Complete genome sequence of Granulicella tundricola type strain MP5ACTX9T, an Acidobacteria from tundra soil

Standards in Genomic Sciences (2014) 9:449-461 DOI:10.4056/sigs.4648353 The Genomic Standards Consortium Complete genome sequence of Granulicella tundricola type strain MP5ACTX9T, an Acidobacteria from tundra soil Suman R. Rawat1, Minna K. Männistö2, Valentin Starovoytov3, Lynne Goodwin4, Matt Nolan5 Loren Hauser6, Miriam Land6, Karen Walston Davenport4, Tanja Woyke5 and Max M. Häggblom1* 1 Department of Biochemistry and Microbiology, Rutgers, The State University of New Jersey, New Brunswick, New Jersey USA 2 Finnish Forest Research Institute, Rovaniemi, Finland 3 Department of Cell Biology and Neuroscience, Rutgers, The State University of New Jersey, Piscataway, New Jersey, USA. 4 Los Alamos National Laboratory, Bioscience Division, Los Alamos, New Mexico, USA 5 DOE Joint Genome Institute, Walnut Creek, California, USA 6 Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA *Correspondence: Max M Häggblom ([email protected]) Keywords: cold adapted, acidophile, tundra soil, Acidobacteria Granulicella tundricola strain MP5ACTX9T is a novel species of the genus Granulicella in subdivision 1 Acidobacteria. G. tundricola is a predominant member of soil bacterial communities, active at low temperatures and nutrient limiting conditions in Arctic alpine tundra. The organism is a cold-adapted acidophile and a versatile heterotroph that hydrolyzes a suite of sugars and complex polysaccharides. Genome analysis revealed metabolic versatility with genes involved in metabolism and transport of carbohydrates, including gene modules encoding for the carbohydrate-active enzyme (CAZy) families for the breakdown, utilization and biosynthesis of diverse structural and storage polysaccharides such as plant based carbon polymers. The genome of G. tundricola strain MP5ACTX9T consists of 4,309,151 bp of a circular chromosome and five mega plasmids with a total genome content of 5,503,984 bp. The genome comprises 4,705 protein-coding genes and 52 RNA genes. Introduction The strain MP5ACTX9T (=ATCC BAA-1859T =DSM 23138T) is the type strain of Granulicella tundricola [tun.dri.co’la. N.L. n. tundra, tundra, a cold treeless region; L. masc. suffix -cola (from L. n. incola) dweller; N.L. n. tundricola tundra dweller] that was isolated from soil at the Malla Nature Reserve, Kilpisjärvi, Finland; 69°01’N, 20°50’E) and described along with other species of the genus Granulicella isolated from tundra soil [1]. Acidobacteria is a phylogenetically and physiologically diverse phylum [2,3], the members of which are ubiquitously found in diverse habitats and are abundant in most soil environments [4,5] including Arctic tundra soils [6,7]. Acidobacteria are relatively difficult to cultivate, as they have slow growth rates. To date only subdivisions 1, 3, 4, 8, 10 and 23 Acidobacteria are defined by taxonomically characterized representatives [8-23] as well as three ‘Candidatus’ taxa [24,25]. The phylogenetic diversity, ubiquity and abundance of this group suggest that they play important ecological roles in soils. The abundance of Acidobacteria correlates with soil pH [26,27] and carbon [28,29], with subdivision 1 Acidobacteria being most abundant in slightly acidic soils. Acidobacteria, including members of the genera Granulicella and Terriglobus, dominate the acidic tundra heaths of northern Finland [26,30-32]. Using selective Granulicella tundricola type strain MP5ACTX9T 450 Standards in Genomic Sciences isolation techniques we have been able to isolate several slow growing and fastidious strains of Acidobacteria [1,11]. On the basis of phylogenetic, phenotypic and chemotaxonomic data, including 16S rRNA, rpoB gene sequence similarity and DNA–DNA hybridization, strain MP5ACTX9T was classified as a novel species of the genus Granulicella [1]. Here, we summarize the physiological features together with the complete genome sequence, annotation and data analysis of Granulicella tundricola strain MP5ACTX9T. Classification and features Within the genus Granulicella, eight species are described with validly published names: G. mallensis MP5ACTX8T,G. tundricola MP5ACTX9T, G. arctica MP5ACTX2T,G. sapmiensis S6CTX5AT isolated from Arctic tundra soil [1] and G. paludicola OB1010T, G. paludicola LCBR1, G. pectinivorans TPB6011T ,G. rosea TPO1014T ,G. aggregans TPB6028T isolated from sphagnum peat bogs [2]. Strain MP5ACTX9T shares 95.5 - 97.2% 16S rRNA gene identity with tundra soil strains G. mallensis MP5ACTX8T (95.5%), G. arctica MP5ACTX2T (96.9%), G. sapmiensis S6CTX5AT (97.2%) and 95.2 – 97.7% identity with the sphagnum bog strains, G. pectinivorans TPB6011T (97.7%), G. rosea TPO1014T (97.2%), %), G. aggregans TPB6028T (96.8%), G. paludicola LCBR1 (95.9%), and G. paludicola strain OB1010T (95.3%), which were isolated from sphagnum peat. Phylogenetic analysis based on the 16S rRNA gene of taxonomically classified strains of family Acidobacteriaceae placed G. rosea type strain T4T (AM887759) as the closest taxonomically classified relative of G. tundricola strain MP5ACTX9T (Table 1, Figure 1). Figure 1. Phylogenetic tree highlighting the position of G. tundricola MP5ACTX9T (shown in bold) relative to the other type strains within subdivision1 Acidobacteria. The maximum likelihood tree was inferred from 1,361 aligned positions of the 16S rRNA gene sequences and derived based on the Tamura-Nei model using MEGA 5 [42]. Bootstrap values >50 (expressed as percentages of 1,000 replicates) are shown at branch points. Bar: 0.01 substitutions per nucleotide position. The corresponding GenBank accession numbers are displayed in parentheses. Strains whose genomes have been sequenced, are marked with an asterisk; G. mallensis MP5ACTX8T (CP003130), G. tundricola MP5ACTX9T (CP002480), T. saanensis SP1PR4T (CP002467), T. roseus KBS63T (CP003379), and A. capsulatum ATCC 51196T (CP001472). Bryobacter aggregatus MPL3 (AM162405) in SD3 Acidobacteria was used as an outgroup. Rawat et al. http://standardsingenomics.org 451 Table 1. Classification and general features of G. tundr ic ola strain MP5ACTX9T MIGS ID Property Term Evidence codea Domain Bacteria TAS [33] Phylum Acidobacteria TAS [34,35 ] Class Acidobacteria TAS [36,37 ] Classification Order Acidobacteriales TAS [37,38 ] Family Acidobacteriaceae TAS [35,39 ] Genus Granulicella TAS [1,40] Species Granulicella tundricola TAS [1] Type strain: MP5ACTX9T (ATCC BAA-1859T = DSM 2 3138T) Gram stain negative TAS [1] Cell shape rod TAS [1] Motility non-motile TAS [1] Sporulation not reported NAS Temperature range 4–28°C TAS [1] Optimum temperature 21–24 °C TAS [1] pH range; Optimum 3.5–6.5; 5 TAS [1] Carbon source D-glucose, maltose, cellobiose, D-fructose, D-galactose, lactose, lactulose, D-mannose, sucrose, trehalose, D-xylose, raffinose, N-acetyl-D-glucosamine, glutamate TAS [1] MIGS-6 Habitat terrestrial, tundra soil TAS [1] MIGS-6.3 Salinity No growth with >1.0% NaCl (w/v) TAS [1] MIGS-22 Oxygen requirement aerobic TAS [1] MIGS-15 Biotic relationship free-living TAS [1] MIGS-14 Pathogenicity non-pathogen NAS MIGS-4 Geog raphic location Malla Nature Reserve, Arctic-alpine tundra, Finland TAS [1] MIGS-5 Sample collection 2006 TAS [1] MIGS-4.1 Latitude 69°01’N TAS [1] MIGS-4.2 Longitude 20°50’E TAS [1] MIGS-4.4 Altitude 700 m TAS [1] a Evidence codes - IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [41]. Granulicella tundricola type strain MP5ACTX9T 452 Standards in Genomic Sciences Morphology and physiology G. tundricola cells are Gram-negative, non-motile, aerobic rods, approximately 0.5 μm wide and 0.5 – 1.8 μm long. Colonies on R2A agar are pink, circular, convex and smooth. Growth occurs at +4 to 28°C and at pH 3.5-6.5 with an optimum at 21-24°C and pH 5 (Fig. 2). Genotypic analyses, including low rpoB gene sequence similarity and phenotypic characteristics clearly distinguished strain MP5ACTX9T from other Granulicella species/strains, leading us to conclude that MP5ACTX9T represents a novel species of the genus Granulicella, for which the name Granulicella tundricola sp. nov. was proposed [1]. Strain MP5ACTX9T hydrolyzed complex to simple carbon substrates [1] which include complex polysaccharides like aesculin, pectin, laminarin, starch and pullulan, but not gelatin, cellulose, lichenan, sodium alginate, xylan, chitosan or chitin. Strain MP5ACTX9T also utilized the following sugars as growth substrates: D-glucose, maltose, cellobiose, D-fructose, D-galactose, lactose, lactulose, Dmannose, sucrose, trehalose, D-xylose, raffinose, Nacetyl-D-glucosamine, glutamate and gluconic acid. Enzyme activities reported for the strain MP5ACTX9T include acid phosphatase, esterase (C4 and C8), leucine arylamidase, valine arylamidase, α-chymotrypsin, trypsin, naphthol-AS-BIphosphohydrolase, αand β-galactosidases, αand β-glucosidases, N-acetylβ-glucosaminidase, βglucuronidase, α-fucosidase and α-mannosidase but negative for alkaline phosphatase and lipase (C14). Strain MP5ACTX9T is resistant to ampicillin, erythromycin, chloramphenicol, neomycin, streptomycin, tetracycline, gentamicin, bacitracin, polymyxin B and penicillin, but susceptible to rifampicin, kanamycin, lincomycin and novobiocin. Chemotaxonomy The major cellular fatty acids in G. tundricola are iso-C15:0 (46.4%), C16:1ω7c (35.0%) and C16:0 (6.6%). The cellular fatty acid composition of strain MP5ACTX9T was similar to that of other Granulicella strains with fatty acids iso-C15:0 and C16:1ω7c being most abundant in all strains. Strain MP5ACTX9T contains MK-8 as the major quinone and also contains 4% of MK-7. Genome sequencing and annotation Genome project history G. tundricola strain MP5ACTX9T was selected for sequencing in 2009 by the DOE Joint Genome Institute (JGI) community sequencing program. The Quality Draft (QD) assembly and annotation were completed on May 24, 2010. The GenBank Date of Release was February 2, 2011. The genome project is deposited in the Genomes On-Line Database (GOLD) [43] and the complete genome sequence of strain MP5ACTX9T is deposited in GenBank (CP002480.1). Table 2 presents the project information and its association with MIGS version 2.0 [44]. Figure 2. Electron micrograph of G. tundricola MP5ACTX9T Rawat et al. http://standardsingenomics.org 453 Table 2. Project information. MIGS ID Property Term MIGS 31 Finishing quality Finished MIGS-28 Libraries used Three libraries, an Illumina GAii shotg un library (GUIX), a 454 Titanium standard library (GTWG, GWTA) and a paired end 454 (GSUN) lib rary MIGS 29 Sequencing platforms 454 Titanium standard, 454 Paired End, Illumina MIGS 31.2 Fold coverage 20×(454), 274X (Illumina) MIGS 30 Assemblers Newbler, VELVET, PHRAP MIGS 32 Gene calling method ProdigaL, GenePRIMP Locus Tag AciX9 Genbank ID CP002480.1 GenBank Date of Release February 2, 2011 GOLD ID Gc01833 BIOPROJECT PRJNA50551, PRJNA47621 Project relevance Environmental, Biogeochemical cycling of Carbon, Biotechnological, GEBA Growth conditions and genomic DNA extraction G. tundricola MP5ACTX9T was cultivated on R2 medium as previously described [1]. Genomic DNA (gDNA) of high sequencing quality was isolated using a modified CTAB method and evaluated according to the Quality Control (QC) guidelines provided by the DOE Joint Genome Institute [45]. Genome sequencing and assembly The finished genome of G. tundricola MP5ACTX9T (JGI ID 4088693) was generated at the DOE Joint genome Institute (JGI) using a combination of Illumina [46] and 454 technologies [47]. For this genome we constructed and sequenced an Illumina GAii shotgun library which generated 42,620,699 reads totaling 3239 Mb, a 454 Titanium standard library which generated 146,119 reads and three paired end 454 libraries with an average insert size of 9.3 kb which generated 178,757 reads totaling 154.3 Mb of 454 data. All general aspects of library construction and sequencing performed at the JGI can be found at the JGI website [45]. The 454 Titanium standard data and the 454 paired end data were assembled with Newbler, version 2.3. Illumina sequencing data was assembled with Velvet, version 0.7.63 [48]. The 454 Newbler consensus shreds, the Illumina Velvet consensus shreds and the read pairs in the 454 paired end library were integrated using parallel phrap, version SPS - 4.24 (High Performance Software, LLC) [49]. The software Consed [50] was used in the finishing process. The Phred/Phrap/Consed software package [51] was used for sequence assembly and quality assessment in the subsequent finishing process. Illumina data was used to correct potential base errors and increase consensus quality using the software Polisher developed at JGI (Alla Lapidus, unpublished). Possible misassemblies were corrected using gapResolution (Cliff Han, unpublished), Dupfinisher [52] or sequencing cloned bridging PCR fragments with sub-cloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR (J-F Cheng, unpublished) primer walks. The final assembly is based on 29.1 Mb of 454 draft data which provides an average 20× coverage of the genome and 975 Mb of Illumina draft data which provides an average 274× coverage of the genome. Genome annotation Genes were identified using Prodigal [53] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [54]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, (COGs) [55,56], and InterPro. These data sources were combined to assert a product description for each predicted protein. Non-coding genes and miscellaneous features were predicted using tRNAscanSE [57], RNAMMer [58], Rfam [59], TMHMM [60], and signalP [61]. Additional gene prediction analysis and functional annotation were performed within the Integrated Microbial Genomes Expert Review (IMG-ER) platform [62]. Granulicella tundricola type strain MP5ACTX9T 454 Standards in Genomic Sciences Genome properties The genome is 5,503,984 bp in size, which includes the 4,309,151 bp chromosome and five plasmids pACIX901 (0.48 Mbp); pACIX902 (0.3 Mbp); pACIX903 (0.19 Mbp), pACIX904 (0.12 Mbp) and pACIX905 (0.12 Mbp), with a GC content of 59.9 mol%. There are 52 RNA genes (Figures 3 and 4, and Table 3). Of the 4,758 predicted genes, 4,706 are protein-coding genes (CDSs) and 163 are pseudogenes. Of the total CDSs, 68.8% represent COG functional categories and 27.5% consist of signal peptides. The distribution of genes into COG functional categories is presented in Figure 3 and Table 4, and Table 5. Table 3. Summary of genome: one chromosome and five plasmids Label Size (Mb) Topology INSDC identifier RefSeq ID Chromosome 4.3 circular CP002480.1 NC_015064.1 Plasmid pACIX901 0.48 circular CP002481.1 NC_015057.1 Plasmid pACIX902 0.3 circular CP002482.1 NC_015065.1 Plasmid pACIX903 0.19 circular CP002483.1 NC_015058.1 Plasmid pACIX904 0.12 circular CP002484.1 NC_015059.1 Plasmid pACIX905 0.12 circular CP002485.1 NC_015060.1 Table 4. Genome statistics. Attribute Value % of Total Genome size (bp) 5,503,984 100 DNA coding (bp) 4,759,459 86.5 DNA G+C (bp) 3,301,098 60.0 DNA scaffolds 6 100 Total genes 4,757 100 Protein coding genes 4,705 98.9 RNA genes 52 1.1 Pseudo genes 163 3. 4 Genes in internal clusters 2 ,395 50.4 Genes with function prediction 2 ,936 61.7 Genes assigned to COGs 3,259 68.5 Genes with Pfam domains 3,504 73.6 Genes with signal peptides 652 13.7 Genes with transmembrane helices 1,108 23. 3 CRISPR repeats 0 - The total is based on either the size of the genome in base pairs or the protein coding genes in the annotated genome. Rawat et al. http://standardsingenomics.org 455 Figure 3. Circular representation of the chromosome of G. tundricola MP5ACTX9T displaying relevant genome features. From outside to center; Genes on forward strand (colored by COG categories), genes on reverse strand (colored by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content and GC skew. Granulicella tundricola type strain MP5ACTX9T 456 Standards in Genomic Sciences Figure 4. Circular representation of the plasmids of G. tundricola MP5ACTX9T displaying relevant genome features. From outside to center; Genes on forward strand (color by COG categories), genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content and GC skew. Order and size from left to right: pACIX901, 0.48 Mbp; pACIX902, 0.3 Mbp; pACIX903, 0.19 Mbp; pACIX904, 0.12 Mbp; pACIX905, 0.12 Mbp. Table 5. Number of genes associated with general COG functional categories. Code Value %age Description J 160 4.45 Translation, ribosomal structure and biogenesis A 2 0.06 RNA processing and modification K 249 6.93 Transcription L 222 6.18 Replication, recombination and repair B 1 0.03 Chromatin structure and dynamics D 33 0.92 Cell cycle control, Cell division, chromosome partitioning V 68 1.89 Defense mechanisms T 212 5.9 Signal transduction mechanisms M 287 7.98 Cell wall/membrane biogenesis N 73 2.03 Cell motility U 123 3. 42 Intracellular trafficking and secretion O 125 3. 48 Posttranslational modification, protein turnover, chaperones C 174 4.84 Energy production and conversion G 248 6.9 Carbohydrate transport and metabolism E 2 34 6.51 Amino acid transport and metabolism F 68 1.89 Nucleotide transport and metabolism H 147 4.09 Coenzyme transport and metabolism I 126 3. 5 Lipid transport and metabolism P 1 37 3. 81 Inorganic ion transport and metabolism Q 91 2.53 Secondary metabolites biosynthesis, transport and catabolism R 446 12.41 General function prediction only S 370 10.29 Function unknown - 1498 31.49 Not in COGs The total is based on the total number of protein coding genes in the genome. Rawat et al. http://standardsingenomics.org 457 Discussion Granulicella tundricola MP5ACTX9T is a tundra soil strain with a genome consisting of a circular chromosome and five mega plasmids ranging in size from 1.1 x 105 to 4.7 x 105 bp for a total genome size of 5.5 Mbp. The G. tundricola genome also contains close to twice as many pseudogenes and a large number of mobile genetic elements as compared to Granulicella mallensis and Terrigobus saanensis, two other Acidobacteria isolated from the same habitat [29]. A large number of genes assigned to COG functional categories for transport and metabolism of carbohydrates (6.9%) and amino acids (6.5%) and involved in cell envelope biogenesis (8%) and transcription (6.9%) were identified. Further genome analysis revealed an abundance of gene modules encoding for functional activities within the carbohydrateactive enzymes (CAZy) families [63,64] involved in breakdown, utilization and biosynthesis of carbohydrates. G. tundricola hydrolyzed complex carbon polymers, including CMC, pectin, lichenin, laminarin and starch, and utilized sugars such as cellobiose, D-mannose, D-xylose and D-trehalose. Genome predictions for CDSs encoding for enzymes such as cellulases, pectinases, alginate lyases, trehalase and amylases are in agreement with biochemical activities in strain MP5ACTX9T. However, the genome of G. tundricola did contain many CDSs encoding for GH18 chitinases although no chitinase activity was detected after 10 dayincubation with chitinazure [29]. In addition, the G. tundricola genome contained a cluster of genes in close proximity to the cellulose synthase gene (bcsAB), which included cellulase (bscZ) (endoglucanase Y) of family GH8, cellulose synthase operon protein (bcsC) and a cellulose synthase operon protein (yhjQ) involved in cellulose biosynthesis. We previously reported on a detailed comparative genome analysis of G. tundricola MP5ACTX9T with other Acidobacteria strains for which finished genomes are available [29]. The data suggests that G. tundricola is involved in hydrolysis and utilization of stored carbohydrates and biosynthesis of exopolysaccharides from organic matter and plant based polymers in the soil. Therefore, G. tundricola may be central to carbon cycling processes in Arctic and boreal soil ecosystems. Acknowledgements The work conducted by the US Department of Energy Joint Genome Institute is supported by the Office of Science of the US Department of Energy Under Contract No. DE-AC02-05CH11231. This work was funded in part by the Ac ademy of Finland and the New Jersey Agricul tural Experiment Station. References 1. Männistö MK, Rawat S, Starovoytov V, Häggblom MM. Granulicella arctica sp. nov., Granulicella mallensis sp. nov., Granulicella sapmiensis sp. nov. and Granulicella tundricola sp. nov., novel Acidobacteria from tundra soil of Northern Finland. Int J Syst Evol Microbiol 2012; 62:20972106. PubMed http://dx.doi.org/10.1099/ijs.0.031864-0 2. Jones RT, Robeson MS, Lauber CL, Hamady M, Knight R, Fierer N. A comprehensive survey of soil acidobacterial diversity using pyrosequencing and clone library analyses. ISME J 2009; 3:442453. PubMed http://dx.doi.org/10.1038/ismej.2008.127 3. Barns SM, Cain EC, Sommerville L, Kuske CR. Acidobacteria phylum sequences in uraniumcontaminated subsurface sediments greatly expand the known diversity within the phylum. Appl Environ Microbiol 2007; 73:3113-31 16. PubMed http://dx.doi.org/10.1128/AEM.02012-06 4. Janssen PH. Identifying the dominant soil bacterial taxa in libraries of 16S rRNA and 16S rRNA genes. Appl Environ Microbiol 2006; 72:17191728. PubMed http://dx.doi.org/10.1128/AEM.72.3.17191728.2006 5. Fierer N, Bradford MA, Jackson RB. Toward an ecological classification of soil bacteria. Ecology 2007; 88:1354-1364. PubMed http://dx.doi.org/10.1890/05-1839 6. Campbell BJ, Polson SW, Hanson TE, Mack MC, Schuur EA. The effect of nutrient deposition on bacterial communities in Arctic tundra soil. Environ Microbiol 2010; 12:1842-1854. PubMed http://dx.doi.org/10.1111/j.14622920.2010.02189.x 7. Chu H, Fierer N, Lauber CL, Caporaso JG, Knight R, Grogan P. Soil bacterial diversity in the Arctic is not fundamentally different from that found in