Regulation of mtloperon promoter of Bacillus subtilis: requirements of its use in expression vectors

Morabbi, Wenzel Marian, Altenbuchner, Altenbuchner Josef

Description

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 Δ ptsG 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.

Sujets

• PTS
• PRD
• Activator
• Catabolite repression

Informations

• Publié par: biomed
• Publié le: 01 janvier 2011
• Nombre de lectures: 8
• Langue: English

Extrait

Morabbi Heravi et al . Microbial Cell Factories 2011, 10 :83 http://www.microbialcellfactories.com/content/10/1/83

R E S E A R C H Open Access Regulation of mtl operon promoter of Bacillus subtilis : Requirements of its use in expression vectors Kambiz Morabbi Heravi, Marian Wenzel and Josef Altenbuchner *

Abstract 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 s 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 Δ ptsG 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. Keywords: PTS, PRD, Activator, Catabolite repression, Mannitol operon

Background promising for industrial applications. However, several For many years, Bacillus subtilis has been vastly investi- bottlenecks exist that limit the expression potential, e.g. gated not only as the model of Gram-positive bacterium, low level of heterologous protein production, instability but also as an expression platform for homologous and of the plasmids, inclusion body formation, and produc- heterologous gene expression. Efficient secretion of tion of extracellular proteases [1]. So far, several expres- enzymes such as proteases, lipases, and amylases along sion systems have been developed in order to overcome with its safe use and completely sequenced genome is the low production categorized to inducer-specific pro- moters, growth-phase promoters and autoinducible pro- moters [2]. Among them, systems based on P spac [3] and I * ns C t o it r u re t s f p ü o r n In d d e u n s c t e r : ie j l o le sef G .a e l n te et n ik b , uc U h ni n v e e r r @ si i t i ä g t .u S n t i u -s t t t u g t a t rt g , a A rt l . l d m e andring31,70569 P xyl [4] have been widely used. Recently, two oth r e Stuttgart, Germany © 2011 Heravi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.