Effects Of Glucose Phosphatase On Cell Wall Characteristics And Environmental Adaptability Of Bacillus Cereus 0-9

As a rigid structure of the outer layer of bacteria,cell wall of bacteria not only plays a key role in maintaining the morphology of bacteria,but also plays an important role in the orderly metabolism of cell wall in the normal growth,differentiation,material transport and even improving the adaptability of bacteria to changes in environmental conditions.Glucose phosphomutase?PGM?is widely present in prokaryoticandeukaryoticcells,catalyzingtheconversionofglucose-6-phosphateinto glucose-1-phosphate,which plays an important role in cell wall and polysaccharide biosynthesis.In the model bacteri?M bacillus subtilis,PGM is involved in the biosynthesis of peptidoglycan and phosphotelic acid.Bacillus cereus widely exists in soil,plant and animal body and body surface and other completely different ecological environment,with good adaptability.At present,the role of PGM in Bacillus cereus has not been further studied.Bacillus cereus 0-9 is a biocontrol bacteri?M isolated from the root of wheat with control effect on wheat sheath blight.In this study,bioinformatics analysis showed that Bacillus cereus 0-9 had a highly homologous coding sequence with the PGM amino acid sequence of Bacillus subtilis,which was named phosman.PCR amplified the flanking sequence of the gene and inserted it into the polyclonal site of the shuttle vector pMAD containing the temperature sensitive replicon,and the gene knockout vector was successf?lly constructed.The knockout vector was transferred into the receptor cells of the 0-9 strain,and the knockout gene was successf?lly constructed by homologous recombination.On this basis,complementary strains of knockout strains were constructed.The phenotypes of phosman's gene strain and its complementary strain were determined under different conditions.The res?lts showed that compared with the wild type 0-9,the mutant's colony morphology,growth,salt stress tolerance and other traits were significantly changed after the deletion of this gene.After gene complementation,the above phenotype of the complementary strain was restored to the level of the wild type.In order to further analyze the function of phosman gene,the gene expression vector was constructed and the gene was induced in e.coli.The enzyme activity of the purified protein showed that the protein encoded by this gene can catalyze the conversion of glucose-6-phosphate into glucose-1-phosphate,indicating that the product encoded by phosman is glucose phosphate mutase?PGM?.The above res?lts indicated that PGM was also present in Bacillus cereus 0-9,and the lack of PGM may affect the phenotypic changes caused by changes in cell wall components.At the same time,it was found that PGM deficiency in Bacillus cereus did not cause significant changes in cell morphology,and the addition of Mg²⁺in the c?lture medi?M co?ld not restore the phenotype of the mutant,suggesting that the effect of PGM deficiency on bacillus cereus was different from that on bacillus subtilis.In order to further analyze the PGM for waxy bacillus the role of cell wall metabolism,using gene knockout UDP homologous recombination method,two coding glucose focal phosphatase gene,respectively to build single gene deletion mutant?gtaB1 and?gtaB2 and double gene deletion mutant?gtaB1?gtaB2,determination of different mutant phenotypes.After the res?lts found that the gene deletion,mutant colony morphology,growth,such as salt stress tolerance traits and?phosman has certain difference,but lies in the synthesis of cell wall of antibiotic susceptibility and?phosman similar,instructions in waxy Bacillus,gtaB gene and phosman gene reg?lation there is a certain relationship,but is like reg?lation pathway in Bacillus subtilis further proved that the determination of several kinds of strains related to cell wall formation of some biological properties such as zeta potential,bacteria's surface charge,bacteria on the property of water,GtaB and phosman genes were found to have an effect on these shapes,indicating that these two genes may be related to the synthesis of cell wall.The viability of different strains in soil was determined by constructing the green fluorescent protein?GFP?labeled strains of 0-9 and its phosman mutants.The res?lts showed that the viability of the phosman mutants in soil was significantly reduced,indicating that the gene phosman affected the survival rate of bacteria in soil.