Studies On The C-Di-AMP Associated Proteins And The Key Enzymes Of 2-Methylcitrate Cycle In Bacillus Thuringiensis

Bacillus thuringiensis(Bt)is a well-known Gram-positive endospore forming insecticidal bacterium.Bt can produce various insecticidal crystal proteins(ICPs),which makes it as the most mainly microorganism used in biological control.The acrystalliferous mutant B.thuringiensis BMB171 possesses very high efficiency of electroporation transformation and favorable plasmid stability,and also is the ideal host strain for the studies of the effect of metabolic pathways on the yield of ICPs and the toxicity of different ICPs through transforming plasmid containing different ICP genes.Our study focused on the signal transduction and metabolic regulation of B.thuringiensis BMB171.1)Functional analysis of the c-di-AMP accociated proteins in B.thuringiensisCyclic di-AMP(c-di-AMP)is a recently discovered bacterial secondary messenger molecule,the intracellular level and turnover of c-di-AMP is mainly regulated by diadenylate cyclases containing DAC domain and c-di-AMP-specific phosphodiesterase containing DHH/DHHA1 or HD domain.By binding to downstream receptors,c-di-AMP can regulate a variety of biological processes,such as to maintain cell wall homeostasis,to help survival of bacteria in low-potassium concentrations,and to regulate central metabolism.Compared with most of bacterial species which contain only one DAC enzyme,the genus Bacillus often encodes three DAC domain-containing proteins:Dis A,Cda A,and Cda S,indicating that c-di-AMP signalling pathway maybe more complex in Bt.So,the investigation of the c-di-AMP accociated proteins will enrich the signal transduction network of B.thuringiensis.Firstly,we reported an efficient enzymatic c-di-AMP synthesis method using a diadenylate cyclase Dis A from Bacillus thuringiensis BMB171(bt Dis A).Under the optimum conditions,100 mg of highly pure c-di-AMP that was harvested in white powder form from a 50 m L enzyme-catalyzed reaction system.Secondly,we demonstrated that Cda S protein from BMB171 is a hexmeric DAC protein that can convert ATP or ADP to c-di-AMP in vitro.Through the N-terminal truncation experiment,we found the N-terminal region from fourth amino acid(Trp)to the ninth amino acid(Glu)is critical for Cda S’s DAC activity.Based on the markerless gene knock-out method,we confirmed that the transcription of cda S is initiated by sporulation-specific sigma factorσ~H and the deletion of cda S significantly delayed the sporulation and the formation of parasporal crystal,which is different with those in B.subtilis.We attempted to delete all the three DAC genes,however we failed to acquire the triple mutant,confirming that c-di-AMP is an essential molecule in B.thuringiensis.Finally,four potential receptor proteins in B.thuringiensis were identified using a c-di-AMP affinity column and relevant experiments are in progress in our laboratory.2)Functional analysis of 2-methylcitrate cycle in B.thuringiensisA conventional pathway for propionyl-Co A metabolism in bacteria and fungi is2-methylcitrate cycle,which includes the dedicated enzymes 2-methylcitrate synthase(Prp C),2-methylcitrate dehydratase(Prp D)and 2-methylisocitrate lyase(Prp B).The genes of the enzymes often locate in the same prp operon.On the transcription level,we found that prp operon is negatively regulated by transcriptional factors Ccp A and Abr B,and positively regualted by transcriptional factor Ccp C.These transcription regulation modes enable prp operon started to be transcribed in stationary phase so that can directly use propionyl-Co A from fatty acid oxidation and protein turnover.prp D and prp B deletion mutants can inhibit sporulation and exhibit cytotoxicity.Several lines of evidence showed that these phenotypes are directly causing by2-methylcitrate or 2-methylisocitrate.Through the analysis of the transcriptome data of BMB171 andΔprp D,we found that activity of Sig F is significantly inhibited in post-transcription level,and then prevent the formation of asymmetric septum.Meanwhile,the largely changed in central metabolic pathways reveals that a severe metabolic disturbance inΔprp D,which could lead to the cell lysis.This would be the reason for the cytotoxicity of 2-methylcitrate.And more importantly,by transforming plasmid containing cry1Ac10 into BMB171andΔprp CDB,we demonstrated that the loss of 2-methylcitrate cycle significantly reduces the yield of ICPs.These results imply that propionyl-Co A metabolism via2-methylcitrate cycle is an important material source for ICPs synthesis in B.thuringiensis.