The protein bands A and B were excised manually and in-gel digested, and then analyzed by LC-MS/MS. MS was analyzed with sequest

software. The lowest Xcorr values of the peptide were set to be 1.9 (+1 charge), 2.2 (+2 charge) and 3.75 (+3 charge), respectively, and ΔCn must be larger than 0.08 (Wang & Yuan, 2005). The matched peptides revealed that the protein A was InhA (Fig. 4b) protein B camelysin (Fig. 4c). To further support the results, shotgun analysis of the sporulated crystal cultures confirmed that the protein of InhA was not Protein Tyrosine Kinase inhibitor expressed in the camelysin-deficient strain. Grass et al. (2004) reported that the molecular mass of metalloproteinase camelysin was 21.569 kDa with a putative signal peptide of 27 amino acids from B. cereus. In the present study, the calY gene encoded a protein with a deduced size of 199 amino acids. signalp 3.0 server (http://www.cbs.dtu.dk/services/SignalP/) analysis showed that the deduced sequence contained a signal peptide. The prediction result revealed that the cleavage sites might be 31/32 (AFF-SD) and 29/30 (TFA-FF). clustalx analysis showed that there was a 99% homology of the camelysin protein between B. cereus and B. thuringiensis as well as homology of their calY gene sequence; the Selleck BTK inhibitor homology between

Bacillus anthracis and B. thuringiensis was 95%. The high degree of homology of camelysin suggested that the genesis of B. thuringiensis camelysin had a close relationship with B. cereus

and B. anthracis, and that it was more closely related to B. cereus. This work demonstrated that the global expression Cediranib (AZD2171) patterns of proteins differed between the wild-type and camelysin-deficient strain as determined by SDS-PAGE (Fig. 4a) associated with MS (Fig. 4b and c). Results of SDS-PAGE and LC-MS/MS suggested that there were many differences after knocking out the calY gene. It was obvious that the InhA was not expressed in the camelysin-deficient strain (Fig. 4a), and that the InhA reappeared in the complementation strain KCTFC (Fig. 4a). Previous studies reported that the inhA promoters of B. thuringiensis were a –35 (TTGAAA) and a –10 (TAAAAT) hexamer, which are highly similar to the σA promoter consensus (TTGACA 17-18N TATAAT) (Grandvalet et al., 2001). Our sequencing results showed that the transcriptional start site and ORF of the inhA gene remained intact after displacing the calY gene. Thus, it is suggested that there is a relationship between camelysin and InhA. InhA was synthesized during the stationary phase (Dalhammar & Steiner, 1984). It was suggested that the inhA transcription might depend on the complex regulatory mechanisms that control later growth development in Bacillus species (Grandvalet et al., 2001). It was previously reported that AbrB and SinR acted as repressors to prevent expression of InhA.