The Function Of SigL Gene In Bacillus Thuringiensis

Bacillus thuringiensis (Bt) has been widely used as bioinsecticidal microbe for its high toxicity, safety and the other merits. Comparing with other Bacillus species, Bt can produce large crystalline parasporal inclusions during sporulation. The process of Cry from beginning expression to formation of crystal protein and sporulation involved in substance and energy metabolism. In the process of formation of crystal protein and sporulation, there may be subtle way in the enzymic regulation and synthesize of metabolism pathway in Bt.σL encoded by sigL plays a very important role to regulate many metabolic pathways in bacteria. In this report, the function of sigL gene in Bt was discussed.We characterized sigL genes cloned from Bt HD-73 strain. The nucleotide sequence of sigL gene showed significant identity with that in the B. cereus group, but their similarity score between HD-73 and B. subtilis 168 was lower, only 43%. The analysis of amino acid sequences indicated that SigL was belonged toσ54 family.To determine the function of sigL of Bt HD-73 strain, the sigL gene deactivation inactivation mutant was obtained and its corresponding complementary strain was also constructed. The growth speed of the sigL mutant and complementary strain are investigated in different nutrient medium and using different amino acids as nitrogen source respectively. The results suggested that the deletion of sigL gene maybe blocked some important metabolic pathways, and sigL mutant could not grow using arginine, proline, valine, isoleucine, glutamine, phenylalanine, methionine and tryptophane as the sole nitrogen source separately. The sigL mutant strain could retard the formation of crystal protein Cry1Ac compared with the host strain HD-73. The amount of active spore in sigL mutant strain was less than that in HD-73 host strain.We found fiveσL-dependent transcriptional activators (AcoR,BkdR,Bt104,LevR,Reg) in Bt 97-27. In order to certify the effect ofσL on metabolic pathway, lacZ gene was fused with the promoters of these transcriptional activator genes respectively, and these recombined genes were expressed in sigL mutant and HD-73 host strain sequentially. Enzymic assay revealed that activity ofβ-galactosidase in sigL mutant strain was much lower than it in host strain, and all the results indicated that these transcriptional activators wereσL-dependent transcriptional activators in Bacillus thuringiensis. The promoters of probably operons which were regulated by these transcriptional activators were -12/-24 promoters. They may be also controlled byσL respectively.The promoter of gabT gene is a -12/-24 promoter. We constructed the promoter of gabT and lacZ fusion expression plasmid. Assay ofβ-galactosidase activity showed that its expression depended on the presence of SigL and transcription of gabT gene was decreased in reg (downstream of gabT gene) mutant and sigL mutant. These results indicated that sigL regulated the GABA pathway and the upstream sequence of gabT were regulated by reg gene.Determination the function of sigL gene will promoter to reveal regulatory metabolism for substance and energy during sporulation and crystallization in Bacillus thuringiensis.