Regulation of mtl operon promoter of Bacillus subtilis: requirements of its use in expression vectors.

BACKGROUND

Several vector systems have been developed to express any gene desired to be studied in Bacillus subtilis. Among them, the transcriptionally regulated promoters involved in carbohydrate utilization are a research priority. Expression systems based on Bacillus promoters for xylose, maltose, and mannose utilization, as well as on the heterologous E. coli lactose promoter, have been successfully constructed. The promoter of the mtlAFD operon for utilization of mannitol is another promising candidate for its use in expression vectors. In this study, we investigated the regulation of the mtl genes in order to identify the elements needed to construct a strong mannitol inducible expression system in B. subtilis.

RESULTS

Regulation of the promoters of mtlAFD operon (P(mtlA)) and mtlR (P(mtlR)) encoding the activator were investigated by fusion to lacZ. Identification of the P(mtlA) and P(mtlR) transcription start sites revealed the σ(A) like promoter structures. Also, the operator of P(mtlA) was determined by shortening, nucleotide exchange, and alignment of P(mtlA) and P(mtlR) operator regions. Deletion of the mannitol-specific PTS genes (mtlAF) resulted in P(mtlA) constitutive expression demonstrating the inhibitory effect of EIICB(Mtl) and EIIA(Mtl) on MtlR in the absence of mannitol. Disruption of mtlD made the cells sensitive to mannitol and glucitol. Both P(mtlA) and P(mtlR) were influenced by carbon catabolite repression (CCR). However, a CcpA deficient mutant showed only a slight reduction in P(mtlR) catabolite repression. Similarly, using P(groE) as a constitutive promoter, putative cre sites of P(mtlA) and P(mtlR) slightly reduced the promoter activity in the presence of glucose. In contrast, glucose repression of P(mtlA) and P(mtlR) was completely abolished in a ΔptsG mutant and significantly reduced in a MtlR (H342D) mutant.

CONCLUSIONS

The mtl operon promoter (P(mtlA)) is a strong promoter that reached a maximum of 13,000 Miller units with lacZ as a reporter on low copy plasmids. It is tightly regulated by just one copy of the mtlR gene on the chromosome and subject to CCR. CCR can be switched off by mutations in MtlR and the glucose transporter. These properties and the low costs of the inducers, i.e. mannitol and glucitol, make the promoter ideal for designing regulated expression systems.