The Structure And Function Analysis Of Gene Cluster Involved In GABA Shunt 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 study on Insecticidal Crystal Proteins (ICPs) is mainly focused on the expression and regulation of cry gene encoding ICPs. However, the research about the molecular mechanism of metabolism regulation involved in crystal protein formation is less. In this report, the structure and function of gene cluster involved inγ-aminobutyric acid (GABA) shunt in Bt were discussed.We characterized gabT and gabD genes cloned from B. thuringiensis strain G03 isolated in China. Both genes were expressed in E. coli BL21 (DE3) strain, and their products were purified by affinity chromatography respectively. By enzymatic assay, GabT protein showed the activity of GABA transaminase, while GabD protein exhibited SSADH activity. The amino acid sequences of two proteins, GABASE and SSADH, showed significant identity with those in the B. cereus group, but their similarity score between G03 and B. subtilis 168 was lower, only 58% and 51%, respectively.To determine the function of gabT and gabD of Bt HD-73 strain, the gabT, gabD gene deleted mutants were obtained by means of gene knock-out respectively, and their corresponding complementary strains were also constructed. The deletions of gabT and gabD gene could not affect the growth of mutant strains in rich culture medium, but the growth of gabT deletion mutant strain was repressed in basic medium (containing 0.2% GABA). The gabT deletion mutant strain could retard the formation of crystal protein Cry1 Ac, comparing with the host strain HD-73. The amounts of active spore both in gabT and gabD deletion mutant strains were all less than that in host strain HD-73.The structure of gab gene cluster in Bt HD-73 is distinctly different from that in E. coli and B. subtilis, but is common in B. cereus group by genomic sequence alignment. It means that the gene cluster involved in GABA shunt is very special in B. cereus group. The result of RT-PCR showed that gabT and gabD were not co-transcribed, in which gabT seperately was transcribed, while gabD and its upstream reg gene were co-transcribed. We had cloned the promoters of gabT and reg gene respectively, and constructed their corresponding lacZ fusion expression vectors. Assay ofβ-galactosidase activity showed that both gabT and reg gene was transcribed by its own promoter. These results indicated that gabT and gabD gene belonged to different transcription units, while gabD and reg gene formed an operon.The plasmid pMADDreg was constructed for knock-out of reg gene, and then introduced into HD-73 strain. Both the growth and the formation of crystal and spore were not affected in the relevant mutant strain HD-73 (Δreg) comparing with the host strain HD-73. However the activity of SSADH was almost lost in the reg deletion mutant strain, and restored in complementary strain HDHFreg. These results indicated that reg gene played a positive regulation role in gabD expression. The reg gene was also confirmed to positively regulate the gabT expression. Reg protein encoded by reg gene maybe a potentialσ⁵⁴-dependent transcriptional activator according to the conserved domian search in NCBI, and it is very conservative in B. cereus group by animo acid sequence alignment. Its function in transcriptional regulation need to be further confirmed.In a word, this study will be helpful to better understand the regulation mechanism and physiological function of GABA shunt, and to further study the mechanism of sporulation and crystal formation in B. thuringiensis.