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High-quality draft genome sequence of Gracilimonas tropica CL-CB462T (DSM 19535T), isolated from a Synechococcus culture
Standards in Genomic Sciences volume 10, Article number: 98 (2015)
Gracilimonas tropica Choi et al. 2009 is a member of order Sphingobacteriales, class Sphingobacteriia. Three species of the genus Gracilimonas have been isolated from marine seawater or a salt mine and showed extremely halotolerant and mesophilic features, although close relatives are extremely halophilic or thermophilic. The type strain of the type species of Gracilimonas, G. tropica DSM19535T, was isolated from a Synechococcus culture which was established from the tropical sea-surface water of the Pacific Ocean. The genome of the strain DSM19535T was sequenced through the Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes project. Here, we describe the genomic features of the strain. The 3,831,242 bp long draft genome consists of 48 contigs with 3373 protein-coding and 53 RNA genes. The strain seems to adapt to phosphate limitation and requires amino acids from external environment. In addition, genomic analyses and pasteurization experiment suggested that G. tropica DSM19535T did not form spore.
The genus Gracilimonas was first established in 2009 , and at the time of writing this paper there are three species that comprise this genus, G. tropica , G. mengyeensis , and G. rosea . They are Gram-negative, catalase- and oxidase-positive, aerobic and facultatively anaerobic and have rod-shaped cells (Fig. 1) [1–3]. In addition, they form endospores except G. mengyeensis . Gracilimonas tropica CL-CB462T (=KCCM 90063T = DSM 19535T ), the type strain of the type species of the genus Gracilimonas , was isolated from a Synechococcus culture which was established from the tropical sea-surface water of the Pacific Ocean . Interestingly, the genus Gracilimonas formed a robust clade together with extremely halophilic or thermophilic bacteria ( Salinibacter ruber and Rhodothermus marinus , respectively). On the contrary, Gracilimonas species show only extremely halotolerant and mesophilic features. Considering the phenotypic diversity within the clade, their comparative genomic analyses could provide a good clue to understand bacterial adaptation to extreme environments based on genomic context. Here we present a summary of the genomic features of G. tropica DSM 19535T , which is the first genome-sequenced type strain from the genus Gracilimonas .
Classification and features
Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed G. tropica DSM19535T is classified into the genus Gracilimonas (Fig. 2). The type strains which were most closely related to strain DSM19535T were Gracilimonas mengyeensis YIM J14T with 16S rRNA sequence similarity of 96.9 %, and Gracilimonas rosea CL-KR2T with a similarity of 96.1 %. Strain DSM19535T is tolerant of high salinity (up to 20 %) with a growth occurring over the range of salinity of 1–20 % (w/v) sea salts (optimum 3–6 %) (Table 1). Growth occurs under either aerobic or facultatively anaerobic conditions. The optimum pH is 7.0–8.0 with a growth range of pH 6–10 (Table 1). The strain was auxotroph for isoleucine and methionine (Table 1). Despite the phylum Bacteroidetes is known to be as a non-spore forming group , the strain was reported to form endospores, together with G. rosea . However, strain DSM19535T could not be asserted to form spore by the genomic analysis (see ‘Insights from the genome sequence’).
By phylogenetic analyses (Fig. 2), the genus Gracilimonas formed a sister clade with the genus Balneola which shows mesophilic features [5, 6]. At an outer branch, the clade with Gracilimonas and Balneola formed a robust clade with the moderate halophilic Fodinibius salinus (Fig. 2). Moreover, at a deeper branch, the clade formed a robust association with a clade that includes the thermophilic genus Rhodothermus  and the genus of extremely halophilic Salinibacter , despite the relatively low (ca. 80 %) similarities between the two clades. Thus, the phylogentically robust clade contains both extremophiles and non-extremophiles.
Auxotrophy for amino acids was examined using a minimal medium (glucose, 2 g; pyruvate, 0.3 g; K2HPO4, 3 g; NaH2PO4, 1 g; NH4Cl, 1 g; MgSO4•7H2O, 0.3 g; 1 ml of Holden’s trace elements ; 1 ml of Balch’s vitamin solution ; 1 L of artificial seawater ) supplemented with 0.3 mM or 3 mM of all amino acids except a focal amino acid. The strain could not grow in minimal medium without supplementation of L-isoleucine and L-methionine. But, the strain did not require other amino acids (L-alanine, L-arginine, L-asparagine, L-aspartate, L-cysteine, L-glutamate, L-glutamine, glycine, L-histidine, L-lysine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and selenocysteine) for growth.
Genome sequencing information
Genome project history
A culture of DSM 19535T (strain CL-CB462T) was selected for sequencing on the basis of its phylogenetic position [12, 13], and is part of the Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes project , a follow-up of the Genomic Encyclopedia of Bacteria and Archaea pilot project , which aims in increasing the sequencing coverage of key reference microbial genomes and to generate a large genomic basis for the discovery of genes encoding novel enzymes . The one thousand microbial genomes-I is the first of the production phases of the Genomic Encyclopedia of Bacteria and Archaea: sequencing a myriad of type strains initiative  and a Genomic Standards Consortium project . The genome project is deposited in the Genomes On Line Database  and the genome sequence is available from GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI) using state of the art sequencing technology . A summary of the project information is presented in Table 2.
Growth conditions and genomic DNA preparation
G. tropica DSM 19535T , was grown in DSMZ medium 514 (Bacto Marine Broth)  at 28 °C. Genomic DNA was isolated from about 0.5 g of cell paste using Jetflex Purification Kit (Epicentre MGP04100) following the standard protocol as recommended by the manufacturer with an additional protease K (50 μl; 21 mg/ml) digest for 60 min. at 58 °C followed by addition of 200 μl Protein Precipitation Buffer after protein precipitation and overnight incubation on ice . DNA was quality controlled according to JGI guidelines and is available through the DNA Bank Network .
Genome sequencing and assembly
The draft genome was generated using Illumina technology . An Illumina Std shotgun library was constructed and sequenced using the Illumina HiSeq 2000 platform which generated 14,058,618 reads totaling 2108.8 Mbp. All general aspects of library construction and sequencing performed at the JGI can be found at . All raw Illumina sequence data was passed through DUK, a filtering program developed at JGI, which removes known Illumina sequencing and library preparation artifacts (Mingkun L, Copeland A, Han J. DUK, unpublished, 2011). Artifact filtered sequence data was then screened and trimmed according to the k–mers present in the dataset (Mingkun L. kmernorm, unpublished, 2011). High–depth k–mers, presumably derived from MDA amplification bias, cause problems in the assembly, especially if the k–mer depth varies in orders of magnitude for different regions of the genome. Reads with high k–mer coverage (>30 × average k–mer depth) were normalized to an average depth of 30×. Reads with an average kmer depth of less than 2× were removed. Following steps were then performed for assembly: (1) normalized Illumina reads were assembled using Velvet version 1.1.04 , (2) 1–3 Kbp simulated paired end reads were created from Velvet contigs using wgsim , (3) normalized Illumina reads were assembled with simulated read pairs using Allpaths–LG (version r41043) . Parameters for assembly steps were: 1) Velvet (velveth: 63 –shortPaired and velvetg: −very clean yes –exportFiltered yes –min contig lgth 500 –scaffolding no –cov cutoff 10), 2) wgsim (−e 0 –1 100 –2 100 –r 0 –R 0 –X 0), 3) Allpaths–LG (PrepareAllpathsInputs: PHRED 64 = 1 PLOIDY = 1 FRAG COVERAGE = 125 JUMP COVERAGE = 25 LONG JUMP COV = 50, RunAllpathsLG: THREADS = 8 RUN = std shredpairs TARGETS = standard VAPI WARN ONLY = True OVERWRITE = True). The final draft assembly contained 48 contigs in 48 scaffolds. The total size of the genome is 3.8 Mbp and the final assembly is based on 457.7 Mbp of Illumina data. Based on a presumed genome size of 5Mbp, the average coverage of the genome was 421 × .
Genes were identified using Prodigal  as part of the DOE-JGI Annotation pipeline [30, 31] followed by a round of manual curation using the JGI GenePRIMP pipeline . The predicted CDSs were translated and used to search the National Center for Biotechnology Information non-redundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes .
The genome of the strain is 3,831,242 bp long and comprises 48 contigs ranging 1177 to 783,752 bp, with an overall GC content of 42.9 % (Table 3). Of the 3426 genes predicted, 3373 were protein coding genes, and 53 were RNA genes. A total of 2413 genes (70.4 %) were assigned a putative function while the remaining ones were annotated as hypothetical or unknown proteins. The distribution of genes into COG functional categories is presented in Table 4. The properties and the statistics of the genome are summarized in Tables 3 and 4.
Insights from the genome sequence
Based on genomic analysis of the metabolic features, G. tropica DSM19535T is predicted to be an auxotroph for L-lysine, L-phenylalanine, L-tyrosine, L-arginine, L-aspartic acid, L-isoleucine, L-proline, and L-methionine. In the auxotroph test, however, the strain was found to be auxotroph only for L-isoleucine and L-methionine (Table 1). This discrepancy might be due to missing annotations of essential genes by incomplete sequencing or presence of unknown genes related with transport and/or assimilation. In addition, despite selenocysteine was one of essential amino acids required for growth by the genomic analysis, the strain could grow in a medium without selenocysteine. Genome analysis also revealed that strain DSM19535T has a copper-containing nitrite reductase gene (nirK) homolog, suggesting that the strain may transform nitrite to nitric oxide (NO) under low oxygen or anoxic conditions. In addition, the strain contains DnrN (nitric oxide-dependent regulator) gene and this may protect cells from nitrosative stress . However, the nitrate, nitric oxide and nitrous oxide reductases involved in denitrification were not found. The strain has an ATP-dependent glutamine synthetase and a NADPH-dependent glutamate-oxoglutarate amidotransferase, and thus can assimilate ammonia into glutamate and glutamine. In the strain, ammonium may be transported by an ammonium transport protein. Genes participating in phosphate metabolism were also identified in the genome of the strain DSM19535T . Inorganic pyrophosphatase catalyzing the conversion of pyrophosphate to phosphate ion, and polyphosphate kinase catalyzing the formation of polyphosphate from ATP were found in the genome. The strain has several genes of Pho regulon (phoH, phoU, phoR and phoB) mediating an adaptive response to inorganic phosphate limitation but not high affinity phosphate binding protein and transporter (pstS and pstACB). In addition, the strain may hydrolyze phosphate groups from many types of organic molecules using alkaline phosphatase.
In the previous study, G. tropica DSM19535T was reported to be able to form spores . The spore-formation is very unusual in the phylum Bacteroidetes . Despite four and five proteins were annotated as stage II sporulation protein E (SpoIIE) and sporulation related domain, respectively, by search against the Pfam database, more than a hundred sporulation-related genes identified in Bacillus subtilis 168T were absent from the genome of strain DSM19535T . Further, the genes found in G. tropica were also found in genomes of phylogenetically close but non-sporulating genera, Balneola vulgaris DSM 17,893 and Salisaeta longa DSM2114. Therefore, further tests to examine spore-formation were conducted in this study. Consistent with the previous study, spore-like spherical cells were found after malachite green staining. However, after pasteurization at 60 °C for 10 and 20 min and 80, 90 and 100 °C for 10 min, re-growth of cells was never observed, suggesting that the coccoid cells may not be endospore. Actually, non-spore but spore-like spherical cells were also found in aging cultures of a variety of non-sporulating bacteria including Salinispira pacifica belonging to the phylum Spirochaetae , Prolinoborus fasciculus belonging to the class Betaproteobacteria and Anaerophaga thermohalophila belonging to the phylum Bacteroidetes [35–37]. The genomic analyses and pasteurization experiment convincingly suggested that the spore-like coccoid cells of G. tropica DSM19535T are not endospores.
The genome of a member belonging to the genus Gracilimonas in the phylum Bacteroidetes is reported here. In addition to detailed information of genome sequencing and annotation, genetic characteristics related with nitrogen and phosphorus utilization could be understood on the basis of genomic analyses. In addition, genomic analyses and pasteurization experiments suggested that G. tropica DSM19535T does not form spores.
Choi DH, Zhang GI, Noh JH, Kim W-S, Cho BC. Gracilimonas tropica gen. nov., sp. nov., isolated from a Synechococcus cultuire. Int J Syst Evol Microbiol. 2009;59:1167–72.
Wang YX, Li YP, Liu JH, Xiao W, Lai YH, Li ZY, et al. Gracilimonas mengyeensis sp. nov., a moderately halophilic bacterium isolated from a salt mine in Yunnan, south-western China. Int J Syst Evol Microbiol. 2013;63:3989–93.
Cho Y, Chung H, Jang GI, Choi DH, Noh JH, Cho BC. Gracilimonas rosea sp. nov., isolated from tropical seawater, and emended description of the genus Gracilimonas. Int J Syst Evol Microbiol. 2013;63:4006–11.
Krieg NR, Ludwig W, Euzéby J, Whitman WBW. Phylum XIV. Bacteroidetes phyl. nov. In: Krieg NR, Staley JT, Brown DR, Hedlund BP, Paster BJ, Ward NL, Ludwig W, Whitman WB, editors. Bergey’s Manual of Systematic Bacteriology, vol. 4. 2nd ed. New York: Springer; 2011. p. 25.
Urios L, Agogué H, Lesongeur F, Stackebrandt E, Lebaron P. Balneola vulgaris gen. nov., sp. nov., a member of the phylum Bacteroidetes from the north-western Mediterranean Sea. Int J Syst Evol Microbiol. 2006;56:1883–7.
Urios L, Intertaglia L, Lesongeur F, Lebaron P. Balneola alkaliphila sp. nov., a marine bacterium isolated from the Mediterranean Sea. Int J Syst Evol Microbiol. 2008;58:1288–91.
Flferdsson GA, Kristjansson JK, Hjorleifsdottir S, Stetter KO. Rhodothermus marinus gen. nov., a thermophilic, halophilic bacterium from submarine hot springs in Iceland. J Gen Microbiol. 1995;134:299–306.
Antón J, Oren A, Benlloch S, Rodríguez-Valera F, Amann R, Rosselló-Mora R. Salinibacter ruber gen. nov., sp. nov., a novel, extremely halophilic member of the Bacteria from saltern crystallizer ponds. Int J Syst Evol Microbiol. 2002;52:485–91.
Holden JF, Takai K, Summit M, Bolton S, Zyskowski J, Baross JA. Diversity among three novel groups of hyperthermophilic deep-sea Thermococcus species from three sites in the northeastern Pacific Ocean. FEMS Microbiol Ecol. 2001;36:51–60.
Balch WE, Wolfe RS. New approach to the cultivation of methanogenic bacteria: 2-mercaptoethanesulfonic acid (HSCoM)-dependent growth of Methanobacterium ruminantiumin a pressurized atmosphere. Appl Environ Microbiol. 1976;32:781–91.
Lyman J, Fleming RH. Composition of sea water. J Mar Res. 1940;3:134–46.
Göker M, Klenk HP. Phylogeny-driven target selection for large-scale genome-sequencing (and other) projects. Stand Genomic Sci. 2013;8:360–74.
Klenk HP, Göker M. En route to a genome-based classification of Archaea and Bacteria? Syst Appl Microbiol. 2010;33:175–82.
Kyrpides NC, Woyke T, Eisen JA, Garrity G, Lilburn TG, Beck BJ, et al. Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes (KMG-I) project. Stand Genomic Sci. 2014;9:1278–84.
Wu D, Hugenholtz P, Mavromatis K, Pukall R, Dalin E, Ivanova NN, et al. A phylogeny-driven Genomic Encyclopaedia of Bacteria and Archaea. Nature. 2009;462:1056–60.
Piao H, Froula J, Du C, Kim TW, Hawley E, Bauer S, et al. Identification of novel biomass-degrading enzymes from microbial dark matter: populating genome sequence space with functional annotation. Biotechnol Bioeng. 2014;111:1550–65.
Kyrpides NC, Hugenholtz P, Eisen JA, Woyke T, Göker M, Parker CT, et al. Genomic Encyclopedia of Bacteria and Archaea: sequencing a myriad of type strains. PLoS Biology. 2014;12:e1001920.
Field D, Sterk P, Kottmann R, De Smet JW, Amaral-Zettler L, Cochrane G, et al. Genomic Standards Consortium projects. Stand Genomic Sci. 2014;9:599–601.
Pagani I, Liolios K, Jansson J, Chen IMA, Smirnova T, Nosrat B, et al. The Genomes OnLine Database (GOLD) v.4: Status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res. 2012;40:D571–9.
Mavromatis K, Land ML, Brettin TS, Quest DJ, Copeland A, Clum A, et al. The fast changing landscape of sequencing technologies and their impact on microbial genome assemblies and annotation. PLoS ONE. 2012;7:e48837.
Anon: List of growth media used at DSMZ. [http://www.dsmz.de/catalogues/catalogue-microorganisms/culture-technology/list-of-media-for-microorganisms.html]. Accessed 24 Sep 2014.
Sakamoto M, Lapidus A, Han J, Trong S, Haynes M, Reddy TBK, et al. High quality draft genome sequence of Bacteroides barnesiae type strain BL2T (DSM 18169T) from chicken caecum. Stand Genomic Sci. 2015;10:48.
Gemeinholzer B, Dröge G, Zetzsche H, Haszprunar G, Klenk HP, Güntsch A, et al. The DNA bank network: the start from a German initiative. Biopreserv Biobank. 2011;9:51–5.
Bennett S. Solexa Ltd. Pharmacogenomics. 2004;5:433–8.
DOE Joint Genome Institute [http://www.jgi.doe.gov]. Accessed 24 Sep 2014.
Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008;18:821–9.
wgsim [https://github.com/lh3/wgsim]. Accessed 24 Sep 2014.
Gnerre S, MacCallum I, Przybylski D, Ribeiro FJ, Burton JN, Walker BJ, et al. High–quality draft assemblies of mammalian genomes from massively parallel sequence data. Proc Natl Acad Sci USA. 2011;108:1513–8.
Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: Prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010;11:119.
Mavromatis K, Ivanova NN, Chen IM, Szeto E, Markowitz VM, Kyrpides NC. The DOE-JGI Standard operating procedure for the annotations of microbial genomes. Stand Genomic Sci. 2009;1:63–7.
Chen IM, Markowitz VM, Chu K, Anderson I, Mavromatis K, Kyrpides NC, et al. Improving microbial genome annotations in an integrated database context. PLoS ONE. 2013;8:e54859.
Pati A, Ivanova N, Mikhailova N, Ovchinikova G, Hooper SD, Lykidis A, et al. GenePRIMP: A Gene Prediction Improvement Pipeline for microbial genomes. Nat Methods. 2010;7:455–7.
Markowitz VM, Mavromatis K, Ivanova NN, Chen IM, Chu K, Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics. 2009;25:2271–8.
Heurlier K, Thomson MJ, Aziz N, Moir JWB. The Nitric Oxide (NO)-sensing repressor NsrR of Neisseria meningitidis has a compact regulon of genes involved in NO synthesis and detoxification. Appl Environ Microbiol. 2008;190:2488–95.
Hania WB, Joseph M, Schumann P, Bunk B, Fiebig A, Spröer C, et al. Complete genome sequence and description of Salinispira pacifica gen. nov., sp. nov., a novel spirochaete isolated form a hypersaline microbial mat. Stand Genomic Sci. 2015;10:7.
Koechlein DJ, Krieg NR. Viable but nonculturable coccoid forms of Prolinoborus fasciculus (Aquaspirillum fasciculus). Can J Microbiol. 1998;44:910–2.
Denger K, Warthmann R, Ludwig W, Schink B. Anaerophaga thermohalophila gen. nov., sp. nov., a moderately thermohalophilic, strictly anaerobic fermentative bacterium. Int J Syst Evol Microbiol. 2002;52:173–8.
Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, et al. Towards a richer description of our complete collection of genomes and metagenomes “Minimum Information about a Genome Sequence “ (MIGS) specification. Nat Biotechnol. 2008;26:541–7.
Field D, Amaral-Zettler L, Cochrane G, Cole J, Dawyndt P, Garrity GM, et al. The Genomic Standards Consortium. PLoS Biology. 2011;9:e1001088.
Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA. 1990;87:4576–9.
Kämper P. Class III. Sphingobacteriia class. In: Krieg NR, Staley JT, Brown DR, Hedlund BP, Paster BJ, Ward NL, Ludwig W, Whitman WB, editors. Bergey’s Manual of Systematic Bacteriology, vol. 4. 2nd ed. New York: Springer-Verlag; 2011. p. 330.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000;25:25–9.
The authors gratefully acknowledge the help of Susanne Schneider for growing cells of DSM 19535T and of Evelyne-Marie Brambilla (both at DSMZ), for DNA extraction and quality control. This work was performed under the auspices of the US Department of Energy’s Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231. This work was also supported in part by a research program (PE99314) of the Korea Institute of Ocean Science and Technology (KIOST), and EAST-1 and the BK21+ project of the Korean Government. This study was supported in part by Russian Ministry of Science Mega-grant no.11.G34.31.0068 (PI. Dr Stephen J O’Brien).
The authors declare that they have no competing interests.
DHC, CA, GIJ, HPK, MG, NCK, AP and BCC drafted the manuscript. AL, JH, TBKR, MH, NI, VM, NR, BT and TW sequenced, assembled and annotated the genome. All authors read and approved the final manuscript.
Dong Han Choi and Chisang Ahn are Co-first author
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Choi, D.H., Ahn, C., Jang, G.I. et al. High-quality draft genome sequence of Gracilimonas tropica CL-CB462T (DSM 19535T), isolated from a Synechococcus culture. Stand in Genomic Sci 10, 98 (2015). https://doi.org/10.1186/s40793-015-0088-8
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