologous a e age nucleo ide iden i y
(O hoANI)/a e age amino acid iden i y (AAI) analyses also e idenced he ele a ed le el
o gene ic ela edness wi hin membe s o his genus and allowed o g oup all he
Salini ib io s ains wi h a ailable genomes in se en sepa a ed species. Genome-scale
a ibu e s udy o he salini ib ios iden i ied ai s ela ed o pola lagellum, acul a i ely
anae obic g ow h and osmo ic esponse, in acco dance o he pheno ypic ea u es
desc ibed o species o his genus.
Keywo ds: Salini ib io,Salini ib io kushne i, comple e genome, phylogenomics, genomics, syn eny, halophilic
bac e ia, hype saline en i onmen s
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de la Haba e al. Compa a i e Genomics o he Genus Salini ib io
INTRODUCTION
Fi s ly desc ibed by Mellado e al. (1996) o accommoda e
he species Vib io cos icola (Smi h, 1938), membe s o he
genus Salini ib io, belonging o he amily Vib ionaceae, class
Gammap o eobac e ia, ha e been isola ed om di e se habi a s,
such as aqua ic hype saline sys ems, b ines, sal ed mea s,
and saline soils (Cham oensaks i e al., 2009;Go i i e al.,
2014;Ven osa, 2015;López-He moso e al., 2018a,b). The
axonomic a ilia ion o he ype and o he 70 ep esen a i e
s ains o his genus has been ecen ly e alua ed using a
Mul i Locus Sequence Analysis (MLSA) (López-He moso
e al., 2017b, 2018a). Fu he mo e, d a genomic sequences
o he ype s ains and only a ew (14) ep esen a i e s ains
has also been used o delinea e Salini ib io species (López-
He moso e al., 2018a,b). As a esul o he a o emen ioned
s udies, he genus Salini ib io is cu en ly composed o
i e species: S. cos icola [con aining wo subspecies, i.e.,
S. cos icola subsp. cos icola (Ga cia e al., 1987;Mellado
e al., 1996) and S. cos icola subsp. alcaliphilus (Romano
e al., 2005)], Salini ib io kushne i (López-He moso e al.,
2018b), S. p o eoly icus [which also includes he o me
S. cos icola subsp. allismo is (Amoozega e al., 2008b;
López-He moso e al., 2018a)], S. sha mensis (Romano e al.,
2011), and Salini ib io siamensis (Cham oensaks i e al.,
2009). Addi ionally, he species “Salini ib io socompensis”
has also been p oposed o include h ee Salini ib io
s ains (Go i i e al., 2014), bu his name has no been
alidly published.
Al hough o y-six Salini ib io genomes a e a ailable in
GenBank da abase, all o hem ep esen d a genomes and
no comple e genome p ojec s ha e been conduc ed wi hin
his genus. Besides, only wo s udies dealing wi h genome
sequence analysis o salini ib ios ha e been published
(Go i i e al., 2014;López-He moso e al., 2017a), bu
hose we e ocused on a ew genomes ( he o me ) o he
analysis only p o ided genome s a is ics ( he la e ), and
in bo h cases using d a genome sequences. On he o he
hand, almos hal o he a ailable Salini ib io genomes a e
no classi ied a he species le el o misnamed (w ongly
designa ed). The ela ionship o Salini ib io species is
no o ally clea o s able acco ding o p e ious s udies
because phylogene ic ees we e based on MLSA o only
up o eigh housekeeping genes (Go i i e al., 2014;López-
He moso e al., 2017b) o he phylogenomic analysis was
conduc ed only wi h a ew o he Salini ib io genomes
(López-He moso e al., 2018a,b).
In his s udy, we cla i y he phylogene ic ela ionships
o exis ing genomes and species and e iew he axonomic
a ilia ion o he s ains included wi hin he genus
Salini ib io using a wide phylogenomic app oach. This
wo k also epo s he i s comple e genome o a species
o he genus Salini ib io,S. kushne i, which was used
as a e e ence o o de he con igs o he d a genomes
o he ype s ains wi hin his genus, as well as o
pe o m a compa a i e analysis wi h all he o he a ailable
Salini ib io genomes.
MATERIALS AND METHODS
Cul u e Condi ions and Genomic DNA
Ex ac ion
The s ain S. kushne i AL184Twas ob ained om ou cul u e
collec ion whe e i was s o ed a −80◦C and i was cul u ed
in liquid SW b o h (whose composi ion in g l−1is: NaCl,
58.5; MgCl2·6H2O, 9.75; MgSO4·7H2O, 15.25; CaCl2, 0.25; KCl,
1.5; NaHCO3, 0.05; NaB , 0.175; and yeas ex ac , 5.0) a
37◦C wi h he pH adjus ed be ween 7.2 and 7.4, acco ding o
López-He moso e al. (2018b). High-quali y genomic DNA was
ex ac ed using he QIAmp DNA Mini Ki (Qiagen) ollowing he
manu ac u e ’s ins uc ions.
Genome Sequencing, Assembly, and
Anno a ion
A single molecule eal- ime sequencing app oach was
accomplished by using PacBio echnologies. Fo ha pu pose, a
PacBio lib a y wi h 10 kbp inse size was cons uc ed and a 350×
sequencing dep h was achie ed. Addi ionally, whole genome
sho gun eads ob ained om an Illumina HiSeq (2 ×100-bp
pai ed-end eads) de ice in an ea lie s udy (López-He moso
e al., 2017a) we e used o ca y ou a hyb id assembly using
SPAdes .3.13.0 (Banke ich e al., 2012). Subsequen ly, he inal
assembly was achie ed using only il e ed by quali y PacBio
eads ( ead leng h a e imming ≥5000 n , polyme ase ead
quali y ≥0.80, and polyme ase ead leng h ≥100) using an
O e lap-Layou -Consensus algo i hm as implemen ed in he
HGAP .2 pipeline (Chin e al., 2013). Do plo s o check o
ci cula i y a con ig ends we e d awn using Gepa d .1.40
so wa e (K umsiek e al., 2007). The esul ing genome was
ci cula ized using oAmos and minimus2 (Somme e al., 2007),
ollowed by Ci cla o (Hun e al., 2015) ools.
All he o y-six Salini ib io d a genomes a ailable in
GenBank da abase we e eco e ed, wi h he excep ion o ha o
S. cos icola subsp. cos icola ATCC 33508T(assembly accession
no. GCA_000390145.1), which p esen ed a suspicious leng h
(4.78 Mb) and is excluded om Re Seq da abase due o i s
low quali y sequence, and ha o S. kushne i AL184T(assembly
accession no. GCA_001995845.1), which was eplaced by he
comple e genome o his s ain achie ed in his s udy (Table 1).
Au oma ic anno a ion o hose d a genomes oge he o he
comple e genome o s ain S. kushne i AL184Tachie ed in his
s udy was pe o med using RAST (O e beek e al., 2014) and
KAAS-KEEG (Mo iya e al., 2007). RNA genes we e de e mined
by means o RNAmme so wa e (Lagesen e al., 2007).
Syn eny Analysis
Mau e genome alignmen p og am (Da ling e al., 2004) was
employed o add ess he issue o genome ea angemen s and
con ig o de ing o he d a genomes by pai wise compa ison
using he comple e genome o S. kushne i AL184Tas a e e ence.
P og essi e Mau e so wa e (Da ling e al., 2010) was used
o mul iple genome alignmen o he p e iously o de ed d a
genomes oge he o he comple e genome o he ype s ains
wi hin he genus in o de o ind locally collinea blocks
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de la Haba e al. Compa a i e Genomics o he Genus Salini ib io
TABLE 1 | Salini ib io genomes a ailable in GenBank da abase used in his s udy, including hei basic s a is ical in o ma ion.
New p oposed s ain designa ion (o iginal
s ain label)
Assembly no. Comple eness
le el
Size (Mb) GC (mol%) Sca olds CDS
Salini ib io cos icola subsp. cos icola LMG 11651TGCA_000565345.1 Con ig 3.38 49.3 202 2332
Salini ib io cos icola subsp. alcaliphilus DSM
16359T
GCA_001996185.1 Con ig 3.38 49.3 248 2949
Salini ib io cos icola AR640 (Salini ib io sp. AR640) GCA_001995405.1 Sca old 3.26 49.3 60 2873
Salini ib io cos icola AR647 (Salini ib io sp. AR647) GCA_001995415.1 Con ig 3.28 49.3 22 2920
Salini ib io cos icola IB643 (Salini ib io sp. IB643) GCA_001996105.1 Con ig 3.12 49.4 91 2739
Salini ib io cos icola MA351 (Salini ib io sp. MA351) GCA_001996065.1 Con ig 3.31 49.4 59 2923
Salini ib io cos icola MA427 (Salini ib io sp. MA427) GCA_001996115.1 Sca old 3.26 49.5 513 2739
Salini ib io cos icola MA440 (Salini ib io sp. MA440) GCA_001996265.1 Con ig 3.42 49.3 102 2996
Salini ib io cos icola MA607 (Salini ib io sp. MA607) GCA_001996245.1 Con ig 3.35 49.2 68 2946
Salini ib io kushne i AL184TCP040021, CP040022 Comple e 3.44 50.7 1 3055
Salini ib io kushne i BNH (Salini ib io sp. BNH) GCA_001722105.1 Con ig 3.48 50.5 45 3073
Salini ib io kushne i HTSP (Salini ib io sp. HTSP) GCA_003390965.1 Sca old 3.39 50.6 121 3007
Salini ib io kushne i IB282 (Salini ib io sp. IB282) GCA_001995685.1 Con ig 3.23 50.7 157 2836
Salini ib io kushne i IB560 GCA_001995915.1 Con ig 3.49 50.5 65 3081
Salini ib io kushne i IB563 GCA_001995785.1 Sca old 3.48 50.5 173 3031
Salini ib io kushne i IC202 GCA_001995805.1 Con ig 3.61 50.5 111 3160
Salini ib io kushne i IC317 GCA_001995875.1 Sca old 3.28 50.9 236 2881
Salini ib io kushne i MA421 GCA_001995635.1 Sca old 3.54 50.4 70 3112
Salini ib io kushne i ML277 GCA_001995865.1 Con ig 3.45 50.4 73 3036
Salini ib io kushne i ML318 GCA_001995885.1 Sca old 3.36 50.7 96 2978
Salini ib io kushne i ML323 (Salini ib io sp. ML323) GCA_001995745.1 Con ig 3.23 50.7 63 2865
Salini ib io kushne i ML328A GCA_001995725.1 Con ig 3.39 50.6 70 3001
Salini ib io kushne i ML331 GCA_001995995.1 Con ig 3.55 50.5 112 3143
Salini ib io kushne i PRJEB21454 (Salini ib io
cos icola PRJEB21454)
GCA_900188555.1 Sca old 3.32 50.5 23 2952
Salini ib io p o eoly icus DSM 19052TGCA_001996165.1 Con ig 3.6 49.8 51 3234
Salini ib io p o eoly icus DSM 8285 GCA_001996225.1 Con ig 3.5 49.9 95 3130
Salini ib io p o eoly icus DV (Salini ib io sp. DV) GCA_001722075.1 Con ig 3.71 49.8 145 3268
Salini ib io p o eoly icus IB574 GCA_001996205.1 Sca old 3.61 49.8 69 3187
Salini ib io p o eoly icus IB872 GCA_001995345.1 Sca old 3.64 49.8 102 3201
Salini ib io p o eoly icus PR5 GCA_001996085.1 Con ig 3.46 50 105 3035
Salini ib io p o eoly icus PR919 GCA_001995355.1 Con ig 3.49 49.9 176 3076
Salini ib io p o eoly icus PR932 GCA_001996125.1 Sca old 3.5 49.9 74 3085
Salini ib io p o eoly icus YCSC6 (Salini ib io sp.
YCSC6)
GCA_002369825.1 Con ig 3.71 49.8 2 3262
Salini ib io sha mensis DSM 18182TGCA_001995985.1 Con ig 3.33 50.3 40 2944
Salini ib io siamensis JCM 14472TGCA_001996005.1 Con ig 3.44 50.4 61 3025
Salini ib io siamensis IB868 (Salini ib io sp. IB868) GCA_001995655.1 Con ig 3.44 50.5 30 3049
Salini ib io siamensis IB870 (Salini ib io sp. IB870) GCA_001995945.1 Con ig 3.44 50.5 42 3036
Salini ib io siamensis KP-1 (Salini ib io sp. KP-1) GCA_000968645.1 Con ig 3.5 50.5 49 3110
Salini ib io siamensis ML198 (Salini ib io sp.
ML198)
GCA_001996015.1 Con ig 3.43 50.5 87 3015
Salini ib io siamensis ML290 (Salini ib io sp.
ML290)
GCA_001995695.1 Sca old 3.52 50.3 36 3128
Salini ib io siamensis PR6 (Salini ib io sp. PR6) GCA_001995955.1 Con ig 3.44 50.4 49 3055
“Salini ib io socompensis” S10B GCA_000565325.1 Con ig 3.35 49.5 252 2310
“Salini ib io socompensis” S34 GCA_000513735.1 Con ig 3.33 49.4 334 ND
“Salini ib io socompensis” S35 GCA_000513715.1 Con ig 3.41 49.5 270 2311
Salini ib io sp. ES.052 GCA_900141775.1 Con ig 3.57 49.3 6 3146
ND, no de e mined.
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de la Haba e al. Compa a i e Genomics o he Genus Salini ib io
(LCBs). Syn eny among di e en genomes was de e mined by
measu ing he alignmen leng h o all he LCBs sha ed be ween
he genomes being compa ed.
Ci cula plo s showing LCBs and links be ween Salini ib io
genomes we e d awn wi h he use o Ci cos .0.69, a ci cula
isualiza ion so wa e (K zywinski e al., 2009). Fu he mo e, his
p og am was u ilized o ep esen he la ge and small ci cula
ch omosomes o S. kushne i AL184T.
Phylogenomic Analysis
All p edic ed p o ein-coding genes and p o eins anno a ed
om each a ailable genome we e sea ched using an all- s.-
all BLAST compa ison using he En eomics collec ion ools
(Rod iguez-R and Kons an inidis, 2016). This analysis allows
us o de ec sha ed ecip ocal bes ma ches (desc ibed as
hose wi h equal o abo e 70% nucleo ide iden i y o 40%
amino acid iden i y) in all pai wise genome compa isons
(co e OGs o p o eins) o he 45 Salini ib io s ains unde
s udy. The single copy co e genes and p o eins, espec i ely,
we e indi idually aligned using MUSCLE (Edga , 2004).
The esul ing nucleo ide and amino acids alignmen s we e
au oma ically immed using imAl .1.4 on “gappyou ” mode
(Capella-Gu ié ez e al., 2009) and, subsequen ly, conca ena ed
o c ea e co e-genome and co e-p o eome alignmen s, and
he phylogenomic ees we e econs uc ed by maximum-
likelihood me hod wi h Fas T eeMP .2.1.8 (P ice e al., 2010)
whe e he b anch suppo was es ima ed by means o he
Shimodai a-Hasegawa es (Shimodai a and Hasegawa, 1999;
Goldman e al., 2000).
Calcula ion o he o hologous a e age nucleo ide iden i y
(O hoANI) among he s udied genomes was accomplished
employing USEARCH 8.1.1861 as implemen ed in O hoANIu
ool (Yoon e al., 2017). When genome pai compa isons
showed less han 80% ANI alues such genomes ha e di e gen
oo much o use nucleo ide le el sea ch and some genes
migh be missing; hus, a e age amino acid iden i y (AAI)
was also de e mined. Mean AAI alues o each genome
pai we e calcula ed using ecip ocal bes hi s ( wo-way AAI)
wi h he app op ia e sc ip in he En eomics collec ion ools
(Rod iguez-R and Kons an inidis, 2016).
Me agenomic Analysis and F agmen
Rec ui men Plo s
To de ec pu a i e no el Salini ib io species using cul u e-
independen app oaches, ou 16S RNA gene amplicon da ase s
epo ing he p esence o salini ib ios in hei espec i e
en i onmen s (Tka c e al., 2011;Zhang e al., 2016, 2017;
C isle e al., 2019) we e ob ained om GenBank and NCBI’s
Sequence Read A chi e da abases (accession no. FN823320-
FN824096, SRP072906, SRP090542, SRP089997, and SRP090529)
o p o ided by he au ho s. Fo hose amplicon da a, ead quali y
il e was pe o med using P inseq (Schmiede and Edwa ds,
2011), and chime a de ec ion and il e ing, OTU’s picking (a 97%
simila i y clus e ing alue), ep esen a i e picking o each OTUs,
and axonomy assignmen we e achie ed using he so wa e
QIIME . 1.9.0 (Capo aso e al., 2010).
Abundance es ima ion o Salini b io ype s ains and
close ela i es in se e al hype saline en i onmen s was
ca ied ou by means o agmen ec ui men using sho gun
me agenomic da abases (Supplemen a y Table 1). To a oid
analysis bias, con igs o each o he genomes we e conca ena ed
and, subsequen ly, he RNA gene sequences we e masked.
Blas n sea ch (wi h he ollowing pa ame e s: leng h o he
alignmen ≥30 n , simila i y >95%, E alue ≤1e-5) was
employed o map he me agenomic eads (p e iously il e ed
o assess hei quali y) agains each genome. The op-bes -
hi eco e ed a e Blas n sea ch was used o cons uc he
ec ui men plo s.
RESULTS AND DISCUSSION
Comple e Genome Sequencing o
Salini ib io kushne i AL184T
The species S. kushne i was p oposed based on 10 isola es, wi h
s ain AL184Tdesigna ed as he ype s ain (López-He moso
e al., 2018b). In despi e o he ac ha s ains belonging
o he genus Salini ib io a e commonly isola ed due o hei
as and easy g ow h on egula labo a o y media, only a ew
species wi hin his genus ha e been desc ibed so a . The
ela i e low numbe o species is in spi e he ac ha di e en
media and cul u e condi ions ( empe a u e, pH, salini y, and
ae obic/anae obic g ow h) we e used o e he ime o isola e
new Salini ib io s ains and o a emp o desc ibe new species
(Huang e al., 2000;Sánchez-Po o e al., 2003;Ca on e al., 2004;
Romano e al., 2005, 2011;Yeon e al., 2005;Amoozega e al.,
2008a,b;Zhu e al., 2008;Cham oensaks i e al., 2009;Xiao e al.,
2009;Al-Mailem e al., 2014;Go i i e al., 2014;Asheng oph,
2017;López-He moso e al., 2017b, 2018b;Le and Yang, 2018). In
any case, he e migh be s ill cul u al biases. The e o e, al hough
he d a genome o he s ain AL184Twas al eady published
elsewhe e (López-He moso e al., 2017a), gi en he po en ial
in e es ha his new species migh ha e o un eil he specia ion
p ocesses wi hin his genus and i s ecological ole, he comple e
genome sequence o s ain AL184Twas ob ained in his s udy.
Fi s ly, a hyb id assembly by using PacBio and Illumina
eads was pe o med wi h he aim o accu a ely es ima ing he
genome size, which we de e mined o be 3,436,949 bp. This
esul was equi ed o achie e he inal assembly based on he
PacBio eads. Acco ding o PacBio ecommenda ions and o
Chin e al. (2013), he PacBio-only de no o assembly is p e e ed
when i is possible o ge a leas 50X co e age. Fo his s ain,
he sequencing dep h ob ained was 350×, which mo i a ed he
choice o a PacBio-only based assembly s a egy. No a single
con ig, bu wo we e ob ained a e he assembly, which was
expec ed since o he membe s o he amily Vib ionaceae ha e
been epo ed o con ain wo ch omosomes (Dikow and Smi h,
2013;Be na dy e al., 2016;Rashid e al., 2016;Kachwamba
e al., 2017). In o de o es his hypo hesis a do plo o each
con ig was ca ied ou o check o e lapping be ween he ends
(Figu e 1). Bo h do plo s showed his o e lapping be ween
he s a and he end o each con ig, indica ing ha s ain
AL184Tcon ains wo ci cula ch omosomes. In addi ion, do
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de la Haba e al. Compa a i e Genomics o he Genus Salini ib io
FIGURE 1 | Do plo s displaying he compa ison o assembled con ig 1 (A) and con ig 2 (B) o Salini ib io kushne i AL184Tagains i sel . The enla ged images o he
do plo s show ha bo h ends o each con ig a e iden ical and, he e o e, demons a e ha hey cons i u e wo ci cula ch omosomes.
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de la Haba e al. Compa a i e Genomics o he Genus Salini ib io
plo esul s we e con i med by BLAST sea ch. The e o e, he wo
ch omosomes we e ci cula ized and, subsequen ly, a linea ized
e sion was ou pu wi h he dnaA gene as he s a ing posi ion
o ch omosome I and a andom gene o ch omosome II. The
empi ical pe -base co e age achie ed was 211× o ch omosome
I and 216× o ch omosome II.
The inal assembly o s ain AL184Tconsis s o wo ci cula
ch omosomes wi h 2,840,906 bp and 602,384 bp, espec i ely
(Figu e 2). The comple eness and con amina ion o he genome
(bo h ch omosomes oge he ) es ima ed by CheckM ool (Pa ks
e al., 2014) was 99.9% and 0.54%, espec i ely, which means
ha i ually he comple e genome wi h a negligible -i any-
amoun o con amina ion was eco e ed. Al hough no essen ial
genes we e ound in ch omosome II, se e al lines o e idence
sugges ha i is a ch omosome a he han a plasmid: (i) DNA
G+C con en was e y simila o bo h ch omosomes (50.8 mol%
o I and 50.1 mol% o II); (ii) he leng h o ch omosome
II is mo e likely o co espond o a ch omosome ins ead o
a plasmid; (iii) he exis ence o genes heo e ically belonging
o he same clus e in di e en ch omosomes, o example he
clus e be ABC, in ol ed in he syn hesis o glycine be aine, was
coded in ch omosome I (genes be A and be B) and ch omosome
II (gene be C); and (i ) as a o emen ioned, he membe s o
he amily Vib ionaceae usually ha e wo ch omosomes (Dikow
and Smi h, 2013;Be na dy e al., 2016;Rashid e al., 2016;
Kachwamba e al., 2017).
Anno a ion o he comple e genome p edic ed 1244 and
233 CDS ansc ibed in a clockwise di ec ion and 1280 and
298 CDS ansc ibed in a coun e clockwise di ec ion o
ch omosomes I and II, espec i ely (Figu e 2). A o al o
28 RNA, 95 RNA, 57 ibosomal p o ein, 51 lagellum and
lagella mo ili y, 5 compa ible solu e syn hesis, 10 compa ible
solu e anspo e , and 4 anae obic espi a ion- ela ed genes
we e iden i ied in ch omosome I, whe eas only 1 RNA, 4
compa ible solu e syn hesis, and 5 compa ible solu e anspo e
genes we e de ec ed in ch omosome II (Figu e 2). This la ge
numbe o RNA genes, which we e clus e ed in nine comple e
ibosomal ope ons (and an addi ional 5S RNA gene loca ed
only 300 bp downs eam wi h espec o one o he RNA
ope ons), migh explain he as g owing a e o salini ib ios
cul u ed in copio ophic labo a o y condi ions (Rolle e al.,
2016). This inding was also obse ed in ela ed axa, such as
Vib io chole ae, ha con ains eigh comple e ibosomal ope ons
(Rodicio and Mendoza, 2004) and i is a common ai o as
ep oduc ion o ganisms (Rolle e al., 2016). I is well-known ha
he di e en 16S RNA genes con ained in he same s ain can be
he e ogeneous up o some ex en ; in he case o s ain AL194T he
nine 16S RNA genes p esen ed 100–98.4% sequence simila i y,
which is, p ac ically, wi hin he cu o alue cu en ly used o
species delinea ion (Kim e al., 2014).
Syn eny Analysis Among Salini ib io
Type S ain Genome Assemblies
Analyses o conse a ion o homologous genes and gene o de
be ween wo o mo e genomes o di e en species (syn eny)
play a pi o al ole in compa a i e genomics (Lee e al., 2018)
and can p o ide insigh s in o e olu iona y p ocesses ha lead
o di e si y, ch omosomal dynamics, and ea angemen a es
be ween species (Bhu ka e al., 2006). Al hough analysis o
syn eny among closely ela ed species is now widely used o
e e y new published genome, his analysis is egula ly pe o med
on assembled sequences ha a e agmen ed, neglec ing he ac
ha mos me hods we e de eloped using comple e genomes
(Liu e al., 2018). He e, we ha e used he comple e genome o
S. kushne i AL184Tas a e e ence o econs uc he agmen ed
genomes o he ype s ains o species o he genus Salini ib io by
o de ing he con igs and assigning hem o ei he ch omosome I
o II (Rissman e al., 2009). Following his s a egy, ch omosome
I o he ype species o he genus Salini ib io,S. cos icola
subsp. cos icola LMG 11651T, would be o med by 90 con igs
(2,513,750 bp), and ch omosome II by 39 con igs (691,570 bp),
while he emaining 73 con igs (174,314) o ha assembly could
no be assigned o ei he ch omosome I o II (Supplemen a y
Figu e 1 and Supplemen a y Table 2), and p esumably ep esen
gene-con en di e ences be ween he wo genomes compa ed.
Fo he ype s ains o he o he species o subspecies o he
genus, ch omosome I was cons i u ed by 63 (2,545,420 bp), 32
(2,869,249 bp), 26 (2,717,996 bp), and 40 (2,729,566 bp) con igs
co esponding o S. cos icola subsp. alcaliphilus DSM 16359T,
S. p o eoly icus DSM 19052T,S. sha mensis DSM 18182T, and
S. siamensis JCM 14472T, espec i ely, and he ch omosome II o
hose s ains was composed o 19 (706,389 bp), 10 (716,104 bp),
6 (589,195 bp), and 12 (688,170 bp) con igs, espec i ely, while
166 (129,848 bp), 9 (18,143 bp), 8 (19,704 bp), and 9 (24,449 bp)
con igs le unassigned, espec i ely (Supplemen a y Figu e 1
and Supplemen a y Table 2).
Re ie ed la ge and small ch omosomes om all he ype
s ains we e u he analyzed o sea ch syn eny segmen s (LCBs).
Figu e 3 shows ha he as majo i y o ch omosomes I and II
can be pai wise aligned o i s espec i e coun e pa in S. kushne i
AL184T, wi h 99.3–95.7% o ch omosome I and 98.2–89.5% o
ch omosome II om S. cos icola subsp. cos icola LMG 11651T,
S. cos icola subsp. alcaliphilus DSM 16359T,S. p o eoly icus DSM
19052T,S. sha mensis DSM 18182T, and S. siamensis JCM 14472T
ma ching a conse ed egion in S. kushne i AL184T. Mul iple
sequence alignmen among he la ge and small ch omosomes
o all he six genomes unde s udy allows iden i ying 87 LCBs
(3,713,502 bp alignmen leng h) o ch omosome I, o which
34 LCBs (3,594,867 bp alignmen leng h) whe e common o
all genomes, and 30 LCBs (1,090,330 bp alignmen leng h)
o ch omosome II, wi h 17 LCBs (1,034,054 bp alignmen
leng h) sha ed among all axa (Figu e 4). Al hough he numbe
o common LCBs o he la ge ch omosome was signi ican ly
smalle han he 306 common LCBs epo ed by Dikow and
Smi h (2013) o a simila compa ison wi hin he amily
Vib ionaceae, he leng hs o he alignmen s we e almos he same,
which means ha ou common LCBs o ch omosome I a e ewe
bu longe , and ac ually span be ween 95.0 and 98.7% o he la ge
ch omosome leng h o he six analyzed s ains.
Conce ning he small ch omosome, he numbe o common
LCBs iden i ied in ou s udy was app oxima ely hal o hose
de ec ed by Dikow and Smi h (2013) o o he membe s o he
amily Vib ionaceae, bu ou alignmen leng h was mo e han
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de la Haba e al. Compa a i e Genomics o he Genus Salini ib io
wice longe . Tha means ha he small ch omosomes o he
six Salini ib io genomes we e much mo e homologized, wi h
pe cen ages be ween 88.7 and 98.5%. These measu emen s we e
made when gaps we e emo ed om he alignmen s. The e o e,
in con as o he s udy o Dikow and Smi h (2013), no signi ican
di e ences in homologiza ion a es be ween la ge and small
Salini ib io ch omosomes could be obse ed. I mus be no ed
ha he s udy o Dikow and Smi h (2013) deal wi h comple e
genomes, while he e o de ed d a genomes we e employed, wha
migh pa ially explain such di e ences.
This highe syn eny in ou Salini ib io genomes s. he esul s
epo ed by Dikow and Smi h (2013) migh be due o he ac
ha he genomes analyzed in his s udy we e mo e closely
ela ed among hem (84.3% a e age O hoANI and 90.0% a e age
FIGURE 2 | G aphical ci cula map o he ch omosome I (A) and cho omosome II (B) o S. kushne i AL184T. F om he ou e o inne ch omosomal ings: (1)
p edic ed CDSs ansc ibed in a clockwise di ec ion; (2) p edic ed CDSs ansc ibed in a coun e clockwise di ec ion; (3) GC con en in a 1,000-bp sliding window; (4)
GC skew (C-G/G+C) in a 1,000-bp sliding window; (5) RNAs ( ed), RNAs (yellow), ibosomal p o eins (blue), lagellum and lagella mo ili y genes (g een),
compa ible solu e syn hesis genes (g ay), compa ible solu e anspo e s (black), and anae obic espi a ion- ela ed genes (pu ple).
FIGURE 3 | Pai wise alignmen o locally collinea blocks (LCBs) be ween la ge (A) and small (B) ch omosomes o S. kushne i AL184Tand hose o S. cos icola
subsp. cos icola LMG 11651T,S. cos icola subsp. alcaliphilus DSM 16359T,S. p o eoly icus DSM 19052T,S. sha mensis DSM 18182T, and S. siamensis JCM
14472T. Blue bands ep esen s LBC >100 Kb and g ay bands LCB <100 Kb.
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de la Haba e al. Compa a i e Genomics o he Genus Salini ib io
FIGURE 4 | Salini ib io la ge (87 LCBs) (A) and small (30 LCBs) (B)
ch omosomes ci cula plo s. Each ci cle ep esen s a genome. F om he ou e
mos ci cle: S. kushne i AL184T,S. cos icola subsp. cos icola LMG 11651T,
S. cos icola subsp. alcaliphilus DSM 16359T,S. p o eoly icus DSM 19052T,
S. sha mensis DSM 18182T, and S. siamensis JCM 14472T. LCBs in he
same colo a e sha ed by all six s ains, wi h he excep ion o LCBs in black
which a e sha ed by less han he six s ains.
AAI) han he Vib ionaceae genomes o he men ioned s udy
(74.1% a e age O hoANI and 70.6% a e age AAI), hus, he
highe he ela edness o he genomes unde s udy he highe
will be he syn eny. To con i m his s a emen , we calcula ed
he co espondence be ween syn eny (measu e as he pe cen age
o aligned genome) and O hoANI and AAI alues among all
pai o he genomes om ype s ains. Howe e , he Pea son’s
coe icien was only 0.21 o O hoANI and 0.25 o AAI,
indica ing a poo bu s ill signi ican co ela ion, which means
ha he conse ed syn eny o Salini ib io genomes is pa ially
due o he high a e age O hoANI/AAI alues among he s udied
genomes, bu also we can hypo hesize a slow specia ion p ocess o
homogeniza ion e en s ha migh be occu ing in salini ib ios, a
s a emen ha needs o be es ed expe imen ally in he u u e.
Phylogenomics o he Genus Salini ib io
Single-copy co e-genome genes and p o eins we e employed
o cons uc a phylogenomic ee in o de o elucida e
he axonomic ela ionship among membe s o he genus
Salini ib io. Maximum-likelihood phylogenies based on he
conca ena ion o 776 genes (777,643 bp alignmen leng h) and
1,637 p o eins (515,359 bp alignmen leng h) yielded wo e y
simila ees wi h high boo s ap suppo , whe e i e di e en
phylog oups and wo phylo ypes can be dis inguished (Figu e 5
and Supplemen a y Figu e 2). Phylog oup 1 co esponds o
S. kushne i s ains, and includes s ains p e iously a ilia ed o
his species (López-He moso e al., 2018b) as well as h ee
o he s ains o iginally named as Salini ib io sp. and ano he
p obably mislabeled s ain ini ially designa ed as S. cos icola.
Phylog oup 2 is o med by S. siamensis JCM 14472Tand o he
i e s ains iden i ied as membe s o his species in a p e ious
MLSA app oach (López-He moso e al., 2017b) as well as one
addi ional s ain labeled as Salini ib io sp. KP-1. Phylog oup 3
ag ees on he monophyle ic g oup de ined by López-He moso
e al. (2018a) o emend he desc ip ion o S. p o eoly icus bu
i also g oups wo addi ional s ains no analyzed by hose
au ho s, which we p o e o belong o he a o emen ioned
species. Phylog oup 4 consis s o he h ee s ains p oposed by
Go i i e al. (2014) as a new Salini ib io species wi h he no -ye
alida ed name “S. socompensis.” Finally, phylog oup 5 exac ly
i s he clus e de ined by López-He moso e al. (2017b) o med
by nine s ains, including he wo subspecies o S. cos icola,
S. cos icola subsp. cos icola and S. cos icola subsp. alcaliphilus.
Ac ually, phylogenomically, he wo subspecies canno be clea ly
di e en ia ed (especially when he ee is cons uc ed using he
conca ena ed co e genes), a maybe he Salini ib io subspecies
ank should be e ised. Fu he mo e, wo Salini ib io s ains
could nei he be included in any o he abo e phylog oups,
no o med a phylog oup hemsel es, so hey we e de ined
as phylo ypes. One o hem consis s o he ype s ain o
S. sha mensis, DSM 18182T, and he o he is an unnamed
Salini ib io s ain, ES.052, isola ed om an in e idal mic obial
ma in Elkho n Slough (Cali o nia), which p obably cons i u es a
new species o Salini ib io no desc ibed ye .
O hoANI alues calcula ed o all- s.-all pai s
(Supplemen a y Figu e 3) con i med ha he a o emen ioned
phylog oups and phylo ypes a e ac ually di e en species o
he genus Salini ib io. The O hoANI alues wi hin each
phylog oup we e always abo e 95%, whe eas, he alues among
phylog oups/phylo ypes we e in all cases a below 95%, he
h eshold alue p oposed o species bounda ies (Rich e and
Rosselló-Mó a, 2009), hus suppo ing ou p oposal o designa e
each phylog oup and phylo ype o a di e en Salini ib io species.
We also p opose o ename he Salini ib io s ains used he e as
ollows: all s ains belonging o phylog oup 1 should be labeled as
S. kushne i, hose o phylog oup 2 as Salini ib io siamensis, and
hose o phylog oup 3 as S. p o eoly icus. S ains o phylog oup 5
should be elabeled as S. cos icola, wi hou indica e he subspecies
o which each s ain is a ilia ed, wi h he excep ion o he ype
s ains o he subspecies. Finally, phylog oup 4 and he wo
phylo ypes will e ain hei ac ual designa ion (Table 1). Fo
some genome pai s (i.e., s ains o phylog oup 3 s. s ains
o phylog oups 4 and 5) he O hoANI alues we e sligh ly
below 80%, and so, hose genomes a e oo di e gen o use
nucleo ide le el compa isons. In consequence, AAI alues we e
es ima ed o all- s.-all genome pai s (Supplemen a y Figu e 3)
con i ming ha phylog oups 3, 4, and 5, as well as he phylo ype
Salini ib io sp. ES.052, cons i u e sepa a e species. Howe e ,
AAI esul s equal o abo e 95% among s ains o phylog oups
1 and 2, and o he phylo ype S. sha mensis DSM 18182T,
sugges ha hey all migh o m a single species. Ne e heless,
hese s ains sha e mo e han 90% ANI in all cases, he e o e,
acco ding o Rod iguez-R and Kons an inidis (2014) o such
closely ela ed s ains ANI o e s a obus esolu ion and should
be used ins ead. Fo ha eason, we sugges main aining ou
p oposal o phylog oups 1 and 2, and phylo ype S. sha mensis
DSM 18182Tas independen species wi hin his genus.
O hologous gene (OGs) clus e analysis based on amino acid
and nucleo ide sequences was pe o med o de ine he pan-
genome o he genus Salini ib io. By using ansla ed amino
acid sequence compa ison o he 45 analyzed genomes, he pan-
genome is composed o 5,570 OGs, o which 2,080 OGs a e
common o all axa (co e-genome), and 3,490 OGs cons i u e
he a iable-genome (Table 2). I he nucleo ide sequences a e
used, hen pan-genome is composed o la ge OGs, 7,462, bu
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de la Haba e al. Compa a i e Genomics o he Genus Salini ib io
FIGURE 5 | Maximum-likelihood phylogenomic ee based on he conca ena ion o 776 single copy co e genes showing he ela ionships among 45 Salini ib io
s ains whose genomes a e a ailable. Boo s ap alues ≥70% a e shown a he nodes. Ba , 0.02 n changes pe posi ion.
he co e-genome dec eases up o 1,211 OGs while he accesso y-
genome ises up o 6,251 OGs (Table 2). The smalle pan-genome
when using p o ein sequences was expec ed due o he ac ha a
lowe cu -o alue (40% s. 70% sequence iden i y) was se o
he clus e ing. Howe e , gi en ha p o ein sequences a e mo e
conse a i e han nucleo ide sequences, i is no su p isingly he
bigge co e-genome ob ained when analyzing ansla ed amino
acid sequences, especially i he genomes unde s udy ha e
di e ged oo much (O hoANI alues <80%). A simila s udy
wi hin he amily Vib ionaceae de ec ed 6,629 OGs o which 1,882
OGs whe e ound in all 11 p o eomes unde s udy (Lilbu n e al.,
2009), bu his smalle co e-genome is p obably a ibu able o
he ac ha he analysis was pe o med on genomes belonging o
di e en gene a. A mo e ecen esea ch only ocused in a single
genus, Vib io, and conduc ed wi h 20 p o eomes (co esponding
o 20 di e en species) yielded a la ge pan-genome o 21,844 OGs,
wi h only 1,630 OGs common o all axa (Lin e al., 2018), which
may be explained by he lowe genomic ela edness (measu ed
by O hoANI/AAI alues) among he Vib io genomes. The e o e,
membe s o he genus Salini ib io appea o ha e highe gene ic
ela edness wi hin he g oup han s ains o he genus Vib io.
These indings a e consis en wi h ou hypo hesis ha he e
should be an homogenizing o ce ac ing on he salini ib ios,
wha could explain he ew numbe o species desc ibed so
a , a puzzling occu ence o a genus so o en isola ed om
hype saline en i onmen s all o e he wo ld (He zog e al.,
2016;Asheng oph, 2017;Fe nández-Delgado e al., 2017;López-
He moso e al., 2017b;Sel a ajan e al., 2017;Le and Yang, 2018;
A ias e al., 2019). Howe e , his hypo hesis awai s expe imen al
es ing in he u u e.
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