Comparative Genomics and Phylogenomic Analysis of the Genus Salinivibrio

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 F on ie s in Mic obiology | www. on ie sin.o g 1Sep embe 2019 | Volume 10 | A icle 2104 micb-10-02104 Sep embe 10, 2019 Time: 15:59 # 2 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 F on ie s in Mic obiology | www. on ie sin.o g 2Sep embe 2019 | Volume 10 | A icle 2104 micb-10-02104 Sep embe 10, 2019 Time: 15:59 # 3 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. F on ie s in Mic obiology | www. on ie sin.o g 3Sep embe 2019 | Volume 10 | A icle 2104 micb-10-02104 Sep embe 10, 2019 Time: 15:59 # 4 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 F on ie s in Mic obiology | www. on ie sin.o g 4Sep embe 2019 | Volume 10 | A icle 2104 micb-10-02104 Sep embe 10, 2019 Time: 15:59 # 5 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. F on ie s in Mic obiology | www. on ie sin.o g 5Sep embe 2019 | Volume 10 | A icle 2104 micb-10-02104 Sep embe 10, 2019 Time: 15:59 # 6 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 F on ie s in Mic obiology | www. on ie sin.o g 6Sep embe 2019 | Volume 10 | A icle 2104 micb-10-02104 Sep embe 10, 2019 Time: 15:59 # 7 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. F on ie s in Mic obiology | www. on ie sin.o g 7Sep embe 2019 | Volume 10 | A icle 2104 micb-10-02104 Sep embe 10, 2019 Time: 15:59 # 8 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 F on ie s in Mic obiology | www. on ie sin.o g 8Sep embe 2019 | Volume 10 | A icle 2104 micb-10-02104 Sep embe 10, 2019 Time: 15:59 # 9 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. F on ie s in Mic obiology | www. on ie sin.o g 9Sep embe 2019 | Volume 10 | A icle 2104