Complete Genome Sequence of Bacillus thuringiensis Serovar finitimus Strain YBT-020

Abstract

Bacillus thuringiensis is a Gram-positive, spore-forming bacterium that forms parasporal crystals at the onset of the sporulation phase of its growth. Here, we report the complete genome sequence of B. thuringiensis serovar finitimus strain YBT-020, whose parasporal crystals consist of Cry26Aa and Cry28Aa crystal proteins and are located between the exosporium and the spore coat and remain adhering to the spore after sporulation.

TEXT

Typically, the parasporal crystals of Bacillus thuringiensis are deposited against the forespore, develop outside the exosporium, and are separated from spores after sporulation (12). However, in a few cases, such as in B. thringiensis serovar finitimus strains (2, 7, 13) and serovar oyamesis strain LBIT-113 (9), the parasporal crystals are located between the exosporium and the spore coat and remain adhering to the spore after sporulation. We have previously described this phenotype as spore-crystal association (SCA) and found that the crystal protein genes themselves (cry26Aa and cry28Aa) and their promoters cannot determine crystal formation inside the exosporium (7). Using a complementation test, the genes determining the SCA phenotype have been isolated from strain YBT-020 (unpublished).

The genome sequence of B. thuringiensis strain YBT-020 was determined at the Beijing Genomics Institute (BGI; Shenzhen, China) with a strategy of Solexa paried-end sequencing technology. Filtered paired-end reads (698.65 Mb in total) were obtained to reach a depth of 122-fold coverage of the genome with an Illumina Solexa GA IIx instrument, and about 96.53% of the reads were assembled into 105 contigs (defined as >500 bp) by using the SOAPdenovo alignment tool (http://soap.genomics.org.cn/index.html#intro2). Gaps between contigs were closed by custom primer walks, long-distance PCR amplification, or inverse PCR amplification followed by DNA sequencing with an ABI 3730 sequencer. tRNA and rRNA genes were identified by tRNAscan-SE (10) and RNAmmer (8), respectively.

The complete genome of B. thuringiensis strain YBT-020 contains three replicons: a circular chromosome (5,355,490 bp) containing 5,477 open reading frames (ORFs) and two circular plasmids, which were named pBMB26 (187,880 bp) and pBMB28 (139,013 bp), carrying 200 and 149 predicted ORFs, respectively. The G+C content of the chromosome is 35.3%, while those of plasmids pBMB26 and pBMB28 are 33.1% and 33.9%, respectively. This genome carries 107 tRNAs and 14 rRNA operons. Plasmid pBMB26 harbors cry26Aa (YBT020_27999) and cry28Aa (YBT020_28569) crystal protein genes, while pBMB28 harbors another cry28Aa (YBT020_29291) gene. The two cry28Aa genes share 99% sequence identity. There are also four truncated crystal protein genes, YBT020_27744, YBT020_27749, YBT020_27819, and YBT020_27994, which are homologous to cry7Aa1, cry8Ka2, cry28Aa1, and cry26Aa1, respectively. All the putative crystal genes are located on plasmid pBMB26.

Based on the phylogenetic trees from the chromosome that were inferred by PTreeRec (3), strain B. thuringiensis YBT-020 was found to be closest to B. thuringiensis Al Hakam (1), B. thuringiensis serovar konkukian 97-27 (4), and B. thuringiensis BMB171 (5), in order of closeness. The chromosome of strain YBT-020 was phylogenetically more closely related to that of B. anthracis Ames (11) than that of B. cereus ATCC 14579 (6).

This is the first genome sequence of B. thuringiensis harboring crystal protein genes, and its availability will facilitate the understanding of evolutionary relationships among B. cereus group organisms.

Nucleotide sequence accession numbers.

The sequences of the B. thuringiensis serovar finitimus strain YBT-020 have been deposited in GenBank under accession numbers CP002508 (chromosome), CP002509 (plasmid pBMB26), and CP002510 (plasmid pBMB28).