Regulation And Expression Of Gab Gene Cluster In Bacillus Thuringiensis

γ-Aminobutyric acid (GABA) is ubiquitous in most prokaryotic and eukaryotic organisms. Preliminary studies had shown that the structure and regulation of GABA (γ-aminobutyric acid) shunt was a new type in Bacillus thuringiensis (Bt). The expression of the gab cluster is regulated by a Sigma54factor by way of the transcription activator GabR. The GABA aminotransferase (GABAAT) encoded by gabT could convert GABA into succinic semialdehyde (SSA), and the latter was further degraded to semialdehyde (SA) by Succinic semialdhyde dehydrogenase (SSADH), encoded by gabD. Further study revealed that transcriptional acitivity of gabTp gene promoter was improved significantly by SSA. In this report, the mechanism of SSA increasing transcriptional acitivity of gabTp gene promoter was discussed.GabR protein purified was used to analyse the binding to promoters of gabT and gabR by EMSA (Electrophoretic mobility shift assay). A clear mobility shift was found for the two promoter probes. But GABA and SSA could not affect the binding of GabR protein and two promoter probes. Footprinting analysis showed that GabR protein binding to gabT regulatory region is a70bp fragment.GabR protein contains three conserved domains, its N terminus is a PAS domain, EBPs is a Sigma54interaction module in the middle region.There is a DNA-binding domain containing a helix-turn-helix sequence motif in its C terminus. β-galactosidase enzymic assay revealed that gabT expression increased when PAS domain deleted. Site-directed mutagenesis of Ser58, Leu72and Glu74, β-galactosidase enzymic assay showed that no significant difference of gabT expression between mutants and wild-type. We Speculated the three amino acid sites may be the key loci SSA affected.The result of RT-PCR showed that the GabR protein was autoregulatory, its transcription was likely to be mediated by two different mechanisms. There must be constitutive low level expression of GabR under all conditions. SigmaL then worked with activated GabR to stimulate additional expression of gabTRD.To determine the relationship among small molecular ligand, transcriptional activator and Sigma factor, we studied the mechanism of transcriptional activity of gabT gene promoter induced by SSA. 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.