Complete genome sequence of Terriglobus saanensis type strain SP1PR4T, an Acidobacteria from tundra soil

S anda ds in Genomic Sciences (2012) 7:59-69 DOI:10.4056/sigs.3036810 The Genomic S anda ds Conso ium Comple e genome sequence o Te iglobus saanensis ype s ain SP1PR4T, an Acidobac e ia om und a soil Suman R. Rawa 1*, Minna K. Männis ö2, Valen in S a o oy o 3, Lynne Goodwin5, Ma Nolan6 Lau en Hause 4, Mi iam Land4, Ka en Wals on Da enpo 5, Tanja Woyke6 and Max M. Häggblom1 1 Depa men o Biochemis y and Mic obiology, Ru ge s, The S a e Uni e si y o New Je sey, New B unswick, New Je sey, 08901-8520, USA 2 Finnish Fo es Resea ch Ins i u e, Ro aniemi, Finland 3 Depa men o Cell Biology and Neu oscience, Ru ge s, The S a e Uni e si y o New Je sey, Pisca away, New Je sey, USA. 4 Oak Ridge Na ional Labo a o y, Oak Ridge, Tennessee, USA 5 Los Alamos Na ional Labo a o y, Bioscience Di ision, Los Alamos, New Mexico, USA 6 DOE Join Genome Ins i u e, Walnu C eek, Cali o nia, USA *Co esponding au ho : Suman R. Rawa ( awa @aesop. u ge s.edu) Keywo ds: cold adap ed, acidophile, und a soil, Acidobac e ia Te iglobus saanensis SP1PR4T is a no el species o he genus Te iglobus. T. saanensis is o ecological in e es because i is a ep esen a i e o he phylum Acidobac e ia, which a e dominan membe s o bac e ial soil mic obio a in A c ic ecosys ems. T. saanensis is a cold- adap ed acidophile and a e sa ile he e o oph u ilizing a sui e o simple suga s and complex polysaccha ides. The genome con ained an abundance o genes assigned o me abolism and anspo o ca bohyd a es including gene modules encoding o ca bohyd a e-ac i e enzyme (CAZyme) amily in ol ed in b eakdown, u iliza ion and biosyn hesis o di e se s uc u al and s o age polysaccha ides. T. saanensis SP1PR4T ep esen s he i s membe o genus Te iglobus wi h a comple ed genome sequence, consis ing o a single eplicon o 5,095,226 base pai s (bp), 54 RNA genes and 4,279 p o ein-coding genes. We in e ha he physiology and me abolic po en ial o T. saanensis is adap ed o allow o esilience o he nu ien - de icien condi ions and luc ua ing empe a u es o A c ic und a soils. In oduc ion S ain SP1PR4T (= DSM 23119 = ATCC BAA- 1853) is he ype s ain o Te iglobus saanensis. I is second o wo alidly asc ibed species o he genus Te iglobus, wi h T. oseus i s isola ed om ag icul u al soils in 2007 [1]. T. saanensis SP1PR4T was isola ed om A c ic und a soil col- lec ed om a wind exposed si e o Saana jeld, no h-wes e n Finland (69°01’N, 20°50’E) [2,3]. The species name saanensis (sa.a.nen' sis. N.L. masc. adj. saanensis) pe ains o Moun Saana in Finland. Acidobac e ia a e ound in di e se soil en i on- men s and a e widely dis ibu ed in A c ic and bo eal soils [4-8]. Howe e , ela i ely li le is s ill known abou hei me abolic po en ial and ecological oles in hese habi a s. Despi e a la ge collec ion o Acidobac e ia 16S RNA gene se- quences in da abases ha ep esen di e se phylo ypes om a ious habi a s, ew ha e been cul i a ed and desc ibed. Acidobac e ia ep esen 26 phylogene ic subdi isions based on 16S RNA gene phylogeny [9] o which subdi isions 1, 3, 4 and 6 a e mos commonly de ec ed in soil en i- onmen s [10]. The abundance o Acidobac e ia has been ound o co ela e wi h soil pH [2,10,11] and ca bon [1,12,13] wi h subdi ision 1 Acidobac e ia being mos abundan in sligh ly acidic soils. The phylogene ic di e si y, ubiqui y and abundance o his g oup sugges ha hey play impo an ecological oles in soils. Te iglobus saanensis ype s ain SP1PR4T 60 S anda ds in Genomic Sciences Ou p e ious s udies on bac e ial communi y p o- iling om A c ic alpine und a soils o no he n Finland ha e shown ha Acidobac e ia domina e in he acidic und a hea hs [2] and a e mul iple eeze- haw cycles [6]. Using selec i e isola ion echniques, including eezing soils a -20°C o 7 days, we ha e been able o isola e se e al slow g owing and as idious s ains o Acidobac e ia. On he basis o phylogene ic, pheno ypic and chemo axonomic da a, including 16S RNA, poB gene sequence simila i y and DNA–DNA hyb idi- za ion, s ain SP1PR4T was classi ied as a no el species o he genus Te iglobus [3]. He e, we summa ize he physiological ea u es oge he wi h he comple e genome sequence and anno a- ion o Te iglobus saanensis SP1PR4T. Classi ica ion and ea u es Wi hin he genus Te iglobus, wo species a e as- c ibed wi h alidly published names, T. saanensis SP1PR4T [3] isola ed om A c ic und a soils and T. oseus KBS 63T (DSM 18391) isola ed om ag icul- u al soils (KBS-LTER si e) [1]. Sea ching he NCBI non- edundan nucleo ide da abase o homology o 16S RNA gene sequence o T. saanensis SP1PR4T iden i ied 10 cul u ed and 20 uncul u ed s ains ha we e unclassi ied, wi h ≥97% 16S RNA se- quence iden i y. Phylogene ic ee based on 16S RNA gene depic ing he posi ion o T. saanensis SP1PR4T ela i e o he o he ype s ains wi hin he amily Acidobac e iaceae is shown in Figu e 1. T. saanensis SP1PR4T is dis inc ly clus e ed in o a sepa a e b anch wi h T. oseus KBS 63T (DQ660892) [1], as i s closes desc ibed ela i e (97.1% 16S RNA sequence iden i y). S ain SP1PR4T showed ~95% 16S RNA gene iden i y o ou s ains in he genus G anulicella isola ed om und a soils, namely “G. und icola” (95.9%), “G. sapmiensis” (95.8%), “G. mallensis” (95.5%) and “G. a c ica” (94.9%) [3,15] (Figu e 1). S ain SP1PR4T g ows a pH 4.5-7.5 wi h an op i- mum a 6.0 and a empe a u es o +4 o +30°C wi h an op imum o +25°C on R2 medium [3]. On R2 aga , s ain SP1PR4T o ms small, ci cula , con- ex colonies wi h a diame e o app oxima ely 1 mm. The pigmen a ies om ligh beige o ligh pink depending on he age o he cul u e. Cells o s ains SP1PR4T a e G am-nega i e, non-spo e- o ming, non-mo ile ae obic ods wi h a leng h o 1.5– 3.0 µm and a diame e o 0.5–0.7 µm. The cell- wall s uc u e in ul a hin sec ions o elec on mi- c og aphs o cells o s ain SP1PR4T demons a es nume ous ou e -memb ane esicles (Table 1, Fig- u e 2). S ain SP1PR4T u ilized ca bon subs a es o g ow h which include cellobiose, D- uc ose, D- galac ose, D-glucose, lac ose, D-mal ose, D- mannose, D- ibose, suc ose, D- ehalose, D-xylose, D-melezi ose, D- a inose and N-ace yl-D- glucosamine. S ain SP1PR4T hyd olyzed polysac- cha ides such as s a ch, pec in, lamina in and aesculin bu no gela in, cellulose, xylan, lichenan, sodium algina e, pullulan, chi osan o chi in. En- zyme ac i i ies o s ain SP1PR4T include chi obiase, ca alase, acid and alkaline phospha- ase, leucine a ylamidase, naph hol-AS-B1- phosphohyd olase, α- and β-galac osidase, α- and β-glucosidase, β-glucu onidase, N-ace yl-β- glucosaminidase, α-mannosidase and α- ucosidase [3,15]. Chemo axonomy The majo cellula a y acids in T. saanensis SP1PR4T a e iso-C15:0 (39.9%), C16:1 ω7c (28.4%), iso-C13:0 (9.8%) and C16:0 (9.8%). The cellula a y acid composi ions o s ain SP1PR4T we e ela- i ely simila o ha o T. oseus DSM 18391T, wi h highe ela i e abundance o iso-C13:0 and a co e- sponding lowe abundance o iso-C15:0 in s ain SP1PR4T [3]. Genome sequencing and anno a ion Genome p ojec his o y S ain SP1PR4T was selec ed o sequencing in 2009 by he DOE Join Genome Ins i u e (JGI) communi y sequencing p og am. The Quali y D a (QD) assembly and anno a ion we e com- ple ed on Augus 6, 2010. The comple e genome was made a ailable on Jan 24, 2011. The genome p ojec is deposi ed in he Genomes On-Line Da a- base (GOLD) [25] and he comple e genome se- quence o s ain SP1PR4T is deposi ed in GenBank. Table 2 p esen s he p ojec in o ma ion and i s associa ion wi h MIGS e sion 2.0 [16]. Rawa e al. h p://s anda dsingenomics.o g 61 Figu e 1. Phylogene ic ee highligh ing he posi ion o T. saanensis SP1PR4T ela i e o he o he ype s ains wi hin he amily Acidobac e iaceae. The maximum likelihood ee was in e ed om 1,359 aligned posi ions o he 16S RNA gene sequences and de i ed using MEGA e sion 5 [14]. Boo s ap alues (exp essed as pe cen ages o 1,000 eplica es) o >50 a e shown a b anch poin s. Ba : 0.02 sub- s i u ions pe nucleo ide posi ion. The s ains ( ype s ain=T) and hei co esponding GenBank acces- sion numbe s a e displayed in pa en heses wi h s ain T. saanensis SP1PR4T shown in bold. B yobac e agg ega us MPL3 (AM162405) was used as ou g oup. T. saanensis SP1PR4T and T. oseus KBS 63T (DSM 18391) genome sequences ha e been e ealed. Figu e 2. Elec on mic og aph o cells o T. saanensis s ain SP1PR4T (ba 0.5 µm). Te iglobus saanensis ype s ain SP1PR4T 62 S anda ds in Genomic Sciences Table 1. Classi ica ion and gene al ea u es o T. saanensis SP1PR4T acco ding o he MIGS ecommenda ions [16]. MIGS ID P ope y Te m E idence codes Domain Bac e ia TAS [17] Phylum Acidobac e ia TAS [18,19] Class Acidobac e ia TAS [20] Classi ica ion O de Acidobac e iales TAS [21,22] Family Acidobac e iaceae TAS [18,23] Genus Te iglobus TAS [1] Species Te iglobus saanensis TAS [3] Type s ain: SP1PR4T G am s ain nega i e TAS [3] Cell shape od TAS [3] Mo ili y non-mo ile TAS [3] Spo ula ion non-spo e o ming TAS [3] Tempe a u e ange 4–30°C TAS [3] Op imum empe a u e 25°C TAS [3] pH ange 4.5-7.5 TAS [3] Op imum pH 6.0 TAS [3] Salini y no epo ed NAS MIGS-22 Oxygen equi emen ae obe TAS [3] Ca bon sou ce cellobiose, D- uc ose, D-galac ose, D-glucose, lac ose, D-mal ose, D-mannose, D- ibose, su- c ose, D- ehalose, D-xylose, D-melezi ose, D- a inose, s a ch, pec in, lamina in and aesculin TAS [3] MIGS-6 Habi a e es ial TAS [3] MIGS-15 Bio ic ela ionship ee-li ing TAS [3] MIGS-14 Pa hogenici y non-pa hogen NAS Biosa e y le el 1 NAS Isola ion und a soil TAS [3] MIGS-4 Geog aphic loca ion Saana jeld, A c ic und a, Finland TAS [3] MIGS-5 Sample collec ion ime 2004-2005 TAS [3] MIGS-4.1 La i ude 69°01’N, TAS [3] MIGS-4.2 Longi ude 20°50’E TAS [3] MIGS-4.3 Dep h no epo ed NAS MIGS-4.4 Al i ude no epo ed NAS *E idence codes - IDA: In e ed om Di ec Assay ( i s ime in publica ion); 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 species, o anecdo al e i- dence). These e idence codes a e om he Gene On ology p ojec [24]. Rawa e al. h p://s anda dsingenomics.o g 63 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 lib a ies, an Illumina GAii sho gun lib a y (GSGY), a 454 Ti anium s anda d lib a y (GSXT, GWTA) and a pai ed end 454 (GSFP) lib a y MIGS 29 Sequencing pla o ms 454 Ti anium s anda d, 454 Pai ed End, Illumina MIGS 31.2 Sequencing co e age 39× (454), 180× (Illumina) MIGS 30 Assemble s Newble , Vel e , Ph ap MIGS 32 Gene calling me hod P odigaL, GenePRIMP Locud Tag AciPR4 INSDC / Re Seq ID CP002467, NC_014963, GenBank Da e o Release Oc obe 7, 2011 GOLD ID Gc01604 NCBI p ojec ID 48971 MIGS 13 Sou ce ma e ial iden i ie ATCC BAA-1853, DSM 23119 P ojec ele ance En i onmen al, Biogeochemical cycling o ca bon, Bio echno- logical, GEBA G ow h condi ions and genomic DNA ex ac ion S ain SP1PR4T was cul i a ed in R2 medium as p e iously desc ibed [3]. Genomic DNA (gDNA) o high sequencing quali y was isola ed using a modi ied CTAB me hod and e alua ed acco ding o he Quali y Con ol (QC) guidelines p o ided by he DOE Join Genome Ins i u e. Genome sequencing and assembly The inished genome o T. saanensis SP1PR4T (JGI ID 4088690) was gene a ed a he DOE Join ge- nome Ins i u e (JGI) using a combina ion o Illumina [26] and 454 echnologies [27]. Fo his genome, an Illumina GAii sho gun lib a y which gene a ed 23,685,130 eads o aling 916 Mb, a 454 Ti anium s anda d lib a y which gene a ed 409,633 eads and a pai ed end 454 lib a y wi h an a e age inse size o 10.8 kb which gene a ed 180,451 eads o aling 157 Mb o 454 da a, we e cons uc ed and sequenced. All gene al aspec s o lib a y cons uc ion and sequencing pe o med a he JGI can be ound a he JGI websi e [28]. The 454 Ti anium s anda d da a and he 454 pai ed end da a we e assembled oge he wi h Newble , e sion 2.3. Illumina sequencing da a was assem- bled wi h Vel e , e sion 0.7.63 [29]. We in eg a ed he 454 Newble consensus sh eds, he Illumina Vel e consensus sh eds and he ead pai s in he 454 pai ed end lib a y using pa allel ph ap, e sion SPS - 4.24 (High Pe o mance So wa e, LLC). The so wa e Consed [30,31] was used in he inishing p ocess. Illumina da a was used o co ec po en ial base e o s and inc ease consensus quali y using he so wa e Polishe de eloped a JGI (Alla Lapidus, unpublished). Possible mis-assemblies we e co ec ed using gapResolu ion (Cli Han, un- published), Dup inishe [32], o sequencing cloned b idging PCR agmen s wi h sub-cloning. Gaps be- ween con igs we e closed by edi ing in Consed, by PCR and by Bubble PCR (J-F Cheng, unpublished) p ime walks. The inal assembly is based on 157 Mb o 454 da a which p o ides an a e age 39× co e age and 916 Mb o Illumina da a which p o- ides an a e age 180× co e age o he genome. Genome anno a ion Genes we e iden i ied using P odigal [33] as pa o he Oak Ridge Na ional Labo a o y genome an- no a ion pipeline, ollowed by a ound o manual cu a ion using he JGI GenePRIMP pipeline [34]. The p edic ed CDSs we e ansla ed and used o sea ch he Na ional Cen e o Bio echnology In- o ma ion (NCBI) non- edundan da abase, UniP o , TIGRFam, P am, PRIAM, KEGG, (COGs) [35,36], and In e P o. 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 [37], RNAMMe [38], R am [39], TMHMM [40], and signalP [41]. Addi ional gene p edic ion anal- ysis and unc ional anno a ion we e pe o med wi hin he In eg a ed Mic obial Genomes Expe Re iew (IMG-ER) pla o m [42]. Te iglobus saanensis ype s ain SP1PR4T 64 S anda ds in Genomic Sciences Genome p ope ies The genome consis s o one ci cula ch omosome o 5,095,226 bp in size wi h a GC con en o 57.3% and consis s o 54 RNA genes (Figu e 3, Table 3). O he 4,333 p edic ed genes, 4,279 a e p o ein- coding genes (CDSs) and 99 a e pseudogenes. O he o al CDSs, 67% ep esen COG unc ional ca - ego ies and 43% consis o signal pep ides. The dis ibu ion o genes in o COG unc ional ca ego- ies is p esen ed in Figu e 3 and Table 4. Discussion Genome analysis o T. saanensis iden i ied a high abundance o genes assigned o COG unc ional ca e- go ies o anspo and me abolism ca bohyd a es (9.5%) and amino acids (7.6%), ene gy con e sion (6.2%), cell en elope biogenesis (9.6%) and an- sc ip ion (9.2%) [15]. This indica es ha he T. saanensis genome encodes o unc ions in ol ed in anspo and u iliza ion o nu ien s, mainly ca bo- hyd a es and amino acids o ene gy p oduc ion and cell biogenesis o main ain cell in eg i y in cold un- d a soils. Fu he genome analysis e ealed an abundance o gene modules o glycoside hyd olas- es, glycosyl ans e ases, polysaccha ide lyases, ca - bohyd a e es e ases, and non-ca aly ic ca bohy- d a e-binding modules wi hin he ca bohyd a e- ac i e enzymes (CAZy [43]) amily in ol ed in b eakdown, u iliza ion and biosyn hesis o ca bohy- d a es [15]. T. saanensis hyd olyzed complex ca bon polyme s, including pec in, lamina in, and s a ch, and u ilized suga s such as cellobiose, D-mannose, D-xylose, D- ehalose and lamina in. This pa allels genome p edic ions o CDSs encoding o enzymes such as pec inases, chi inases, algina e lyases, ehalase and amylases. T. saanensis was unable o hyd olyze ca boxyme hyl cellulose (CMC) on pla e assays and lacked CDSs encoding o cellulases in- ol ed in cellulose hyd olysis. Howe e , he T. saanensis genome con ained a BcsZ gene encoding o an endocellulase (GH8) as pa o a bac e ial cel- lulose syn hesis (bcs) ope on in ol ed in cellulose biosyn hesis in se e al species. This ope on consis s o clus e s o genes in close p oximi y o he BcsZ gene which includes a cellulose syn hase gene (bcsAB), a cellulose syn hase ope on p o ein (bcsC) and a cellulose syn hase ope on p o ein (yhj) [15]. In addi ion, he T. saanensis genome encoded o a la ge numbe o gene modules ep esen ing glycosyl ans e ases (GTs) in ol ed in ca bohyd a e biosyn- hesis which include cellulose syn hase (UDP- o ming), α- ehalose phospha e syn hase [UDP- o ming], s a ch glucosyl ans e ase, ce amide β- glucosyl ans e ase in ol ed in biosyn hesis o cellu- lose, ehalose, s a ch, hopanoid, and capsula / ee exopolysaccha ide (EPS) [15]. This sugges s ha T. saanensis is in ol ed in hyd olysis o lignocellulosic soil o ganic ma e , u iliza ion o s o ed ca bohy- d a es and biosyn hesis o exopolysaccha ides. The e o e, we su mise ha T. saanensis may be cen- al o ca bon cycling p ocesses in A c ic and bo eal soil ecosys ems. Table 3. Genome s a is ics A ibu e Value % o To al Genome size (bp) 5,095,226 100% DNA coding (bp) 4,578,206 89.9% DNA G+C (bp) 2,921,371 57.3% Numbe o eplicons 1 100% To al genes 4,334 100% RNA genes 54 1.3% RNA ope ons 1 - P o ein coding genes 4,180 98.8% Pseudo genes 99 2.3% Genes wi h unc ion p edic ion 3,203 73.9% Genes in pa alog clus e s 2,220 51.2% Genes assigned o COGs 3,170 73.2% Genes wi h P am domains 3,108 71.7% Genes wi h signal pep ides 1,867 43.1% Genes wi h ansmemb ane helices 1,082 25% CRISPR epea s 0 - Rawa e al. h p://s anda dsingenomics.o g 65 Figu e 3. G aphical ep esen a ion o ci cula map o he ch omosome o T. saanensis s ain SP1PR4T displaying el- e an genome ea u es. F om ou side o 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. Te iglobus saanensis ype s ain SP1PR4T 66 S anda ds in Genomic Sciences Table 4. Numbe o genes associa ed wi h gene al COG unc ional ca ego ies. Code Value %age Desc ip ion J 163.0 4.6 T ansla ion, ibosomal s uc u e and biogenesis A 2.0 0.1 RNA p ocessing and modi ica in K 293.0 8.3 T ansc ip ion L 142.0 4.0 Replica ion, ecombina ion and epai B 0.0 0.0 Ch oma in s uc u e and dynamics D 24.0 0.7 Cell cycle con ol, Cell di ision, ch omosome pa i ioning Y 0.0 0.0 Nuclea s uc u e V 98.0 2.8 De ense mechanisms T 174.0 4.9 Signal ansduc ion mechanisms M 307.0 8.7 Cell wall/memb ane biogenesis N 56.0 1.6 Cell mo ili y Z 2.0 0.1 Cy oskele on W 0.0 0.0 Ex acellula s uc u es U 113.0 3.2 In acellula a icking and sec e ion O 122.0 3.4 Pos ansla ional modi ica ion, p o ein u no e , chape ones C 196.0 5.5 Ene gy p oduc ion and con e sion G 303.0 8.6 Ca bohyd a e anspo and me abolism E 243.0 6.9 Amino acid anspo and me abolism F 69.0 2.0 Nucleo ide anspo and me abolism H 134.0 3.8 Coenzyme anspo and me abolism I 116.0 3.3 Lipid anspo and me abolism P 134.0 3.8 Ino ganic ion anspo and me abolism Q 85.0 2.4 Seconda y me aboli es biosyn hesis, anspo and ca abolism R 443.0 12.5 Gene al unc ion p edic ion only S 323.0 9.1 Func ion unknown - 1163. 0 26.8 No in COGs Acknowledgemen s The wo k conduc ed by he US Depa men o Ene gy Join Genome Ins i u e is suppo ed by he O ice o Science o he US Depa men o Ene gy Unde Con ac No. DE-AC02-05CH11231. 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