Skip to content
1887

Microbiology Society logo Microbiology

Volume 160, Issue 1

Regulation of the mannitol utilization genes: promoter structure and transcriptional activation by the wild-type regulator (MtlR) and its mutants Free

Abstract

Expression of mannitol utilization genes in is directed by P, the promoter of the operon, and P, the promoter of the MtlR activator. MtlR contains phosphoenolpyruvate-dependent phosphotransferase system (PTS) regulation domains, called PRDs. The activity of PRD-containing MtlR is mainly regulated by the phosphorylation/dephosphorylation of its PRDII and EIIB-like domains. Replacing histidine 342 and cysteine 419 residues, which are the targets of phosphorylation in these two domains, by aspartate and alanine provided MtlR-H342D C419A, which permanently activates P. In the -H342D C419A mutant, P was active, even when the operon was deleted from the genome. The -H342D C419A allele was expressed in an strain lacking enzyme I of the PTS. Electrophoretic mobility shift assays using purified MtlR-H342D C419A showed an interaction between the MtlR double-mutant and the Cy5-labelled P and P DNA fragments. These investigations indicate that the activated MtlR functions regardless of the presence of the mannitol-specific transporter (MtlA). This is in contrast to the proposed model in which the sequestration of MtlR by the MtlA transporter is necessary for the activity of MtlR. Additionally, DNase I footprinting, construction of P-P hybrid promoters, as well as increasing the distance between the MtlR operator and the −35 box of P revealed that the activated MtlR molecules and RNA polymerase holoenzyme likely form a class II type activation complex at P and P during transcription initiation.

  • Received:
  • Accepted:
  • Published Online:
Funding
This study was supported by the:
  • the German Academic Exchange Service (DAAD)
© Microbiology Society
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.071233-0
2014-01-01
2025-01-16
Loading full text...

Full text loading...

/deliver/fulltext/micro/160/1/91.html?itemId=/content/journal/micro/10.1099/mic.0.071233-0&mimeType=html&fmt=ahah

References

  1. Altenbuchner J., Viell P., Pelletier I. ( 1992). Positive selection vectors based on palindromic DNA sequences. Methods Enzymol 216:457–466 [View Article][PubMed]
     [Google Scholar]
  2. Arnaud M., Vary P., Zagorec M., Klier A., Debarbouille M., Postma P., Rapoport G. ( 1992). Regulation of the sacPA operon of Bacillus subtilis: identification of phosphotransferase system components involved in SacT activity. J Bacteriol 174:3161–3170[PubMed]
     [Google Scholar]
  3. Baba T., Ara T., Hasegawa M., Takai Y., Okumura Y., Baba M., Datsenko K. A., Tomita M., Wanner B. L., Mori H. ( 2006). Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:0008 [View Article][PubMed]
     [Google Scholar]
  4. Bouraoui H., Ventroux M., Noirot-Gros M. F., Deutscher J., Joyet P. ( 2013). Membrane sequestration by the EIIB domain of the mannitol permease MtlA activates the Bacillus subtilis mtl operon regulator MtlR. Mol Microbiol 87:789–801 [View Article][PubMed]
     [Google Scholar]
  5. Bradford M. M. ( 1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 [View Article][PubMed]
     [Google Scholar]
  6. Browning D. F., Busby S. J. ( 2004). The regulation of bacterial transcription initiation. Nat Rev Microbiol 2:57–65 [View Article][PubMed]
     [Google Scholar]
  7. Crutz A. M., Steinmetz M., Aymerich S., Richter R., Le Coq D. ( 1990). Induction of levansucrase in Bacillus subtilis: an antitermination mechanism negatively controlled by the phosphotransferase system. J Bacteriol 172:1043–1050[PubMed]
     [Google Scholar]
  8. Deutscher J., Galinier A., Martin-Verstraete I. ( 2002). Carbohydrate uptake and metabolism. Bacillus Subtilis and its Closest Relatives: from Genes to Cells129–150 Sonenshein A. L., Hoch J. A., Losick R. Washington, DC: American Society for Microbiology; [CrossRef]
     [Google Scholar]
  9. Deutscher J., Francke C., Postma P. W. ( 2006). How phosphotransferase system-related protein phosphorylation regulates carbohydrate metabolism in bacteria. Microbiol Mol Biol Rev 70:939–1031 [View Article][PubMed]
     [Google Scholar]
  10. Fujita Y. ( 2009). Carbon catabolite control of the metabolic network in Bacillus subtilis. . Biosci Biotechnol Biochem 73:245–259 [View Article][PubMed]
     [Google Scholar]
  11. Greenberg D. B., Stülke J., Saier M. H. Jr ( 2002). Domain analysis of transcriptional regulators bearing PTS regulatory domains. Res Microbiol 153:519–526 [View Article][PubMed]
     [Google Scholar]
  12. Guérout-Fleury A. M., Frandsen N., Stragier P. ( 1996). Plasmids for ectopic integration in Bacillus subtilis. . Gene 180:57–61 [View Article][PubMed]
     [Google Scholar]
  13. Harwood C. R., Cutting S. M. ( 1990). Molecular Biological Methods for Bacillus New York: Wiley;
     [Google Scholar]
  14. Heravi K. M., Wenzel M., Altenbuchner J. ( 2011). Regulation of mtl operon promoter of Bacillus subtilis: requirements of its use in expression vectors. Microb Cell Fact 10:83 [View Article][PubMed]
     [Google Scholar]
  15. Hoffmann J., Bóna-Lovász J., Beuttler H., Altenbuchner J. ( 2012). In vivo and in vitro studies on the carotenoid cleavage oxygenases from Sphingopyxis alaskensis RB2256 and Plesiocystis pacifica SIR-1 revealed their substrate specificities and non-retinal-forming cleavage activities. FEBS J 279:3911–3924 [View Article][PubMed]
     [Google Scholar]
  16. Joyet P., Derkaoui M., Poncet S., Deutscher J. ( 2010). Control of Bacillus subtilis mtl operon expression by complex phosphorylation-dependent regulation of the transcriptional activator MtlR. Mol Microbiol 76:1279–1294 [View Article][PubMed]
     [Google Scholar]
  17. Joyet P., Bouraoui H., Aké F. M., Derkaoui M., Zébré A. C., Cao T. N., Ventroux M., Nessler S., Noirot-Gros M. F. & other authors ( 2013). Transcription regulators controlled by interaction with enzyme IIB components of the phosphoenolpyruvate: sugar phosphotransferase system. Biochim Biophys Acta 1834:1415–1424 [View Article][PubMed]
     [Google Scholar]
  18. Lee D. J., Minchin S. D., Busby S. J. ( 2012). Activating transcription in bacteria. Annu Rev Microbiol 66:125–152 [View Article][PubMed]
     [Google Scholar]
  19. Lindner C., Galinier A., Hecker M., Deutscher J. ( 1999). Regulation of the activity of the Bacillus subtilis antiterminator LicT by multiple PEP-dependent, enzyme I- and HPr-catalysed phosphorylation. Mol Microbiol 31:995–1006 [View Article][PubMed]
     [Google Scholar]
  20. Lindner C., Hecker M., Le Coq D., Deutscher J. ( 2002). Bacillus subtilis mutant LicT antiterminators exhibiting enzyme I- and HPr-independent antitermination affect catabolite repression of the bglPH operon. J Bacteriol 184:4819–4828 [View Article][PubMed]
     [Google Scholar]
  21. Lopian L., Nussbaum-Shochat A., O’Day-Kerstein K., Wright A., Amster-Choder O. ( 2003). The BglF sensor recruits the BglG transcription regulator to the membrane and releases it on stimulation. Proc Natl Acad Sci U S A 100:7099–7104 [View Article][PubMed]
     [Google Scholar]
  22. Luria S. E., Adams J. N., Ting R. C. ( 1960). Transduction of lactose-utilizing ability among strains of E. coli and S. dysenteriae and the properties of the transducing phage particles. Virology 12:348–390 [View Article][PubMed]
     [Google Scholar]
  23. Martin-Verstraete I., Débarbouillé M., Klier A., Rapoport G. ( 1992). Mutagenesis of the Bacillus subtilis “-12, -24” promoter of the levanase operon and evidence for the existence of an upstream activating sequence. J Mol Biol 226:85–99 [View Article][PubMed]
     [Google Scholar]
  24. Michel J. F., Millet J. ( 1970). Physiological studies on early-blocked sporulation mutants of Bacillus subtilis. . J Appl Bacteriol 33:220–227 [View Article][PubMed]
     [Google Scholar]
  25. Miller J. H. ( 1972). Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  26. Motejadded H., Altenbuchner J. ( 2007). Integration of a lipase gene into the Bacillus subtilis chromosome: Recombinant strains without antibiotic resistance marker. Iranian J Biotechnol 5:105–109
     [Google Scholar]
  27. Reizer J., Bachem S., Reizer A., Arnaud M., Saier M. H. Jr, Stülke J. ( 1999). Novel phosphotransferase system genes revealed by genome analysis - the complete complement of PTS proteins encoded within the genome of Bacillus subtilis . Microbiology 145:3419–3429[PubMed] [CrossRef]
     [Google Scholar]
  28. Rothe F. M., Wrede C., Lehnik-Habrink M., Görke B., Stülke J. ( 2013). Dynamic localization of a transcription factor in Bacillus subtilis: the LicT antiterminator relocalizes in response to inducer availability. J Bacteriol 195:2146–2154 [View Article][PubMed]
     [Google Scholar]
  29. Sambrook J., Fritsch E. F., Maniatis T. ( 1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  30. Sanger F., Nicklen S., Coulson A. R. ( 1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74:5463–5467 [View Article][PubMed]
     [Google Scholar]
  31. Schnetz K., Stülke J., Gertz S., Krüger S., Krieg M., Hecker M., Rak B. ( 1996). LicT, a Bacillus subtilis transcriptional antiterminator protein of the BglG family. J Bacteriol 178:1971–1979[PubMed]
     [Google Scholar]
  32. Sonenshein A. L. ( 2007). Control of key metabolic intersections in Bacillus subtilis. . Nat Rev Microbiol 5:917–927 [View Article][PubMed]
     [Google Scholar]
  33. Stülke J., Martin-Verstraete I., Zagorec M., Rose M., Klier A., Rapoport G. ( 1997). Induction of the Bacillus subtilis ptsGHI operon by glucose is controlled by a novel antiterminator, GlcT. Mol Microbiol 25:65–78 [View Article][PubMed]
     [Google Scholar]
  34. Stülke J., Arnaud M., Rapoport G., Martin-Verstraete I. ( 1998). PRD–a protein domain involved in PTS-dependent induction and carbon catabolite repression of catabolic operons in bacteria. Mol Microbiol 28:865–874 [View Article][PubMed]
     [Google Scholar]
  35. Sun T., Altenbuchner J. ( 2010). Characterization of a mannose utilization system in Bacillus subtilis. . J Bacteriol 192:2128–2139 [View Article][PubMed]
     [Google Scholar]
  36. Titok M. A., Chapuis J., Selezneva Y. V., Lagodich A. V., Prokulevich V. A., Ehrlich S. D., Jannière L. ( 2003). Bacillus subtilis soil isolates: plasmid replicon analysis and construction of a new theta-replicating vector. Plasmid 49:53–62 [View Article][PubMed]
     [Google Scholar]
  37. Tobisch S., Stülke J., Hecker M. ( 1999). Regulation of the lic operon of Bacillus subtilis and characterization of potential phosphorylation sites of the LicR regulator protein by site-directed mutagenesis. J Bacteriol 181:4995–5003[PubMed]
     [Google Scholar]
  38. Tortosa P., Declerck N., Dutartre H., Lindner C., Deutscher J., Le Coq D. ( 2001). Sites of positive and negative regulation in the Bacillus subtilis antiterminators LicT and SacY. Mol Microbiol 41:1381–1393 [View Article][PubMed]
     [Google Scholar]
  39. Watanabe S., Hamano M., Kakeshita H., Bunai K., Tojo S., Yamaguchi H., Fujita Y., Wong S. L., Yamane K. ( 2003). Mannitol-1-phosphate dehydrogenase (MtlD) is required for mannitol and glucitol assimilation in Bacillus subtilis: possible cooperation of mtl and gut operons. J Bacteriol 185:4816–4824 [View Article][PubMed]
     [Google Scholar]
  40. Wenzel M., Altenbuchner J. ( 2013). The Bacillus subtilis mannose regulator, ManR, a DNA-binding protein regulated by HPr and its cognate PTS transporter ManP. Mol Microbiol 88:562–576 [View Article][PubMed]
     [Google Scholar]
  41. Yamamoto H., Serizawa M., Thompson J., Sekiguchi J. ( 2001). Regulation of the glv operon in Bacillus subtilis: YfiA (GlvR) is a positive regulator of the operon that is repressed through CcpA and cre. . J Bacteriol 183:5110–5121 [View Article][PubMed]
     [Google Scholar]
  42. Yanisch-Perron C., Vieira J., Messing J. ( 1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119 [View Article][PubMed]
     [Google Scholar]
/content/journal/micro/10.1099/mic.0.071233-0
Loading
Regulation of the Bacillus subtilis mannitol utilization genes: promoter structure and transcriptional activation by the wild-type regulator (MtlR) and its mutants
Microbiology 160, 91 (2014); https://doi.org/10.1099/mic.0.071233-0
/content/journal/micro/10.1099/mic.0.071233-0
/content/journal/micro/10.1099/mic.0.071233-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error