Study On Cellular Inclusions Formation By S-layer Proteins Among Bacillus Thuringiensis

This thesis focused on the research of S-layer(surface layer) of Bacillus thuringiensis.S-layer,also called crystalline bacterial cell surface layer,is a layer with two-dimensional lattice structure.It forms the outermost component of many archaeobacteria and eubacteria.Its functions have been proposed to be involved in cell integrity and shape maintenance,molecule or ion traps in cell adhesion,surface recognition,a factor of virulence,or a kind of murein hydrolase and protection from predacious attack by Bdellovibrio.The results are summarized as following:1.The S-layer protein resembling as CTC2 of Bt.str.CTC has been proved that it can not only locate on the surface of the cell,but serve as some structural and functional parts inside of the cell during sporulation.2.Based on the previous research results in our lab,it is first reported that the proteinaceous mass(or termed as "inclusions") of the Bt.str.CTC,accumulated during sporulation,was not a typical crystal protein encoded by cry gene,but a proteinaceous inclusion coded by slp gene.Furthermore,the strains,with the so-called parasporal crystal encoded by S-layer gene during sporulation,are widely distributed and account for 22.6%among the Bt.species.All these strains have provided a new aspect for research of Bt nontoxic strains.All the above results show that a novel group of inclusions encoded by S-layer gene of Bt has been discovered.3.During the further discovery,the formation of the inclusions coded by S-layer protein gene from Bt str.CTC is related to the dissolved oxygen concentration(DOC). The Bt str.CTC can accumulate the inclusion while growing on the condition of 2.0 mg/L DOC during sporulation.The ctc₂ gene is also testified to code for the inclusions by the method of Western blot,RT-PCR and polypeptide sequencing by mass spectrometry.4.A new method basing on the electro-poration to transfer the heterogenous gene into the wild type strains among the B.cereus group has been constructed.The method is followed by the following procedures:adding suitable glycine or lysozyme during the cells culture,washing the cells with SH buffer,desalting the plasmid donor, heat-treatment at the temperature of 42℃after the electric pulse.5.The role of S-layer of Bacillus thuringiensis strain CTC has been discussed. The genes,ctc₁ and ctc₂,responsible for the surface layer(S-layer) protein synthesis in Bt str.CTC,were mutated through the method of double crossover and resulted in Bt str.BMB0549.The results show that the S-layer proteins might function as some characteristics adaption to the basic environment.