Identification And Characterization Of An Osmolarity Inducible Promoter Newly Isolated From Bacillus Subtilis

Section1:An osmolarity sensitive promoter fragment, P23423, isolated from Bacillus subtilis was characterized. The expression of β-galactosidase (β-Gal) driven by P23423was regulated by osmolarity both in Escherichia coli and B. sublilis.1The cloned promoter was characterized by quantitatively determining pSI23423-driven β-Gal production in E. coli and B. subtilis. In both E. coli and B. subtilis, the β-Gal productions from pSI23423were higher in LBON medium supplemented with2.5%NaCl than that in LBON medium.2The β-Gal production driven by the pSI23423cultivated in LBON medium with2.5%NaCl reached maximum (2525.36mill U) at36h in E. coli, which was almost4.12times of that in LBON medium. These results further confirmed that the cloned promoter in pSI23423is osmolarity-sensitive in driving expression of β-Gal in E. coli. The p-Gal production from the pSI23423in B. subtilis was lower than that in E. coli. However, the P-Gal production from pSI23423cultivated in LBON medium with2.5%NaCl was4.05fold higher than that in the medium without NaCl in B. subtilis. This implied that the cloned promoter in pSI23423might be a potential osmoregulated element in the genetic evolution of E. coli and B. subtilis.Section2:Sequence analysis and predication of the putative promoter1Sequence analysis (Figure2A) indicates that the inserted fragment upstream of the reporter gene bgaB in pSI23423is about898-bp.2Two conserved prokaryotic promoter regions were found in the cloned fragment. To find the authentic promoter, the two probable promoters P23S and P24S were sub-cloned into the promoter probe vector. The trend of β-Gal production from the pSI23is similar with that from pSI23423both in E. coli and B. subtilis. The β-Gal production from the pSI23reached3122.44and294.25mill U/mL when supplemented with2.5%NaCl in E. coli and B. subtilis, respectively, which was higher than that from the pSI23423. Whereas, no β-Gal activity was detected from the pSI24either induced or not induced by salt. Therefore, the promoter driven inducible expression of β-Gal in this study is achieved using the P23S.Section3:Detection of the potential promoter activity of P23S fragment1To examine the expression efficiency of the reconstructed promoter system for cloned genes, the coding region of bgaB was used as a reporter gene and cloned into the expression vector, pOMH. β-Gal from the pOMH-bgaB in E. coli under the2.5%salt inducible conditions revealed a distinct band with a molecular mass of approximately66kDa, corresponding to the molecular weight of β-Gal, while a relatively weak band was visible from that under no salt inducible conditions. These results confirmed that the β-Gal was efficiently expressed under salt induction.2The bioB gene, which is involved in biotin biosynthesis of E. coli, and the vgb gene coding for Vitreoscilla haemoglobin, were inserted downstream of P23S in the pOMH, respectively. As expected, these two genes were successfully expressed by this promoter system in E. coli under2.5%salt inducible conditions. Only relatively weak target protein bands were visible under no salt inducible conditions, further demonstrating the efficiency of the osmolarity-inducible promoter system developed in this study.Thus, this study provided a simple and low-cost inducible expression system for the expression of cloned genes.