- Open Access
Complete genome sequence of the gliding freshwater bacterium Fluviicola taffensis type strain (RW262T)
Standards in Genomic Sciences volume 5, pages21–30 (2011)
Fluviicola taffensis O’Sullivan et al. 2005 belongs to the monotypic genus Fluviicola within the family Cryomorphaceae. The species is of interest because of its isolated phylogenetic location in the genome-sequenced fraction of the tree of life. Strain RW262T forms a monophyletic lineage with uncultivated bacteria represented in freshwater 16S rRNA gene libraries. A similar phylogenetic differentiation occurs between freshwater and marine bacteria in the family Flavobacteriaceae, a sister family to Cryomorphaceae. Most remarkable is the inability of this freshwater bacterium to grow in the presence of Na+ ions. All other genera in the family Cryomorphaceae are from marine habitats and have an absolute requirement for Na+ ions or natural sea water. F. taffensis is the first member of the family Cryomorphaceae with a completely sequenced and publicly available genome. The 4,633,577 bp long genome with its 4,082 protein-coding and 49 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.
Strain RW262T (= DSM 16823 = NCIMB 13979) is the type strain of the species Fluviicola taffensis, which is the type species of the monotypic genus Fluviicola , affiliated with the family Cryomorphaceae . The genus name is derived from the Latin words fluvius, meaning ‘river’ and -cola meaning ‘inhabitant, dweller’, yielding the Neo-Latin word Fluviicola, the river dweller [1,3]. The species epithet is derived from the Neo-Latin word taffensis, referring to the place where the type strain has been isolated, the river Taff (Wales, UK) [1,3]. The family Cryomorphaceae belongs to the class Flavobacteria which contains many species that probably play an integral role for the flow of carbon and energy in the marine environment .
Flavobacteria are the major decomposers of high-molecular-mass organic matter in sea water . Phylogenetically the family Cryomorphaceae is located between the families Flavobacteriaceae and Bacteroidaceae  and currently comprises the genera Brumimicrobium, Cryomorpha and Crocinitomix , Owenweeksia , Wandonia , Fluviicola  and Lishizhenia . The family Cryomorphaceae exhibits the greatest degree of phenotypic similarity to the family Flavobacteriaceae  and includes species with a mostly rod-like to filamentous morphology; cells are usually non-motile or move by gliding and often contain carotenoid pigments [1,2]. All members of the Cryomorphaceae are strictly aerobic or facultatively anaerobic (fermentative) with a chemoheterotrophic metabolism [1,2] and often have complex growth requirements for sea water salts, organic compounds as sole nitrogen sources, yeast extract and vitamins for growth . To date no further isolates of F. taffensis have been reported. Here we present a summary classification and a set of features for F. taffensis RW262T, together with the description of the complete genomic sequencing and annotation.
Classification and features
A representative genomic 16S rRNA sequence of F. taffensis RW262T was compared using NCBI BLAST  under default settings (e.g., considering only the high-scoring segment pairs (HSPs) from the best 250 hits) with the most recent release of the Greengenes database  and the relative frequencies of taxa and keywords (reduced to their stem ) were determined, weighted by BLAST scores. The most frequently occurring genera were Brumimicrobium (62.9%) and Fluviicola (37.1%) (3 hits in total). Among all other species, the one yielding the highest score was ‘Brumimicrobium mesophilum’ (DQ660382), which corresponded to an identity of 92.1% and an HSP coverage of 58.0%. (Note that the Greengenes database uses the INSDC (= EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification.) The most frequently occurring keywords within the labels of all environmental samples which yielded hits were ‘lake’ (9.1%), ‘tin’ (3.4%), ‘microbi’ (2.5%), ‘depth’ (2.0%) and ‘tract’ (1.7%) (247 hits in total). The most frequently occurring keywords within those labels of environmental samples which yielded hits of a higher score than the highest scoring species were ‘lake’ (9.2%), ‘tin’ (3.8%), ‘microbi’ (2.3%), ‘depth’ (2.0%) and ‘tract’ (1.8%) (169 hits in total). The most frequent keyword ‘lake’ may reflect the freshwater origin of strain RW262T, whereas the keywords ‘tin’ and ‘depth’ may allude to some until now unrecognized ecological features of F. taffensis.
Figure 1 shows the phylogenetic neighborhood of F. taffensis in a 16S rRNA based tree. The sequences of the two identical 16S rRNA gene copies in the genome differ by two nucleotides from the previously published 16S rRNA sequence (AF493694), which contains one ambiguous base call.
Strain RW262T is strictly aerobic, Gram-negative, motile by gliding and flexirubin-pigmented . Cells are flexible rods with rounded ends (Figure 2), 0.4–0.5 µm in diameter and 1.5–5.7 µm in length, with rare longer filaments of up to 51 µm in length . Growth occurs at 4ºC and 20ºC, but not in the presence of Na+ ions . Growth of strain RW262T at 4 ºC is only weak, so that F. taffensis should not be considered to be psychrotolerant like the other members of the family [1,2]. Strain RW262T is capable of DNA hydrolysis , is catalase positive but oxidase negative, able to catalyze the hydrolysis of arginine, aesculin or starch, whereas it weakly hydrolyzes gelatine . It is negative for nitrate and nitrite reduction; indole production; β-galactosidase, urease and xylanase activity; hydrolysis of agar, arginine, aesculin and starch; and acid production from carbohydrates . The strain is not able to utilize glucose, arabinose, mannose, mannitol, N-acetylglucosamine, maltose, gluconate, caprate, adipate, malate, citrate or phenyl acetate . However, within the genome are several genes for utilization of complex organic carbon compounds. The strain is resistant to chloramphenicol (10 µg), streptomycin (10 µg), and kanamycin (30 µg) but susceptible to penicillin G (10 units), ampicillin (10 µg), rifampicin (5 µg) and tetracycline (10 µg) .
The predominant cellular acid of strain RW262T was the branched-chain saturated fatty acid iso-C15:0 (44.2%) . Unsaturated branched-chain fatty acids, straight-chain saturated and mono-unsaturated fatty acids occur only in lower amounts: C14:0 (3.2%), C15:0 (7.5%), C16:0 (3.0%), iso-C15:1 ω10c (11.8%), iso-C16:1 ω12c (4.9%). Lipopolysaccharide hydroxy fatty acids constitute 20.4% of the total cellular fatty acids, mainly composed of iso-C17:0 3-OH (12.3%), iso-C15:0 3-OH (4.2%) and iso-C15:0 2-OH (3.5%) .
Genome sequencing and annotation
Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position , and is part of the Genomic Encyclopedia of Bacteria and Archaea project . The genome project is deposited in the Genome On Line Database  and the complete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.
Growth conditions and DNA isolation
F. taffensis RW262T, DSM 16823, was grown in DSMZ medium 948 (Oxoid nutrient medium)  at 28°C. DNA was isolated from 0.5–1 g of cell paste using JetFlex Genomic DNA Purification kit (GENOMED 600100) following the standard protocol as recommended by the manufacturer, but with additional 20 µl proteinase K incubation (one hour) at 58° for improved cell lysis. DNA is available through the DNA Bank Network .
Genome sequencing and assembly
The genome was sequenced using a combination of Illumina and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website . Pyrosequencing reads were assembled using the Newbler assembler (Roche). The initial Newbler assembly consisting of 51 contigs in one scaffold was converted into a phrap  assembly by making fake reads from the consensus, to collect the read pairs in the 454 paired end library. Illumina GAii sequencing data (801.4 Mb) was assembled with Velvet  and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. The 454 draft assembly was based on 164.9 Mb 454 draft data and all of the 454 paired end data. Newbler parameters are -consed -a 50 -l 350 -g -m -ml 20. The Phred/Phrap/Consed software package  was used for sequence assembly and quality assessment in the subsequent finishing process. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with gapResolution , Dupfinisher , or sequencing clones bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F. Chang, unpublished). A total of 161 additional reactions and shatter libraries were necessary to close gaps and to raise the quality of the finished sequence. Illumina reads were also used to correct potential base errors and increase consensus quality using a software Polisher developed at JGI . The error rate of the completed genome sequence was less than 1 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 374.0 × coverage of the genome. The final assembly contained 232,904 pyrosequence and 44,902,395 Illumina reads.
Genes were identified using Prodigal  as part of the Oak Ridge National Laboratory genome annotation pipeline, 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 (NCBI) non-redundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes - Expert Review (IMG-ER) platform .
The genome consists of a 4,633,577 bp long chromosome with a G+C content of 36.5% (Table 3 and Figure 3). Of the 4,131 genes predicted, 4,082 were protein-coding genes, and 49 RNAs; 49 pseudogenes were also identified. The majority of the protein-coding genes (55.0%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.
O’Sullivan LA, Rinna J, Humphreys G, Weightman AJ, Fry JC. Fluviicola taffensis gen. nov., sp. nov., a novel freshwater bacterium of the family Cryomorphaceae in the phylum ‘Bacteroidetes’. Int J Syst Evol Microbiol 2005; 55:2189–2194. PubMed doi:10.1099/ijs.0.63736-0
Bowman JP, Mancuso Nichols C, Gibson JAE. Algoriphagus ratkowskyi gen. nov., sp. nov., Brumimicrobium glaciale gen. nov., sp. nov., Cryomorpha ignavagen. nov., sp. nov. and Crocinitomix catalasitica gen. nov., sp. nov., novel flavobacteria isolated from various polar habitats. Int J Syst Evol Microbiol 2003; 53:1343–1355. PubMed doi:10.1099/ijs.0.02553-0
Euzéby JP. List of bacterial names with standing in nomenclature: A folder available on the internet. Int J Syst Bacteriol 1997; 47:590–592. PubMed doi:10.1099/00207713-47-2-590
Kirchman DL. The ecology of Cytophaga-Flavobacteria in aquatic environments. FEMS Microbiol Ecol 2002; 39:91–100. PubMed
Cottrell MT, Kirchman DL. Natural assemblages of marine proteobacteria and members of the Cytophaga-Flavobacter cluster consuming low — and high-molecular-weight dissolved organic matter. Appl Environ Microbiol 2000; 66:1692–1697. PubMed doi:10.1128/AEM.66.4.1692-1697.2000
Lau KWK, Ng CYM, Ren J, Lau SCL, Qian PY, Wong PK, Lau TC and WU M. Owenweeksia hongkongensis gen. nov., sp. nov., a novel marine bacterium of the phylum ‘Bacteroidetes’. Int J Syst Evol Microbiol 2005; 55:1051–1057. PubMed doi:10.1099/ijs.0.63155-0
Lee DH, Choi EK, Moon SR, Ahn S, Lee YS, Jung JS, Jeon CO, Whang KS, Kahng HY. Wandonia haliotis gen. nov., sp. nov., a marine bacterium of the family Cryomorphaceae, phylum Bacteroidetes. Int J Syst Evol Microbiol 2010; 60:510–514. PubMed doi:10.1099/ijs.0.012674-0
Lau KWK, Ren J, Wai NLM, Qian PY, Wong PK, Wu M. Lishizhenia caseinilytica gen. nov., sp. nov., a marine bacterium of the phylum Bacteroidetes. Int J Syst Evol Microbiol 2006; 56:2317–2322. PubMed doi:10.1099/ijs.0.64415-0
Bernardet JF, Nakagawa Y, Holmes B. Proposed minimal standards for describing new taxa of the family Flavobacteriaceae and emended description of the family. Int J Syst Evol Microbiol 2002; 52:1049–1070. PubMed doi:10.1099/ijs.0.02136-0
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215:403–410. PubMed
DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol 2006; 72:5069–5072. PubMed doi:10.1128/AEM.03006-05
Porter MF. An algorithm for suffix stripping. Program: electronic library and information systems 1980; 14:130–137.
Lee C, Grasso C, Sharlow MF. Multiple sequence alignment using partial order graphs. Bioinformatics 2002; 18:452–464. PubMed doi:10.1093/bioinformatics/18.3.452
Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 2000; 17:540–552. PubMed
Stamatakis A, Hoover P, Rougemont J. A rapid bootstrap algorithm for the RAxML web-servers. Syst Biol 2008; 57:758–771. PubMed doi:10.1080/10635150802429642
Hess PN, De Moraes Russo CA. An empirical test of the midpoint rooting method. Biol J Linn Soc Lond 2007; 92:669–674. doi:10.1111/j.1095-8312.2007.00864.x
Pattengale ND, Alipour M, Bininda-Emonds ORP, Moret BME, Stamatakis A. How many bootstrap replicates are necessary? Lect Notes Comput Sci 2009; 5541:184–200. doi:10.1007/978-3-642-02008-7_13
Swofford DL. PAUP*: Phylogenetic Analysis Using Parsimony (*and Other Methods), Version 4.0 b10. Sinauer Associates, Sunderland, 2002.
Liolios K, Chen IM, Mavromatis K, Tavernarakis N, Hugenholtz P, Markowitz VM, Kyrpides NC. The Genomes On Line Database (GOLD) in 2009: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res 2010; 38:D346–D354. PubMed doi:10.1093/nar/gkp848
Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ, Angiuoli SV, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol 2008; 26:541–547. PubMed doi:10.1038/nbt1360
Garrity G. NamesforLife. BrowserTool takes expertise out of the database and puts it right in the browser. Microbiol Today 2010; 37:9.
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; 87:4576–4579. PubMed doi:10.1073/pnas.87.12.4576
Garrity GM, Holt JG. The Road Map to the Manual. In: Garrity GM, Boone DR, Castenholz RW (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 1, Springer, New York, 2001, p. 119–169.
Ludwig W, Euzeby J, Whitman WG. Draft taxonomic outline of the Bacteroidetes, Planctomycetes, Chlamydiae, Spirochaetes, Fibrobacteres, Fusobacteria, Acidobacteria, Verrucomicrobia, Dictyoglomi, and Gemmatimonadetes. http://www.bergeys.org/outlines/Bergeys_Vol_4_Outline.pdf
Garrity GM, Holt JG. 2001. Taxonomic outline of the Archaea and Bacteria, p. 155–166. In: Garrity GM, Boone DR, Castenholz RW (ed), Bergey’s Manual of Systematic Bacteriology, 2nd ed, vol. 1. Springer, New York.
BAuA. 2010, Classification of bacteria and archaea in risk groups. http://www.baua.de TRBA 466, p. 89.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. Gene Ontology: tool for the unification of biology. Nat Genet 2000; 25:25–29. PubMed doi:10.1038/75556
Klenk HP, Göker M. En route to a genome-based classification of Archaea and Bacteria? Syst Appl Microbiol 2010; 33:175–182. PubMed doi:10.1016/j.syapm.2010.03.003
Wu D, Hugenholtz P, Mavromatis K, Pukall R, Dalin E, Ivanova NN, Kunin V, Goodwin L, Wu M, Tindall BJ, et al. A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea. Nature 2009; 462:1056–1060. PubMed doi:10.1038/nature08656
List of growth media used at DSMZ: http://www.dsmz.de/microorganisms/media_list.php
Gemeinholzer B, Dröge G, Zetzsche H, Haszprunar G, Klenk HP, Güntsch A, Berendsohn WG, Wägele JW. The DNA Bank Network: the start from a German initiative. Biopreservation and Biobanking 2011; 9:51–55. doi:10.1089/bio.2010.0029
JGI website. http://www.jgi.doe.gov
The Phred/Phrap/Consed software package. http://www.phrap.com
Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 2008; 18:821–829. PubMed doi:10.1101/gr.074492.107
Han C, Chain P. Finishing repeat regions automatically with Dupfinisher. In: Proceeding of the 2006 international conference on bioinformatics & computational biology. Arabnia HR, Valafar H (eds), CSREA Press. June 26–29, 2006: 141–146.
Lapidus A, LaButti K, Foster B, Lowry S, Trong S, Goltsman E. POLISHER: An effective tool for using ultra short reads in microbial genome assembly and finishing. AGBT, Marco Island, FL, 2008.
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. PubMed doi:10.1186/1471-2105-11-119
Pati A, Ivanova NN, Mikhailova N, Ovchinnikova G, Hooper SD, Lykidis A, Kyrpides NC. GenePRIMP: a gene prediction improvement pipeline for prokaryotic genomes. Nat Methods 2010; 7:455–457. PubMed doi:10.1038/nmeth.1457
Markowitz VM, Ivanova NN, Chen IMA, Chu K, Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics 2009; 25:2271–2278. PubMed doi:10.1093/bioinformatics/btp393
We would like to gratefully acknowledge the help of Helga Pomrenke (DSMZ) for growing F. taffensis cultures. This work was performed under the auspices of the US Department of Energy Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396, UT-Battelle and Oak Ridge National Laboratory under contract DE-AC05-00OR22725, as well as German Research Foundation (DFG) INST 599/1-2.