The Function Of PlcR In Bacillus Anthracis

PlcR(Phospholipase C Regulator) is a very important pleiotropic regulator present in Bacillus cereus and B.thuringiensis. It can activate the expression of many genes encoding virulence factors, such as phospholipase, protease and hemolysin. B. anthracis, B. thuringiensis and B. cereus are members of the B. cereus group. They share high genetic similarity. But there is a nonsense mutation in all B. anthracis strains, making the gene dysfunctional. To study the function of PlcR in B. anthracis, we constructed a recombinant expression plasmids pBE2A-plcR、 pBE2A-FplcRpapR、 pBE2A-plcRpapR、 pBE2A-papR, using the B.cereus genome as a PCR template, and introduced each of them into the B. anthracis strain A16R and B17DD. Then analyzed the Phenotype, the level of genes transcription and proteomics research. The results showed that transformation of B. anthracis with plasmid pBE2A-plcR carrying the native B.cereus plcR gene active the expression of sphingomyelinase gene, but did not activate expression of hemolysin genes of B. anthracis. When we expressed the fusion protein PlcRPapR in B. anthracis, both hemolysin gene and sphingomyelinase gene were activated in A16R strain and B17DD strain. But the hemolysin is less stable in A16R strain than in B17DD strain. And the strain both A16R and B17DD with the plasmid pBE2A-plcRpapR and pBE2A-papR did not have hemolysin neither lecithin. The expression of PlcR is regulated by itself. The N terminal of PlcR is more conservative, belongs the DNA-binding domain. Sequence analysis of the promoter region of the PlcR regulated genes revealed the presence of a highly conserved palindromic region (TATGNANNNNTNCATA). This highly conserved sequence is presumably the specific recognition target for PlcR activation. Use the conservative sequence we found92proteins which may be regulated by PlcR. The proteomics study on pBE2A/A16R、pBE2A-plcR/A16R、 pBE2A-FplcRpapR/A16R、 pBE2A-plcRpapR/A16R、 pBE2A-papR/A16R by two-dimensional electrophoresis we found four proteins which might be regulated by PlcR. The RT-PCR results shows that the relative expression of sph gene and plc gene were in upward trends when hemolytic activity and sphingomyelin activity increased. The relative expression of atxA gene is negative correlation with spore formation rate. Though it can not effect the spore formation, the spore formation rate is desend when we introduced the pBE2A-plcR、 pBE2A-FplcRpapR into A16R strain.