| Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)is a key enzyme in the process of glucose metabolism,catalyzing the reversible transformation of glyceraldehyde 3-phosphoglyceraldehyde to 1,3-diphosphoglycerate.Studies have shown that cells glyceraldehyde-3-phosphate dehydrogenase also involved in a variety of other physiological metabolism,for example the catalytic function of microtubule polymerization,promoting DNA damage repair,modifying protein phosphorylation,acceleration cell apoptosis between the membrane and membrane fusion.Biofilm was a highly organized structure containing microorganism and their secreted extracellular matrix,which is composed by a large number of highly organized and systematic membrane-like polymers by secreting extracellular matrices such as polysaccharides,fibrin and lipid proteins.Biofilm,as a kind of self-protection mechanism of microorganism against adverse environment,is the main survival way of microorganism in the natural environment,and plays an important role in the biological bacteria.Many reports indicate that biofilm formation contributes to the colonization and activity of bacteria in plant tissues.The biofilm containing extracellular protein,extracellular polysaccharide and nucleic acid,and the relationship between extracellular polysaccharide production and glyceraldehyde-3-phosphate dehydrogenase widely involving in glucose metabolism remains to be elucidated.Bacillus cereus 0-9 is an endophytic bacterium belonging to a gram-positive bacterium isolated and screened from the roots of healthy wheat at the jointing stage.This strain had the ability to form biofilm,and the influence of preventing and controlling wheat sheath blight was up to 82.86%,which has potential application value.In order to explore the regulatory mechanism of GAPDH involved in the formation of biofilm in endophyte 0-9,three genes related to GAPDH were analyzed based on bioinformatics:gapA,gapB and gapN,and three defective strains were constructed by using the principle of homologous recombination crossover.The results showed that after the gapB knockout,the biofilm formation of the mutant significantly decreased,while the formation of the biofilm of AgapA and AgapN were not significantly different from wild type.Complementary strains of two complementary vectors of gapB were found that the biofilm formation of complementary strains recovered and had no different from wild type 0-9.The results showed that the gene gapB was involved in biofilm formation in B.cereus 0-9 under the conditions of determination.Through construction of GapB protein expressing strain,the purified protein His6-GapB was obtained by inducing and purifying.By measuring the activity of purified protein,it was found that the protein had the activity of glyceraldehyde-3-phosphate dehydrogenase with two coenzyme factors,and the encoding protein of the gene gapB was a glyceraldehyde-3-phosphate dehydrogenase.The transcription factor SinR and its antagonist Sinl regulate biofilm formation in Bacillus cereus.To analyze whether SinI/SinR system influenced the biofilm formation through a similar mechanism in Bacillus cereus 0-9.The biofilm formation was determined by constructing ?sinI and ?sinR defective strains.The study found that SinR negatively regulated biofilm formation and Sinl regulated biofilm formation by inhibiting SinR activity.Quantitative-time PCR was used to determine the expressions of tasA,calY and sipW in 0-9 and AsinR.It was found that the expression of tasA,calY and sipW decreased after the sinR gene was deleted.This results showed that SinI/SinR system regulated biofilm formation in B.cereus 0-9.In order to analyze whether gapB deletion in B.cereus 0-9 affected the biofilm formation through SinI/SinR system,we constructed the ?sinl?gapB and ?sinR?gapB double defection strains and complementary strains AsiI?gapB::gapB,?sinR?gapB::gapB and ?sinl?gapB::sinI,?sinR?gapB::sinR,It was found that the character of double defective strain was consistent with that of AgapB strain.The character of double defective strain were found to a certain extent using the complementary strains of gene gapB.On the contrary,the recovery of the character could not be found by the complementary strains of gene sinl and sinR.The expression of gapB in 0-9 and AsinR strains was determined by fluorescence quantitative PCR and the expression of GapB in 0-9 and AsinR strains was determined by Western blot.The results showed that the influence of gapB on biofilm formation was not determined by SinI/SinR system,the gene gapB controled bacterial colony morphology and extracellular protein production.Automated curve growth analyzer was used to determine the basic growth of the defective strains in the absence of carbon source and additional primary carbon sources(glucose and glycerol)and secondary carbon sources(pyruvic acid).It was found that the defective strain ?gapB was in a non growth state in the condition of no carbon source and adding secondary carbon source,whereas the defective strain AgapB restored a substantially growing state in the case of the addition of a primary carbon source.The results showed that B.cereus 0-9,GapB mainly involved in the formation of glyceraldehyde-3-phosphate rather than the degradation of glyceraldehyde-3-phosphate under the condition of determination.The pgm gene expression in B.cereus 0-9 and ?gapB was determined by real-time quantitative PCR.The expression level of the gene pgm in the defective strain AgapB was found to decrease.It was shown that in B.cereus 0-9,GapB involved in biofilm formation through the gluconeogenesis pathway affecting the production of glucose.The presence of bacteria on the surface of wheat roots was determined by the suspension of bacteria from a nutrient solution containing different carbon sources and irrigation of wheat seedling The results showed that the colonization ability of the strain AgapB was significantly reduced in B.cereus 0-9.It indicated that glyceraldehyde-3-phosphate dehydrogenase GapB affects the colonization of wheat roots in B.cereus 0-9. |
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