Complete genome sequence of the halophilic and highly halotolerant Chromohalobacter salexigens type strain (1H11 T)

S anda ds in Genomic Sciences (2011) 5:379-388 DOI:10.4056/sigs.2285059 The Genomic S anda ds Conso ium Comple e genome sequence o he halophilic and highly halo ole an Ch omohalobac e salexigens ype s ain (1H11T) Alex Copeland1, Ka hleen O’Conno 2, Susan Lucas1, Alla Lapidus1, Ke ie W. Be y1, John C. De e 1,3, Tijana Gla ina Del Rio1, Nancy Hammon1, Eileen Dalin1, Hope Tice1, Sam Pi - luck1, Da id B uce1,3, Lynne Goodwin1,3, Cli Han1,3, Roxanne Tapia1,3, Elizabe h Saund- e s1,3, Je emy Schmu z3, Thomas B e in1,4 F ank La ime 1,4, Mi iam Land1,4, Lo en Hause 1,4, Ca men Va gas5, Joaquin J. Nie o5, Nikos C. Ky pides1, Na alia I ano a1, Ma kus Göke 6, Hans-Pe e Klenk6*, Laszlo N. Csonka2*, and Tanja Woyke1 1 DOE Join Genome Ins i u e, Walnu C eek, Cali o nia, USA 2 Depa men o Biological Sciences, Pu due Uni e si y, Wes La aye e, Indiana, USA 3 Los Alamos Na ional Labo a o y, Bioscience Di ision, Los Alamos, New Mexico, USA 4 Oak Ridge Na ional Labo a o y, Oak Ridge, Tennessee, USA 5 Depa men o Mic obiology and Pa asi ology, Uni e si y o Se ille, Spain 6 Leibniz Ins i u e DSMZ – Ge man Collec ion o Mic oo ganisms and Cell Cul u es, B aunschweig, Ge many *Co esponding au ho s: [email protected], [email protected] due.edu Keywo ds: ae obic, chemoo gano ophic, G am-nega i e, mo ile, mode a ely halophilic, halo ole an , ec oine syn hesis, Halomonadaceae, Gammap o eobac e ia, DOEM 2004 Ch omohalobac e salexigens is one o nine cu en ly known species o he genus Ch omoha- lobac e in he amily Halomonadaceae. I is he mos halo ole an o he so-called ‘mod- e a ely halophilic bac e ia’ cu en ly known and, due o i s s ong eu yhaline pheno ype, i is an es ablished model o ganism o p oka yo ic osmoadap a ion. C. salexigens s ain 1H11T and Halomonas elonga a a e he i s and he second membe s o he amily Halomonada- ceae wi h a comple ely sequenced genome. The 3,696,649 bp long ch omosome wi h a o al o 3,319 p o ein-coding and 93 RNA genes was sequenced as pa o he DOE Join Genome Ins i u e P og am DOEM 2004. In oduc ion S ain 1H11T (= DSM 3043 = ATCC BAA-138 = CECT 5384) is he ype s ain o he species Ch o- mohalobac e salexigens [1], which is one o cu - en ly nine species in he genus Ch omohalobac e [1,2]. The genus name was de i ed om he G eek wo ds ch oma, colo , hals halos, sal , and he Neo- La in bac e , od, meaning he colo ed sal od. The species epi he o igina ed om he La in wo ds sal salis, sal , and exigo, o demand; sal - demanding [3]. S ain 1H11T was o iginally iso- la ed in 1974 in Bonai , Ne he lands An illes, om sal e ns con aining 18.6% sal , and was ini ially published as a s ain belonging o he species Ha- lomonas elonga a [4]. In 2001, A ahal e al. ans- e ed he s ain o he genus Ch omohalobac e [2] as he ype s ain o he hen no el species C. salexigens [1] ollowing de ailed pheno ypic, geno- ypic, and phylogene ic analyses. C. salexigens is known o i s e y b oad salini y ange [1] and o i s ole as a model o ganism o p oka yo ic os- mosadap a ion [5-7], e.g. he syn hesis o ec oines (ec oine and hyd oxyec oine) o cell s ess p o- ec ion [8,9]. He e we p esen a summa y classi i- ca ion and cha ac e is ics o C. salexigens 1H11T, oge he wi h he desc ip ion o he comple e ge- nomic sequencing and anno a ion. Classi ica ion and ea u es The sequences o he i e iden ical 16S RNA genes o s ain 1H11T we e compa ed using NCBI BLAST [10] unde de aul se ings (e.g., conside ing only he high-sco ing segmen pai s (HSPs) om he bes 250 hi s) wi h he mos ecen elease o he G een- genes da abase [11] and he ela i e equencies o axa and keywo ds ( educed o hei s em [12]) we e de e mined and weigh ed by BLAST sco es. Ch omohalobac e salexigens ype s ain (1H11T) 380 S anda ds in Genomic Sciences The mos equen ly occu ing gene a we e Halo- monas (50.7%), Ch omohalobac e (46.3%), 'Hae e ehalobac e ' (1.7%), Bacillus (0.8%) and Pseudomonas (0.5%) (214 hi s in o al). Fo 16 hi s o sequences om membe s o he C. salexigens spe- cies, he a e age iden i y wi hin HSPs was 99.9% and he a e age co e age by HSPs was 97.9%. Fo 22 hi s o sequences om o he membe s o he ge- nus Ch omohalobac e , he a e age iden i y wi hin HSPs was 98.2% and he a e age co e age by HSPs was 98.6%. Among all o he species, he one yielding he highes sco e was Ch omohalobac e ma ismo - ui (X87222), which co esponded o an iden i y o 99.9% and an HSP co e age o 100.0%. (No e ha he G eengenes da abase uses he INSDC (= EMBL/NCBI/DDBJ) anno a ion, which is no an au- ho i a i e sou ce o nomencla u e o classi ica- ion.) The highes -sco ing en i onmen al sequence was EU799899 ('I 's all anking aqua ic Newpo Ha bo RI clone 1C227569'), which showed an iden i y o 100.0% and an HSP co e age o 100.0%. The mos equen ly occu ing keywo ds wi hin he labels o en i onmen al samples which yielded hi s we e 'soil' (12.1%), 'lake' (3.6%), 'salin' (3.0%), 'ag icul u ' (2.9%) and 'alkalin, chang, lood, o me , mexico, excoco' (2.6%) (36 hi s in o al). The mos equen ly occu ing keywo d wi hin he labels o en i onmen al samples which yielded hi s o a high- e sco e han he highes sco ing species was 'aqua , ha bou , newpo , ank' (25.0%) (2 hi s in o al). These keywo ds i easonably well wi h he ecolog- ical and physiological p ope ies epo ed o s ain 1H11T in he o iginal desc ip ion [1]. Figu e 1 shows he phylogene ic neighbo hood o C. salexigens in a 16S RNA based ee. The sequences o he i e iden ical 16S RNA gene copies in he ge- nome di e by wo nucleo ides om he p e iously published 16S RNA sequence (AJ295146), which con ains h ee ambiguous base calls. Figu e 1. Phylogene ic ee highligh ing he posi ion o C. salexigens ela i e o he ype s ains o he o h- e species wi hin he genus and he ype species o he o he gene a wi hin he amily Halomonadaceae. The ee was in e ed om 1,440 aligned cha ac e s [13,14] o he 16S RNA gene sequence unde he maximum likelihood (ML) c i e ion [15]. Roo ing was done ini ially using he midpoin me hod [16] and hen checked o i s ag eemen wi h he cu en classi ica ion (Table 1). The b anches a e scaled in e ms o he expec ed numbe o subs i u ions pe si e. Numbe s adjacen o he b anches a e suppo alues om 1,000 ML boo s ap eplica es [17] (le ) and om 1,000 maximum pa simony boo s ap eplica es [18] ( igh ) i la ge han 60%. Lineages wi h ype s ain genome sequencing p ojec s egis e ed in GOLD [19] a e labeled wi h one as e isk, hose also lis ed as 'Comple e and Published' wi h wo as e isks [20]. Copeland e al. h p://s anda dsingenomics.o g 381 Cells o C. salexigens s ain 1H11T a e s aigh o sligh ly cu ed ods, 0.7 o 1.0 by 2 o 3 µm in size (Figu e 2) wi h squa ed ends and occu singly o in pai s [1,4]. Cells o s ain 1H11T s ain G am- nega i e, a e mo ile wi h pola lagella, s ic ly ae obic, and a e non-spo e- o ming [1,4]. Ca bon and ni ogen sou ce u iliza ion and biochemis y o he s ain we e epo ed by A ahal e al. [1]. A pa - ial cha ac e iza ion o he ca bon-sou ce u iliza- ion by he o ganism has also been p esen ed by Csonka e al. [36], who epo ed ha he s ain can deg ade a numbe o a oma ic compounds, includ- ing benzoa e, p o oca echua e, 4-hyd oxybenzoa e, and oluene. C. salexigens 1H11T is a halophile, which acco ding o he classi ica ion p oposed by Kushne [37], is on he bo de line be ween "mode a e" halophiles ( hose g owing op imally be ween 2.9 -14.5% NaCl) and "ex eme" halophiles ( hose g owing op imally be ween 8.7 -23.2% NaCl). In addi ion, i displays ex ao dina ily high halo ole ance (con- side ed as he abili y o li e and su i e unde high sal concen a ions), and is able o g ow a sal concen a ions o e 17.4% and 32% in de- ined and complex media, espec i ely. Howe e , bo h he minimum NaCl equi emen and he up- pe limi o NaCl ole ance a e dependen on g ow h medium and empe a u e. The o ganism can ole a e highe NaCl concen a ions in LB o in o he complex media han in de ined media. In de ined media, halo ole ance is enhanced by os- mop o ec an s, such as glycine be aine o i s p e- cu so , choline [4,6,33]. In he complex medium SW (‘sea wa e ’), which is ou inely used o g ow- ing his ype o mic oo ganism, s ain 1H11T g ows op imally a 7.5 o 10% (w/ ) NaCl, wi h g ow h occu ing o e he ange o 0.9% o 25% NaCl [1]. In casein medium, which was ini ially used o s ain isola ion, g ow h occu s in he p esence o 32% sola sal s [4]. In SW medium con aining 10% (w/ ) o al sal s, C. salexigens 1H11T can g ow a a pH ange om 5 o 10, wi h an op imum a pH 7.5 [1]. In he same medium, he empe a u e ange o g ow h is 15 – 45°C, wi h an op imum a 37°C [1]. In he s anda d de- ined medium M63, supplemen ed wi h glucose as he sole ca bon sou ce, g ow h is op imal a 8.7 o 11.6% NaCl bu occu s o e he ange o 2.9% NaCl o a maximum o 19% NaCl [6]. In e es ingly, C. salexigens 1H11T exhibi s maximal g ow h a e in glucose-M63 wi h only 1.8% (0.3M) NaCl in he p esence o high concen a ions o sal s o o he ino ganic ions, including K+, Rb+, NH4+, B -, NO3-, o SO4- [38]. Howe e , i is an open ques ion whe he his s ain is unique among halophiles in being able o use o he ino ganic ions in addi ion o Na+ and Cl- o maximal g ow h a e. Chemo axonomy Da a on he s uc u e o he cell wall, a y acids lipid composi ion, quinones and pola lipids a e no a ailable. Figu e 2. Ligh mic oscopic image o C. salexigens 1H11T Ch omohalobac e salexigens ype s ain (1H11T) 382 S anda ds in Genomic Sciences Table 1. Classi ica ion and gene al ea u es o C. salexigens acco ding o he MIGS ecommenda ions [21]. MIGS ID P ope y Te m E idence code Cu en classi ica ion Domain Bac e ia TAS [22] Phylum P o eobac e ia TAS [23] Class Gammap o eobac e ia TAS [24,25] O de Oceanospi illales TAS [24,26] Family Halomonadaceae TAS [27-31] Genus Ch omohalobac e TAS [2,32] Species Ch omohalobac e salexigens TAS [1] Type s ain 1H11 TAS [1,4] G am s ain nega i e TAS [1] Cell shape od-shaped TAS [1] Mo ili y mo ile TAS [1] Spo ula ion none TAS [1] Tempe a u e ange mesophilic, 15–45°C TAS [1] Op imum empe a u e 37°C TAS [1] Salini y halophilic and halo ole an . Salini y ange om 0.9 o 32% (w/ ) NaCl in ich media, 2.9% o 19% (0.5 M o 3.75M) NaCl in minimal media; halo ole ance inc eased by osmo- p o ec an s; halo ole ance dec eases a high empe a u e. TAS [1,4,6,33] MIGS-22 Oxygen equi emen uses O2 and NO3- as elec on accep o s; does no g ow e men a i ely TAS [4] Ca bon sou ce a ious o ganic acids, alcohols, suga s, and a oma ic compounds TAS [1] Ene gy me abolism chemoo gano ophic NAS MIGS-6 Habi a sal e n, esh wa e TAS [4] MIGS-15 Bio ic ela ionship ee li ing TAS [1] MIGS-14 Pa hogenici y none NAS Biosa e y le el 1 TAS [34] Isola ion sola sal acili y, concen a ion mo e han 10% NaCl TAS [1] MIGS-4 Geog aphic loca ion Bonai e, Ne he lands An illes TAS [1] MIGS-5 Sample collec ion ime June 1974 TAS [4] MIGS-4.1 La i ude 12.25 NAS MIGS-4.2 Longi ude -68.26 NAS MIGS-4.3 Dep h su ace NAS MIGS-4.4 Al i ude sea le el NAS E idence codes - TAS: T aceable Au ho S a emen (i.e. a di ec epo exis s in he li e a u e); NAS: Non- aceable Au ho S a emen (i.e. no di ec ly obse ed o he li ing, isola ed sample, bu based on a gene ally accep ed p ope y o he spe- cies, o anecdo al e idence). These e idence codes a e om o he Gene On ology p ojec [35]. Genome sequencing and anno a ion Genome p ojec his o y This o ganism was selec ed o sequencing on he basis o he DOE Join Genome Ins i u e P og am DOEM 2004. The genome p ojec is deposi ed in he Genomes On Line Da abase [19] and he com- ple e genome sequence is deposi ed in GenBank. Sequencing, inishing and anno a ion we e pe - o med by he DOE Join Genome Ins i u e (JGI). A summa y o he p ojec in o ma ion is shown in Table 2. S ain his o y The his o y o s ain 1H11T begins wi h R.H. V eeland, who deposi ed he o ganism in he DSMZ open collec ion, whe e cul u es o he s ain a e main ained eeze d ied as well as in liquid ni ogen (since 1984). The s ain used o he p ojec was p o ided by he Ca men Va gas – Joa- quín Nie o lab in Se ille (Spain), who acqui ed i om he DSMZ. Copeland e al. h p://s anda dsingenomics.o g 383 Table 2. Genome sequencing p ojec in o ma ion MIGS ID P ope y Te m MIGS-31 Finishing quali y Finished MIGS-28 Lib a ies used Th ee genomic Sange lib a ies: 4 kb pUC, 8kb pMCL200 and osmid pcc1Fos lib a ies. MIGS-29 Sequencing pla o ms ABI3730 MIGS-31.2 Sequencing co e age 11.5 × Sange MIGS-30 Assemble s Ph ap MIGS-32 Gene calling me hod C i ica complemen ed wi h he ou pu o Glimme INSDC ID CP000285 GenBank Da e o Release Ap il 16, 2006 GOLD ID Gc00371 NCBI p ojec ID 12636 Da abase: IMG 637000075 MIGS-13 Sou ce ma e ial iden i ie DSM 3043 P ojec ele ance Bio emedia ion, Bio echnology, En i onmen al G ow h condi ions and DNA isola ion The cul u e o s ain 1H11T, DSM 3043, used o p epa e genomic DNA (gDNA) o sequencing was g own in LB medium wi h 1 M NaCl. DNA was ex- ac ed as desc ibed by O’Conno and Zusman [39]. The pu i y, quali y and size o he bulk gDNA p epa a ion we e assessed by JGI acco ding o DOE-JGI guidelines. Genome sequencing and assembly The genome was sequenced using a combina ion o 4 kb, 8 kb and osmid DNA lib a ies. All gene al as- pec s o lib a y cons uc ion and sequencing can be ound a he JGI websi e [40]. D a assemblies we e based on 44,750 o al eads. The Ph ed/Ph ap/Consed so wa e package was used o sequence assembly and quali y assessmen [41]. A e he sho gun s age, eads we e assembled wi h pa allel ph ap (High Pe o mance So wa e, LLC). Possible mis-assemblies we e co ec ed wi h Dup- inishe o ansposon bombing o b idging clones (Epicen e Bio echnologies, Madison, WI) [42]. Gaps be ween con igs we e closed by edi ing in Consed, cus om p iming, o PCR ampli ica ion (Roche Applied Science, Indianapolis, IN). A o al o 920 addi ional eac ions, 14 sha e and 18 ans- poson bomb lib a ies we e needed o close gaps and o aise he quali y o he inished sequence. The e o a e o he comple ed genome sequence is less han 1 in 100,000. Toge he all lib a ies p o- ided 11.5 × co e age o he genome. Genome anno a ion Genes we e iden i ied using wo gene modeling p og ams, Glimme [43] and C i ica [44] as pa o he Oak Ridge Na ional Labo a o y genome anno- a ion pipeline. The wo se s o gene calls we e combined using C i ica as he p e e ed s a call o genes wi h he same s op codon. Genes speci- ying ewe han 80 amino acids ha we e p e- dic ed by only one o he gene calle s and had no Blas hi in he KEGG da abase a ≤1e-05, we e dele ed. Au oma ed anno a ion was ollowed by a ound o manual cu a ion o elimina e ob ious o e laps. The p edic ed CDSs we e ansla ed and used o sea ch he Na ional Cen e o Bio echnol- ogy In o ma ion (NCBI) non- edundan da abase, UniP o , TIGRFam, P am, PRIAM, KEGG, COG, and In e P o da abases. These da a sou ces we e combined o asse a p oduc desc ip ion o each p edic ed p o ein. Non-coding genes and miscel- laneous ea u es we e p edic ed using RNAscan- SE [45], TMHMM [46], and signalP [47]. Genome p ope ies The genome consis s o a 3,696,649 bp long ch o- mosome wi h a 63.9% G+C con en (Figu e 3 and Table 3). O he 3,412 pu a i e genes, 3,319 a e p o ein-coding, and 93 speci y RNAs; 21 pseudo- genes we e also iden i ied. The majo i y o he p o ein-coding genes (76.8%) we e assigned a pu a i e unc ion while he emaining ones we e anno a ed as encoding hypo he ical p o eins. The dis ibu ion o genes in o COGs unc ional ca ego- ies is p esen ed in Table 4. Ch omohalobac e salexigens ype s ain (1H11T) 384 S anda ds in Genomic Sciences Figu e 3. G aphical ci cula map o he genome. F om ou side o he cen e : Genes on o wa d s and (colo by COG ca ego ies), Genes on e e se s and (colo by COG ca ego ies), RNA genes ( RNAs g een, RNAs ed, o he RNAs black), GC con en , GC skew. Insigh s in o he genome The publica ion o genome sequence s ain 1H11T is p eceded by some publica ions ha we e based on d a e sions o he sequence o on publicly a ailable genome sequence and anno a ion. O en e al. [48] ound ha he p edic ed isoelec ic poin s o pe iplasmic p o eins o C. salexigens 1H11T a e signi ican ly mo e acidic han hose o o hologous p o eins in mesophilic bac e ia, and hey sugges ed ha his ea u e may con ibu e o he halophilic cha ac e is ics o 1H11T. Analysis o he genomic sequence indic ed ha he o ganism has all o he enzymes o he Embden-Meye ho glycoly ic pa hway, hexose monophospha e shun , and TCA cycle bu seemed o lack he s anda d uc ose-1,6-bisphospha e phospha ase o he glu- coneogene ic pa hway [36]. K ejcík e al. p e- dic ed he ise hiona e o ma ion om au ine based on he genome sequence [49]. A es e al. ecen ly p esen ed a genome-scale econs uc ion o a me abolic ne wo k o s ain 1H11T ocusing on he up ake and accumula ion o indus ially impo an o ganic osmoly es such as ec oine and be aine [5]. Copeland e al. h p://s anda dsingenomics.o g 385 Table 3. Genome S a is ics A ibu e Value % o To al Genome size (bp) 3,696,649 100.00% DNA coding egion (bp) 3,333,410 90.17% DNA G+C con en (bp) 2,362,597 63.91% Numbe o eplicons 1 Ex ach omosomal elemen s 0 To al genes 3,412 100.00% RNA genes 93 2.73% RNA ope ons 5 P o ein-coding genes 3,319 97.27% Pseudogenes 21 0.62% Genes wi h unc ion p edic ion 2,621 76.82% Genes in pa alog clus e s 402 11.78% Genes assigned o COGs 2,842 83.29% Genes assigned P am domains 2,928 85.81% Genes wi h signal pep ides 689 20.19% Genes wi h ansmemb ane helices 828 24.27% CRISPR epea s 5 Table 4. Numbe o genes associa ed wi h he gene al COG unc ional ca ego ies Code alue %age Desc ip ion J 166 5.2 T ansla ion, ibosomal s uc u e and biogenesis A 1 0.0 RNA p ocessing and modi ica ion K 251 7.8 T ansc ip ion L 114 3.5 Replica ion, ecombina ion and epai B 1 0.0 Ch oma in s uc u e and dynamics D 34 1.1 Cell cycle con ol, cell di ision, ch omosome pa i ioning Y 0 0.0 Nuclea s uc u e V 33 1.0 De ense mechanisms T 152 4.7 Signal ansduc ion mechanisms M 184 5.7 Cell wall/memb ane biogenesis N 81 2.5 Cell mo ili y Z 0 0.0 Cy oskele on W 0 0.0 Ex acellula s uc u es U 77 2.4 In acellula a icking and sec e ion, and esicula anspo O 122 3.8 Pos ansla ional modi ica ion, p o ein u no e , chape ones C 207 6.4 Ene gy p oduc ion and con e sion G 227 7.1 Ca bohyd a e anspo and me abolism E 324 10.1 Amino acid anspo and me abolism F 81 2.5 Nucleo ide anspo and me abolism H 152 4.7 Coenzyme anspo and me abolism I 110 3.4 Lipid anspo and me abolism P 175 5.4 Ino ganic ion anspo and me abolism Q 76 2.4 Seconda y me aboli es biosyn hesis, anspo and ca abolism R 385 12.0 Gene al unc ion p edic ion only S 269 8.4 Func ion unknown - 570 16.7 No in COGs Ch omohalobac e salexigens ype s ain (1H11T) 386 S anda ds in Genomic Sciences Acknowledgemen s The wo k conduc ed by he U.S. Depa men o Ene gy Join Genome Ins i u e was suppo ed by he O ice o Science o he U.S. Depa men o Ene gy unde Con- ac No. DE-AC02-05CH11231. 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