Taxogenomics of the Genus Cyclobacterium: Cyclobacterium xiamenense and Cyclobacterium halophilum as Synonyms and Description of Cyclobacterium plantarum sp. nov.

mic oo ganisms A icle Taxogenomics o he Genus Cyclobac e ium: Cyclobac e ium xiamenense and Cyclobac e ium halophilum as Synonyms and Desc ip ion o Cyclobac e ium plan a um sp. no . Azadeh Shahinpei 1, Mohammad Ali Amoozega 1, Leila Mi eizi 1, Mahdi Mosh aghi Nikou 2, An onio Ven osa 3,* and C is ina Sánchez-Po o 3 1Ex emophiles Labo a o y, Depa men o Mic obiology, Facul y o Biology and Cen e o Excellence in Phylogeny o Li ing O ganisms, College o Science, Uni e si y o Teh an, 1417414418 Teh an, I an; [email p o ec ed] (A.S.); [email p o ec ed] (M.A.A.); [email p o ec ed] (L.M.) 2Mic oo ganisms Bank, I anian Biological Resou ce Cen e (IBRC), ACECR, 1551916111 Teh an, I an; [email p o ec ed] 3Depa men o Mic obiology and Pa asi ology, Facul y o Pha macy, Uni e si y o Se illa, 41012 Se illa, Spain; [email p o ec ed] *Co espondence: [email p o ec ed]; Tel.: +34-954-556-765 Recei ed: 5 Ap il 2020; Accep ed: 20 Ap il 2020; Published: 23 Ap il 2020   Abs ac : The genus Cyclobac e ium belongs o he phylum Bac e oide es and includes eigh species. Ou s udy, based on he genomic pa ame e s in silico DNA–DNA hyb idiza ion (GGDC), a e age nucleo ide iden i y (O hoANI), and a e age amino acid iden i y (AAI), con i med ha all cu en species o Cyclobac e ium belong o his genus and cons i u e a cohe en phylogenomic g oup, bu wi h species o ming wo sepa a e b anches. In addi ion, he genome-based analyses e ealed ha Cyclobac e ium xiamenense and Cyclobac e ium halophilum a e membe s o he same species. Besides, we ca ied ou a axonomic cha ac e iza ion o he new s ain GBPx2 T , isola ed om he halophy ic plan Salico nia sp. Analysis o i s 16S RNA gene sequence showed he highes sequence simila i y (97.5%) o Cyclobac e ium lianum HY9 T . Pe cen ages o GGDC and O hoANI be ween s ain GBPx2 T and species o he genus Cyclobac e ium we e lowe han he h eshold alue o species delinea ion. The DNA G+C con en was 43.0 mol%. The pola lipids included phospha idyle hanolamine as well as one uniden i ied phospholipid and ou uniden i ied lipids, and i s majo cellula a y acids we e iso-C 15:0 and summed ea u e 3 (C 16:1ω 7c and/o iso-C 15:0 2-OH). The only quinone p esen was menaquinone 7. Based on a combina ion o pheno ypic, chemo axonomic, and phylogenomic ea u es, he GBPx2 T s ain ep esen s a no el species o he genus Cyclobac e ium, o which he name Cyclobac e ium plan a um sp. no . is p oposed. The ype s ain o Cyclobac e ium plan a um is GBPx2 T (=IBRC-M 10634T=LMG 28551T). Keywo ds: Cyclobac e ium;Cyclobac e ium xiamenense;Cyclobac e ium halophilum;Cyclobac e ium plan a um; axogenomics; bac e ial axonomy; halophilic bac e ia; new species 1. In oduc ion The genus Cyclobac e ium is he ype genus o he amily Cyclobac e iaceae, o de Cy ophagales, wi hin he class Cy ophagia, in he phylum Bac e oide es [ 1 ]. This genus was o iginally desc ibed by Raj and Maloy [ 2 ] and has been emended h ee imes [ 3 – 5 ]. Cyclobac e ium ma inum ( ype species) was ini ially desc ibed as Mic ocyclus ma inus ( e e ing o i s ib ioid bac e ial cell mo phology ha exhibi s a closed ing-like mo phology) [ 6 ], and was eclassi ied as Flec obacillus ma inus [ 7 ]. Mic oo ganisms 2020,8, 610; doi:10.3390/mic oo ganisms8040610 www.mdpi.com/jou nal/mic oo ganisms Mic oo ganisms 2020,8, 610 2 o 16 Recen ly, he genus Cyclobac e ium has been es ablished as a sepa a e genus and species om he genus Flec obacillus, which includes i s eshwa e coun e pa s [ 2 ]. The genus Cyclobac e ium comp ises eigh species wi h alid published names: Cyclobac e ium ma inum ( ype species) [ 2 ], Cyclobac e ium amu skyense [ 8 ], Cyclobac e ium lianum [ 3 ], Cyclobac e ium qasimii [ 9 ], Cyclobac e ium caenipelagi [ 4 ], Cyclobac e ium jeungdonense [ 10 ], Cyclobac e ium xiamemense [ 5 ], and Cyclobac e ium halophilum [ 11 ]. The e is also ano he species, “Cyclobace ium sediminis”, which was desc ibed in 2017, bu i s name has no been alida ed o da e [ 12 ]. These species ha e been isola ed om di e en saline habi a s such as seawa e , ma ine sedimen s, soil om sola sal e ns, agg ega es o he alga Chlo ella au o ophica, o om sea cucumbe [ 2 – 5 , 8 – 11 ]. Fu he mo e, 16S RNA gene sequences ela ed o his genus ha e also been epo ed by cul u e-dependen and/o cul u e-independen s udies in di e en habi a s such as a soda saline c a e lake [ 13 ], mic obial ma s om An a c ic lakes [ 14 ], o om an alkaline, cold habi a in G eenland [ 15 ]. The species o his genus include G am-s ain-nega i e, cu ed ing-like o ho seshoe-shaped bac e ia. These species a e non- lagella ed, non-mo ile, ae obic, and he e o ophic, and hei colonies a e pigmen ed pink o o ange/ ed. The majo o sole espi a o y quinone is MK-7. They a e psych o ole an o mesophilic and halo ole an o mode a ely halophilic. The G+C con en o hei DNA anges om 33.7 o 48.1 mol% and hei majo cellula a y acids (>10%) a e iso-C 15:0 and summed ea u e 3 (comp ising C16:1ω7c and/o C16:1ω6c) [2–5,8–11]. In 2010, s ain GBPx2 T was isola ed om Salico nia sp., a halophy ic plan , in he Gomishan we land, I an. This s ain was a ilia ed o he genus Cyclobac e ium bu di e ed in some pheno ypic and phylogene ic ea u es om he Cyclobac e ium species. We p opose ha i ep esen s a no el species o he genus Cyclobac e ium. Genome-based analysis was pe o med, using he genome sequences o he ype s ains o he species o Cyclobac e ium and hose o species o he amily Cyclobac e iaceae a ailable in da abases, in o de o ca y ou a axogenomic s udy and de e mine in de ail he phylogenomic ela ionships among species o he genus Cyclobac e ium and o he membe s o he amily. 2. Ma e ials and Me hods 2.1. Bac e ial S ains S ain GBPx2 T was isola ed om Salico nia sp., a halophy ic plan o he Gomishan we land, an alkaline, halassohaline, coas al-ma ine we land loca ed along he eas e n sho e o he Caspian Sea in I an. The we land wa e con ains 30–50 g dissol ed sal s pe li e , and he a e age pH is 8.8 [ 16 ]. The geog aphic coo dina es o he sampling loca ion we e 37 ◦ 03 0 64.2 00 N 054 ◦ 01 0 90.4 00 E. Plan s we e sampled om sou heas e n he we land in No embe 2010. The no el s ain was isola ed om a halophy ic plan by using se ial dilu ions: 10 g o he sample was weighed and added o 90 mL o s e ile 3.0% (w/ ) NaCl solu ion. Se ial dilu ions we e pla ed on Ma ine Aga 2216 (MA; Di co) and incuba ed a 30 ◦ C o wo weeks. The colony o he s ain was subsequen ly pu i ied h ee imes by pla ing on he same medium. I was main ained on he same medium and also a − 80 ◦ C in MA medium wi hou aga and supplemen ed wi h 37.8% (w/ ) glyce ol. The ype s ains o h ee Cyclobac e ium species we e ob ained om he I anian Biological Resou ce Cen e (IBRC) and used as e e ence s ains. These we e Cyclobac e ium lianum IBRC-M 10422 T , Cyclobac e ium jeungdonense IBRC-M 11102 T , and Cyclobac e ium halophilum IBRC-M 10761 T . They we e cul u ed ollowing he ecommenda ions o he cul u e collec ion. 2.2. Taxophylogenomic Cha ac e iza ion 2.2.1. DNA Ex ac ion, Pu i ica ion, and Sequencing DNA was ex ac ed ollowing he p o ocol o Ma mu [ 17 ]. The DNA quali y was checked by (0.8%) aga ose gel elec opho esis. The quan i ica ion o he ex ac ed DNA was de e mined by spec opho ome y (DeNo ix DS-11 FX, DeNo ix Technologies, Wilming on, DA, USA) and luo ome y (Qubi 3.0 Fluo ome e , The mo ishe Scien i ic, Wal ham, MA, USA). PCR p oduc s we e pu i ied using Mic oo ganisms 2020,8, 610 3 o 16 he comme cial ki MEGAquick-spinTM Plus (IN RON Bio ecnology, Labo aq, Se illa, Spain). Sequencing o he 16S RNA PCR p oduc s was ca ied ou by Mac ogene (Sangdaewon-dong, Gyeonggi-do, Sou h Ko ea) using he Sange me hod and he p ime s 16F27 (5 0 -AGAGTTTGATCMTGGCTCAG-3 0 ), 16R343 (5 0 -ACTGCTGCCTCCCGTA-3 0 ), 16F530 (5 0 -GTGCCAGCAGCCGCGG-3 0 ), and 16R1488 (5 0 -CGGTTACCTTGTTAGGACTTCACC-3 0 ) [ 18 ], and he genome o s ain GBPx2 T was sequenced using he Illumina No aSeq 6000 pla o m (No ogene Eu ope, Camb idge, UK). 2.2.2. Phylogene ic Analysis Based on 16S RNA Gene Sequence Compa ison The pa ial 16S RNA gene was ampli ied using he uni e sal p ime pai s 16F27 and 16R1488 [ 19 ]. The PCR p oduc s we e isualized on 1% aga ose gel. The o wa d and e e se sequences we e assembled by using Ch omas P o 1.7.7 (Technelysium P y L d., Sou h B isbane, Aus alia). The 16S RNA gene sequence o s ain GBPx2 T was ob ained and used o BLAST sea ches in GenBank and phylogene ic analysis. The iden i ica ion o phylogene ic neighbo s and calcula ion o pai wise 16S RNA gene sequence simila i y we e achie ed using he EzBioCloud se e (h ps://www.ezbiocloud. ne /) [ 20 ] and he alignmen s we e pe o med by CLUSTAL-X [ 21 ]. E olu iona y dis ances be ween aligned 16S RNA gene sequences o s ain GBPx2 T wi h he mos closely ela ed ype s ains we e calcula ed using he Jukes–Can o model, and phylogene ic ees we e econs uc ed by he neighbou -joining [ 22 ], minimum-e olu ion [ 23 ], and maximum-likelihood [ 24 ] me hods using he MEGA e sion 6 p og am [ 25 ]. Boo s ap analysis was ca ied ou o e alua e he ee opology by pe o ming esampling 1000 imes [ 26 ]. The GenBank/EMBL/DDBJ accession numbe o he 16S RNA gene sequence o s ain GBPx2 T is MG457806. The 16S RNA gene sequences o he e e ence ype s ains used o he phylogene ic compa ison we e ob ained om GenBank da abase and hei accession numbe s a e shown in Figu e 1. 2.2.3. Genome Assembly and Anno a ion The no o assembly o he eads o he genome o s ain GBPx2 T was pe o med using Spades 3.13.0 [ 27 ]. The quali y o inal con igs was assessed by bioin o ma ics ools CheckM 1.0.5 [ 28 ] and Quas 2.3 [ 29 ]. The genome sequence was anno a ed using he NCBI P oka yo ic Genome Anno a ion Pipeline (PGAP) [ 30 ]. The genome o s ain GBPx2 T was deposi ed in GenBank/EMBL/DDBJ unde he accession numbe JAANYN00000000. 2.2.4. Phylogenomic Compa a i e Analysis Fo he phylogenomic compa a i e analysis we used genomes a ailable om GenBank da abase. The cha ac e is ics o he genomes and hei accession numbe s o he ype s ains o species o he genus Cyclobac e ium a e shown in Table 1. The quali y o hese genome sequences was in acco dance wi h he ecommended minimal s anda ds o he use o genome da a o he axonomy o p oka yo es [ 31 ]. To de e mine he co e-genome, he En eomics [32] ool was used. To iden i y clus e s o o hologous genes (OGs), an all- e sus-all BLAST sea ch based on p o ein-coding gene anno a ed sequences o s ain GBPx2 T and all ype species o he gene a included in he amily Cyclobac e iaceae a ailable in da abases was ca ied ou . Those OGs sha ed among all axa and p esen in a single copy pe genome we e selec ed. They we e aligned wi h MUSCLE . 3.8.31 [ 33 ] and subsequen ly conca ena ed. A maximum-likelihood ee was cons uc ed using Fas T ee . 2.1.9 [ 34 ] wi h he JTT eplacemen ma ix [ 35 ] unde he CAT app oxima ion (single a e o each si e) wi h 20 a e ca ego ies. Local suppo alues we e es ima ed wi h he Shimodai a–Hasegawa es [36]. Mic oo ganisms 2020,8, 610 4 o 16 Table 1. Gene al ea u es o he genomes o he ype s ains o species o he genus Cyclobac e ium. Fea u e 1 2 3 4 5 6 7 8 Size (bp) 6,169,285 6,158,829 5,662,104 5,792,371 5,675,162 6,221,270 6,291,928 5,784,474 Con igs 37 1 30 41 31 1 202 98 Genome co e age 193X 101X 175X 100X 247X 30X 240X 100X G+C(mol%) 43.0 38.3 48.4 44.0 45.5 38.1 38.8 48.5 N50 (bp) 547,880 6,158,829 350,204 381,560 266,214 6,221,273 107,474 137,064 To al genes 4943 4833 4689 4646 4736 4981 5997 4595 P o ein coding genes 4818 4715 4635 4534 4687 4868 5958 4474 RNA 6 12 5 7 5 9 4 7 RNA 41 39 38 40 38 39 35 39 Accession numbe JAANYN000000000 CP012040 FNZH00000000 WMCD00000000 FRCY00000000 NC_015914 ATNM00000000 WIOK00000000 S ains: 1, S ain GBPx2 T ; 2, Cyclobac e ium amu skyense KCTC 12363 T ; 3, Cyclobac e ium halophilum IBRC-M 10761 T ; 4, Cyclobac e ium jeungdinense KCTC 23150 T ; 5, Cyclobac e ium lianum CGMCC 1.6102T; 6, Cyclobac e ium ma inum DSM 745T; 7, Cyclobac e ium qasimii M12-11BT; 8, Cyclobac e ium xiamenense CGMCC 1.12432T. Mic oo ganisms 2020,8, 610 5 o 16 2.2.5. In Silico DNA–DNA Hyb idiza ion (GGDC), A e age Nucleo ide Iden i y (ANI), and A e age Amino Acid Iden i y (AAI) The genomic pa ame e s o in silico DNA–DNA hyb idiza ion (GGDC), a e age nucleo ide iden i y (O hoANI), and a e age amino acid iden i y (AAI) among s ain GBPx2 T , he ype s ains o species o he genus Cyclobac e ium, and he ype species o he amily Cyclobac e iaceae a ailable om da abases we e de e mined. GGDC was calcula ed by he bioin o ma ic ool Genome- o-Genome Dis ance Calcula o (GGDC e sion 2.1) a ailable om he Leibniz Ins i u e DSMZ [ 37 ]. The O hoANI was calcula ed wi h ChunLab’s O hologous A e age Nucleo ide Iden i y Tool (OAT) [ 38 ]. Fo he es ima ion o he AAI, he Compa eM p og am (h ps://gi hub.com/dpa ks1134/Compa eM) was used. 2.3. Pheno ypic Cha ac e iza ion Cell mo phology and mo ili y we e examined using an Olympus BX51 mic oscope equipped wi h phase-con as op ics wi h cells om exponen ially g owing cul u es. G am s aining was pe o med by he Bu ke me hod [ 39 ]. Mo ili y was de e mined by he we -moun me hod [ 39 ]. Colony mo phology was obse ed on MA aga medium unde op imal g ow h condi ions a e incuba ion a 25 ◦ C o wo days. To de e mine he empe a u e and pH anges o g ow h, b o h cul u es o MA medium we e incuba ed a 0, 4, 10, 15, 20, 25–37 (a in e als o 1.0 ◦ C), 40, and 45 ◦ C and a pH 5–10 a in e als o 0.5 pH uni s; he bu e s sodium ace a e/ace ic acid (pH 5.0–6.0), T is/HCl (pH 6.5–8.5), and glycine/sodium hyd oxide (pH 9.0–10.0) we e added a a concen a ion o 50 mM. The equi emen s o NaCl o g ow h we e de e mined in media con aining 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.5, 10.0, 12.5, and 15.0% (w/ ) NaCl. Liquid cul u es we e incuba ed on a shaking incuba o a 150 pm and g ow h a es we e de e mined by moni o ing he inc ease in he op ical densi y (OD) a 600 nm (The moSpec onics Spec onic 20D+). Ca alase and oxidase es s, ni a e and ni i e educ ion, hyd olysis o aesculin, and p oduc ion o indole and H 2 S we e ca ied ou as ecommended by Smibe and K ieg [ 40 ], using media wi h 5% (w/ ) NaCl. Hyd olysis ac i i y o Tween 20, 40, and 80 was de ec ed as desc ibed by Gu i é ez and Gonz á lez [ 41 ]. Hyd olysis o gela in, casein, y osine, and s a ch, and ac i i y o u ease and DNase we e de e mined as desc ibed by Ma a e al. [ 42 ]. The anae obic g ow h o he s ain was es ed in he p esence o ni a e by adding 0.1% (w/ ) KNO 3 o he medium wi h 5% (w/ ) NaCl in illed s oppe ed ubes in an anae obic chambe [ 43 ]. Acid p oduc ion om ca bohyd a es was es ed in unbu e ed medium and was de e mined by measu ing he ini ial and inal pH o he medium. The cul u e was conside ed posi i e o acid p oduc ion i he pH dec eased by a leas 1 uni . Tes s o he u iliza ion o di e en compounds as he sole sou ce o ca bon and ene gy we e pe o med as ecommended by Ven osa e al. [44]. 2.4. An imic obial Suscep ibili y An imic obial suscep ibili y es s we e pe o med on Muelle –Hin on aga plus 5% (w/ ) ma ine sal s [ 44 ] seeded wi h a bac e ial suspension (in 5% [w/ ] sal s) con aining 1.5 × 10 6 c. .u. mL −1 using discs (HiMedia) imp egna ed wi h a ious an imic obial compounds. The pla es we e incuba ed a 25 ◦ C o 48 h and he inhibi ion zone was in e p e ed acco ding o he manu ac u e ’s manual. The ollowing an imic obial compounds we e used: amoxicillin (30 µ g), ampicillin (10 µ g), baci acin (10 µ g), ca benicillin (100 µ g), ce adine (30 µ g), ce azidime (30 µ g), cephalo hin (30 µ g), chlo amphenicol (30 µ g), e y h omycin (15 µ g), gen amicin (10 µ g), kanamycin (5 µ g), nalidixic acid (30 µ g), neomycin (30 µ g), ni o u an oin (300 µ g), no obiocin (5 µ g), penicillin G (10 U) polymyxin B (300 U), i ampicin (5 µg), s ep omycin (10 µg), and e acycline (30 µg). 2.5. Chemo axonomic Cha ac e iza ion Cell biomass o a y acids, isop enoid quinones, and pola lipids analyses was ob ained by cul i a ion on MA medium a pH 8 and 25 ◦ C. Cells we e ha es ed in he mid-exponen ial g ow h Mic oo ganisms 2020,8, 610 6 o 16 phase de e mined spec opho ome ically wi h an op ical densi y a 600 nm (OD 600 ). The whole-cell a y acids composi ion o s ain GBPx2 T was de e mined acco ding o he s anda d p o ocol o he Mic obial Iden i ica ion Sys em (MIDI, Ve sion 6.1; Iden i ica ion Lib a y TSBA40 4.1; Mic obial ID). Ex ac s we e analyzed using a Hewle Packa d model HP6890A gas ch oma og aph equipped wi h a lame-ioniza ion de ec o as desc ibed by Kämp e and K oppens ed [ 45 ]. Fa y acids peaks we e iden i ied using he TSBA40 da abase. The pola lipids and espi a o y quinones o s ain GBPx2 T we e analyzed as desc ibed by G o h e al. [46]. 3. Resul s and Discussion 3.1. Phylogene ic Analysis Based on 16S RNA Gene Sequence Compa ison The 16S RNA gene sequence compa a i e analysis o s ain GBPx2 T (1438 n ) showed he highes simila i y o Cyclobac e ium lianum HY9 T ,Cyclobac e ium jeungdonense HMD3055 T ,Cyclobac e ium xiamenense KD51 T , and Cyclobac e ium halophilum IBRC-M 10761 T wi h 97.5%, 96.7%, 96.2%, and 96.2% sequence simila i y, espec i ely, and alues lowe han 92.3% wi h species o o he gene a, such as Belliella o Fon ibac e . These pe cen ages we e ob ained by he EzBioCloud ool and indica e ha s ain GBPx2Tis a membe o he genus Cyclobac e ium. The 16S RNA gene sequence phylogene ic analysis using he maximum-likelihood algo i hm showed he posi ion o he no el s ain wi hin he genus Cyclobac e ium (Figu e 1). The phylogene ic posi ion was also con i med in ees gene a ed using he minimum-e olu ion and neighbou -joining algo i hms. Mic oo ganisms 2020, 8, x FOR PEER REVIEW 6 o 16 Figu e 1. Maximum-likelihood phylogene ic ee based on he 16S RNA gene sequence compa ison, showing he ela ionships be ween s ain GBPx2T and membe s o he amily Cyclobac e iaceae. Filled ci cles indica e nodes ha we e also ob ained in ees based on minimum-e olu ion and maximum- likelihood algo i hms. Boo s ap alues ( o 1000 eplica es) o e 70% a e shown a he nodes. The sequence accession numbe s a e shown in pa en hesis. Ba , 2% es ima ed sequence di e gence. The sequence o Pa apedobac e soli DCY14T (EF151805) was used as ou g oup. This phylogene ic ee shows ha he genus Cyclobac e ium is no monophyle ic; he species o his genus a e g ouped in o wo clea ly di e en ia ed b anches suppo ed wi h 100% alues o boo s ap. On he one hand, C. xiamenense KD51T, C. halophilum GASx41T, C. jeungdonense HMD3055T, C. lianum HY9T, and he new isola e GBPx2T appea g ouped, and on he o he hand C. ma inum LMG 13164T, C. qasimii M12-11BT, C. caenipelagi HD-17T, and C. amu skyense KMM 6143T a e clus e ed. To de e mine he ela ionship be ween hese wo clus e s, a phylogenomic compa a i e analysis be ween hem and also wi h membe s o o he gene a o he amily Cyclobac e iaceae was pe o med. 3.2. Phylogenomic Compa a i e Analysis We ca ied ou phylogenomic compa a i e analysis and ob ained he co e-genome ee, based on 1309 single-copy ansla ed genes o s ain GBPx2T, he genomes a ailable o he ype s ains o Cyclobac e ium species (Table 1), and he genomes o all ype species o he gene a o he amily Cyclobac e iaceae a ailable in da abases (Figu e 2). This analysis shows ha s ain GBPx2T cons i u es a axon which is su icien ly di e en om he o he species o Cyclobac e ium so as o be conside ed as a new species. Fu he , as occu ed in he phylogene ic ee based on he 16S RNA, he species o he genus Cyclobac e ium appea ed g ouped in wo di e en b anches. Finally, his phylogenomic ee showed a close phylogene ic ela ionship be ween Cyclobac e ium xiamenense CGMCC 1.12432T and Cyclobac e ium halophilum IBRC-M 10761T, wo species ha we e desc ibed almos simul aneously in 2014 [5,11], and so hey we e no conside ed o a compa ison be ween hem. Besides, he genomes o hese wo species a e only now a ailable o compa ison and he cu en compa a i e da a show in his s udy e ealed ha bo h a e membe s o he same species. Figu e 1. Maximum-likelihood phylogene ic ee based on he 16S RNA gene sequence compa ison, showing he ela ionships be ween s ain GBPx2 T and membe s o he amily Cyclobac e iaceae. Filled ci cles indica e nodes ha we e also ob ained in ees based on minimum-e olu ion and maximum-likelihood algo i hms. Boo s ap alues ( o 1000 eplica es) o e 70% a e shown a he nodes. The sequence accession numbe s a e shown in pa en hesis. Ba , 2% es ima ed sequence di e gence. The sequence o Pa apedobac e soli DCY14T(EF151805) was used as ou g oup. This phylogene ic ee shows ha he genus Cyclobac e ium is no monophyle ic; he species o his genus a e g ouped in o wo clea ly di e en ia ed b anches suppo ed wi h 100% alues o boo s ap. On he one hand, C. xiamenense KD51 T ,C. halophilum GASx41 T ,C. jeungdonense HMD3055 T ,C. lianum HY9 T , and he new isola e GBPx2 T appea g ouped, and on he o he hand C. ma inum LMG 13164 T , Mic oo ganisms 2020,8, 610 7 o 16 C. qasimii M12-11B T ,C. caenipelagi HD-17 T , and C. amu skyense KMM 6143 T a e clus e ed. To de e mine he ela ionship be ween hese wo clus e s, a phylogenomic compa a i e analysis be ween hem and also wi h membe s o o he gene a o he amily Cyclobac e iaceae was pe o med. 3.2. Phylogenomic Compa a i e Analysis We ca ied ou phylogenomic compa a i e analysis and ob ained he co e-genome ee, based on 1309 single-copy ansla ed genes o s ain GBPx2 T , he genomes a ailable o he ype s ains o Cyclobac e ium species (Table 1), and he genomes o all ype species o he gene a o he amily Cyclobac e iaceae a ailable in da abases (Figu e 2). This analysis shows ha s ain GBPx2 T cons i u es a axon which is su icien ly di e en om he o he species o Cyclobac e ium so as o be conside ed as a new species. Fu he , as occu ed in he phylogene ic ee based on he 16S RNA, he species o he genus Cyclobac e ium appea ed g ouped in wo di e en b anches. Finally, his phylogenomic ee showed a close phylogene ic ela ionship be ween Cyclobac e ium xiamenense CGMCC 1.12432 T and Cyclobac e ium halophilum IBRC-M 10761 T , wo species ha we e desc ibed almos simul aneously in 2014 [ 5 , 11 ], and so hey we e no conside ed o a compa ison be ween hem. Besides, he genomes o hese wo species a e only now a ailable o compa ison and he cu en compa a i e da a show in his s udy e ealed ha bo h a e membe s o he same species. Mic oo ganisms 2020, 8, x FOR PEER REVIEW 7 o 16 Figu e 2. Phylogenomic ee based on he co e o hologous ansla ed genes o s ain GBPx2T, ype species o Cyclobac e ium, and ype species o he gene a o he amily Cyclobac e iaceae ob ained om he genomes a ailable in da abases, based on he maximum-likelihood algo i hm. This ee was ob ained a e he alignmen o 1309 sha ed o hologous single-copy ansla ed genes o hese genomes. Boo s ap alues highe han 70% a e indica ed a b anch-poin s. Ba , 0.1 subs i u ions pe amino acid posi ion. 3.3. in silico DNA–DNA Hyb idiza ion (GGDC), ANI, and AAI Values In o de o con i m ha s ain GBPx2T was indeed a new axon and he ela ionship be ween C. xiamenense and C. halophilum and he wo clus e s o he genus Cyclobac e ium, a e age nucleo ide iden i y (O hoANI), a e age amino acid iden i y (AAI), and in silico DNA–DNA hyb idiza ion (GGDC) o he s ain GBPx2T and membe s o he amily Cyclobac e iaceae we e calcula ed. GGDC pe cen ages abo e o equal o 70% indica e ha he s ains can be assigned o he same species, and alues unde 70% indica e ha he s ains belong o di e en species [47–49]. GGDC alues we e equal o lowe han 35% be ween s ain GBPx2T and species o he genus Cyclobac e ium (Table 2), p o ing ha his s ain cons i u es a new species. In addi ion, he GGDC alue o 81.6% which was de e mined be ween C. xiamenense CGMCC 1.12432T and C. halophilum IBRC-M 10761T, which was highe han he h eshold pe cen age o 70% o species delinea ion, shows ha bo h species belong o he same axon [31,37]. Wi h espec o he GGDC alues be ween he o he membe s o his amily, all we e lowe han 70%, showing ha all o hem can be conside ed di e en axa a he species le el. O hoANI pe cen ages calcula ed be ween s ain GBPx2T and species o he genus Cyclobac e ium anged om 71.8% o 79.2% (Table 2), lowe han he h eshold alue o species delinea ion (95%– 96%) [31,38,49,50], showing ha s ain GBPx2T belongs o a di e en species. Values be ween 67.2% and 69.9% wi h he ype species o he o he gene a o he amily Cyclobac e iaceae we e ob ained. Fu he , he O hoANI alue o 97.8% be ween C. xiamenense CGMCC 1.12432T and C. halophilum IBRC-M 10761T showed again ha bo h species cons i u ed a single axon. An al e na i e o GGDC and ANI o mo e dis an ly ela ed genomes is he AAI. In his case, o con i m ha s ain GBPx2T and all species o Cyclobac e ium we e well assigned o his genus, he AAI pe cen ages be ween hem we e calcula ed. The AAI alues be ween each o he we e in he ange o 72.2%–97.9% (Table 3). These alues we e abo e he h eshold conside ed o species o he same Pleomo pho ib io ma inusSW125T(QCXY01000001) Luna imonas lona ensis AK24T(AQHR00000000) Echinicola paci ica DSM 19836T(ARDN00000000) Algo iphagus a kowskyi DSM 22686T(MSSV00000000) Belliella bal ica DSM 15883T(CP003281) Rhodonellum psych ophilum GCM71T(ARMB00000000) Indibac e alkaliphilus LW1T(ALWO00000000) Mongoliibac e ube DSM 27929T(PRJNA402522) Cecembia lona ensisLW9T(AMGM00000000) Ma ini adiussaccha oly icusAK6T (AMZY0000000) Aqui lexum bal icum DSM 16537T(LT838813) Ni i alea halalkaliphila LW7T(AJYA00000000) 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 0.10 Cyclobac e ium jeungdonese KCTC 12363T(WMCD00000000) Cyclobac e ium halophilum IBRC-M 10761T(FNZH00000000) Cyclobac e ium xiamenense CGMCC 1.12432T(WIOK00000000) Cyclobac e ium lianum CGMCC 1.6102T(FRCY00000000) Cyclobac e ium plan a um GBPx2T (JAANYN000000000) Cyclobac e ium ma inum DSM 745T(NC_015914) Cyclobac e ium amu skyense KCTC 12363T (CP_012040) Cyclobac e ium quasimii M12-11BT(ATNM00000000) Figu e 2. Phylogenomic ee based on he co e o hologous ansla ed genes o s ain GBPx2 T , ype species o Cyclobac e ium, and ype species o he gene a o he amily Cyclobac e iaceae ob ained om he genomes a ailable in da abases, based on he maximum-likelihood algo i hm. This ee was ob ained a e he alignmen o 1309 sha ed o hologous single-copy ansla ed genes o hese genomes. Boo s ap alues highe han 70% a e indica ed a b anch-poin s. Ba , 0.1 subs i u ions pe amino acid posi ion. 3.3. in silico DNA–DNA Hyb idiza ion (GGDC), ANI, and AAI Values In o de o con i m ha s ain GBPx2 T was indeed a new axon and he ela ionship be ween C. xiamenense and C. halophilum and he wo clus e s o he genus Cyclobac e ium, a e age nucleo ide iden i y (O hoANI), a e age amino acid iden i y (AAI), and in silico DNA–DNA hyb idiza ion (GGDC) o he s ain GBPx2Tand membe s o he amily Cyclobac e iaceae we e calcula ed. Mic oo ganisms 2020,8, 610 8 o 16 GGDC pe cen ages abo e o equal o 70% indica e ha he s ains can be assigned o he same species, and alues unde 70% indica e ha he s ains belong o di e en species [ 47 – 49 ]. GGDC alues we e equal o lowe han 35% be ween s ain GBPx2 T and species o he genus Cyclobac e ium (Table 2), p o ing ha his s ain cons i u es a new species. In addi ion, he GGDC alue o 81.6% which was de e mined be ween C. xiamenense CGMCC 1.12432 T and C. halophilum IBRC-M 10761 T , which was highe han he h eshold pe cen age o 70% o species delinea ion, shows ha bo h species belong o he same axon [ 31 , 37 ]. Wi h espec o he GGDC alues be ween he o he membe s o his amily, all we e lowe han 70%, showing ha all o hem can be conside ed di e en axa a he species le el. O hoANI pe cen ages calcula ed be ween s ain GBPx2 T and species o he genus Cyclobac e ium anged om 71.8% o 79.2% (Table 2), lowe han he h eshold alue o species delinea ion (95%–96%) [ 31 , 38 , 49 , 50 ], showing ha s ain GBPx2 T belongs o a di e en species. Values be ween 67.2% and 69.9% wi h he ype species o he o he gene a o he amily Cyclobac e iaceae we e ob ained. Fu he , he O hoANI alue o 97.8% be ween C. xiamenense CGMCC 1.12432 T and C. halophilum IBRC-M 10761Tshowed again ha bo h species cons i u ed a single axon. An al e na i e o GGDC and ANI o mo e dis an ly ela ed genomes is he AAI. In his case, o con i m ha s ain GBPx2 T and all species o Cyclobac e ium we e well assigned o his genus, he AAI pe cen ages be ween hem we e calcula ed. The AAI alues be ween each o he we e in he ange o 72.2%–97.9% (Table 3). These alues we e abo e he h eshold conside ed o species o he same genus (65%) [ 50 – 52 ], so we can a i m ha all species belong o he genus Cyclobac e ium. I is ema kable o highligh ha AAI alues be ween C. ma inum DSM 745 T ,C. qasimii M12-11B T , and C. amu skyense KMM 6143 T we e highe (83.5% o 87.5%) as compa ed o wi h o he species o Cyclobac e ium (72.2%–73.5%), and lowe han 66.2% wi h espec o species o he es o gene a o he amily Cyclobac e iaceae. Simila esul s we e obse ed in he o he g oup o species o he genus Cyclobac e ium ha appea g ouped in he 16S RNA phylogene ic ee (Figu e 1) and also in he co e-genome ee (Figu e 2). This g oup included C. xiamenense KD51 T ,C. halophilum IBRC-M 10761 T ,C. jeungdonense HMD3055 T ,C. lianum CGMCC 1.6102 T , and he new isola e GBPx2 T . AAI alues be ween hem anged om 77.2% o 73.5%. Wi h espec o he o he species o he genus Cyclobac e ium he AAI anged be ween 72.2% and 73.5%, and alues anged be ween 61.6% o 68.1% wi h ega d o he es o he gene a o he amily Cyclobac e iaceae. All hese da a showed ha he pe cen ages o species o Cyclobac e ium we e always highe han 65% and hus hey a e membe s o he same genus, al hough he e was a highe simila i y be ween he espec i e membe s o he wo phylog oups. The e o e, we conclude ha he genus Cyclobac e ium is monophyle ic wi hin he amily, bu once di e en ia ed, i is di ided in o wo clea ly sepa a ed g oups, as obse ed p e iously in bo h he 16S RNA and co e-genome ees (Figu es 1and 2). On he o he hand, he alue o 97.9% con i ms ha C. xiamenense and C. halophilum a e membe s o he same axon, as was desc ibed by Kons an inidis e al. [ 51 ] who es ablished he h eshold AAI ange o 95%–100% o s ains o he same species. Mic oo ganisms 2020,8, 610 9 o 16 Table 2. Pe cen ages o GGDC and O hoANI be ween s ain GBPx2Tand membe s o he amily Cyclobac e iaceae. Mic oo ganisms 2020, 8, x FOR PEER REVIEW 9 o 16 Table 2. Pe cen ages o GGDC and O hoANI be ween s ain GBPx2T and membe s o he amily Cyclobac e iaceae. Pe cen ages o simila i y 100 90–99 80–89 70–79 60–69 50–59 40–49 30–39 20–29 10–19 0–9 O hoANI 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1 100 78.1 80.0 71.8 71.4 71.2 70.6 70.5 69.3 68.5 68.5 68.2 69.7 68.8 68.7 69.0 68.4 67.8 69.1 67.8 2 26.9 100 82.4 72.0 71.1 71.4 70.6 70.5 68.8 68.1 68.3 68.4 69.6 68.6 68.7 69.0 68.6 67.7 68.9 67.6 3 31.3 44.4 100 72.1 71.1 71.5 70.7 70.6 69.0 68.2 68.2 68.1 69.3 68.8 68.7 69.0 68.6 67.6 69.1 67.6 4 14.3 14.6 14.5 100 79.2 76.0 74.3 74.1 69.9 69.1 68.4 67.7 69.0 69.0 68.7 68.7 68.9 68.0 69.0 67.5 5 14.1 13.8 14.1 35.0 100 74.1 73.5 73.4 69.6 69.2 68.0 67.4 69.3 68.6 68.2 68.4 68.5 67.9 68.9 67.2 6 14.2 14.3 14.4 20.8 17.8 100 76.6 76.4 69.3 69.2 68.0 67.5 68.6 68.8 68.3 68.2 68.7 67.9 68.7 67.3 7 13.9 13.9 14 17.4 16.9 24.2 100 97.8 69.4 69.3 68.1 67.1 68.1 68.3 67.9 67.9 68.2 68.0 68.5 67.6 8 14.0 13.9 14.1 17.4 16.7 23.9 81.6 100 69.2 69.2 68.0 67.1 68.3 68.2 68.1 68.2 68.1 67.7 68.7 67.6 9 12.9 12.8 12.8 13 13 12.9 12.9 12.9 100 69.6 67.3 67.2 68.5 68.5 68.6 68.4 68.7 67.7 69.0 67.1 10 12.9 12.8 12.9 12.9 12.8 12.9 13.1 13.0 13.1 100 68.0 67.4 68.8 68.3 68.9 68.3 69.2 68.1 68.9 67.8 11 12.7 12.8 12.8 12.7 12.7 12.7 12.7 12.7 12.7 12.7 100 68.7 69.5 68.6 68.9 69.0 69.0 68.3 69.0 68.1 12 12.7 12.7 12.7 12.6 12.5 12.6 12.6 12.6 12.6 12.6 12.7 100 69.7 69.0 68.7 68.9 68.6 68.0 69.0 67.5 13 12.8 12.8 12.9 12.8 12.9 12.8 12.7 12.7 12.8 12.8 12.8 13.0 100 71.7 72.5 71.7 71.5 69.7 72.4 69.3 14 12.8 12.8 12.8 12.7 12.7 12.7 12.7 12.7 12.7 12.7 12.8 12.8 13.6 100 70.9 70.7 70.8 70.0 71.7 68.1 15 12.8 12.8 12.8 12.8 12.8 12.7 12.8 12.8 12.7 12.9 12.7 12.8 14.1 13.4 100 75.7 72.7 70.1 72.5 68.4 16 12.8 12.7 12.8 12.7 12.7 12.7 12.8 12.8 12.7 12.7 12.9 12.8 13.6 13.2 21.2 100 72.2 69.9 72.4 68.2 17 12.7 12.7 12.7 12.7 12.7 12.8 12.7 12.7 12.8 12.8 12.9 12.7 13.4 13.2 14.5 14.1 100 70.6 72.5 69.1 18 12.7 12.7 12.6 12.7 12.6 12.7 12.7 12.6 12.7 12.7 12.8 12.7 12.9 12.9 13.1 13.2 12.8 100 70.9 68.1 19 12.8 12.7 12.7 12.8 12.9 12.7 12.7 12.7 12.8 12.8 12.8 12.7 13.6 13.2 13.8 13.8 13.9 13.3 100 68.4 20 12.7 12.7 12.7 12.7 12.6 12.7 12.7 12.7 12.6 12.7 12.8 12.7 13.1 12.8 12.8 12.8 13.1 12.7 12.8 100 S ains: 1, Cyclobac e ium ma inum DSM 745T; 2, Cyclobac e ium qasimii M12-11BT; 3, Cyclobac e ium amu skyense KCTC 12363T; 4, S ain GBPx2T; 5, Cyclobac e ium lianum CGMCC 1.6102T; 6, Cyclobac e ium jeungdinense KCTC 23150T; 7, Cyclobac e ium xiamenense CGMCC 1.12432T; 8, Cyclobac e ium halophilum IBRC-M 10761T; 9, Pleomo pho ib io ma inus SW125T; 10, Luna imonas lona ensis AK24T; 11, Echinicola paci ica DSM 19836T; 12, Algo iphagus a kowskyi DSM 22686T; 13, Belliella bal ica DSM 15883T; 14, Rhodonellum psych ophilum GCM71T; 15, Indibac e alkaliphilus LW1T; 16, Mongoliibac e ube DSM 27929T; 17, Cecembia lona ensis LW9T; 18, Ma ini adius saccha oly icus AK6T; 19, Aqui lexum bal icum DSM 16537T; 20, Ni i alea halalkaliphila LW7T. ANI: a e age nucleo ide iden i y. GGDC S ains: 1, Cyclobac e ium ma inum DSM 745 T ; 2, Cyclobac e ium qasimii M12-11B T ; 3, Cyclobac e ium amu skyense KCTC 12363 T ; 4, S ain GBPx2 T ; 5, Cyclobac e ium lianum CGMCC 1.6102 T ; 6, Cyclobac e ium jeungdinense KCTC 23150 T ; 7, Cyclobac e ium xiamenense CGMCC 1.12432 T ; 8, Cyclobac e ium halophilum IBRC-M 10761 T ; 9, Pleomo pho ib io ma inus SW125 T ; 10, Luna imonas lona ensis AK24 T ; 11, Echinicola paci ica DSM 19836 T ; 12, Algo iphagus a kowskyi DSM 22686 T ; 13, Belliella bal ica DSM 15883 T ; 14, Rhodonellum psych ophilum GCM71 T ; 15, Indibac e alkaliphilus LW1 T ; 16, Mongoliibac e ube DSM 27929 T ; 17, Cecembia lona ensis LW9 T ; 18, Ma ini adius saccha oly icus AK6 T ; 19, Aqui lexum bal icum DSM 16537 T ; 20, Ni i alea halalkaliphila LW7T. ANI: a e age nucleo ide iden i y. Mic oo ganisms 2020,8, 610 16 o 16 47. Kim, M.; Oh, H.S.; Pa k, S.C.; Chun, J. 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