Conjugative Transfer of Insecticidal Plasmid pHT73 from Bacillus thuringiensis to B. anthracis and Compatibility of This Plasmid with pXO1 and pXO2

Abstract

Bacillus anthracis, the etiologic agent of anthrax, is genetically close to and commonly shares a giant gene pool with B. cereus and B. thuringiensis. In view of the human pathogenicity and the long persistence in the environment of B. anthracis, there is growing concern about the effects of genetic exchange with B. anthracis on public health. In this work, we demonstrate that an insecticidal plasmid, pHT73, from B. thuringiensis strain KT0 could be efficiently transferred into two attenuated B. anthracis strains, Ba63002R (pXO1 ؉ pXO2 ؊ ) and Ba63605R (pXO1 ؊ pXO2 ؉ ), by conjugation in liquid medium in the laboratory, with transfer rates of 2.3 ؋ 10 ؊4 and 1.6 ؋ 10 ؊4 CFU/donor, respectively. The B. anthracis transconjugants containing both pHT73 and pXO1 or pXO2 could produce crystal protein Cry1Ac encoded by plasmid pHT73 and had high toxicity to Helicoverpa armigera larvae. Furthermore, the compatibility and stability of pHT73 with pXO1/pXO2 were demonstrated. The data are informative for further investigation of the safety of B. thuringiensis and closely related strains in food and in the environment.In the Bacillus cereus sensu lato family, the classification of three species, B. thuringiensis, B. cereus, and B. anthracis, is based mainly on the presence of different functional plasmids in the clusters (26). B. thuringiensis is an insect pathogen, harboring plasmids encoding insecticidal proteins with toxicity against insects of the orders Diptera, Lepidoptera, and Coleoptera and nematodes (30); B. cereus is a ubiquitous food spoilage bacterium associated with two forms of human food poisoning (diarrheal and emetic syndromes), and the cereulide toxin (which causes the emetic syndrome) is encoded on a large virulence plasmid, pCERE01 (also named pCER270; Ϸ270 kb) (15); B. anthracis is the active agent of anthrax, and its virulence is attributed mainly to the presence of plasmids pXO1 (Ϸ182 kbp) and pXO2 (Ϸ95 kbp) since curing any of the plasmids attenuates the strain (5).Recent studies demonstrated that the genetic backbones of both the pXO1 and pXO2 plasmids are not restricted only to B. anthracis but rather can be found in isolates of the related species B. cereus and B. thuringiensis as well (14,24,33). It was reported that B. cereus strain G9241, isolated from a patient with life-threatening pneumonia, carried a specific protective antigen gene (pagA) on plasmid pBCXO1 and a gene encoding a capsule made mainly of putative polysaccharide on plasmid pBC218 (14, 18). In further studies, the specific pXO1 fragments and pXO2 cap genes were detected in B. cereus strains 03BB102 and 03BB108 (13) and a backbone similar to the common backbone of pXO2 was found in a B. thuringiensis subsp. konkukian strain (serotype H34) isolated from a wounded soldier (12). These findings indicate that B. anthracis and related species may share a large gene pool and that genetic exchange among these species, followed by diversification, may be a major cause for their genomic plasticity and evolution.The vegetative cells of the ...

The resolution and regeneration of a cointegrate plasmid reveals a model for plasmid evolution mediated by conjugation and oriT site‐specific recombination

“…B. thuringiensis, which is characterized by the production of insecticidal crystal proteins (ICP), has been widely used as a biopesticide for controlling agricultural pests (Rosas-Garcia, 2009). Most ICP-coding genes are located on large plasmids (> 70 kb) (Berry et al, 2002;Yuan et al, 2010;He et al, 2011). So far, only two exceptions have been reported.…”

Modular Genetic Architecture of the Toxigenic Plasmid pIS56-63 Harboring cry1Ab21 in Bacillus thuringiensis subsp. thuringiensis strain IS5056

“…The high conservation among the origin of replication and replication protein of pIS56-63 and other B. cereus s.l. plasmids, including pXO1-like plasmids, suggests that these toxigenic elements replicate by a conserved theta mechanism (Yuan et al, 2010). In summary, we described the complete nucleotide sequence and annotation of the toxigenic pIS56-63 plasmid that harbors the cry1Ab21 entomotoxin, one of the 14 plasmids harbored by B. thuringiensis IS5056 (Murawska et al, 2013;Swiecicka et al, 2008).…”

“…Plasmids are among the largest genetic elements capable of being mobilized among bacterial populations, and in many instances their acquisition can confer selective advantages to recipients (Toussaint et al, 2002). In B. cereus sensu lato conjugation plays a key role in genes dispersion among members of the group (Van der Auwera et al, 2005;Yuan et al, 2010). Characteristics of M3-reg, the regulatory module.…”

“…Likewise, genes encoding the entomotoxins of B. thuringiensis reside on large extrachromosomal elements (Barry et al, 2002;Murawska et al, 2013). Therefore the acquired specific phenotypic features, peculiar pathogenicity, and capabilities in horizontal gene transfer (HGT) are largely a result of genes harbored on plasmids (Yuan et al, 2010).…”

Evolutionary aspects of toxin-producing bacteria

“…Selective pressure and HGT, combined with recombination and shuffling between toxin genes (resulting in domain swapping) and sequence divergence, has yielded a wide range of host specificities for these insecticidal toxins [235,[285][286][287]. The genes encoding the crystal protein toxins, for example, are frequently clustered on different transmissible plasmids or transposable elements [235,[285][286][287][288], and conjugation between different strains has been observed in the soil environment and within insect guts [289][290][291][292]. Individual toxins have insecticidal activity only against a limited range of insect species (i.e., usually only within certain insect orders).…”