1887

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.

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2014-01-01
2019-08-18
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References

  1. Altenbuchner J. , Viell P. , Pelletier I. . ( 1992; ). Positive selection vectors based on palindromic DNA sequences. . Methods Enzymol 216:, 457–466. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [PubMed]
    [Google Scholar]
  6. Browning D. F. , Busby S. J. . ( 2004; ). The regulation of bacterial transcription initiation. . Nat Rev Microbiol 2:, 57–65. [CrossRef] [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. . In Bacillus Subtilis and its Closest Relatives: from Genes to Cells, pp. 129–150. Edited by 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. [CrossRef] [PubMed]
    [Google Scholar]
  10. Fujita Y. . ( 2009; ). Carbon catabolite control of the metabolic network in Bacillus subtilis. . Biosci Biotechnol Biochem 73:, 245–259. [CrossRef] [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. [CrossRef] [PubMed]
    [Google Scholar]
  12. Guérout-Fleury A. M. , Frandsen N. , Stragier P. . ( 1996; ). Plasmids for ectopic integration in Bacillus subtilis. . Gene 180:, 57–61. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [PubMed]
    [Google Scholar]
  18. Lee D. J. , Minchin S. D. , Busby S. J. . ( 2012; ). Activating transcription in bacteria. . Annu Rev Microbiol 66:, 125–152. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [PubMed]
    [Google Scholar]
  35. Sun T. , Altenbuchner J. . ( 2010; ). Characterization of a mannose utilization system in Bacillus subtilis. . J Bacteriol 192:, 2128–2139. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [PubMed]
    [Google Scholar]
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