Near-Complete Genome Sequence of the Cellulolytic Bacterium Bacteroides (Pseudobacteroides) cellulosolvens ATCC 35603.

We report the single-contig genome sequence of the anaerobic, mesophilic, cellulolytic bacterium, Bacteroides cellulosolvens. The bacterium produces a particularly elaborate cellulosome system, wherein the types of cohesin-dockerin interactions are opposite of other known cellulosome systems: cell-surface attachment is thus mediated via type-I interactions, whereas enzymes are integrated via type-II interactions.

GENOME ANNOUNCEMENT

The cellulosome is one of the most efficient systems known to biodegrade plant cell-wall polysaccharides and cellulosic wastes. This multi-enzyme extracellular complex incorporates multiple hydrolytic enzymes onto the bacterial cell-surface through dockerin modules that tightly bind to scaffoldin proteins via complementary cohesin modules (1–3). Additional carbohydrate-binding modules (CBM) attach the entire enzymatic complex to the cellulosic substrate (4). The biodegrading activity of cellulosomes has been studied extensively in related cellulolytic bacteria, such as Clostridium (Ruminiclostridium) thermocellum, Acetivibrio cellulolyticus, Clostridium (Ruminiclostridium) clariflavum, Clostridium (Ruminiclostridium) cellulolyticum, Clostridium cellulovorans, Clostridium (Ruminiclostridium) papyrosolvens, and Ruminococcus flavefaciens (5).

Bacteroides cellulosolvens ATCC 35603 (DSM 2933) is a cellulolytic bacterium, originally isolated from sewage sludge (6, 7) in co-culture with Clostridium saccharolyticum. Initially classified as a Gram-negative bacterium, analysis of the 16S RNA indicated that B. cellulosolvens, like A. cellulolyticus, is a member of the phylogenetically diverse clostridial assemblage (8, 9). Recently, B. cellulosolvens was renamed Pseudobacteroides cellulosolvens (10).

B. cellulosolvens was selected for its ability to grow under mesophilic, anaerobic conditions, and the bacterium was able to bind and degrade crystalline cellulose to cellobiose and glucose (11–13). Its cellulose-degrading activity was shown to be cell-associated (14), and elaborate cellulolytic cell-surface structures were subsequently demonstrated (15, 16). Cellulosome-like complexes were further identified in the bacterium (17), supported by the recognition of the major scaffoldin protein (CipBc, later renamed ScaA) (18, 19), which includes eleven type-II cohesin domains, a family-3a CBM, and a C-terminal dockerin domain. Its scaffoldin was shown to interact with a family-48 glycoside hydrolase (18), and the crystal structure of its type-II cohesin was determined (20).

The genome is reported as a large contig of 6,878,816 bp, translated into 5,897 predicted proteins. Sequencing was performed using PacBio RS-II technology and data from four SMRT cells was assembled using SMRTanalysis v2.2 (HGAP3 protocol). The initial assembly generated three contigs at ~65× raw read coverage, which were joined using Geneious R8 (21) and then validated by PCR and Sanger sequencing (22). Illumina reads (at ~200× coverage) also confirmed contiguity. The ends of the single contig were unable to be joined experimentally or in silico, possibly as a result of a misassembly or active mobile genetic element. Active transposase systems have been shown to interfere with closure previously (23). Therefore, the genome is reported as near-complete assembly. Gene prediction and annotation were performed as described previously (24, 25).

Intriguingly, the types of cohesin-dockerin interaction in B. cellulosolvens are reversed from those of all other known cellulosome systems, whereby cell-surface attachment of noncatalytic scaffoldins in B. cellulosolvens is mediated via type-I interactions, whereas the enzymes are integrated via type II-interactions (19, 26, 27). The genome codes for 75 cohesin modules (mostly type-II cohesins), packaged in more than two-dozen scaffoldins, and over 200 dockerin-containing proteins, including glycoside hydrolases, carbohydrate esterases, and polysaccharide lyases. Thus, B. cellulosolvens scaffoldins represent the largest noncatalytic cellulosomal subunits known to date, indicating the presence of a particularly elaborate cellulosome system.

Nucleotide sequence accession numbers.

This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number LGTC00000000. The version described in this paper is version LGTC01000000.