Research Of Co-Existance Of The S-Layer Proteins And The Crystal Protein And Its Application

A surface layer protein was revealed in B. thuringiensis CTC. Protein alignment showed that the S-layer protein might participate formation of the crystalline-like inclusion. Further detection by immunnoprecipitation, immune fluorescence, immune electron microscopy and gene defection proved that, the 100 kDa inclusion protein CTC and the 100 kDa S-layer protein were the same proteins. Another surface protein, CTC2, was discovered, which is around 80 kDa. Gene sequencing and sequence blast suggested that this gene had high identity with ctc gene. Expression and transcription phase were also analyzed in this study.The surface layer protein is one of the most widely distributed crystalline vehicle structures surrounding the outmost cell surface of bacteria. Research relating surface layer protein has been significantly concerned. However, the function of the surface layer protein was not clear up to date. In this work, the physiological function of the surface layer protein in sustaining cellular morphology, cell propagation, resistance to alkalescence and plasmid transmission were studied in B. thuringiensis CTC, using the S-layer genes mutated B. thuringiensis bacterium Tr5. The results showed that, when the S-layer proteins were defected, morphology of bacterium Tr5 was seriously affected. When cultivated in liquid medium, growth rate of the mutant Tr5 was slowed, whereas when cultivated on plate, growth rate was increased. The S-layer proteins might also function in generating new offspring that could adapt to highly basic environment. In addition, the S-layer proteins probably assist the bacterium resist to allothogenic plasmid DNA.Two genes, ctc and ctc2, responsible for surface layer (S-layer) protein synthesis in Bacillus thuringiensis CTC, were mutated and resulted in B. thuringiensis Tr5. In order to synthesize and express the N-Acyl-homoserine lactonase (AHL-lactonase) on cell surface and in the extracellular space of B. thuringiensis, the aiiA_4Q7 gene (an AHL-lactonase gene from B. thuringiensis 4Q7), which confers the ability to inhibit plant soft rot disease in B. thuringiensis 4Q7, was fused with the upstream sequence of the ctc gene, which in turn is essential for S-layer protein secretion and anchoring on the cell surface. The resulting fusion gene, slh-aiiA, was expressed in B. thuringiensis Tr5 to avoid competition for the extracellular space with the native S-layer protein. Our results indicate that B. thuringiensis Tr5 containing the fusion gene slh-aiiA displayed high extracellular AHL-degrading activity. To detect the effect of the surface displayed AHL expressed in different B. thuringiensis, the ICP3A promoted endocellular AHL lactonase AiiA_4Q7 was studied; nine B. thuringiensis recombinant strains expressing single endocellular or extracellular AiiA protein, or coexpressing the two proteins, were analyzed in Tr5 and the other two crystal defected strains BMB171 and 407. The results showed that, when compared with the recombinant strains expressing single endocellular AiiA_4Q7 protein, the strains expressing extracellular SLH-AiiA protein could effectively inhibit soft rot disease caused by E. carotovora. Under the experimental conditions, the Tr5 derivatives expressing SLH-AiiA displayed best effects. When compared with wild type B. thuringiensis strains, the ability of the constructed strain to inhibit soft rot disease caused by Erwinia carotovora SCG1 was markedly increased. The mechanism of the inhibition ability of the SLH-AiiA protein was also studied. These findings provide evidence for a significant advance in our ability to inhibit soft rot disease caused by E. carotovora.