Revisiting the Taxonomy of the Genus Arcobacter: Getting Order From the Chaos

micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 1 ORIGINAL RESEARCH published: 04 Sep embe 2018 doi: 10.3389/ micb.2018.02077 Edi ed by: Ma ha E. T ujillo, Uni e sidad de Salamanca, Spain Re iewed by: John Phillip Bowman, Uni e si y o Tasmania, Aus alia Ja ie Pascual, Deu sche Sammlung on Mik oo ganismen und Zellkul u en (DSMZ), Ge many *Co espondence: Jesus L. Romalde [email p o ec ed] Ma ía J. Figue as ma iajose. igue as@u .ca Special y sec ion: This a icle was submi ed o E olu iona y and Genomic Mic obiology, a sec ion o he jou nal F on ie s in Mic obiology Recei ed: 15 Feb ua y 2018 Accep ed: 14 Augus 2018 Published: 04 Sep embe 2018 Ci a ion: Pé ez-Ca aluña A, Salas-Massó N, Diéguez AL, Balboa S, Lema A, Romalde JL and Figue as MJ (2018) Re isi ing he Taxonomy o he Genus A cobac e : Ge ing O de F om he Chaos. F on . Mic obiol. 9:2077. doi: 10.3389/ micb.2018.02077 Re isi ing he Taxonomy o he Genus A cobac e : Ge ing O de F om he Chaos Alba Pé ez-Ca aluña1, Nu ia Salas-Massó1, Ana L. Diéguez2, Sabela Balboa2, Albe o Lema2, Jesús L. Romalde2*and Ma ia J. Figue as1* 1Depa amen de Ciències Mèdiques Bàsiques, Facul a de Medicina, Ins i u d’In es igació Sani à ia Pe e Vi gili, Uni e si a Ro i a i Vi gili, Reus, Spain, 2Depa amen o de Mic obiología y Pa asi ología, CIBUS-Facul ad de Biología, Uni e sidade de San iago de Compos ela, San iago de Compos ela, Spain Since he desc ip ion o he genus A cobac e in 1991, a o al o 27 species ha e been desc ibed, al hough some species ha e shown 16S RNA simila i ies below 95%, which is he cu -o ha usually sepa a es species ha belong o di e en gene a. The objec i e o he p esen s udy was o eassess he axonomy o he genus A cobac e using in o ma ion de i ed om he co e genome (286 genes), a Mul ilocus Sequence Analysis (MLSA) wi h 13 housekeeping genes, as well as di e en genomic indexes like A e age Nucleo ide Iden i y (ANI), in silico DNA–DNA hyb idiza ion (isDDH), A e age Amino-acid Iden i y (AAI), Pe cen age o Conse ed P o eins (POCPs), and Rela i e Synonymous Codon Usage (RSCU). The s udy included a o al o 39 s ains ha ep esen all he 27 species included in he genus A cobac e oge he wi h 13 s ains ha a e po en ially new species, and he analysis o 57 genomes. The di e en phylogene ic analyses showed ha he A cobac e species g ouped in o ou clus e s. In addi ion, A. leki hoch ous and he candida us species ‘A. aqua icus’ appea ed, as did A. ni o igilis, he ype species o he genus, in sepa a e b anches. Fu he mo e, he genomic indices ANI and isDDH no only con i med ha all he species we e well- de ined, bu also he cohe ence o he clus e s. The AAI and POCP alues showed in a-clus e anges abo e he espec i e cu -o alues o 60% and 50% desc ibed o species belonging o he same genus. Pheno ypic analysis showed ha ce ain es combina ions could allow he di e en ia ion o he ou clus e s and he h ee o phan species es ablished by he phylogene ic and genomic analyses. The o igin o he s ains showed ha each o he clus e s emb aced species eco e ed om a common o ela ed en i onmen . The esul s ob ained enable he di ision o he cu en genus A cobac e in a leas se en di e en gene a, o which he names A cobac e ,Aliia cobac e gen. no ., Pseudoa cobac e gen. no ., Haloa cobac e gen. no ., Malacobac e gen. no ., Poseidonibac e gen. no ., and Candida e ‘A coma inus’ gen. no . a e p oposed. Keywo ds: A cobac e ,Aliia cobac e gen. no ., Pseudoa cobac e gen. no ., Haloa cobac e gen. no ., Malacobac e gen. no ., Poseidonibac e gen. no ., axonomic c i e ia F on ie s in Mic obiology | www. on ie sin.o g 1Sep embe 2018 | Volume 9 | A icle 2077 micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 2 Pé ez-Ca aluña e al. Re isi ing he Taxonomy o he Genus A cobac e INTRODUCTION The genus A cobac e was c ea ed by Vandamme e al. (1991) o accommoda e G am-nega i e, cu ed-shaped bac e ia belonging o wo species Campylobac e c yae ophila (now A cobac e c yae ophilus) and Campylobac e ni o igilis (now A. ni o igilis), conside ed a ypical campylobac e s due o hei abili y o g ow a lowe empe a u es (15◦C–30◦C) and wi hou mic oae ophilic condi ions (Vandamme e al., 1991). The la e species was selec ed as he ype species o he new genus (Vandamme e al., 1991). One yea la e he genus was enla ged wi h he addi ion o wo new species, A. ski owii wi h an animal o igin being isola ed om abo ed o ine, po cine and bo ine e uses, and om lambs wi h dia hea, and A. bu zle i, which was eco e ed om cases o human and animal dia hea (Vandamme e al., 1992). Ano he wo new species we e inco po a ed in o he genus in 2005. A. halophilus was isola ed om wa e om a hype saline lagoon in Hawaii (Donachie e al., 2005), and A. ciba ius was isola ed om b oiled ca casses in Belgium (Hou e al., 2005). These species we e assigned o he genus A cobac e on he basis o he 16S RNA gene simila i y (94% and 95% o A. ni o igilis wi h A. halophilus and A. ciba ius, espec i ely). Howe e , hese alues a e equal, o e en below, he cu -o o 95% o genus de ini ion (Rosselló-Mo a and Amann, 2001;Ya za e al., 2008, 2014;Tindall e al., 2010). F om 2009 onwa d, new species we e being desc ibed yea - by-yea , eaching a o al numbe o 27 in 2017. In some o hese desc ip ions, he simila i y o he 16S RNA gene was he decisi e cha ac e o axonomic assigna ion a genus le el, al hough phylogeny based on housekeeping genes ( poB i s and hen gy B and hsp60) was also included as addi ional, mo e disc imina o y ools o he species (Collado e al., 2009a, 2011;De Sme e al., 2011). Using his app oach, A. mollusco um,A. ellisii,A. de lu ii, o A. bi al io um we e de ined, among o he s (Collado e al., 2009a, 2011;Figue as e al., 2011a,b;Le ican e al., 2012), which showed 16S RNA simila i ies anging om 91.1 o 94.7%, no suppo ing hei common a ilia ion. On he o he hand, he mos closely ela ed species, which showed a simila i y o 99.1% we e A. ellisii and A. de lu ii (Collado e al., 2011), gi ing e idence o he i s ime o he poo esolu ion o he 16S RNA gene o sepa a ing closely ela ed species in he genus A cobac e . Howe e , he phylogene ic analysis based on he conca ena ed sequences o gy B, poB, and cpn60 genes, oge he wi h he DNA–DNA hyb idiza ion esul s, clea ly suppo ed he exis ence o hese wo di e en ia ed axa (Figue as e al., 2011a). Also in 2011, A. ophia um was disco e ed om he in es inal ac o heal hy a ening pigs, which in e es ingly showed he closes simila i ies (≥97.4%) wi h he o he species also eco e ed om humans o animals, i.e., A. c yae ophilus,A. he eius,A. ciba ius, o A. ski owii (De Sme e al., 2011;Figue as e al., 2014;Van den Abeele e al., 2014). In 2013, he species A. cloacae and A. suis we e desc ibed, using a Mul ilocus Sequence Analysis (MLSA) app oach including i e housekeeping genes (Le ican e al., 2013) o he i s ime. Simul aneously, and due o he highes 16S RNA gene simila i y wi h A. ma inus (95.5%), he species A. anae ophilus was inco po a ed o he genus (Sasi-Jyo hsna e al., 2013). Howe e , his species showed a ypical cha ac e is ics, including lack o mo ili y and obliga e anae obic me abolism, which led o he o iginal desc ip ion o he genus A cobac e being emended (Sasi-Jyo hsna e al., 2013). The mos ecen ly desc ibed species om shell ish a e A. leki hoch ous,A. halio is, and A. canalis (Diéguez e al., 2017;Tanaka e al., 2017;Pé ez-Ca aluña e al., 2018a). The i s one included se e al isola es eco e ed om scallop la ae and om ank seawa e o a No wegian ha che y (Diéguez e al., 2017), he second species came om an abalone o Japan (Tanaka e al., 2017) and he hi d om oys e s subme ged in a wa e channel con amina ed wi h was ewa e (Pé ez-Ca aluña e al., 2018a). Howe e , Diéguez e al. (2018) e idenced ha he species A. halio is is a la e he e o ypic synonym o A. leki hoch ous. Addi ionally, he low 16S RNA gene simila i y o A. leki hoch ous wi h he known A cobac e species (91.0–94.8%) ound in he A. leki hoch ous desc ip ion made Diéguez e al. (2017) sugges ha ce ain species migh belong o o he gene a and ecommend ha a p o ound e ision o he genus migh cla i y he axonomy. On he o he hand, adding 2.5% NaCl o he en ichmen medium and subcul u ing on ma ine aga , Salas-Massó e al. (2016) ecognized se en po en ial new species om wa e and shell ish (mussels and/o oys e s), and eco e ed new isola es o A. halophilus and A. ma inus o which only he ype s ains had been known. In addi ion, du ing he cha ac e iza ion o he mos ecen ly desc ibed species A. canalis (Pé ez-Ca aluña e al., 2018a) and when ying o de ine he se en men ioned new species, we obse ed ha he A cobac e species o med se e al di e en clus e s dis an enough o suspec hey migh co espond o di e en gene a, in ag eemen wi h Diéguez e al. (2017). The e a e clea c i e ia o desc ibing new bac e ial species (Tindall e al., 2010;Figue as e al., 2011a,b). Howe e , he desc ip ion o a genus is usually based on a cu -o o <95% simila i y in he 16S RNA gene sequence, and a G+C (% mol) con en di e ing by mo e han 10% (Rosselló-Mo a and Amann, 2001;Ya za e al., 2008;Tindall e al., 2010;Ya za e al., 2014). Nowadays, genomic da a like he A e age Nucleo ide Iden i y (ANI) and he in silico DNA–DNA hyb idiza ion (isDDH) a e used o de ine bac e ial species, al hough ha e no ye been ully explo ed o delinea ing gene a (Kons an inidis and Tiedje, 2005; Go is e al., 2007;Rich e and Rosselló-Mó a, 2009;Qin e al., 2014;Chun e al., 2018). A pe cen age o A e age Amino-acid Iden i y (AAI) anging om 60 o 80% be ween he compa ed genomes o species o s ains and a Pe cen age o Conse ed P o eins (POCPs) abo e 50% has been p oposed i hey a e o belong o he same genus (Kons an inidis and Tiedje, 2005;Qin e al., 2014). Finally, he Rela i e Synonymous Codon Usage (RSCU) has also been used by some au ho s o in e e olu iona y and ecological links among bac e ial species (Ma e al., 2015;Fa ooqi e al., 2016). Ve y ecen ly, Wai e e al. (2017) ca ied ou a compa a i e genomic analysis o he class Epsilonp o eobac e ia. Using 16S and 23S RNA, 120 single-copy ma ke p o eins and AAI analysis hey p oposed i s eclassi ica ion as he new phylum Epsilonbac e aeo a. In ha s udy, Wai e e al. (2017) also p oposed a eclassi ica ion o he genus A cobac e as a new F on ie s in Mic obiology | www. on ie sin.o g 2Sep embe 2018 | Volume 9 | A icle 2077 micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 3 Pé ez-Ca aluña e al. Re isi ing he Taxonomy o he Genus A cobac e Family A cobac e aceae, wi hin he class Campylobac e ia, o de Campylobac e ales. One weakness o his s udy, speci ically ega ding he genus A cobac e , is ha only se en alida ed species we e included in he analysis. The new amily he e o e comp ised only he genus A cobac e . Howe e , hese indings also suppo he need o a cla i ica ion o he axonomy o he cu en genus A cobac e . The ise o genome sequencing has d ama ically changed he landscape o sys ema ics o p oka yo es, imp o ing di e en aspec s such as he iden i ica ion o species, he unc ional cha ac e iza ion o esol ing axonomic g oups, and he esolu ion o he phylogeny o highe axa (Whi man, 2015). I seems clea ha he inco po a ion o genomics in o he axonomy will boos i s c edibili y p o iding ep oducible, eliable, highly in o ma i e means o in e phylogene ic ela ionships among p oka yo es, and a oiding un eliable me hods and subjec i e di icul - o- eplica e da a (Chun and Rainey, 2014;Chun e al., 2018). Wi hin his mode n axonomy con ex , he objec i e o he p esen s udy was o eassess he axonomy o he known and newly ecognized A cobac e species by using a MLSA o 13 housekeeping genes, he whole genome sequences and he de i ed genomic analysis. The la e analysis included ANI, isDDH, AAI, POCP, and RSCU o all A cobac e ype s ains. In addi ion, phylogenies based on 16S and 23S RNA gene sequences we e also pe o med wi h compa a i e pu poses. The new axonomic c i e ia we e s able when including whole genome sequences o a second s ain o each species o o unassigned sequences ob ained om he public da abases. MATERIALS AND METHODS Bac e ial S ains All 27 alid species included in he genus A cobac e ha e been s udied. They a e ep esen ed by 39 s ains, and 13 s ains ha a e po en ially new species (Table 1). Fu he mo e, 50 genomes o A cobac e s ains iden i ied a species le el we e in es iga ed, 39 o which we e ob ained in ou labo a o y (27 om known species and 13 om po en ially new species) and he o he s om he public da abases1,2. Fi e genomes ha had been deposi ed as A cobac e sp. in he da abases we e also included in he s udy. I he e was mo e han one s ain o a known A cobac e species, wo ep esen a i e genomes o each species we e included in he analysis. The only excep ions we e: A. ac icola (Pa k e al., 2016) and A. paci icus (Zhang e al., 2015), whose axonomic posi ions we e only in e ed by he phylogene ic analysis o he 16S RNA gene sequences published in hei species desc ip ions, oge he wi h a MLSA o h ee housekeeping genes (a pA, gy B, and poB) o A. paci icus (Zhang e al., 2015;Pa k e al., 2016). The s ains conside ed po en ially new species, and named he ea e as ‘candida e species,’ had been ecognized wi h an MLSA analysis o i e housekeeping genes (a pA, gy A, gy B, hsp60, and poB) (da a no shown). 1h ps://www.ncbi.nlm.nih.go /genome/ 2h ps://gold.jgi.doe.go / Cul u ing o genome sequencing was ca ied ou ei he on blood aga (DIFCO, Mad id, Spain) o ma ine aga (Scha lau, Sen mena , Spain) a 30◦C in ae obiosis o 24–72 h, depending on he equi emen s. DNA was ex ac ed using Easy-DNATM gDNA Pu i ica ion ki (In i ogen, Mad id, Spain) ollowing he manu ac u e ’s ins uc ions. The in eg i y o he DNA was e alua ed by elec opho esis o 10 µl o he sample in a 1.5% aga ose gel. The o al amoun o DNA was quan i ied using Qubi TM wi h he dsDNA B oad Range Assay ki (In i ogen). Pai ed-end lib a ies we e cons uc ed wi h 50 ng o DNA using Nex e a DNA Lib a y P epa a ion Ki (Illumina, Lisbon, Po ugal) and sequenced wi h MiSeq pla o m (Illumina). Sequencing gene a ed 2 ×300 bp pai ed-end eads. Clean eads we e assembled wi h SPAdes (Nu k e al., 2013) and he CGE assemble (La sen e al., 2012) in o de o selec he be e assembly. Be o e deposi ing he genomes in he NCBI da abase, FASTA iles we e sc eened o euka yo ic and p oka yo ic sequences using BLASTn, and o adap o s wi h VecSc een s andalone so wa e3. The i e housekeeping genes used in he i s MLSA analysis (a pA, gy A, gy B, hsp60, and poB) we e ex ac ed om each genome and compa ed wi h he Sange sequences o hese genes ob ained o iginally o he iden i ica ion o he s ain. The exis ence o a single and iden ical copy o hese genes con i med ha he genomes we e no con amina ed and belonged o he co ec s ain. Finally, con igs we e dele ed i hey had less han 200 bp. The genomes we e deposi ed in he GenBank da abase and Table 1 lis s he accession numbe s. The 55 genomes we e anno a ed wi h a local ins alla ion o P okka 1.2 (Seemann, 2014) using an e- alue o 1e-06. The anno a ion was pe o med wi h P okka, wi h he p edic ion ools P odigal 2.6 (Hya e al., 2010) and ARAGORN 1.2 (Lasle and Canback, 2004). The p edic ion ool Ba nap 0.64included in P okka 1.2 was used o he anno a ion o RNA genes. Coding sequences (CDS) we e anno a ed, combining he Rapid Anno a ion Subsys ems Technology (RAST) (O e beek e al., 2014) using he classic RAST scheme and he Anno a ion Tools o PATRIC se e (Wa am e al., 2017). The cha ac e is ics o each genome (i.e., N50, numbe o con igs, numbe o CDS, G+C con en ) we e ob ained om NCBI anno a ions. Analysis o Housekeeping Genes, Ribosomal Genes, and Co e Genome Thi een housekeeping genes (a pA, a pD, dnaA, dnaJ, dnaK, sZ, gy A, hsp60, adA, ecA, poB, poD, and s ) we e ob ained om he genomes using BLASTn sea ch. Sequence simila i ies o housekeeping genes we e de e mined using he MegAlign p og am (DNASTARR , Madison, WI, Uni ed S a es). Genes we e aligned using Clus alW (La kin e al., 2007) and phylogenies based on indi idual genes and on he conca ena ed sequences was cons uc ed wi h MEGA e sion 6.0 (Tamu a e al., 2013) using he Neighbo -Joining (NJ) and Maximum-Likelihood (ML) algo i hms. 3 p:// p.ncbi.nlm.nih.go /blas /demo/ 4h p://www. icbioin o ma ics.com/so wa e.ba nap.sh ml F on ie s in Mic obiology | www. on ie sin.o g 3Sep embe 2018 | Volume 9 | A icle 2077 micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 4 Pé ez-Ca aluña e al. Re isi ing he Taxonomy o he Genus A cobac e The phylogene ic analysis o he co e genome was assessed wi h he Roa y so wa e (Page e al., 2015) using 80% as cu -o o he BLASTp sea ch. The co e genome alignmen was ex ac ed wi h he la e so wa e and he phylogeny was in e ed using Spli sT ee e sion 4.14.2 as desc ibed in Sawabe e al. (2007) using Spli sT ee e sion 4.14.2, wi h a neighbo ne d awing and Jukes- Can o co ec ion (Bandel and D ess, 1992;Huson and B yan , 2005). Fu he mo e, he 16S and 23S RNA genes o each genome we e ob ained using RNamme (Lagesen e al., 2007). In some cases, 16S RNA gene sequences we e ob ained in ou labo a o ies by Sange sequencing o om he GenBank. The simila i y o he 16S RNA genes was calcula ed using MegAlign e sion 7.0.0 (DNASTARR , Madison, WI, Uni ed S a es). Phylogene ic ees we e econs uc ed wi h MEGA e sion 6.0 (Tamu a e al., 2013) also using he NJ and ML algo i hms. Alignmen s ob ained o bo h genes we e isually analyzed in o de o localize signa u e sequences o s ains o g oups o s ains. Genomic Indices In o de o ensu e he co ec assigna ion a species le el o each analyzed genome, he ANI and he isDDH we e calcula ed be ween all he genomes (Kons an inidis and Tiedje, 2005; Rich e and Rosselló-Mó a, 2009;Qin e al., 2014). The ANIb was calcula ed using JSpeciesWS (Rich e e al., 2016), he esul ing ma ix was clus e ed and isualized using ggplo 2 2.2.1 package (Wickham, 2009) and he isDDH was calcula ed wi h he GGDC so wa e using esul s ob ained wi h he o mula 2 (Meie -Kol ho e al., 2013). Two o he indices (AAI and POCP) desc ibed o genus classi ica ion (Kons an inidis and Tiedje, 2005;Luo e al., 2014;Qin e al., 2014) we e calcula ed among he genomes ha co esponded o he ype s ains o he accep ed species and he e e ence s ains o he candida e species. The AAI was calcula ed wi h he Lycoming College Newman Lab AAI Calcula o 5using he Sequence-Based Compa ison Tools ou pu ile om RAST (O e beek e al., 2014). The POCP was de e mined as desc ibed by Qin e al. (2014) using he ollowing pa ame e s o conside a pep ide as a conse ed p o ein: an e- alue lowe han 1e-5 and an iden i y pe cen age highe han 40% om an aligned egion highe han 50%. Finally, he RSCU was compu ed using he Codon Adap a ion Index (CAI) de eloped by Sha p and Li (1987) h ough he CAIcal web-se e (Puigbò e al., 2008). S a is ical di e ences in he RSCU we e assessed by a mul inomial eg ession app oach using he R so wa e en i onmen (R Co e Team, 2015). The p incipal componen analysis (PCA) was pe o med by he R so wa e en i onmen (R Co e Team, 2015, and isualized using ggplo 2 2.2.1 and gg o i y 0.4.4 (Wickham, 2009;Ho ikoshi and Tang, 2015;Tang e al., 2016) o pca3d 0.10 (Weine , 2017) packages. Pheno ypic Analysis and Me abolic In e ence Pheno ypic cha ac e iza ion o each desc ibed species was ob ained om his s udy, om he o iginal desc ip ions o om 5h p://lyco s01.lycoming.edu/∼newman/AAI/ he summa y published by On e al. (2017). Fo he po en ially new A cobac e species, he pheno ype was cha ac e ized ollowing he ecommended minimal s anda ds desc ibed o new axa o he amily Campylobac e aceae (U sing e al., 1994; On e al., 2017) and wi h complemen a y es s used in he desc ip ion o o he A cobac e species (Le ican e al., 2013). In e ence o he me abolic ou es om he genome sequences was pe o med wi h he so wa e package T ai a (Mic obial T ai Analyze ) (Weimann e al., 2016), using he p o ein coding genes iles ob ained wi h P okka 1.2 (Seemann, 2014). T ai a so wa e is based on pheno ypic da a ex ac ed om he Global In ec ious Disease and Epidemiology Online Ne wo k (GIDEON) and Be gey’s Sys ema ic Bac e iology. The so wa e uses wo p edic ion models: he phypa classi ie , which p edic s he p esence/absence o p o eins ound in he pheno ype o 234 bac e ial species; and he phypa +PGL classi ie , which uses he same in o ma ion as he phypa combined wi h he in o ma ion o he acquisi ion and loss o p o ein amilies and pheno ypes du ing e olu i e e en s. A o al o 67 ai s a ailable wi hin he so wa e, ela ed o oxygen equi emen , enzyma ic ac i i ies, p o eolysis, an ibio ic esis ance, mo phology and mo ili y and he use o di e en ca bon sou ces, we e es ed and he combined esul s o he wo p edic o s we e analyzed using a hea map. RESULTS AND DISCUSSION S ains and Genomes All he 27 species cu en ly included in he genus A cobac e and 13 candida e species ha e been in es iga ed in he p esen s udy, which has analyzed 55 genomes, 16 o hem om he public da abases and 39 sequenced in his s udy (Tables 1,2). I was no possible o analyze he genomes om A. ac icola and A. paci icus because we we e unable o ge he ype s ains o he species. The con igs ob ained and he N50 alues complied wi h he ecen ly p oposed minimal s anda ds o he use o genomes in axonomic s udies (Chun e al., 2018). The genome size anged om 1.81 Mb o A. ski owii F28 o 3.60 Mb o A. leki hoch ous CECT 8942T(Table 2). The G+C con en anged om 26.1% in A. mollusco um CECT 7696T o 34.9% in ‘A. aqua icus’ W112-28. The G+C alues ag ee wi h he ange om 24.6% (which co esponded o he ype s ain o A. anae ophilus) o 31% indica ed o he genus A cobac e in he ecen emended desc ip ion by Sasi-Jyo hsna e al. (2013). In e es ingly, 26 genomes (47.3%) showed he p esence o Clus e ed Regula ly In e spaced Sho Palind omic Repea s (CRISPRs) and CRISPR- associa ed genes, ela ed wi h he immune esponse o he bac e ia. Taxonomic and Phylogene ic Analysis Simila i ies in he 16S RNA gene sequences among ype and ep esen a i e s ains o he di e en A cobac e species (all he 27 species cu en ly included in he genus and he 13 new candida e species) showed a wide ange o alues (Supplemen a y Tables S1, S4). They anged om 90.8% (obse ed be ween A. anae ophilus and A. aecis) o 99.9% (be ween A. bu zle i and ‘A. lacus’). The lowe ange o F on ie s in Mic obiology | www. on ie sin.o g 4Sep embe 2018 | Volume 9 | A icle 2077 micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 5 Pé ez-Ca aluña e al. Re isi ing he Taxonomy o he Genus A cobac e TABLE 1 | S ains used in his s udy, sou ce o isola ion and accession numbe s o he a ailable genomes. Species S ain Sou ce Acc. No. Genome Species S ain Sou ce Acc. No. Genome A. ac icola KCTC 52212TSeawa e NAaA. my ili T234 Seawa e PDJW00b A. anae ophilus DSM 24636TEs ua ine sedimen PDKO00bA. ni o igilis DSM7299TMa shland plan NC014166c IR-1 U si a aqui e NZ_JXXG00cA. paci icus DSM 25018TSeawa e NAa A. aquima inus CECT 8442TMedi e anean Sea NXIJ00bA. ski owii LMG 6621TDia heic lamb NXIC00b A. bi al io um CECT 7835TMussels PDKM00bF28 Wild pig PDJT00b F118-4 Mussels PDKL00bA. suis CECT 7833TPo k mea NREO00b A. bu zle i RM4018THuman (Clinical) NC_009850cA. he eius LMG 24486TAbo ed pig oe us LLKQ01c ED1 Mic obial uel cell NC_017187cDU22 Duck cloaca LCUJ01c A. canalis F138-33 Oys e PNCeNWVW01bA. ophia um LMG 25534TPigle eces PDKD00b SH-4D_Col1 Unknown FUYO00cCECT 7650 Chicken cloacal swab PDJS00b A. ciba ius LMG 21996TB oile , skin NZ_JABW00cA. ene upis CECT 7836TClams NREP00b A. cloacae CECT 7834TSewage NXII00bA cobac e sp. L Mic obial uel cell NC_017192c F26 Mussels PDJZ00bAF1028 Human eces JART01c A. c yae ophilus LMG 24291TAbo ed bo ine oe us NXGK00bCAB Ma ine Go0012496d A. de lu ii CECT 7697TSewage NXIH00bLA11 Ma ine BDIR01c A. eb onensis CECT 8441TMussels PDKK00bLPB0137 En i onmen al CP019070c CECT 8993 Seawa e PDKJ00b A. ellisii CECT 7837TMussels NXIG00b‘A. aqua icus’ W112-28 F eshwa e PNCePDKN00b A. aecis LMG 28519THuman sep ic ank NZ_JARS00c‘A. caeni’ RW17-10 Recycled was ewa e MUXE00b A. halophillus DSM 18005THype saline lagoon PDJY00b‘A. hispanicus’ FW-54 Was ewa e PDKI00b F166-45 Oys e PNCePDJY00b‘A. lacus’ RW43-9 Recycled was ewa e MUXF00b A. lan hie i LMG 28516TPig manu e JARU01c‘A. medi e aneus’ F156-34 Mussels Al acs Bay NXIE00b LMG 28517 Dai y ca le manu e JARV01c‘A. mi oungae’ 9An Cloaca elephan seal PDKH00b A. leki hoch ous CECT 8942TG ea scallop la ae NZ_MKCO00b‘A. nep unis’ F146-38 Mussels Al acs Bay PDKG00b LMG 28652 Abalon PZYW00c‘A. po cinus’ LMG 24487TAbo ed pig oe us LCUH01c A. ma inus CECT 7727TSeawa e NXAO01b‘A. pon icus’ F161-33 Cockle Al acs Bay PDKF00b F140-37 Clams Al acs Bay NWVX01b‘A. salis’ F155-33 Oys e PNCePDKE00b A. mollusco um CECT 7696TMussels NZ_NXFY00b‘A. iscosus’ F142-34gMussels PNCePDKC00b F91 Mussels PDJX00b‘A. i o iensis’ FW59gWas ewa e PDKB00b A. my ili CECT 7386TMussels NXID00bA cobac e sp. F2176 Mussels PDJV00b aGenome no a ailable; bGenome sequenced in his s udy; cGenome ob ained om NCBI da abase; dGenome ob ained om JGI Gold a abase; ePNC means PobleNou Channel, which is a eshwa e channel hea ily (geome ic mean o E. coli coun s 4.1 ×104c. .u./100ml) con amina ed wi h was ewa e whe e shell ish we e exposed o 72h (Salas-Massó e al., 2016, 2018). This s ain was ob ained om F.J. Ga cía om he Labo a o io Cen al de Ve e ina ia de Alge e, MAGRAMA, Mad id, Spain; gThese s ains we e eco e ed a he Facul y o Pha macy, Uni e si y o he Basque Coun y (UPV-EHU), Vi o ia-Gas eiz, Spain, by R. Alonso, I. Ma inez-Malaxe xeba ia and A. Fe nández-As o ga. simila i y (90.8%) is due o he ac ha hose species, as occu ed wi h o he s, we e assigned wi hin he genus based on he p emise ha 16S RNA gene simila i y was highe wi h any ype s ain o A cobac e han wi h o he axa. Howe e , in some cases being below he 95% cu -o alue o genus delimi a ion (Rosselló-Mo a and Amann, 2001;Ya za e al., 2008; Tindall e al., 2010;Figue as e al., 2011a,b). I is in e es ing o poin ou ha 16S RNA gene sequence simila i ies among A. ni o igilis, he ype species o he genus, and he o he desc ibed species anged om 93.2% (wi h A. he eius) o 95.9% (wi h A. ene upis). Fu he mo e, A. ni o igilis showed highe simila i ies han he h eshold alue o 95% wi h only se en species (A. ac icola, ‘A. caeni,’ A. cloacae,A. de lu ii, A. ellisii, A. suis, and A. ene upis) ou o he 27 accep ed species. In any case, om he analysis o he simila i ies in he 16S RNA gene sequences among he A cobac e species i is clea ha his gene has limi ed alue and ha o he app oaches a ailable in he genomic e a o axonomy a e needed o hei s udy. Phylogene ic analysis based on he co e genome made up o 286 genes (Figu e 1 and Supplemen a y Table S5) and also on he conca ena ed sequences o 13 housekeeping genes o he ep esen a i e A cobac e s ains (Figu e 2) e ealed ha he A cobac e species could be g ouped in o 4 majo monophyle ic clus e s. Clus e 1, comp ised se en alida ed species: A. bu zle i,A. ciba ius,A. c yae ophilus,A. lan hie i, A. ski owii,A. he eius, and A. ophia um, oge he wi h A. aecis (species desc ibed bu no alida ed ye ) and i e candida e axa ‘A. hispanicus,’ ‘A. lacus,’ ‘A. mi oungae,’ ‘A. po cinus,’ and ‘A. i o iensis’ (Figu e 1). Clus e 2 emb aced he species A. aquima inus,A. cloacae,A. de lu ii,A. ellisii, A. suis, and A. ene upis, as well as he non- alida ed A. ac icola and he candida us ‘A. caeni.’ Clus e 3 included i e species, A. canalis,A. halophilus,A. ma inus,A. mollusco um, F on ie s in Mic obiology | www. on ie sin.o g 5Sep embe 2018 | Volume 9 | A icle 2077 micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 6 Pé ez-Ca aluña e al. Re isi ing he Taxonomy o he Genus A cobac e TABLE 2 | Genome cha ac e is ics and anno a ion esul s. Sou ce o whole genome sequences as indica ed in Table 1. Species No. Con igs N50 (Kb) CDS (To al) CDS (Coding) RNA Genes RNAs ncRNAs CRISPR A ays G+C (%) Size (Mb) A. anae ophilus DSM 24636T40 186 2,938 2,922 45 40 2 1 29.9 2.98 A. anae ophilus IR1 7 1,179 3,360 3,024 61 47 2 3 30.2 3.25 ‘A. aqua icus’ W112-28T20 370 2,500 2,487 55 45 3 0 34.9 2.53 A. aquima inus CECT 8442T68 75 2,473 2,463 46 42 2 0 26.6 2.46 A. bi al io um CECT 7835T179 461 2,786 2,728 50 41 3 0 28.2 2.75 A. bi al io um F118-4 26 209 2,652 2,652 47 38 3 0 28.1 2.71 A. bu zle i RM4018T1 – 2,261 2,256 71 54 2 0 27.0 2.34 A. bu zle i ED1 1 – 2,151 2,145 71 54 2 0 27.1 2.26 ‘A. caeni RW17-10T59 123 2,357 2,337 58 51 3 0 27.1 2.42 A. canalis CECT8984T50 166 2,733 2,720 53 48 2 1 27.3 2.78 A. canalis SH-4D_Col1 69 72 2,716 2,663 63 52 2 1 27.1 2.82 A. ciba ius LMG 21996T44 119 2,156 2,110 68 46 2 0 27.1 2.20 A. cloacae CECT 7834T135 135 2,826 2,795 58 51 2 3 26.8 2.78 A. cloacae F26 40 218 2,470 2,459 53 44 2 1 26.9 2.51 A. c yae ophilus LMG 24291T91 54 2,092 2,081 49 40 3 0 27.2 2.06 A. de lu ii CECT 7697T80 166 2,921 2,894 57 49 2 2 26.3 2.94 A. eb onensis CECT 8441T103 188 3,089 3,072 47 39 3 1 29.2 3.15 A. eb onensis W129-34 126 217 3,206 3,171 46 40 3 2 29.2 3.23 A. ellisii CECT 7837T135 177 2,875 2,840 64 52 2 1 26.9 2.80 A. aecis LMG 28519T55 127 2,429 2,376 76 53 2 1 27.2 2.50 A. halophilus DSM 18005T111 56 2,677 2,660 54 46 3 2 27.4 2.75 A. halophilus F166-45 90 56 2,879 2,864 59 51 2 2 27.0 2.96 ‘A. hispanicus’ FW54T76 148 2,228 2,207 46 40 3 1 26.4 2.21 ‘A. lacus’ RW43-9T24 295 2,194 2,182 47 40 2 0 26.8 2.22 A. lan hie i LMG 28516T29 466 2,223 2,190 73 52 3 1 26.7 2.29 A. lan hie i AF1581 24 353 2,199 2,186 88 57 3 0 26.8 2.26 A. leki hoch ous CECT 8942T436 343 3,628 3,316 88 75 3 0 28.6 3.61 A. leki hoch ous LMG 28652 82 343 3,499 3,330 61 55 3 0 28.2 3.50 A. ma inus CECT 7727T162 54 2,809 2,781 55 50 2 0 27.0 2.87 A. ma inus F140-37 76 67 2,725 2,652 59 48 2 0 27.0 2.78 ‘A. medi e aneus’ F156-34T29 689 2,769 2,750 47 41 3 1 27.3 2.83 ‘A. mi oungae’ 9An T35 363 1,868 1,847 46 41 2 1 28.1 1.84 A. mollusco um CECT 7696T117 121 2,746 2,736 58 49 3 6 26.1 2.76 (Con inued) F on ie s in Mic obiology | www. on ie sin.o g 6Sep embe 2018 | Volume 9 | A icle 2077 micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 7 Pé ez-Ca aluña e al. Re isi ing he Taxonomy o he Genus A cobac e TABLE 2 | Con inued Species No. Con igs N50 (Kb) CDS (To al) CDS (Coding) RNA Genes RNAs ncRNAs CRISPR A ays G+C (%) Size (Mb) A. mollusco um F91 240 150 2,951 2,889 71 58 3 2 26.3 2.89 A. my ili CECT 7386T126 70 2,950 2,934 58 48 3 1 26.3 2.97 A. my ili T234 145 37 2,735 2,723 54 48 3 0 26.4 2.77 ‘A. nep unis’ F146-38T36 267 2,627 2,614 57 45 3 0 27.1 2.65 A. ni o igilis DSM 7299T1 – 3,101 3,086 69 55 2 1 28.4 3.19 ‘A. pon icus’ F161-33 24 597 2,632 2,621 46 36 3 0 28.1 2.74 ‘A. po cinus’ LMG 24487T70 123 2,186 2,112 47 41 2 0 27.0 2.14 ‘A. salis’ F155-33T153 169 2,932 2,904 50 43 3 0 29.0 2.93 A. ski owii LMG 6621T62 306 2,029 2,006 48 42 2 2 27.7 1.97 A. ski owii F28 110 40 1,911 1,897 46 41 2 0 27.8 1.81 A. suis CECT 7833T122 142 2,646 2,613 57 52 2 0 27.3 2.62 A. he eius LMG 24486T2 1,039 1,896 1,883 57 46 2 3 27.0 1.91 A. he eius DU22 19 252 2,006 1,983 47 42 2 1 26.8 2.01 A. ophia um CECT 7650 37 152 1,911 1,894 48 37 3 0 28.0 1.90 A. ophia um LMG 25534T266 86 2,167 2,071 49 41 3 0 29.4 2.00 A. ene upis CECT 7836T234 182 3,319 3,267 64 52 2 0 28.0 3.28 ‘A. iscosus’ F142-34T82 65 2,772 2,756 55 48 3 1 26.6 2.79 ‘A. i o iensis’ FW59T144 179 2,617 2,570 53 46 2 0 27.4 2.58 A cobac e sp. CAB 367 20 3,596 3,392 NA 31 NA NA 28.2 3.48 A cobac e sp. F2176 99 178 3,212 3,186 67 57 2 0 28.1 3.27 A cobac e sp. LA11 53 229 3,006 2,961 49 43 3 0 27.9 3.10 A cobac e sp. LPB0137 1 – 2,731 2,698 85 64 2 0 27.7 2.87 A cobac e sp. La1 – 2,847 2,834 73 56 2 1 26.6 2.95 A cobac e sp. AF1028b46 148 2,336 2,285 71 51 2 1 27.2 2.41 aGenome sequenced in his s udy; bGenome ob ained om NCBI da abase; cGenome ob ained om JGI Gold da abase. Ou esul s show ha hese s ains belong o he species. dA. de lu ii and eA. aecis. F on ie s in Mic obiology | www. on ie sin.o g 7Sep embe 2018 | Volume 9 | A icle 2077 micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 8 Pé ez-Ca aluña e al. Re isi ing he Taxonomy o he Genus A cobac e and A. my ili, oge he wi h wo candida es, ‘A. nep unis’ and ‘A. iscosus.’ Finally, Clus e 4 included he species A. anae ophilus,A. bi al io um, and A. eb onensis, as well as he candida es ‘A. medi e aneus,’ ‘A. pon icus,’ and ‘A. salis.’ The spli decomposi ion ne wo k analysis o he co e genome showed ha he species A. leki hoch ous CECT 8942Tand A. ni o igilis DSM 7299Tappea ed as o phan species. Fu he mo e, wi h his analysis he candida us ‘A. aqua icus’ W112-28 also appea ed in a sepa a e b anch nea o A. ni o igilis DSM 7299T. On he o he hand, bo h analyses, MLSA and co e genome, con i med he exis ence o wo sub-clus e s in Clus e 1 (again A. bu zle i and ‘A. lacus’ we e loca ed in he mos dis an b anch wi hin he clus e ), and also wo subg oups could be obse ed in Clus e 4, one comp ising he species A. anae ophilus and A. eb onensis, and he o he including he es o species wi hin his clus e (Figu es 1,2). All he clus e s and sub-clus e s showed a simila i y in he conca ena ed sequences o he 13 housekeeping genes highe han 85% (Figu e 2). Phylogenies based on he 16S and 23S RNA gene sequences, unde aken wi h he NJ and ML app oachese ealso cons uc ed wi h compa a i e pu poses. 16S RNA based ee showed also he ou majo clus e s al hough less de ined (Supplemen a y Figu e S1A). Species wi hin Clus e 1, showed 16S RNA gene sequence simila i ies anging om 96.1 o 99.9%. Clus e 2 yielded simila i ies among species o he 16S RNA gene be ween 96.7 and 99.6%, whe eas wi hin Clus e 3 anged be ween 93.0 and 99.1%. Finally, Clus e 4 included species wi h a ange o 16S RNA sequence simila i y om 94.0 o 99.5%. Wi h he excep ion o Clus e 3, simila i y alues wi hin he clus e s (>94–95%) we e wi hin he classical bounda ies o genus assigna ion in bac e ial axonomy (Rosselló-Mo a and Amann, 2001;Ya za e al., 2008, 2014;Tindall e al., 2010;Figue as e al., 2011a,b). Ou esul s ag ee wi h hose om a ecen s udy by Ya za e al. (2014), who in es iga ed 568 axa and desc ibed a h eshold in 16S RNA sequence iden i y o 94.5% o genus delinea ion. Simila g oups and opology, wi h only mino di e ences, we e ob ained when he 23S RNA gene sequences we e used o analyze he phylogeny o he genus (Supplemen a y Figu e S2). In his analysis, he ecen ly desc ibed species A. ac icola, and A. paci icus could no be included because o he una ailabili y o he ype s ains and/o whole genome sequences. The same ou majo clus e s o med in he 23S RNA gene phylogene ic ee, and he species A. leki hoch ous and A. ni o igilis appea ed also as o phan species (Supplemen a y Figu e S2). Wi hin Clus e 1 wo subg oups could also be ob ained, di e en ia ing he species A. bu zle i and ‘A. lacus’ om he es o he species. Simila ly, he species A. anae ophilus and A. eb onensis o med a di e en ia ed subg oup in Clus e 4. The isual analysis o he alignmen s ob ained wi h he sequences o he 16S and 23S RNA genes allowed he localiza ion o signa u e mo i s, especially in he 16S RNA gene, o he di e en clus e s es ablished in he phylogene ic analysis. In hese sequences, a o al o 16 loca ions we e ound, p esen ing nucleo ide combina ions cha ac e is ic o he clus e s (Supplemen a y Figu e S3). Some o hese mo i s we e loca ed in helix egions as in e ac ions wi h p o eins o he ibosomal 30S subuni , such as helix 21 ( egion V4) o helix 28/44 ( egion V9), and he e o e had a conside able le el o p o ec ion agains mu a ions (Adilakshmi e al., 2008;Ki aha a e al., 2012). The e a e some s udies on he p esence o signa u e egions wi h axonomic/phylogene ic implica ions in he ibosomal genes (Ma ínez-Mu cia e al., 1992, 2007;Ue e al., 2011;ˇ Reháko á e al., 2014;Ma ínez-Mu cia and Lamy, 2015). Some egions wi h signa u e mo i s de ec ed in he p esen s udy ha e also shown implica ions o phylogene ic analysis in cyanobac e ia, including egions H15, H17, H21, H22-H23, H41, and H44 (ˇ Reháko á e al., 2014). A ee was also cons uc ed weigh ing such posi ions (Supplemen a y Figu e S1B), which allowed a be e de ini ion o he main clus e s obse ed wi h he whole 16S RNA sequences al hough, as expec ed, di e en ia ion among species wi hin each clus e was lowe . Two sub-clus e s we e obse ed in Clus e 1, whe e he species A. bu zle i and ‘A. lacus’ g ouped in o a well-di e en ia ed b anch wi h espec o he o he species in he clus e (Supplemen a y Figu e S1B). In his analysis, A. paci icus was clea ly loca ed in he Clus e 3, whe eas in Clus e 4, A anae ophilus was he bo de line species, while A. eb onensis and ‘A. medi e aneus’ we e loca ed in an independen b anch (Supplemen a y Figu e S1B). The e o e, he signa u e mo i s desc ibed he e migh be a new ool o iden i ica ion o he di e en clus e s and/o genus. Genomic Indices The esul s o he calcula ions o he ANI and he isDDH among he 36 s udied genomes a e gi en in he Supplemen a y Table S2 and Supplemen a y Figu e S4. The esul s o he ANI and isDDH calcula ions showed ha he genomes g ouped in o he same clus e s obse ed by he analyses o he MLSA o he 13 housekeeping and co e genes (Figu es 1,2). Ranges o ANI wi hin each clus e we e om 75.2 o 95.4%, whe eas isDDH alues we e be ween 19.5 and 65.4% (Figu e 2 and Table 3). These esul s con i m he phylogene ic analysis o he 13 new candida e species because all o hem showed ANI and isDDH alues o <96% and <70%, espec i ely, which a e he cu -o alues p oposed o he delinea ion o new species (Kons an inidis and Tiedje, 2005;Go is e al., 2007; Rich e and Rosselló-Mó a, 2009;Figue as e al., 2017). As discussed in o he s udies, he ANI and isDDH indices p o ided eliable in o ma ion o he delinea ion o A cobac e species and a e also included in he minimal guidelines o de ine species using genomes (Whi educk-Lé eillée e al., 2015, 2016;Figue as e al., 2017;Chun e al., 2018). Al hough hose indices a e no conside ed use ul o delimi ing gene a, each o he ou clus e s showed alues ha anged be ween 75.2 and 81.8% as hei lowes ANI, which migh be he sui able ange o sepa a ing di e en , closely ela ed gene a. These alues a e ela i ely simila o hose epo ed by Qin e al. (2014) ha ound 68–82% in e species ANI alues among he gene a ha hey s udied. Values o ANI ob ained o he candida e species ‘A. aqua icus’ we e lowe han he o he esul s, om 70.0% wi h A. c yae ophilus LMG 24291T o 71.9% wi h A. bi al io um CECT 7835Tand mo e in line wi h he Qin e al. (2014) esul s o 68% (Supplemen a y Table S2). In he case o he isDDH he lowe alues among species in he same clus e anged be ween 19.5 and 24.8%, and again hese migh be he le els associa ed o di e en gene a. F on ie s in Mic obiology | www. on ie sin.o g 8Sep embe 2018 | Volume 9 | A icle 2077 micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 9 Pé ez-Ca aluña e al. Re isi ing he Taxonomy o he Genus A cobac e FIGURE 1 | Spli decomposi ion ne wo k cons uc ed wi h he conca ena ed sequences o 284 co e genes om he genomes o 36 ype and ep esen a i e s ains o A cobac e . Scale ba , base subs i u ions pe si e. Wi h he aim o con i ming i he clus e s obse ed migh ep esen di e en gene a, as sugges ed by he phylogene ic analyses, he simila i y indices AAI and POCP we e also calcula ed (Supplemen a y Table S3). In ag eemen wi h he 60–80% AAI ha ha e been desc ibed o species belonging o he same genus (Kons an inidis and Tiedje, 2005) all ou clus e s showed lowe anges o be ween 67.6 o 80.3% (Table 3). All he clus e s also complied wi h he POCP p oposed o genus sepa a ion abo e 50% (Luo e al., 2014;Qin e al., 2014) because as shown in Table 3 all clus e s showed he lowes alues om 67.0 o 75.4%. I is widely known ha synonymous codon usage a ies among o ganisms and ha i is ela ed o di e ences in G+C con en , eplica ion s and skew, o gene exp ession (Suzuki e al., 2008;Fa ooqi e al., 2016). The in e ac ion o hese ac o s may a y among species depending on hei e olu iona y p ocess (Ma e al., 2015). I has also been sugges ed ha he ex en o codon usage bias plays a ole in he adap a ion o p oka yo ic o ganisms o hei en i onmen s and li es yles (Bo zman and Ma gali , 2011). To analyze he o e all codon usage ends o he A cobac e species, he equencies o he di e en codons we e ob ained om he whole genomes and he RSCU was compu ed using he CAI, which is a use ul ool o es ima ing codon usage bias (Ma e al., 2015;Fa ooqi e al., 2016). A i s inding was ha all he A cobac e species p esen ed a p e e en ial use o he codons inishing in A o T (Supplemen a y Figu e S5), which migh be expec ed due o hei low G+C% con en . The cha ac e is ic pa e n showed by A. aqua icus is no ewo hy (Supplemen a y Figu e S5), which suppo s i s di e en ia ion om he o he species in Clus e 3 as well as i s unique axonomy. Such di e ence was he only s a is ically signi ican (p<0.05) in he mul inomial eg ession analysis ca ied ou . Nex , he codon usage ends we e analyzed by PCA o e eal possible e olu iona y ela ionships. In e es ingly, di e en g oups o s ains could be obse ed in he h ee- dimensional g aphic (Figu e 3), which co ela ed wi h hose clus e s es ablished in he di e en phylogene ic analyses, as shown abo e. As epo ed p e iously o di e en species o Mycoplasma (Ma enda e al., 2005;Ma e al., 2015), PCA p o ides an addi ional pa hway o in es iga e he e olu iona y di ec ion o he A cobac e species. In addi ion, simila i ies in he synonymous codon usage pa e ns migh e lec simila li es yles (pa hogenic s. non-pa hogenic) and adap a ion o ce ain en i onmen s (ma ine wa e , shell ish, e c.). Me abolic In e ence and Pheno ypic Analysis Phylogene ic and genomic analysis con i med he exis ence o ou clus e s among he alida ed and candida e A cobac e F on ie s in Mic obiology | www. on ie sin.o g 9Sep embe 2018 | Volume 9 | A icle 2077 micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 16 Pé ez-Ca aluña e al. Re isi ing he Taxonomy o he Genus A cobac e Desc ip ion o Malacobac e canalis comb. no . Basonym: A cobac e canalis Pé ez-Ca aluña e al., 2018b. The desc ip ion is he same gi en by Pé ez-Ca aluña e al. (2018b). The ype s ain is F138-33T(= CECT 8984T= LMG 29148T). Desc ip ion o Malacobac e mollusco um comb. no . Basonym: A cobac e mollusco um Figue as e al., 2011a. The desc ip ion is he same gi en by Figue as e al. (2011a). The ype s ain is F98-3T(= CECT 7696T= LMG 25693T). Desc ip ion o Malacobac e paci icus comb. no . Basonym: A cobac e paci icus Zhang e al., 2015. The desc ip ion is he same gi en by Zhang e al. (2015). The ype s ain is SW028T(= DSM 25018T = JCM 17857T= LMG 26638T). Desc ip ion o Haloa cobac e gen. no . Haloa cobac e (Ha.lo.a .co.bac’ e , G . n. halo, sal ; N.L. masc. n. A cobac e , a bac e ial gene ic name; N.L. masc. n. Haloa cobac e , A cobac e sal lo ing). G am-nega i e, cells a e od shaped and mo ile. Cell size 0.1– 0.5 µm in diame e and 0.9–2.5 µm in leng h. Oxidase posi i e and ca alase a iable among species. Halophilic, g ow h can be ob ained wi hin he ange o 0.5% ( a iable among species) and up o 4% NaCl. G ow h occu s a 15–42◦C. G ow h a 37◦C in mic oae ophilic condi ions o a 42◦C in anae obiosis a iable among species. Ca bohyd a es a e no e men ed. Some species may educe ni a e o ni i e. Nega i e o he hyd olysis o u ea (wi h he excep ion o H. eb onensis). Some species may hyd olyze indoxyl ace a e. G ow h does no occu in he p esence o oxgall (1% w / ol) (wi h he excep ion o H. mollusco um) o 2,3,5- iphenyl e azolium chlo ide (0.04%, w / ol). No g ow h on CCDA. Some species may g ow in he p esence o glycine (1% w / ol) o sa anin (0.05% w / ol). Sensi i e o ce ope azone (64 mg/l). Range o DNA G+C con en is 27.3–29.9 mol%. The ype species is Haloa cobac e bi al io um. Desc ip ion o Haloa cobac e bi al io um comb. no . Basonym: A cobac e bi al io um Le ican e al., 2012. The desc ip ion is he same gi en by Le ican e al. (2012). The ype s ain is F4T(= CECT 7835T= LMG 26154T). Desc ip ion o Haloa cobac e anae ophilus comb. no . Basonym: A cobac e anae ophilus Sasi-Jyo hsna e al., 2013. The desc ip ion is he same gi en by Sasi-Jyo hsna e al. (2013). The ype s ain is JC84T(= KCTC 15071T= MTCC 10956T= DSM 24636T). Desc ip ion o Haloa cobac e eb onensis comb. no . Basonym: A cobac e eb onensis Le ican e al., 2015. The desc ip ion is he same gi en by Le ican e al. (2015). The ype s ain is F128-2T(= CECT 8441T= LMG 27922T). Desc ip ion o Poseidonibac e gen. no . Poseidonibac e (Po.se.i.do.ni.bac’ e , G . n. Poseidon, God o he sea; G . n. bac e , od; N.L. masc. n. Poseidonibac e e e ing o he ma ine habi a o his bac e ia). G am-nega i e, cells a e od shaped and mo ile. Oxidase and ca alase posi i e. Halophilic, no g ow h can be ob ained wi hou seawa e o he addi ion o combined ma ine sal s o he medium. G ow h occu s a 15◦C–25◦C, bu no a 37◦C o 42◦C. Range o pH o g ow h is 6–8. Ca bohyd a es a e no e men ed. Reduce ni a e o ni i e. Nega i e o he hyd olysis o indoxyl ace a e and u ea. G ow h occu s in he p esence o sa anin (0.05% w / ol), and 2,3,5- iphenyl e azolium chlo ide (0.04%, w / ol), bu no in he p esence o glycine (1% w / ol) sensi i e o ce ope azone (30 µg). Possess ubiquinone MK-6 as a espi a o y quinone. DNA G+C con en is 28.7 mol%. The ype species is Poseidonibac e leki hoch ous. Desc ip ion o Poseidonibac e leki hoch ous comb. no . Basonym: A cobac e leki hoch ous Diéguez e al., 2017. The desc ip ion is he same gi en by Diéguez e al. (2017). The ype s ain is LFT1.7T(= CECT 8942T= DSM 100870T). AUTHOR CONTRIBUTIONS MF and JR designed he wo k. AP-C, NS-M, and AD pe o med he pheno ypic and phylogene ic expe imen s. AP-C and SB ca ied ou he genome sequencing and analysis. AP-C, AL, and JR pe o med he bioin o ma ic wo k. JR, MF, AP-C, and AD w o e he pape . FUNDING This wo k was suppo ed in pa by G an s JPIW2013-69095- C03-03 om he Minis e io de Economía y Compe i i idad (MINECO), AQUAVALENS o he Se en h F amewo k P og am (FP7/2007-2013) g an ag eemen 311846 om he Eu opean Union and AGL2013-42628-R and AGL2016-77539-R (AEI/FEDER UE) om he Agencia Es a al de In es igación (Spain). ACKNOWLEDGMENTS The au ho s hank D . F. J. Ga cía (Labo a o io Cen al de Ve e ina ia de Alge e, MAGRAMA, Mad id, Spain) and D s. R. Alonso, I. Ma inez-Malaxe xeba ia, and A. Fe nandez-As o ga [Facul y o Pha macy, Uni e si y o he Basque Coun y (UPV- EHU), Vi o ia-Gas eiz, Spain], o kindly p o iding some o he F on ie s in Mic obiology | www. on ie sin.o g 16 Sep embe 2018 | Volume 9 | A icle 2077 micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 17 Pé ez-Ca aluña e al. Re isi ing he Taxonomy o he Genus A cobac e A cobac e s ains. AP-C hanks Ins i u d’In es igació Sani à ia Pe e Vi gili (IISPV) o he Ph.D. ellowship and NS-M hanks he Uni e si a Ro i a i Vi gili (URV), he Ins i u de Rece ca i Tecnologia Ag oalimen à ia (IRTA) and he Banco San ande o he Ph.D. ellowship. SUPPLEMENTARY MATERIAL The Supplemen a y Ma e ial o his a icle can be ound online a : h ps://www. on ie sin.o g/a icles/10.3389/ micb. 2018.02077/ ull#supplemen a y-ma e ial REFERENCES Adilakshmi, T., Bellu , D. L., and Woodson, S. A. (2008). Concu en nuclea ion o 16S olding and induced i in 30S ibosome assembly. Na u e 455, 1268–1272. doi: 10.1038/na u e07298 Bandel , H. J., and D ess, A. W. M. 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The use, dis ibu ion o ep oduc ion in o he o ums is pe mi ed, p o ided he o iginal au ho (s) and he copy igh owne (s) a e c edi ed and ha he o iginal publica ion in his jou nal is ci ed, in acco dance wi h accep ed academic p ac ice. No use, dis ibu ion o ep oduc ion is pe mi ed which does no comply wi h hese e ms. F on ie s in Mic obiology | www. on ie sin.o g 19 Sep embe 2018 | Volume 9 | A icle 2077