Complete genome sequence of Chitinophaga pinensis type strain (UQM 2034T)
- Tijana Glavina Del Rio1, 6,
- Birte Abt2, 6,
- Stefan Spring2, 6,
- Alla Lapidus1, 6,
- Matt Nolan1, 6,
- Hope Tice1, 6,
- Alex Copeland1, 6,
- Jan-Fang Cheng1, 6,
- Feng Chen1, 6,
- David Bruce1, 3, 6,
- Lynne Goodwin1, 3, 6,
- Sam Pitluck1, 6,
- Natalia Ivanova1, 6,
- Konstantinos Mavromatis1, 6,
- Natalia Mikhailova1, 6,
- Amrita Pati1, 6,
- Amy Chen4, 6,
- Krishna Palaniappan4, 6,
- Miriam Land1, 5, 6,
- Loren Hauser1, 5, 6,
- Yun-Juan Chang1, 5, 6,
- Cynthia D. Jeffries1, 5, 6,
- Patrick Chain2, 3, 6,
- Elizabeth Saunders3, 6,
- John C. Detter1, 3, 6,
- Thomas Brettin1, 3, 6,
- Manfred Rohde6, 7,
- Markus Göker2, 6,
- Jim Bristow1, 6,
- Jonathan A. Eisen1, 6, 8,
- Victor Markowitz4, 6,
- Philip Hugenholtz1, 6,
- Nikos C. Kyrpides1, 6,
- Hans-Peter Klenk2, 6 and
- Susan Lucas1, 6
© The Author(s) 2010
Published: 28 February 2010
Chitinophaga pinensis Sangkhobol and Skerman 1981 is the type strain of the species which is the type species of the rapidly growing genus Chitinophaga in the sphingobacterial family ‘Chitinophagaceae’. Members of the genus Chitinophaga vary in shape between filaments and spherical bodies without the production of a fruiting body, produce myxospores, and are of special interest for their ability to degrade chitin. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a member of the family ‘Chitinophagaceae’, and the 9,127,347 bp long single replicon genome with its 7,397 protein-coding and 95 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.
Strain UQM 2034T (DSM 2588 = ATCC 43595 = KCTC 3412) is the type strain of the species Chitinophaga pinensis and was first described in 1981 by Sangkhobol and Skerman . In 1981, strain UQM 2034T was described as a long, filamentous, gliding microorganism isolated from an infusion of litter from the base of a pine tree in Alderley, Brisbane, Australia . In 1999, the phylogenetic position of C. pinensis was determined. The comparison of 16S rRNA sequences revealed Flexibacter filimoris as the most closely related bacterium .
In 2006 Kämpfer et al. reclassified F. sancti, F. filiformis, F. japonensis and Cytophaga arvensicola to the monospecific genus Chitinophaga and proposed C. skermanii sp. nov. . In recent years the number of newly described species belonging to the genus Chitinophaga increased. Two additional new Chitinophaga species were described in 2007, C. ginsengisegetis sp. nov. and C. ginsengisoli sp. nov. isolated from soil of a ginseng field in South Korea . In the same year Kim and Jung described the new species C. terrae sp. nov . In 2009, three additional Chitinophaga species were described: C. niabensis sp. nov. , C. niastensis sp. nov , and C. rupis sp. nov . Here we present a summary classification and a set of features for C. pinensis UQM 2034T, together with the description of the complete genomic sequencing and annotation.
Classification and features
The most similar 16S rRNA gene sequences from cultivated strains that are stored in GenBank originate from isolates belonging to different species of the genus Chitinophaga: C. sancti, C. filiformis and C. ginsengisoli with 96–97% sequence similarity; all of them were isolated from soil samples. In metagenomic surveys of environmental samples only 16S rRNA genes with sequence similarity values below 92% to C. pinensis were detected, indicating that members of this species are not abundant in the so far genomically screened habitats (status July 2009).
Classification and general features of C. pinensis UQM 2034T according to the MIGS recommendations 
Species Chitinophaga pinensis
Type strain UQM 2034
up to 1.5% NaCl
acid production from glucose, lactose and sucrose
Alderley, Brisbane, Australia
Sample collection time
in 1981 or before
Strain UQM 2034T produces acid from glucose, lactose, and sucrose. Chitin, casein and gelatin are hydrolyzed, whereas according to Sangkhobol and Skerman (1981) cellulose, starch, alginate, and agar are not hydrolyzed . Nitrate is not reduced to nitrite. C. pinensis UQM 2034T produces urease and is catalase and oxidase positive [1,3]. UQM 2034T is susceptible to tetracycline, streptomycin, and chloramphenicol and resistant to neomycin, kanamycin, penicillin G, and erythromycin . C. pinensis UQM 2034T is able to lyse Staphylococcus aureus cells but not cells of Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis .
The fatty acid profile of strain UQM 2034T revealed C15:0 iso (30.4%) and C16:1ω5c (33.2%) as the major fatty acids and C17:0 iso-3-OH (11.5%) and C15:0 iso-3-OH (3.1%) as the major hydroxyl fatty acids. MK-7 is the predominant menaquinone . The polar lipid composition has not been analyzed, but phosphatidylethanolamine is reported for C. rupis .
Genome sequencing and annotation
Genome project history
Genome sequencing project information
Two Sanger libraries: 8kb pMCL200 and fosmid pcc1 Fos. One 454 pyrosequence standard library.
ABI3730, 454 GS FLX
8.9× Sanger; 17.4× pyrosequence
Gene calling method
Genbank Date of Release
August 08, 2009
NCBI project ID
Source material identifier
Tree of Life, GEBA
Growth conditions and DNA isolation
C. pinensis UQM 2034T, DSM 2588, was grown in DSMZ medium 67 (CY-Medium)  at 22°C. DNA was isolated from 0.5–1 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany), with a modified protocol for cell lysis (st/LALMP), as described in Wu et al. .
Genome sequencing and assembly
The genome was sequenced using a combination of Sanger and 454 sequencing platforms. All general aspects of library construction and sequencing performed at the JGI can be found at the JGI website (http://www.jgi.doe.gov/). 454 Pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 2,046 overlapping fragments of 1,000 bp and 9,925 of them entered into the final assembly as pseudo-reads. The sequences were assigned quality scores based on Newbler consensus q-scores with modifications to account for overlap redundancy and to adjust inflated q-scores. A hybrid 454/Sanger assembly was made using the parallel phrap assembler (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher or transposon bombing of bridging clones . Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification. A total of 882 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence. The error rate of the completed genome sequence is 0.01 in 100,000 nucleotides. Together all sequence types provided 26.3× coverage of the genome. The final assembly contains 91,161 Sanger and 876,658 pyrosequencing 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 (http://geneprimp.jgi-psf.org/) . The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes - Expert Review (http://img.jgi.doe.gov/er) platform .
% of Total
Genome size (bp)
DNA Coding region (bp)
DNA G+C content (bp)
Number of replicons
Genes with function prediction
Genes in paralog clusters
Genes assigned to COGs
Genes assigned Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Number of genes associated with the general COG functional categories
Translation, ribosomal structure and biogenesis
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, mitosis and meiosis
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Not in COGs
Insights from genome sequence
The predominant characteristic feature of C. pinensis is the ability to degrade chitin, a β-1,4-glycosidic linked homopolymer of N-acetyl-D-glucosamine and one of the most abundant polysaccharides in nature. It is a component of fungal cell walls and of arthropod exoskeletons. Chitin is degraded by chitinases (EC 220.127.116.11); endochitinases randomly cleave within the chitin molecule and exochitinases hydrolyze diacetylchitobiose from the end of a chitin chain. Diacetylchitobiose is further degraded to N-acetylglucosamine by the action of N-acetylglucosaminidases (EC 18.104.22.168). These glycosidic bond hydrolyzing enzymes were grouped in glycoside hydrolase (GH) families based on amino acid sequence similarities (http://www.cazy.org) . For the C. pinensis genome 169 glycoside hydrolases belonging to 49 different GH families are predicted; 18 of the predicted glycoside hydrolases belong to GH family 43 which contains xylosidases, xylanases, arabinanases, arabinofuranosidases and galactosidases.
Because of the chitin degrading ability of C. pinensis a great number of chitinases was expected to be encoded in the genome. According to the CAZY-database, exochitinases and endochitinases belong to GH families 18, 19 and 48. As estimated, there were several glycoside hydrolases predicted, which may be involved in chitin degradation; five members of GH family 18 (Cpin_2184, Cpin_2186, Cpin_2580, Cpin_3805, Cpin_3919) and three members of GH family 19 (Cpin_5850, Cpin_5553, Cpin_5898). The comparison of the amino acid sequence from these chitinase candidates to the databank BlastP indicated no homologs according to the whole length of the proteins. However, similarities to the known GH family domains were observed.
The search for N-acetylglucosaminidases (EC 22.214.171.124) in the genome of C. pinensis revealed gene Cpin_3944 which encodes a protein with a GH family 20 domain. The predicted GH family 20 domain resembles the well characterized catalytic domain of a N-acetylglucosaminidase from Serratia marcescens . Further N-acetylhexoaminidases of C. pinensis are encoded by the genes Cpin_1798, Cpin_4994 and Cpin_1915.
A second way to degrade chitin was described by Davis and Eveleigh in 1984 . First, the chitin molecule is deacetyliated by deacetylases (EC 126.96.36.199), afterwards chitobiose is released from chitosan by the action of chitosanases (EC 188.8.131.52), finally chitobiose is hydrolyzed by glucosaminidases (EC 184.108.40.206) and glucosamine molecules are released.
One putative chitin deacetylase is encoded in the genome of C. pinensis. The deduced amino acid sequence of Cpin_6813 shows a GH family 19 domain and a C-terminal deacetylase domain. Chitosanases that are responsible for the hydrolysis of chitosan are mainly found in GH family 46 but also occur in GH families 5 and 18. In C. pinensis, no GH family 46 members were observed, but the presence of nine GHs belonging to family 5 and five members of GH family 18 are predicted. One of these glycoside hydrolases might have a chitosanase function. It remains unclear which pathway C. pinensis uses for the degradation of chitin and whether the predicted functions of the proteins match the real functions.
We would like to gratefully acknowledge the help of Birgit Merkhoffer for growing C. pinensis cultures and Susanne Schneider for DNA extraction and quality analysis (both at DSMZ). 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, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396, as well as German Research Foundation (DFG) INST 599/1-1.
- Sangkhobol V, Skerman VBD. Chitinophaga, a new genus of chitinolytic myxobacteria. Int J Syst Bacteriol 1981; 31:285–293.View ArticleGoogle Scholar
- Sly LI, Taghavi M, Fegan M. Phylogenetic position of Chitinophaga pinensis in the Flexibacter-Bacteroides-Cytophaga phylum. Int J Syst Bacteriol 1999; 49:479–481. PubMedView ArticlePubMedGoogle Scholar
- Kämpfer P, Young CC, Sridhar KR, Arun AB, Lai WA, Shen FT, Rekha PD. Transfer of [Flexibacter] sancti, [Flexibacter] filiformis, [Flexibacter] japonensis and [Cytophaga] arvensicola to the genus Chitinophaga and description of Chitinophaga skermanii sp. nov. Int J Syst Evol Microbiol 2006; 56:2223–2228. PubMed doi:10.1099/ijs.0.64359-0View ArticlePubMedGoogle Scholar
- Lee HG, An DS, Im WT, Liu QM, Na JR, Cho DH, Jin CW, Lee ST, Yang DC. Chitinophaga ginsengisegetis sp. nov. and Chitinophaga ginsengisoli sp. nov., isolated from soil of a ginseng field in South Korea. Int J Syst Evol Microbiol 2007; 57:1396–1401. PubMed doi:10.1099/ijs.0.64688-0View ArticlePubMedGoogle Scholar
- Kim MK, Jung HY. Chitinophaga terrae sp. nov., isolated from soil. Int J Syst Evol Microbiol 2007; 57:1721–1724. PubMed doi:10.1099/ijs.0.64964-0View ArticlePubMedGoogle Scholar
- Weon HY, Yoo SH, Kim YJ, Son JA, Kim BY, Kwon SW, Koo BS. Chitinophaga niabensis sp. nov. and Chitinophaga niastensis sp. nov., isolated from soil. Int J Syst Evol Microbiol 2009; 59:1267–1271. PubMed PubMed doi:10.1099/ijs.0.004804-0View ArticlePubMedGoogle Scholar
- Lee DW, Lee JE, Lee SD. Chitinophaga rupis sp. nov., isolated from soil. Int J Syst Evol Microbiol 2009; 59:2830–2833. PubMed doi:10.1099/ijs.0.011163-0View ArticlePubMedGoogle Scholar
- Lee C, Grasso C, Sharlow MF. Multiple sequence alignment using partial order graphs. Bioinformatics 2002; 18:452–464. PubMed doi:10.1093/bioinformatics/18.3.452View ArticlePubMedGoogle Scholar
- Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 2000; 17:540–552. PubMedView ArticlePubMedGoogle Scholar
- Stamatakis A, Hoover P, Rougemont J. A Rapid Bootstrap Algorithm for the RAxML Web Servers. Syst Biol 2008; 57:758–771. PubMed doi:10.1080/10635150802429642View ArticlePubMedGoogle Scholar
- 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/gkp848PubMed CentralView ArticlePubMedGoogle Scholar
- 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/nbt1360PubMed CentralView ArticlePubMedGoogle Scholar
- Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms. Proposal for the domains Archaea and Bacteria. Proc Natl Acad Sci USA 1990; 87:4576–4579. PubMed doi:10.1073/pnas.87.12.4576PubMed CentralView ArticlePubMedGoogle Scholar
- Garrity GM, Holt JG. Taxonomic Outline of the Archaea and Bacteria. In: Garrity GM, Boone DR, Castenholz RW (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 1, Springer, New York, 2001, p. 155–166Google Scholar
- Biological Agents. Technical rules for biological agents www.baua.de TRBA 466.
- 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. The Gene Ontology Consortium. Nat Genet 2000; 25:25–29. PubMed doi:10.1038/75556PubMed CentralView ArticlePubMedGoogle Scholar
- Wu D, Hugenholtz P, Mavrommatis K, Pukall R, Dalin E, Ivanova N, Kunin V, Goodwin L, Wu M, Tindall BJ, et al. A phylogeny-driven genomic encyclopedia of Bacteria and Archaea. Nature 2009; 462:1056–1060. PubMed doi:10.1038/nature08656PubMed CentralView ArticlePubMedGoogle Scholar
- Sims D, Brettin T, Detter JC, Han C, Lapidus A, Copeland A, Glavina Del Rio T, Nolan M, Chen F, Lucas S, et al. Complete genome of Kytococcus sedentarius type strain (541T). Stand Genomic Sci 2009; 1:12–20. doi:10.4056/sigs.761PubMed CentralView ArticlePubMedGoogle Scholar
- Hyatt D, Chen G-L, LoCascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Genomics (In press)Google Scholar
- Pati A, Ivanova N, Mikhailova, N, Ovchinikova G, Hooper SD, Lykidis A, Kyrpides NC. GenePRIMP: A Gene Prediction Improvement Pipeline for microbial genomes. Nature Methods (In press).Google Scholar
- Markowitz VM, Mavromatis K, 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/btp393View ArticlePubMedGoogle Scholar
- Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B. The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res 2009; 37:D233–D238. PubMed doi:10.1093/nar/gkn663PubMed CentralView ArticlePubMedGoogle Scholar
- Prag G, Papanikolau Y, Tavlas G, Vorgias CE, Petratos K, Oppenheim AB. Structures of chitobiase mutants complexed with the substrate di-N-acetyl-D-glucosamine: the catalytic role of the conserved acidic pair, aspartate 539 and glutamate 540. J Mol Biol 2000; 300:611–617. PubMed doi:10.1006/jmbi.2000.3906View ArticlePubMedGoogle Scholar
- Davis B, Eveleigh DE. Chitosanases: occurrence, production and immobilization. In: Zikalis JP (ed.), Chitin, Chitosan and Related Enzymes. Academic Press 1984;161–179.Google Scholar
- List of growth media used at DSMZ: http://www.dsmz.de/microorganisms/media_list.php