Regulation And Expression Of Gab Gene Cluster In Bacillus Thuringiensis

Bacillus thuringiensis (Bt) is an insecticidal microorganism widely commercialized in the world.Comparing with other Bacillus species, Bt can produce large crystalline parasporal inclusions during sporulation. GABA shunt is ubiquitous in most prokaryotic and eukaryotic organisms.σL and GabR control the expression of gab gene cluster which involved in GABA shunt.GABA transaminase (GABASE) was encoded by gabT and Succinic semialdhyde dehydrogenase (SSADH) was encoded by gabD involved in GABA shunt. In this report, the effection and mechanism of gab gene cluster involved inγ-aminobutyric acid (GABA) shunt in Bt were discussed.In order to certify the effect to transcription of gab gene cluster by GABA and SSA. lacZ gene was fused with the promoters of gabT gene,gabR gene and sigL gene respectively, and these recombined genes were expressed in HD-73 host strain.β-galactosidase enzymic assay revealed that transcriptional activity of gabT gene promoter was improved significantly by GABA and SSA, and induced by SSA was more stronger. But neither GABA nor SSA could effect transcriptional activity of gabR gene and sigL gene promoter.And the gabTP-lacZ fusion plasmid was transformated into gabT mutant,gabR mutant,gabD mutant and sigL mutant. When induced by GABA,β-galactosidase enzymic assay revealed that transcriptional activity of gabT gene promoter was more stronger in gabT mutant and gabD mutant than in HD73 host strain.And when induced by SSA, transcriptional activity of gabT gene promoter was significant enhanced in gabD mutant. The results showed that accumulation of GABA and SSA enhanced the inducement. On the other hand, transcriptional activity of gabT gene promoter was abolished in gabR mutant and sigL mutant. This revealed thatσL and GabR control the expression of gab gene cluster directly.The result of RT-PCR showed that gabT gene and gabD gene were co-transcribed when by induced by GABA and SSA, and 5'RACE determined the transcription start point of gabT gene. We characterized gabR and sigL genes cloned from B. thuringiensis strain HD73 isolated in China. Both gabR gene, 1.3 kb encoding a protein with 55 kDa, and gabD gene, 1.3 kb encoding a protein with 50 kDa were expressed in E. coli BL21 (DE3) strain, and GabR protein was purified by Ni+ column.To determine the function of gab gene cluster and sigL gene of Bt HD73, HD73 host strain was compared with mutants in different nutrient medium. Both the growth and the formation of crystal and spore were compared. The results showed that abolished of GABA shunt effected neither the growth nor formation of crystal, but reduced product of spore. On the other hand, the growth and the formation of crystal and spore were reduced significantly in sigL mutant. The cry1AcP-lacZ fusion plasmid was transformated into sigL mutant to reveal influence of Cry protein by sigL gene was not due to transcription.The results revealed that there was a inseparable relation between metabolic pathway controlled by sigL gene and formation of crystal in Bt.