Complete Genome Sequences for the Anaerobic, Extremely Thermophilic Plant Biomass-Degrading Bacteria Caldicellulosiruptor hydrothermalis, Caldicellulosiruptor kristjanssonii, Caldicellulosiruptor kronotskyensis, Caldicellulosiruptor owensensis, and Caldicellulosiruptor lactoaceticus

Abstract

The genus Caldicellulosiruptor contains the most thermophilic, plant biomass-degrading bacteria isolated to date. Previously, genome sequences from three cellulolytic members of this genus were reported (C. saccharolyticus, C. bescii, and C. obsidiansis). To further explore the physiological and biochemical basis for polysaccharide degradation within this genus, five additional genomes were sequenced: C. hydrothermalis, C. kristjanssonii, C. kronotskyensis, C. lactoaceticus, and C. owensensis. Taken together, the seven completed and one draft-phase Caldicellulosiruptor genomes suggest that, while central metabolism is highly conserved, significant differences in glycoside hydrolase inventories and numbers of carbohydrate transporters exist, a finding which likely relates to variability observed in plant biomass degradation capacity.

Members of the genus Caldicellulosiruptor are asporogenic, plant biomass-degrading, hydrogen-generating members of the order Clostridiales (13, 17). The genus is globally distributed: species have been isolated from terrestrial geothermal hot springs in Russia (15, 18, 20), Iceland (3, 16), Yellowstone National Park in the United States (9), and New Zealand (17) and, in one case, from solar-heated mud flats in Owens Lake, CA (11). With optimal growth temperatures ranging from 70 to 78°C, the genus Caldicellulosiruptor contains the most thermophilic microorganisms capable of biological cellulose hydrolysis known. While 16S rRNA phylogeny indicates a very close relationship within the eight species studied thus far (94.8% to 99.4% identity), microbiological analysis indicated that the genus is more physiologically divergent than previously thought (2). Three complete genome sequences are currently available for this genus, those of C. saccharolyticus (19), C. bescii (12), and C. obsidiansis (5), all of which are capable of crystalline cellulose hydrolysis. To further probe the plant biomass-degrading capacity among Caldicellulosiruptor species, genome sequences of five additional members of this genus, including some hemicellulolytic but less cellulolytic members, were completed: those of C. hydrothermalis, C. kristjanssonii, C. kronotskyensis, C. owensensis, and C. lactoaceticus (draft phase). This also provides additional geographical diversity for examining Caldicellulosiruptor physiology.

All members of the Caldicellulosiruptor genus have similarly sized chromosomes, approximately 2.4 to 2.97 Mb. Their genomes are also A+T rich, ranging from 35 to 36% G+C. Similarly to C. bescii (4, 12), C. kristjanssonii also possesses an extrachromosomal element, the 15.9-kb plasmid pCALKR01. There are no similarities, however, between pATHE01/pATHE02 and pCALKR01, suggesting that there are no ubiquitous Caldicellulosiruptor plasmids. The other six genomes do not contain any extrachromosomal elements, although C. hydrothermalis possesses an inverted region that could exist outside the chromosome.

Sequencing strategy.

All general aspects of library construction and sequencing can be found at http://www.jgi.doe.gov/. The genomes of Caldicellulosiruptor species were sequenced at the U.S. Department of Energy Joint Genome Institute (JGI) using a combination of Illumina (1) and 454 (14) technologies similar to the sequencing strategy for C. obsidiansis (5). Sequencing data were assembled either with VELVET (21) or by being converted into a Phrap assembly. The Phred/Phrap/Consed software package was used for sequence assembly and quality assessment (6-8) in the finishing process. Illumina data were used to correct base errors and increase consensus quality using Polisher software (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), Dupfinisher (10), or sequencing of cloned bridging PCR fragments. Gaps between contigs were closed by editing in Consed, by PCR, and by Bubble PCR primer walks. The creation of additional reactions and shatter libraries was necessary to close gaps and to raise the quality of the finished sequences. Final assemblies are based on a minimum of 30× coverage of the genome.

Nucleotide accession numbers.

GenBank accession numbers for Caldicellulosiruptor genomes announced here are as follows: C. hydrothermalis, CP002219; C. kristjanssonii, CP002326; C. kronotskyensis, CP002330; C. lactoaceticus, AEKD00000000; and C. owensensis, CP002216.