Function Analysis Of Quorum Sensing Response Regulator NprR Gene From Bacillus Thuringensis

Bacteria use cell-cell signaling for detecting population density changing, and controling the gene expression in response to cell idensity, the process is called Quorum Sensing. Quorum Sensing regulates a range of processes in bacteria, such as extracelluar enzyme production, antibiotic production, biofilm formation, development and so on. In Gram-positive bacterium Bacillus subtilis, QS use rap-phr signaling cassette to stimulate sporulation and genetic competence. Pottathil and Lazazzera were previously identified nprR-phr like in Bacillus.thuringiensis, which was as putitive rap-phr signaling cassette, but the role of Quorum Sensing response regulator NprR remains unclear in spore and crystal protein formaiton.We cloned nprR and phr like genes in HD-73, and analysis the nprR and phr like by SMART and signalP, The nprR gene inactivation mutant HD73(ΔnprR) was obtained by homologous recombination. In our reaserch, the growth curves of HD-73 and HD73(ΔnprR) showed that the deletion of nprR gene didn't affect on the growth.β-galactosidase assay of cry1Ac′-lacZ gene fusion and SDS-PAGE in both HD-73 and HD73(ΔnprR) strains were performed to analyse the effect of nprR gene deletion on expression of cry1Ac gene.β-galactosidase assay of nprR′-lacZ in both LB and SM medium showed nprR gene in B. thuringiensis was initially transcripted at T0 (end of exponential growth phase) and keeping expression in stationary phase.β-galactosidase assay of cry1Ac′-lacZ and SDS-PAGE indicated that expression of cry1Ac gene in HD73(ΔnprR) was stronger than that in HD-73 during transition phase and early stationary phase. However, Cry expressed product between HD-73 and HD73(ΔnprR) in LB medium has no significant difference when crystal and spore were released HD73(ΔnprR) increase 2.48-fold the concentration of free spores than HD-73. Bio-assay showed no significant difference on insecticidal activity between the two strains。The chemically synthesized putitive signal peptides VKSLK,LKESS,LKEGS,IKYSN and SKPDT were added to cultures during the transition phase in different consentration, they didn't affect the expression of cry1Ac′-lacZ, the trundcated peptide SNPDIYG made the expression of cry1Ac gene in HD73(ΔnprR) stronger than that in HD-73. The results indicated the processing for extracelluar signal peptide was different in Bt and Bs.In this study, we first researched on Quroum Sensing response regulator in Bt, the deletion of nprR gene could accelerate the rate of sporulation, and had weak influence in the expression of cry gene. Our research results provide a new way for shorting the fermentation time, and valuble experimental material for in-depth research.