| Bacillus thuringiensis is a soil bacterium which can produce spores and lots of insecticidal proteins in a form of parasporal crystal during the stationary phase. The metabolic regulatory mechanism of parasporal crystal protein is one of the hotspots in the study of Bacillus thuringiensis, and breakthrough progresses have been made in the formation mechanism of the spore, but there is still no a reasonable and clear explanation in metabolic regulatory mechanism of crystal proteins.CodY is a global regulatory factor of Gram-positive bacteria, regulating more than 200 genes and most of them are expressed in the stationary phase, many virulence genes in pathogenic microorganisms were also included. But little attention has been paid to the function of CodY in Bacillus thuringiensis. The recombinant strain of over-expressed CodY protein and the mutant strain of knockouted cody were constructed to study the mechanism of CodY in Bacillus thuringiensis in our research work. We tried to explain the relationship of CodY protein and parasporal crystal proteins and other metabolic regulations as well, which will lay the foundation for the construction of the entire metabolic regulatory networks in Bacillus thuringiensis.1. According to complete genome sequences of Bacillus thuringiensis BMB171 and CT-43 which have been published by our laboratory, and the not fully released genome sequence of Bacillus thuringiensis YBT-1520, bioinformatics analysis of CodY protein was made. It showed that the similarity of CodY in Bacillus thuringiensis and Bacillus cereus group is over 99%, but a great difference was occurred when compared with Bacillus subtilis, and the difference of upstream and downstream of cody gene is also remarkable.2. pHT1K-Plip-TS was used to construct a recombinant vector pHT1K-PlipcodyTS of overexpressing CodY protein in our experiment. The recombinant vector was transformed into the wild strain Bacillus thuringiensis YBT-881 for over-expressing the CodY protein. The recombinant strain YBT-881-L1 was authenticated, then we identified the characteristics of YBT-881-L1, no significant differences were observed in the growth curve and the parasporal crystal morphology between genetically engineered strain YBT-881-L1 and the wild strain YBT-881. However, the SDS-PAGE and mass spectrometry analysis showed that both of strains contained the same Cryl protein, but the silenced cry2Ac4 gene in the wild strain YBT-881 was activated in YBT-881-L1, and produced lots of Cry2Ac protein. It showed that the sequence similarity of cry2Ac and cry2Ac4 gene was over 99%, which made the genetically engineered strain YBT-881-L1 containing both of Cryl and Cry2A crystal proteins. And further study was required to explain the reason why the expression of Cry2Ac protein was induced.3. In this study, a temperature-sensitive carrier was used to construct knockout vector named pBMB0413, and a cody deleted strain was constructed by homologous recombination in the Bacillus thuringiensis BMB171. The result of PCR amplification, Western blot and RT-PCR analyses showed that CodY protein was not expressed in the mutant strain. The characteristics of this mutant strain were identified, and it showed that the mutant can not form complete normal spores, the content of PHB reached the highest value at 12 h, and PHB was not degraded but maintained at a stable level in stationary phase. While in the BMB171, the PHB reached the highest value at 8 h to 12 h, and sharply declined in stationary phase. The reasons might be that PHB is a storage material of carbon, when the strains lacking nutrition, they need to consume PHB, and at the same time the formation of spores need lots of energy material as well. Therefore, the PHB was harply declined. While the mutant could not form normal spores, so the PHB could not be degraded and utilized. |
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