04 Fakultät Energie-, Verfahrens- und Biotechnik
Permanent URI for this collectionhttps://elib.uni-stuttgart.de/handle/11682/5
Browse
3 results
Search Results
Item Open Access Construction of a super-competent Bacillus subtilis 168 using the PmtlA-comKS inducible cassette(2015) Rahmer, Regine; Morabbi Heravi, Kambiz; Altenbuchner, JosefCompetence is a physiological state that enables Bacillus subtilis 168 to take up and internalize extracellular DNA. In practice, only a small subpopulation of B. subtilis 168 cells becomes competent when they enter stationary phase. In this study, we developed a new transformation method to improve the transformation efficiency of B. subtilis 168, specially in rich media. At first, different competence genes, namely comK, comS, and dprA, were alone or together integrated into the chromosome of B. subtilis 168 under control of mannitol-inducible PmtlA promoter. Overexpression of both comK and comS increased the transformation efficiency of B. subtilis REG19 with plasmid DNA by 6.7-fold compared to the wild type strain 168. This transformation efficiency reached its maximal level after 1.5 h of induction by mannitol. Besides, transformability of the REG19 cells was saturated in the presence of 100 ng dimeric plasmid or 3000 ng chromosomal DNA. Studying the influence of global regulators on the development of competence pointed out that important competence development factors, such as Spo0A, ComQXPA, and DegU, could be removed in REG19. On the other hand, efficient REG19 transformation remained highly dependent on the original copies of comK and comS regardless of the presence of PmtlA-comKS. Finally, novel plasmid-free strategies were used for transformation of REG19 based on Gibson assembly.Item Open Access Regulation of the mannitol utilization genes in Bacillus subtilis(2013) Morabbi Heravi, Kambiz; Mattes, Ralf (Prof. Dr.)Bacillus subtilis takes up mannitol by a phosphoenolpyruvate-dependent phosphotransferase system (PTS). The mannitol utilization system is encoded by the mtlAFD operon consisting of mtlA (encoding membrane-bound EIICBMtl), mtlF (encoding phosphocarrier EIIAMtl), and mtlD (encoding mannitol 1-phosphate dehydrogenase). This operon is activated by MtlR whose coding gene is located approx. 14.4 kb downstream of the operon. The regulation of the mannitol utilization genes in B. subtilis was studied by fusion of the promoters of mtlAFD (PmtlA) and mtlR (PmtlR) to lacZ as a reporter gene. Both the PmtlA and PmtlR were inducible by mannitol and glucitol, while glucose reduced their activities. The promoter strength of PmtlA was about 4.5-fold higher than that of PmtlR. Identification of the transcription start sites of PmtlA and PmtlR revealed that both of these promoters contain a sigma A-type promoter structure. The promoter -35 and -10 boxes in PmtlA were TTGTAT and TAACAT and in PmtlR TTGATT and TATATT, respectively. Catabolite responsive elements (cre) were detected in the sequences of PmtlA and PmtlR overlapping the -10 boxes. Shortening the mRNA 5’untranslated region (5’UTR) increased the PmtlA activity, whereas PmtlR activity was decreased by shortening of its mRNA 5’UTR. Alignment of the -35 upstream sequences of PmtlA and PmtlR revealed the putative MtlR binding site. This sequence comprised a similar incomplete inverted repeat in both the PmtlA and PmtlR sequences (TTGNCACAN4TGTGNCAA). This sequence was encompassed by two 11 bp distal and proximal flanking sequences. Construction of PmtlA-PlicB hybrid promoters and shortening of the 5’-end of PmtlA indicated the probable boundaries of putative MtlR binding site in PmtlA. Increasing the distance between the putative MtlR binding site and -35 box lowered the PmtlA maximal activity, although PmtlA remained inducible by mannitol. PmtlA became inactive by disruption of the TTGNCACAN4TGTGNCAA sequence. In contrast, manipulation of the distal and proximal flanking sequences only reduced the maximal activity of PmtlA, whereas PmtlA remained highly inducible. These flanking sequences contained AT-rich repeats similar to the consensus sequence of alpha CTD binding sites. Regulation of PmtlA and PmtlR was investigated by deletion of mtlAF, mtlF, mtlD, and mtlR. Deletion of the mtlAF genes rendered PmtlA and PmtlR constitutive showing the inhibitory effect of EIICBMtl and EIIAMtl (PTS transporter components) on MtlR in the absence of mannitol. The constitutive activity of PmtlA was increased by the deletion of mtlF. In contrast, the deletion of mtlAFD showed a significant reduction in the PmtlA constitutive activity. Disruption of mtlD made B. subtilis sensitive to mannitol in a way that addition of mannitol or glucitol to the bacterial culture ended in cell lysis. Besides, PmtlA and PmtlR were similarly induced by glucitol and mannitol in a mtlD::erm mutant. Also, deletion of mtlR rendered PmtlA and PmtlR uninducible by mannitol or glucitol. In contrast, deletion of the glucitol utilization genes had no influence on the inducibility of PmtlA or PmtlR by glucitol. The PmtlA activity was drastically reduced in ptsH-H15A (HPr-H15A) mutant similar to the delta mtlR mutant. The mutation of histidine 289 in the PRDI domain of MtlR to alanine reduced the activity of PmtlA, whereas the PmtlA activity in the mtlR H230A mutant was almost similar to wild type. In contrast, mutation of the PRDII domain of MtlR to H342D mainly relieved PmtlA from glucose repression. Moreover, MtlR double mutant H342D C419A which was produced in E. coli was shown to be active in vitro. These results represent the positive regulation of MtlR via phosphorylation of the PRDII domain by HPr(H15~P). Also, dephosphorylation of the domains EIIBGat- and EIIAMtl-like of MtlR by EIIAMtl and EIICBMtl transporter components causes activation. The PmtlA activity was repressed in the presence of glucose and fructose, while sucrose and mannose had no influence on the PmtlA activity. Therefore, catabolite repression of PmtlA and PmtlR were studied by CcpA-dependent carbon catabolite repression mutants, such as ptsH-S46A, delta crh, delta hprK, and delta ccpA. Induction of PmtlA and PmtlR in these mutants did not result in a complete loss of catabolite repression. Therefore, the catabolite responsive elements (cre sites) of PmtlA and PmtlR were investigated. Using a constitutive promoter, PgroE, it was shown that the cre sites of PmtlA and PmtlR were weakly functional. In contrast, deletion of the glucose PTS transporter, encoded by ptsG, resulted in a complete loss of glucose repression in PmtlA and PmtlR. Thus, the main glucose repression of mannitol PTS function at the posttranslational level in a HPr-mediated manner via MtlR-H342 and at transcriptional level by CcpA-dependent carbon catabolite repression.Item Open Access Regulation of mtl operon promoter of Bacillus subtilis: requirements of its use in expression vectors(2011) Morabbi Heravi, Kambiz; Wenzel, Marian; Altenbuchner, JosefSeveral 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. Regulation of the promoters of Bacillus subtilis mtlAFD operon (PmtlA) and mtlR (PmtlR) encoding the activator were investigated by fusion to lacZ. Identification of the PmtlA and PmtlR transcription start sites revealed the sigma A like promoter structures. Also, the operator of PmtlA was determined by shortening, nucleotide exchange, and alignment of PmtlA and PmtlR operator regions. Deletion of the mannitol-specific PTS genes (mtlAF) resulted in PmtlA constitutive expression demonstrating the inhibitory effect of EIICBMtl and EIIAMtl on MtlR in the absence of mannitol. Disruption of mtlD made the cells sensitive to mannitol and glucitol. Both PmtlA and PmtlR were influenced by carbon catabolite repression (CCR). However, a CcpA deficient mutant showed only a slight reduction in PmtlR catabolite repression. Similarly, using PgroE as a constitutive promoter, putative cre sites of PmtlA and PmtlR slightly reduced the promoter activity in the presence of glucose. In contrast, glucose repression of PmtlA and PmtlR was completely abolished in a ptsG deletion mutant and significantly reduced in a MtlR (H342D) mutant.