Complete genome sequence of Bacillus thuringiensis subsp. kurstaki strain HD73.

Bacillus thuringiensis is a Gram-positive bacterium that produces intracellular protein crystals toxic to a wide variety of insect larvae. We report the complete genome sequence of Bacillus thuringiensis subsp. kurstaki strain HD73 from the Centre OILB (Institut Pasteur, France), which belongs to serotype 3ab and is toxic to lepidopteran larvae.

GENOME ANNOUNCEMENT

Bacillus thuringiensis is a Gram-positive bacterium that produces intracellular protein crystals (cry) toxic to a wide variety of insect larvae and is the most commonly used biological pesticide (1). Many B. thuringiensis strains contain multiple cry genes, harbored in plasmids (2). Bacillus thuringiensis subspecies kurstaki strain HD73 (B. thuringiensis HD73), toxic to lepidopteran larvae (serotype 3ab) (3), contains large self-transmissible plasmids, including pHT73 and pAW63 (4). This strain was obtained from the Centre OILB (Institut Pasteur, France). This strain was provided as the B. thuringiensis subspecies kurstaki type strain and was also designated the B. thuringiensis subspecies kurstaki KT0 strain (5). Only one endotoxin gene, the cry1Ac gene, was found to be harbored in pHT73 (6–8), and this gene was considered to be a typical example of a sporulation-dependent crystal gene because the cry1A-like promoter is controlled by sigma E and sigma K during sporulation (9, 10). However, weak transcription of this promoter was detected in the nonsporulating cell (11).

Here, we determined the whole genome sequence to obtain the genomic information. Genomic DNA was isolated from B. thuringiensis HD73. Genome sequencing was performed with 454 GS-FLX titanium (Roche Applied Science) and Illumina GAII (Illumina, United States) platforms. A total of 167,017 high-quality Roche 454 reads with an average read length of 412 bp were produced, providing about 12-fold coverage of the genome, while the Illumina reads provide 370-fold coverage with 21.7 million reads of 100 bp (with insert size 8,000 bp). After preprocessing, Roche 454 reads were assembled into contigs with Newbler, version 2.6, and then scaffolded with Illumina mate-pair reads using SSPACE (12). A 40-kb fosmid library was constructed and subjected to bidirectional end sequencing of 2,261 clones. The gaps were closed by PCR amplification and primer walking. The open reading frames (ORFs) were identified by using Glimmer version 3.02 (13). The tRNAs and rRNAs were predicted using tRNAscan-SE (14) and RNAmmer (15), respectively. The functions of encoding genes were annotated by using NCBI nr, COG (clusters of orthologous groups) (16), KEGG (17), and InterProScan (18).

The genome of B. thuringiensis HD-73 contains 8 replicons, including a circular chromosome and 7 plasmids. The chromosome was 5.6 Mb in length with a GC content of 31.4%, containing 5,892 protein-encoding genes, 104 tRNA, and 36 rRNA-encoding operons. Approximately 32.1% of all coding sequences (a total of 1,894) were assigned to COGs, and 1,282 CDSs can be annotated into the 1109 KEGG orthology system by using KAAS (19). The 7-plasmid length ranges from 8 kb to 77 kb, with GC content from 29.73% to 34.66%, carrying a total of 235 ORFs. Besides the self-transmissible plasmid pHT73, our data revealed another large plasmid, pHT77, in addition to those revealed in a previous study (4), because the size similarity makes it difficult to distinguish these plasmids by gel electrophoresis (pHT77 of 76,490 bp and pHT73 of 77,351 bp). The genome sequence provides insights into plasmid conjugation, spore formation, crystal formation, virulence factor interaction with insect host, and evolution of B. thuringiensis.

Nucleotide sequence accession numbers.

The annotated chromosome and plasmids have been deposited in GenBank under the accession numbers: CP004069 (chromosome), CP004070 (pHT73), CP004071 (pHT77), CP004073 (pHT11), CP004074 (pHT8_1), CP004075 (pHT8_2), and CP004076 (pHT7).