Induced Regulation Mechanism Of Branched-chain Amino Acids And Acetoin Metabolic Pathways In Bacillus Thuringiensis

Bacillus thurigiensis belongs to the Bacillus cereus group.It is a spore-forming gram-positive bacterium.Insecticidal crystal proteins are produced during the process of spore formation.Insects and coleoptera of various insects and their larvae have toxic and bacteric idal activity.Because of their non-toxic killing effect on insects,natural enemies,non-toxic effects on humans and animals,and no pollution to the environment,etc.It is widely used in forestry.Previous studies in our laboratory found that there are eight enhancer binding proteins(EBPs)in Bacillus thurigiensis HD73 strain,which regulate a variety of metabolic pathways,including gamma-aminobutyric acid metabolism,lysine metabolism,and sarcosine metabolism.bkd gene cluster is regulated by Bkd R,which probaly related to the metabolism of branched-chain amino acids(leucine,isoleucine,and valine).aco gene cluster is regulated by Aco R,which probaly related to acetoin metabolic pathway.The degradation products of branched-chain amino acids and acetoin eventually enter the tricarboxylic acid cycle.Here,we studied induced regulation mechanism of branched-chain amino acids and acetoin metabolic pathways in Bacillus thurigiensis HD73 strain.The main results are as follows:Through ?-galactosidase activity analysis and gel block(EMSA)method,it was clarified that in SSM and LB medium,the transcription activity of the bkd gene cluster promoter Pptb is not induced by branched-chain amino acids,but in M9 In the basic medium,branched-chain amino acids(leucine,isoleucine,and valine)can induce the transcriptional activity of P ptb.The transcriptional activity of the bkd gene cluster is directly regulated by Bkd R.The results of ?-galactosidase activity measurement showed that 3-hydroxybutanone can induce the transcription activity of the aco gene cluster promoter PacoA.PacoA-induced transcriptional activity is controlled by Sigma54 and positively regulated by Aco R.Through the methods of EMSA,DNase I footprinting and ?-galactosidase activity analysis,it is clear that the HTH domain of Aco R is combined with a 30 bp palindrome upstream of aco A;the GAF domain plays a role in the process of sensing 3-hydroxybutanone signal.Through sequence analysis,?-galactosidase activity determination analysis and EMSA method,it is clear that glucose inhibits the transcription of bkd gene cluster and aco gene cluster,Ccp A plays a negative regulatory role in this process.By clarifying the induced regulation mechanism of branched-chain amino acids and acetoin metabolic pathways,it will be enriched the metabolic network regulated by EBPs.It will provide a theoretical basis for the development of a new generation of engineering b acteria from the perspective of metabolism.