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

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. 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

Deletion of Rap-phosphatases for quorum sensing control in Bacillus and its effect on surfactin production

The complex regulatory network in Bacillus, known as quorum sensing, offers many opportunities to modify bacterial gene expression and hence to control bioprocesses. One target regulated by this mechanism is the activity of the PsrfA promoter, which is engaged in the formation of lipopeptide surfactin. It was hypothesised that deletion of rapC, rapF and rapH, encoding for prominent Rap-phosphatases known to affect PsrfA activity, would enhance surfactin production. Therefore, these genes were deleted in a sfp + derivative of B. subtilis 168 with subsequent evaluation of quantitative data. Up to the maximum product formation of the reference strain B. subtilis KM1016 after 16 h of cultivation, the titers of the rap deletion mutants did not exceed the reference. However, an increase in both product yield per biomass Y P/X and specific surfactin productivity q surfactin was observed, without any considerable effect on the ComX activity. By extending the cultivation time, a 2.7-fold increase in surfactin titer was observed after 24 h for strain CT10 (ΔrapC) and a 2.5-fold increase for CT11 (ΔrapF) compared to the reference strain KM1016. In addition, Y P/X was again increased for strains CT10 and CT11, with values of 1.33 g/g and 1.13 g/g, respectively. Interestingly, the effect on surfactin titer in strain CT12 (ΔrapH) was not as distinct, although it achieved the highest promoter activity (PsrfA-lacZ). The data presented support the possibility of involving the quorum sensing system of Bacillus in bioprocess control as shown here on the example of lipopeptide production.</p

Regulation der Mannitol-Abbaugene in Bacillus subtilis

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.Bacillus subtilis nimmt Mannitol mit Hilfe eines Phosphoenolpyruvat-abhängigem Phosphotransferasesystems auf. Dieses wird durch das mtlAFD-Operon codiert, welches aus mtlA (codiert das Membran-gebundene EIICBMtl), mtlF (codiert das Phosphorylgruppen-übertragende EIIAMtl) und mtlD (codiert die Mannitol-1-phosphat-Dehydrogenase) besteht. Das Operon wird durch MtlR aktiviert, dessen Gen ca. 14,4 kb stromabwärts des Operons lokalisiert ist. Die Regulation der Gene für die Mannitol-Verwertung in B. subtilis wurde mit Hilfe des Reportergens lacZ untersucht, welches an die Promotoren vom mtlAFD (PmtlA) sowie mtlR (PmtlR) fusioniert wurde. Sowohl PmtlA als auch PmtlR wurden durch Mannitol und Glucitol induziert, während Glucose die Promotoraktivitäten reprimierte. Im Vergleich zu PmtlR zeigte PmtlA eine ca. 4,5-fach höhere Promotorstärke. Die Analyse der Transkriptionsinitiations-Sequenzen durch Transkriptionsstartbestimmung von PmtlA und PmtlR ergab, dass es sich um Sigma A-abhängige Promotoren handelt. Die -35 und -10 Promotorsequenzen von PmtlA und PmtlR wurden als TTGTAT und TAACAT bzw. TTGATT und TATATT identifiziert. In beiden Promotoren wurden cre- Sequenzen (catabolite responsive element) gefunden, welche mit den -10 Regionen überlappen. Eine Verkürzung der 5‘-untranslatierten Region (5’UTR) von PmtlA erhöhte dessen Aktivität, wohingegen die Aktivität von PmtlR durch die Verkürzung der 5’UTR reduziert wurde. Durch Sequenzalignment der stromaufwärts von PmtlA und PmtlR gelegenen Sequenzen konnte die mutmaßliche MtlR-Bindestelle identifiziert werden. Diese Bindestelle ist in beiden Sequenzen durch eine unvollständig ausgeprägte, invertierte Wiederholungssequenz (TTGNCACAN4TGTGNCAA) charakterisiert, welche ihrerseits von jeweils zwei 11 bp langen Sequenzen flankiert wird. Die Regulation von PmtlA und PmtlR wurde mit Hilfe von mtlAF-, mtlF-, mtlD- und mtlR-Deletionen in B. subtilis untersucht. Die Deletion von mtlAF führte zu einer konstitutiven Aktivität von PmtlA und PmtlR, wodurch der inhibitorische Effekt von EIICBMtl und EIIAMtl (Untereinheiten des PTS-Transporters) auf MtlR bei Abwesenheit von Mannitol gezeigt werden konnte. Die konstitutive Aktivität von PmtlA wurde durch die Deletion von mtlF noch verstärkt. Im Gegensatz dazu verursachte die Deletion von mtlAFD eine signifikante Reduktion der konstitutiven Aktivität von PmtlA. Die Mutation von mtlD resultierte in einer Sensitivität von B. subtilis gegenüber Mannitol und Glucitol. Kultivierung in Gegenwart dieser Zucker führte zu einer Lyse der Zellen. In der mtlD::erm Mutante wurden PmtlA und PmtlR durch Glucitol und Mannitol gleichermaßen induziert. Ferner führte die Deletion von mtlR zu einer Uninduzierbarkeit von PmtlA und PmtlR mit Mannitol und Glucitol, wohingegen die Deletion der Gene für die Glucitol-Verwertung keinen Einfluss auf die Induzierbarkeit von PmtlA und PmtlR mit Glucitol hatte. Wie auch in der Delta mtlR Mutante war die Aktivität von PmtlA in der ptsH-H15A (HPr-H15A) Mutante drastisch reduziert. Die Mutation des Histidin 289 in der PRDI-Domäne von MtlR zu einem Alanin reduzierte die Aktivität von PmtlA, während die Aktivität von PmtlA in der mtlR-H230A-Mutante ähnlich wie die des Wildtyps war. Im Gegensatz dazu führte die Mutation H342D der PRDII-Domäne von MtlR zu einer verminderten Glucose-Repression von PmtlA. Eine MtlR Doppelmutante H342D C419A wurde in E. coli produziert und ihre Aktivität in einem Gelmobilityshiftassay in vitro bestätigt. Diese Ergebnisse zeigen, dass MtlR einer, durch die Phosphorylierung der PRDII-Domäne durch HPr(H15~P) stattfindenden, positiven Regulation unterliegt. Ferner kann eine Aktivierung von MtlR durch eine Dephosphorylierung der EIIBGat- und EIIAMtl-ähnlichen Domänen durch die EIIAMtl und EIICBMtl Transporter-Untereinheiten erreicht werden. Glucose und Fructose reprimieren die Aktivität von PmtlA wohingegen Saccharose und Mannose keinen Einfluss auf die PmtlA-Aktivität haben. Daher wurde die Kohlenstoff-Katabolit-Repression von PmtlA und PmtlR in CcpA-abhängigen Mutanten, wie ptsH-S46A, Delta crh, Delta hprK und Delta ccpA, untersucht. Da eine Induktion von PmtlA und PmtlR in den Mutanten nicht zu einem vollständigen Verlust der Katabolit-Repression führte, wurden die cre-Sequenzen (catabolite responsive element) der beiden Promotoren untersucht. Mit Hilfe des konstitutiven Promotors PgroE wurde gezeigt, dass die cre-Sequenzen von PmtlA und PmtlR nur schwach funktionsfähig waren. Im Gegensatz dazu führte die Deletion des Glucose-PTS-Transporters, welcher von ptsG codiert wird, zu einem vollständigen Verlust der Glucose-Repression von PmtlA und PmtlR. Folglich wird die Repression des Mannitol-PTS durch Glucose hauptsächlich posttranslational durch HPr via MtlR-H342 und auf transkriptionaler Ebene durch eine CcpA-abhängige Kohlenstoff-Katabolit-Repression vermittelt

Transcriptional Regulation of the Vanillate Utilization Genes (vanABK Operon) of Corynebacterium glutamicum by VanR, a PadR-Like Repressor

Heravi KM, Lange J, Watzlawick H, Kalinowski J, Altenbuchner J. Transcriptional Regulation of the Vanillate Utilization Genes (vanABK Operon) of Corynebacterium glutamicum by VanR, a PadR-Like Repressor. Journal of Bacteriology. 2015;197(5):959-972.Corynebacterium glutamicum is able to utilize vanillate, the product of lignin degradation, as the sole carbon source. The vanillate utilization components are encoded by the vanABK operon. The vanA and vanB genes encode the subunits of vanillate O-demethylase, converting vanillate to protocatechuate, while VanK is the specific vanillate transporter. The vanABK operon is regulated by a PadR-type repressor, VanR. Heterologous gene expression and variations of the vanR open reading frame revealed that the functional VanR contains 192 residues (21 kDa) and forms a dimer, as analyzed by size exclusion chromatography. In vivo, ferulate, vanillin, and vanillate induced P-vanABK in C. glutamicum, while only vanillate induced the activity of PvanABK in Escherichia coli lacking the ferulate catabolic system. Differential scanning fluorimetry verified that vanillate is the only effector of VanR. Interaction between the PvanABK DNA fragment and the VanR protein had an equilibrium dissociation constant (KD) of 15.1 +/- 1.7 nM. The VanR-DNA complex had a dissociation rate constant (K-d) of (267 +/- 23) x 10(-6) s(-1), with a half-life of 43.5 +/- 3.6 min. DNase I footprinting localized the VanR binding site at P-vanABK, extending from +9 to +45 on the coding strand. Deletion of the nucleotides +18 to +27 inside the VanR binding site rendered P-vanABK constitutive. Fusion of the T7 promoter and the wild-type VanR operator, as well as its shortened versions, indicated that the inverted repeat AACTAACTAA(N-4) TTAGGTATTT is the specific VanR binding site. It is proposed that the VanR-DNA complex contains two VanR dimers at the VanR operator

Phosphosugar stress in Bacillus subtilis: Intracellular accumulation of mannose 6-phosphate derepresed the glcR-phoC operon from repression by GlcR

Bacillus subtilis phosphorylates sugars during or after their transport into the cell. Perturbation in the conversion of intracellular phosphosugars to the central carbon metabolites and accumulation of phosphosugars can impose stress on the cells. In this study, we investigated the effect of phosphosugar stress on B. subtilis Preliminary experiments indicated that the non-matabolizable analogs of glucose were unable to impose stress on B. subtilis In contrast, deletion of manA encoding mannose 6-phosphate isomerase (responsible for conversion of mannose 6-phosphate to fructose 6-phosphate) resulted in growth arrest and bulged cell shape in the medium containing mannose. Besides, an operon encoding a repressor (GlcR) and a haloic acid dehalogenase (HAD)-like phosphatase (PhoC; previously YwpJ) were upregulated. Integration of the P glcR -lacZ cassette into different mutational backgrounds indicated that P glcR is induced when (i) a manA-deficient strain is cultured with mannose or (ii) when glcR is deleted. GlcR represses the transcription of glcR-phoC by binding to the σA-type core elements of P glcR Electrophoretic mobility shift assay showed no interaction between mannose 6-phosphate (or other phosphosugars) and the GlcR-P glcR DNA complex. PhoC was an acid phosphatase mainly able to dephosphorylate glycerol 3-phosphate and ribose 5- phosphate. Mannose 6-phosphate was only weakly dephosphorylated by PhoC. Since deletion of glcR and phoC alone or in combination had no effect on the cell during phosphosugar stress, it is assumed that the derepression of glcR-phoC is a side effect of phosphosugar stress in B. subtilisIMPORTANCEBacillus subtilis has different stress response systems to cope with external and internal stressors. Here, we investigated how B. subtilis deals with the high intracellular concentration of phosphosugars as an internal stressor. The results indicated the derepression of an operon consisting of a repressor (GlcR) and a phosphatase (PhoC). Further analysis revealed that this operon is not a phosphosugar stress response system. The substrate specificity of PhoC may indicate a connection between the glcR-phoC operon and pathways in which glycerol 3-phosphate and ribose 5-phosphate are utilized, such as membrane biosynthesis and teichoic acid elongation

Surfactin Shows Relatively Low Antimicrobial Activity against Bacillus subtilis and Other Bacterial Model Organisms in the Absence of Synergistic Metabolites

Surfactin is described as a powerful biosurfactant and is natively produced by Bacillus&nbsp;subtilis in notable quantities. Among other industrially relevant characteristics, antimicrobial properties have been attributed to surfactin-producing Bacillus isolates. To investigate this property, stress approaches were carried out with biotechnologically established strains of Corynebacterium glutamicum, Bacillus&nbsp;subtilis, Escherichia coli and Pseudomonas putida with the highest possible amounts of surfactin. Contrary to the popular opinion, the highest growth-reducing effects were detectable in B. subtilis and E.&nbsp;coli after surfactin treatment of 100 g/L with 35 and 33%, respectively, while P. putida showed no growth-specific response. In contrast, other antimicrobial biosurfactants, like rhamnolipids and sophorolipids, showed significantly stronger effects on bacterial growth. Since the addition of high amounts of surfactin in defined mineral salt medium reduced the cell growth of B.&nbsp;subtilis by about 40%, the initial stress response at the protein level was analyzed by mass spectrometry, showing induction of stress proteins under control of alternative sigma factors &sigma;B and &sigma;W as well as the activation of LiaRS two-component system. Overall, although surfactin is associated with antimicrobial properties, relatively low growth-reducing effects could be demonstrated after the surfactin addition, challenging the general claim of the antimicrobial properties of surfactin