The Functional Genes Of Sugar Phosphate Sensor System In Bacillus Cereus

Bacillus cereus is gram-positive, soil and spore-forming bacteria. When allowed access to mammalian tissues it is an opportunistic pathogen that may cause severe local or systemic infections. So many scientists focus on study virulence related genes in Bacillus cereus. We have previously developed an IVET system to identify B. cereus ATCC 14579 strain genes specifically expressed during oral infection of Galleria mellonella. One of these genes (spsA with unknown function) is specifically expressed in the larval gut during the early stage of infection. Analysis of the five gene cluster related to spsA indicated that SpsA is part of a new sugar phosphate sensor system composed of a two-component system (TCS) encoded by spsR and spsK, (localized upstream spsA) and two additional downstream genes, spsB and spsC. spsRK and spsABC are separate transcriptional units. Searches for gut-related compounds inducing spsA transcription identified glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) as activation signals. Based on this progress, we analyze the function of Sps system in this study.The analysis of amino acid sequences of sps genes indicated that spsA gene encode a hypothetical protein and a signal peptide localized at N-terminal. spsB gene encode ABC transporter substrate-binding protein and a signal peptide localized at N-terminal. spsC gene encode phosphoglycerate transporter and it belongs to major facilitator superfamily. Its amino acid sequence containing 12 transmembrane domains. Its DNA sequence is similar to glpT gene (identify is 43%) which encode glycerol-3-phosphate transporter and uhpT gene (identify is 31%) which encode glucose-6-phosphate in E.coli. spsRK encode two component system and its amino acid sequence containing typical histidine kinase and response regulator domains. This gene cluster is conserved in B. cereus group bacteria, several strains of gut commensals and some pathogenic Clostridia species.To determine the function of sps system, we constructed sps single mutant, double mutant and multiple mutant and their complemented strains and overexpresion strains. We analyzed the activation of spsA promoter B1 in these mutant, complemented and overexpression strains in vivo and in vitro. The results showed that the SpsRK TCS was essential for transcription of spsABC and both SpsA and SpsB were necessary for this activation. SpsC was essential for the sugar-6-phosphate uptake. Work with gfp-transcriptional fusions showed that the five genes were required for host-activated expression in vivo. The activation of B1 promoter in vitro was the same as in vivo. Although the in vitro conditions used were very simple compared to the environment in vivo in the midgut, the in vitro experiments mimicked what was observed in vivo. This suggests that the major environmental factor responsible for the activation of the system in vivo has been identified.Direct interaction between purified SpsA and SpsB was demonstrated by legend overlay and cross-linking. SpsA protein was localized at surface by western blot.We also analyzed transcription of sps system. We found SpsR, CcpA and CodY putative DNA binding domain in B1 promoter sequence. Deletion of these three DNA binding domain respectively showed that the activation of B1 promoter was abolished or decreased. In spsRK mutant activation of B1 promoter was abolished. It indicated that B1 promoter was positively regulated by SpsRK. B1 promoter was positively regulated by CcpA in exponential growth phase and was repressed by CcpA in stationary phase. spsRK promoter was negatively regulated by CcpA in exponential growth phase. It indicated that CcpA regulates sps system by different mechanism. We analyzed the activation of different size fragments of B1 promoter region. The results indicatedthat there might be other DNA binding domain in B1 promoter region. This sugar phosphate sensor and transport system appears to have a novel regulatory mechanism, involving five factors that may be important for host adaptation in pathogenic Bacillus group and Clostridia bacteria. Our findings provide new insights into the function of two-component sensor systems in host-pathogen interactions, and in particular in the gut.