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 x 10(-4) and 1.6 x 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.

FIG. 1.

Plasmid profiles of donor, recipient, and transconjugant strains. Lanes: 1, KT0; 2, Ba63002R; 3, KBa63002R; 4, Ba63605R; and 5, KBa63605R. Chr, chromosomal products.

FIG. 2.

Results from PCR amplification to detect the relative gene markers in donor KT0 and B. anthracis recipient and transconjugant strains. (A and B) Lanes: M, DL3000; 1, KT0 (erm); 2, KT0 (cry1Ac); 3, Ba16 (cap); 4, Ba16 (pag); and 5, Ba16 (Ba813). (A) Lanes: 6, Ba63002R (cap); 7, Ba63002R (pag); 8, Ba63002R (Ba813); 9, Ba63002R (erm); 10, Ba63002R (cry1Ac); 11, KBa63002R (cap); 12, KBa63002R (pag); 13, KBa63002R (Ba813); 14, KBa63002R (erm); 15, KBa63002R (cry1Ac); and 16, control. (B) Lanes: 6, Ba63605R (cap); 7, Ba63605R (pag); 8, Ba63605R (Ba813); 9, Ba63605R (erm); 10, Ba63605R (cry1Ac); 11, KBa63605R (cap); 12, KBa63605R (pag); 13, KBa63605R (Ba813); 14, KBa63605R (erm); 15, KBa63605R (cry1Ac); and 16, control.

FIG. 3.

RAPD patterns of donor, recipient, and transconjugant strains. Lanes: M, marker; 1, KT0; 2, Ba63002R; 3, KBa63002R; and 4, Ba63605R.

FIG. 4.

Ultrathin electron micrographs of crystals produced in the following donor and transconjugant strains: Ba62002R (A); KBa62002R (B); Ba63605R (C); KBa63605R (D); and KT0 (E and F).