04 Fakultät Energie-, Verfahrens- und Biotechnik
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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.