Analysis Related To Pathogenicity And Genetically Modification Of Bacillus Cereus Group Strains

Bacillus cereus, B. thuringiensis, B. mycoides, B. pseudomycoides, B. anthracisand B. weihenstephanensis are all belong to B. cereus group. B. thuringiensis and B.cereus share a high level of chromosomal similarity and are phenotypieally similarexcept that B. thuringiensis has plasmid encoded cry genes expressing insecticidalcrystal proteins (Cry toxins) during the sporulation phase. B. thuringiensis is aworldwide used pesticide and plays an important role for controlling many kinds ofharmful insects. Withthe widely release of B. thuringiensisin environment, thefamily relationship and the highly similarity even in eytotoxieity between B.thuringiensis and B. cereus result in the concern for its potential risk.Twenty-six B. cereus group strains were surveyed for the presence of enterotoxingenes and other toxin genes related to pathogenicity of B. cereus group strains. PCRdetection results indicated the presence of the pleiotropie virulence regulator PlcRencoding gene and B. anthracis plasmid pXO1-ORF₁₀₁-like fragments in most B.cereus group strains. At least one gene of each of the two protein complexes Hb1 andNhe was detected in 73ï¼…of the B. cereus group strains and in 83ï¼…of the B.thuringiensis strains.The B. anthracis plasmid pXO1-ORF₁₀₁-like fragments in 12 B. cereus groupstrains were sequenced and analyzed. Sequence alignment of tbe pXO1-ORF₁₀₁ fromthree B. anthracis and pXO1-ORF₁₀₁-like fragments from other 12 B. cereus groupisolates indicated high identity and the presence of subgroup- and strain-specific SNPsamong these fragments. Based on the SNP profile, the analyzed B. cereus groupstrains could be assigned to two major genetic clusters-cereus cluster andthuringiensis cluster. Two pXO1-ORF₁₀₁-like fragments from a B. cereus B-4ac and acommercial B. thuringiensis kurstaki HD1 were cloned, sequenced and expressed inEscherichia coli, respectively. Toxicity assays revealed that the product encoded bythe pXO1-ORF₁₀₁-like fragment had no impact on Vero cells, suggesting that thisORF probably not contribute to enterotoxic activity. Considering the potential risk of B. thuringiensis, we need to look for somemeans of constructing or selecting B. thuringiensis strains for biological control withlow expression of B. cereus-like enterotoxic traits. At least two ways can beperformed: 1): to knock out some factors related with the pathogenicity; 2) to transferCry toxin encoding plasmids by conjugation to a number of B. cereus group bacteria,including bacteria known to have low enterotoxic activity.We studied the transfer of two B. thuringiensis plasmids by conjugation among B.cereus group strains and the expression of toxins in different transconjugants.The plasmid pHT73, containing cry1Ac and tagged with an erythromycinresistance gene, was transferred from B. thuringiensis subsp, kurstaki KT0 to severalB. cereus group strains, including recipients with a known low enterotoxic activity.The study demonstrated that pHT73 can be transferred to B. thuringiensis subsp.kurstaki, subsp, darmstadiensis, Bacillus cereus and Bacillus mycoides strains.SDS-PAGE and phase contrast microscopy showed that the transconjugantscontaining plasmid pHT73 could express Cry1Ac toxin and produce bipyramidalcrystalline inclusion bodies during sporulation.Two antibiotic resistance tagged pBtoxis were transferred by conjugation toother B. cereus group strains. A lepidopteran active B. thuringiensis subspecieskurstaki and a B. cereus strain received the plasmid pBtoxis with a low transfer rate ofabout 10^-8transconjugants/recipient. The resulting B. thuringiensis subspecieskurstaki transconjugant was active to both lepidopteran and dipteran targets and the B.cereus transconjugant was active against dipteran insects. Phase contrast microscopyshowed that the B. cereus transconjugants could produce only round crystallineinclusion bodies while B. thuringiensis subspecies kurstaki transconjugant couldproduce both round and bipyramidal crystals during sporulation. However, none ofthe experiment showed any indications of mobilizing abilities of pBtoxis. But thetransposon Tn5401 and helper plasmid pAW63 can mobilize or help the transfer ofpBtoxis. The limited number of strains, which could receive and maintain pBtoxisusing a conjugational helper plasmid, indicates a very narrow host range of the B.thuringiensis subsp, israelensis pBtoxis plasmid and a lower transfer ability thanpHT73.On the other hand, we knocked out the plcR gene of Btk HD1, Bti AND672 andBc CIP 5832, and found the inactivation has neither effect on the expression of threemutants nor the hemolytic activity of the mutants of HD1 and CIP 5832. But it changed the hemolytic activity of AND672 from incomplete hemolytic to nohemolytic at all. Further effect of this inactivation and if this method can be used as auseful engineering improving tool need to be studied.