• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Feb 2010; 192(3): 901–902.
Published online Nov 30, 2009. doi:  10.1128/JB.01450-09
PMCID: PMC2812471

Genome Sequence of the Cellulosome-Producing Mesophilic Organism Clostridium cellulovorans 743B[down-pointing small open triangle]

Abstract

Clostridium cellulovorans 743B was isolated from a wood chip pile and is an anaerobic and mesophilic spore-forming bacterium. This organism degrades native substrates in soft biomass such as corn fiber and rice straw efficiently by producing an extracellular enzyme complex called the cellulosome. Here we report the genome sequence of C. cellulovorans 743B.

The biotechnological potential of polysaccharolytic enzymes has resulted in the isolation and characterization of a large number of anaerobic, Gram-positive, spore-forming bacteria, the majority of which have been allocated to the genus Clostridium. Among clostridia, the cellulosomes produced by Clostridium species are particularly designed for efficient degradation of plant cell wall polysaccharides. The component parts of the multicomponent complex are integrated by virtue of a unique family of integrating modules, the cohesins and the dockerins, whose distribution and specificity dictate the overall cellulosome architecture (3). The cellulosome system in Clostridium cellulovorans 743B (ATCC 35296) has been studied extensively for the last 20 years and has resulted in providing basic information about mesophilic cellulosomes. This organism was isolated from a wood chip pile and is an anaerobic spore-forming bacterium whose optimal growth temperature is 37°C (9). It has the ability to utilize cellulose, xylan, pectin, cellobiose, glucose, fructose, galactose, and mannose as carbon sources for growth. Its fermentation products include H2, CO2, acetate, butyrate, formate, lactate, and ethanol. When it is grown in the presence of cellulose, electron micrographs have shown that large protuberances are present on its cell surface (4), while small or no protuberances are evident when cells are grown in the presence of glucose or cellobiose (5).

We sequenced a total length of 101,749,598 bp and analyzed 381,514 reads by Genome Sequencer FLX 454./Roche sequencing (8) (GS-FLX version) to highly oversample the genome (20× coverage) and generated 123,892 paired-end sequence tags, to enable the assembly of all tags using the GS De Novo Assembler version 1.1.03.24 (Roche Diagnostics) and the Genome Analyzer II and sequencing kit 36-Cycle Run (Illumina). Finally, we assembled 30 scaffolds (sets of 601 ordered and oriented contigs; total length of 5,123,527 bp) to generate approximately 5.1 Mbp of nearly contiguous Clostridium botulinum E3 strain Alaska E43 (accession no. NC_010723) complete genome sequence. We analyzed a number of predicted genes included in the C. cellulovorans genome using CRITICA (version 1.05b) (2) and Glimmer 2 (version 2.10) (6) to find regions in proteins with known functions. We annotated and classified according to Gene Ontology (GO) (1). In silico Molecular Cloning Genomic Edition ver. 3.0.26 software (In Silico Biology, Co., Ltd., Japan) was used for individual genomic analysis.

The C. cellulovorans 743B (ATCC 35296) genome consists of 5,123,527 bp. A total of 4,220 polypeptide-encoding open reading frames (ORFs) were identified using CRITICA, while 4,297 ORFs were identified using Glimmer 2. The number of ORFs identical between CRITICA and Glimmer 2 was 2,773. Sixty-three tRNAs and 33 anticodons were also identified using tRNAscan-SE (7). In comparison of the genome sizes among cellulosomal clostridia such as Clostridium cellulolyticum H10 (4.07 Mbp) (GenBank accession no. CP001348) and Clostridium thermocellum ATCC 27405 (3.84 Mbp) (GenBank accession no. CP000568), the C. cellulovorans genome was over 1 Mbp larger than the other genomes. Moreover, the number of predicted genes (4,220 by CRITICA) in the C. cellulovorans genome was the largest among them. On the other hand, the G+C content in C. cellulovorans was 31.1%, similar to that (30.9%) in Clostridium acetobutylicum ATCC 824 (GenBank accession no. AE001437), while the G+C contents in C. cellulolyticum and C. thermocellum were 37.7% and 39.0%, respectively.

A protein BLAST search against the database of clusters of orthologous groups (COGs) of proteins indicated that 4,171 genes were annotated by 4,220 predicted coding sequences using CRITICA while 4,098 genes were identified by 4,297 predicted coding sequences using Glimmer 2. On the other hand, a protein BLAST search against the NCBI nr database indicated that 4,184 genes were annotated by 4,220 predicted coding sequences using CRITICA while 4,071 genes were identified by 4,297 predicted coding sequences using Glimmer 2. Interestingly, 57 cellulosomal genes were found in the C. cellulovorans genome and coded for not only carbohydrate-active enzymes but also lipases, peptidases, and proteinase inhibitors. Moreover, two novel genes encoding a scaffolding protein were found in the genome. Thus, by examining genome sequences from multiple Clostridium species, comparative genomics offers new insight into genome evolution and the way in which natural selection molds functional DNA sequence evolution. Our analysis, coupled with the genome sequence data, will provide a road map for constructing enhanced cellulosome-producing Clostridium strains for industrial applications such as biofuel production.

Nucleotide sequence accession number.

The C. cellulovorans genome sequence and annotation data have been deposited in GenBank under accession number SRA008356.

Acknowledgments

We thank Sumitomo Corporation, Tokyo, Japan, for financial support of this work. Bioinformatics work was performed under the auspices of the Dragon Genomics Center, Takara Bio Co. Ltd, Japan.

Footnotes

[down-pointing small open triangle]Published ahead of print on 30 November 2009.

REFERENCES

1. Ashburner, M., et al. 2000. Gene Ontology: tool for the unification of biology. Nat. Genet. 25:25-29. [PMC free article] [PubMed]
2. Badger, J. H., and G. J. Olsen. 1999. CRITICA: coding region identification tool invoking comparative analysis. Mol. Biol. Evol. 16:514-524. [PubMed]
3. Bayer, E. A., R. Lamed, B. White, and H. J. Flint. 2008. From cellulosomes to cellulosomics. Chem. Rec. 8:364-377. [PubMed]
4. Blair, B. G., and K. L. Anderson. 1998. Comparison of staining techniques for scanning electron microscopic detection of ultrastructural protuberances on cellulolytic bacteria. Biotech. Histochem. 73:107-113. [PubMed]
5. Blair, B. G., and K. L. Anderson. 1999. Regulation of cellulose-inducible structures of Clostridium cellulovorans. Can. J. Microbiol. 45:242-249. [PubMed]
6. Delcher, A. L., D. Harmon, S. Kasif, O. White, and S. L. Salzberg. 1999. Improved microbial gene identification with GLIMMER. Nucleic Acids Res. 27:4636-4641. [PMC free article] [PubMed]
7. Lowe, T. M., and S. R. Eddy. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25:955-964. [PMC free article] [PubMed]
8. Margulies, M., et al. 2005. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437:376-380. [PMC free article] [PubMed]
9. Sleat, R., R. A. Mah, and R. Robinson. 1984. Isolation and characterization of an anaerobic, cellulolytic bacterium, Clostridium cellulovorans sp. nov. Appl. Environ. Microbiol. 48:88-93. [PMC free article] [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...