Complete Genome Sequence of the Cellulolytic Thermophile Clostridium thermocellum DSM1313

Abstract

Clostridium thermocellum DSM1313 is a thermophilic, anaerobic bacterium with some of the highest rates of cellulose hydrolysis reported. The complete genome sequence reveals a suite of carbohydrate-active enzymes and demonstrates a level of diversity at the species level distinguishing it from the type strain ATCC 27405.

TEXT

Clostridium thermocellum is a thermophilic, anaerobic bacterium of both fundamental and applied significance. The bacterium is highly cellulolytic, degrading cellulose through the action of a tethered, multienzyme complex called a cellulosome. The cellulosome of C. thermocellum is the best-characterized cellulase complex and serves as a paradigm for cellulolytic microorganisms (6, 7). In fermenting cellulose, C. thermocellum produces ethanol and organic acids as primary products. The fermentative capability of the bacterium has been the focus of several decades of research, owing to its potential to be used commercially in the consolidated bioprocessing of lignocellulosic material to ethanol and other products (6, 8). Recent advances in the development of genetic tools for C. thermocellum DSM1313 have enabled scientists to modify the cellulosome and primary metabolism of the bacterium (10, 11). The complete genome sequence of Clostridium thermocellum DSM 1313 expands the knowledge base for thermophilic, cellulolytic bacteria; enables comprehensive comparisons of closely related microbes; and facilitates further genetic engineering of the microbe.

The genome of Clostridium thermocellum was sequenced at the Joint Genome Institute (JGI) using a combination of Illumina (1) and 454 (9) technologies. An Illumina GAii shotgun library with reads of 36 bases, a 454 Titanium draft library with an average read length of 324.1 ± 200.9 bases, and a paired-end 454 library with an average insert size of 7.2 kb were generated for this genome. All general aspects of library construction and sequencing performed at the JGI can be found at http://www.jgi.doe.gov/. Illumina sequencing data were assembled with Velvet (12), and the consensus sequences were shredded into 1.5-kb overlapped fake reads and assembled together with the 454 data. Draft assemblies were based on 179.3 Mb of 454 draft data and all of the 454 paired-end data. Newbler parameters are -consed-a 50-l 350-g-m-ml 20.

The initial Newbler assembly contained 84 contigs in two scaffolds. We converted the initial 454 assembly into a Phrap assembly by making fake reads from the consensus, collecting the read pairs in the 454 paired-end library. The Phred/Phrap/Consed software package was used for sequence assembly and quality assessment (2, 3, 4) in the following finishing process. Illumina data were used to correct potential base errors and increase consensus quality using Polisher software developed at JGI (A. Lapidus, unpublished data). After the shotgun stage, reads were assembled with parallel Phrap (High Performance Software, LLC). Possible misassemblies were corrected with gapResolution (C. Han, unpublished data) or Dupfinisher (5) or by sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR, and by bubble PCR primer walks. A total of 227 additional reactions and 4 shatter libraries were necessary to close gaps and to raise the quality of the finished sequence.

Nucleotide sequence accession number.

The final annotated genome of C. thermocellum DSM1313 has been deposited in GenBank under accession number CP002416.