Contribution Of L-lactate Metabolism To The Alkaline Adaptation Of Bacillus Thuringiensis Regulated By Crp Protein

Bacillus thuringiensis（B.thuringiensis,Bt）,a gram-positive bacterium,has been re-garded as one of the most widely used bio-pesticides due to its specific toxicity against insects.The alkaline environment in the midgut of Lepidoptera insect,which is among the major target of Bt,is necessary for Bt to exert its insecticidal activity.To investigate the alkaline adaptation of Bt is of importance.Here we explored the effection of L-lactate on the alkaline adaptation of Bt,and the regulation of Crp to the L-lactate metabolism.Previous study found that Bt HD73 stopped growth when alkali was added into the cul-ture,and the bacteria restored growth after adaptation.In this study,the restored growth pH of Bt HD73 strain maintained the range from 8.9 to 9.1,when added different contration of Na OH.Restored growth period was prolonged while the concentration of Na OH added were increased,indicating that alkaline adaptation is important for restored growth of Bt HD73strain.Previous study found that the transcriptional activity of ldh2 and crp genes were up-regulated significantly in alkaline condition.It was further verified byβ-galactosidase activ-ity detection,using lac Z fusions shuttle vector with promoter of both ldh2 and crp.5`-RACE assay showed that the transcriptional start site of ldh2 gene was located on the 11^th bp‘G’,upstream of the start codon of ldh2 gene.There are two putative Crp binding sites in the ldh2promoter region.β-galactosidase activity of ldh2 promoter in the deletion muatnt of crp gene,HD（△crp）strain were lower than HD73 strain.The expression and purification of Crp protein were completed in this research,and result of electrophoretic mobility shift assays（EMSA）showed that c AMP-Crp complex can directly bind to the ldh2 promoter region in vitro.Based on these data,it can be deduced that Crp regulates the transcription of ldh2 gene directly.Measurements of L-lactate in the culture media showed that under alkaline condition,the concentration of L-lactate secreted by HD（△ldh2）and HD（△crp）strains were signifi-cantly less than that secreted by HD73 strain.The growth curves under alkaline condition showed that the restored growth ability of HD（△ldh2）and HD（△crp）were both weaker than that of wild-type strain.These results indicated that L-lactate metabolism involved in the alkaline adaptation of Bt.In addition,we compared the toxicity of Bt HD73 and HD（△ldh2）and HD（△crp）by in vivo experiment using the mixture of crystal and spore.The insecticidal activity of wild type strain was higher than mutants significantly.All the results suggests that alkaline adaptation plays a vital role in the process of Bt infection.