First Complete Genome Sequence of Clostridium sporogenes DSM 795T, a Nontoxigenic Surrogate for Clostridium botulinum, Determined Using PacBio Single-Molecule Real-Time Technology.

The first complete genome sequence of Clostridium sporogenes DSM 795(T), a nontoxigenic surrogate for Clostridium botulinum, was determined in a single contig using the PacBio single-molecule real-time technology. The genome (4,142,990 bp; G+C content, 27.98%) included 86 sets of >1,000-bp identical sequence pairs and 380 tandem repeats.

GENOME ANNOUNCEMENT

Clostridium sporogenes is an anaerobic spore-forming bacterium that causes food spoilage (1, 2). C. sporogenes is widely used as a nontoxigenic surrogate for Clostridium botulinum in the validation of food sterilization because of its physiological and phylogenetic similarity to C. botulinum and nontoxigenicity (2–6).

A draft sequence of C. sporogenes DSM 795T has been determined using 454, Illumina, and Sanger technologies in 16 contigs (GenBank accession number JFBQ00000000) (total 4,106,665 bp; average G+C content, 27.8%) (A. Poehlein, R. Karin, S. M. Koenig, R. Daniel, and P. Duerre, submitted for publication) (7, 8). These contigs are disconnected at tandem repeat or low G+C regions. Here, we report the first complete genome sequence of C. sporogenes DSM 795T determined using the PacBio single-molecule real-time (SMRT) technology (9).

The genomic DNA of C. sporogenes DSM 795T, originally isolated from soil in 1908, was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) (10). It was purified using a PowerClean DNA cleanup kit (MoBio, Carlsbad, CA), followed by a 20-kb library construction for P5-C3 chemistry. After >7-kb size selection using BluePippin (Sage Science, Beverly, MA), 8 SMRT cells from the libraries were sequenced using the PacBio RS II platform (Pacific Biosciences, Menlo Park, CA) with 180-min movies. De novo assembly was performed using the hierarchical genome assembly process 2 (HGAP2) workflow (11). A single circular contig representing a chromosome was obtained (4,142,990 bp; average G+C content, 27.98%).

The complete genome sequence of C. sporogenes DSM 795T included 86 sets of >1,000-bp identical sequence pairs (4,911-bp maximum) and 380 tandem repeats (369 bp × 8.5 copies maximum). Tandem repeats were identified using Tandem Repeats Finder (12). Recently, a sequence of C. sporogenes NCIMB 10696T, which originated from the same strain (McClung 2004T) as C. sporogenes DSM 795T, has been determined using 454, Illumina, and Sanger technologies (CP009225) (http://www.straininfo.net/strains/7982) (4,141,984 bp; average G+C content, 28.00%) (13). We found three marked differences between the sequences of DSM 795T and NCIMB 10696T. First, in a 39-bp tandem region, DSM 795T carried 25.5 copies (1,156,066 to 1,157,028), whereas 10696T carried 20.5 copies (1,156,066 to 1,156,839). Second, in a 312-bp tandem region, DSM 795T carried 5.9 copies (3,502,125 to 3,503,970), whereas 10696T carried 4.9 copies (3,501,430 to 3,502,963). Third, DSM 795T had a 501-bp extra region (2,040,199 to 2,040,699) that could be inserted in 10696T (between 2,040,006 and 2,040,007). On DSM 795T sequencing, the PacBio RS II platform produced extra-long reads with an average of 3,959 bp and a maximum of 35,904 bp, and large numbers of reads completely covered those regions: 290 reads for the first, 191 reads for the second, and 359 reads for the third. This result suggests that the number of tandem repeats is underestimated in the 10696T sequence. The SMRT technology provides power for genome sequencing with multikilobase extra-long reads and unbiased G+C coverage (11, 14, 15) for assessing structural variations such as variable number tandem repeat.

Nucleotide sequence accession number.

The complete genome sequence of C. sporogenes DSM 795T was deposited in DDBJ/ENA/GenBank under the accession number CP011663.