1887

Abstract

The exopolysaccharide galactoglucan promotes the establishment of symbiosis between the nitrogen-fixing Gram-negative soil bacterium 2011 and its host plant alfalfa. The transcriptional regulator ExpG activates expression of galactoglucan biosynthesis genes by direct binding to the , / and promoter regions. ExpG is a member of the MarR family of regulatory proteins. Analysis of target sequences of an ExpG(His) fusion protein in the promoter regions resulted in the identification of a binding site composed of a conserved palindromic region and two associated sequence motifs. Association and dissociation kinetics of the specific binding of ExpG(His) to this binding site were characterized by standard biochemical methods and by single-molecule spectroscopy based on the atomic force microscope (AFM). Dynamic force spectroscopy indicated a distinct difference in the kinetics between the wild-type binding sequence and two mutated binding sites, leading to a closer understanding of the ExpG–DNA interaction.

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2005-01-01
2020-01-24
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References

  1. Alekshun, M. N. & Levy, S. B. ( 1999 ). The mar regulon: multiple resistance to antibiotics and other toxic chemicals. Trends Microbiol 7, 410–413.[CrossRef]
    [Google Scholar]
  2. Astete, S. G. & Leigh, J. A. ( 1996 ). mucS, a gene involved in activation of galactoglucan (EPS II) synthesis gene expression in Rhizobium meliloti. Mol Plant–Microbe Interact 9, 395–400.[CrossRef]
    [Google Scholar]
  3. Bartels, F. W., Baumgarth, B., Anselmetti, D., Ros, R. & Becker, A. ( 2003 ). Specific binding of the regulatory protein ExpG to promoter regions of the galactoglucan biosynthesis gene cluster of Sinorhizobium meliloti – a combined molecular biology and force spectroscopy investigation. J Struct Biol 143, 145–152.[CrossRef]
    [Google Scholar]
  4. Becker, A., Rüberg, S., Küster, H., Roxlau, A. A., Keller, M., Ivashina, T., Cheng, H. P., Walker, G. C. & Pühler, A. ( 1997 ). The 32-kilobase exp gene cluster of Rhizobium meliloti directing the biosynthesis of galactoglucan: genetic organization and properties of the encoded gene products. J Bacteriol 179, 1375–1384.
    [Google Scholar]
  5. Bertram-Drogatz, P. A., Rüberg, S., Becker, A. & Pühler, A. ( 1997 ). The regulatory protein MucR binds to a short DNA region located upstream of the mucR coding region in Rhizobium meliloti. Mol Gen Genet 254, 529–538.[CrossRef]
    [Google Scholar]
  6. Bertram-Drogatz, P. A., Quester, I., Becker, A. & Pühler, A. ( 1998 ). The Sinorhizobium meliloti MucR protein, which is essential for the production of high-molecular-weight succinoglycan exopolysaccharide, binds to short DNA regions upstream of exoH and exoY. Mol Gen Genet 257, 433–441.[CrossRef]
    [Google Scholar]
  7. Bisswanger, H. ( 1994 ). Enzymkinetik: Theorie und Methoden. Weinheim: VCH.
  8. 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]
    [Google Scholar]
  9. Calvo, J. M. & Matthews, R. G. ( 1994 ). The leucine-responsive regulatory protein, a global regulator of metabolism in Escherichia coli. Microbiol Rev 58, 466–490.
    [Google Scholar]
  10. Casse, F., Boucher, C., Hulliot, J. S., Michel, M. & Dénarié, F. ( 1979 ). Identification and characterization of large plasmids in Rhizobium meliloti using agarose gel electrophoresis. J Bacteriol 113, 229–242.
    [Google Scholar]
  11. Cohen, S. P., Hachler, H. & Levy, S. B. ( 1993 ). Genetic and functional analysis of the multiple antibiotic resistance (mar) locus in Escherichia coli. J Bacteriol 175, 1484–1492.
    [Google Scholar]
  12. Egland, P. G. & Harwood, C. S. ( 1999 ). BadR, a new MarR family member, regulates anaerobic benzoate degradation by Rhodopseudomonas palustris in concert with AadR, an Fnr family member. J Bacteriol 181, 2102–2109.
    [Google Scholar]
  13. Evans, E. & Ritchie, K. ( 1997 ). Dynamic strength of molecular adhesion bonds. Biophys J 72, 1541–1555.[CrossRef]
    [Google Scholar]
  14. Fried, M. & Crothers, D. M. ( 1981 ). Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res 9, 6505–6525.[CrossRef]
    [Google Scholar]
  15. Glazebrook, J. & Walker, G. C. ( 1989 ). A novel exopolysaccharide can function in place of the calcofluor-binding exopolysaccharide in nodulation of alfalfa by Rhizobium meliloti. Cell 56, 661–672.[CrossRef]
    [Google Scholar]
  16. Gonzalez, J. E., Reuhs, B. L. & Walker, G. C. ( 1996 ). Low molecular weight EPS II of Rhizobium meliloti allows nodule invasion in Medicago sativa. Proc Natl Acad Sci U S A 93, 8636–8641.[CrossRef]
    [Google Scholar]
  17. Hansma, H. G. & Laney, D. E. ( 1996 ). DNA binding to mica correlates with cationic radius: assay by atomic force microscopy. Biophys J 70, 1933–1939.[CrossRef]
    [Google Scholar]
  18. Henikoff, S., Haughn, G. W., Calvo, J. M. & Wallace, J. C. ( 1988 ). A large family of bacterial activator proteins. Proc Natl Acad Sci U S A 85, 6602–6606.[CrossRef]
    [Google Scholar]
  19. Her, G. R., Glazebrook, J., Walker, G. C. & Reinhold, V. N. ( 1990 ). Structural studies of a novel exopolysaccharide produced by a mutant of Rhizobium meliloti strain Rm1021. Carbohydr Res 198, 305–312.[CrossRef]
    [Google Scholar]
  20. Hutter, J. L. & Bechhoefer, J. ( 1993 ). Calibration of atomic-force microscope tips. Rev Sci Instrum 7, 1868–1873.
    [Google Scholar]
  21. Keller, M., Roxlau, A., Weng, W. M., Schmidt, M., Quandt, J., Niehaus, K., Jording, D., Arnold, W. & Pühler, A. ( 1995 ). Molecular analysis of the Rhizobium meliloti mucR gene regulating the biosynthesis of the exopolysaccharides succinoglycan and galactoglucan. Mol Plant–Microbe Interact 8, 267–277.[CrossRef]
    [Google Scholar]
  22. Komeda, H., Kobayashi, M. & Shimizu, S. ( 1996 ). Characterization of the gene cluster of high-molecular-mass nitrile hydratase (H-NHase) induced by its reaction product in Rhodococcus rhodochrous J1. Proc Natl Acad Sci U S A 93, 4267–4272.[CrossRef]
    [Google Scholar]
  23. Lane, D., Prentki, P. & Chandler, M. ( 1992 ). Use of gel retardation to analyze protein-nucleic acid interactions. Microbiol Rev 56, 509–528.
    [Google Scholar]
  24. Lloret, J., Martin, M., Oruezabal, R. I., Bonilla, I. & Rivilla, R. ( 2002 ). MucR and mucS activate exp genes transcription and galactoglucan production in Sinorhizobium meliloti EFB1. Mol Plant–Microbe Interact 15, 54–59.[CrossRef]
    [Google Scholar]
  25. Long, S. R. ( 2001 ). Genes and signals in the Rhizobium–legume symbiosis. Plant Physiol 125, 69–72.[CrossRef]
    [Google Scholar]
  26. Lysetska, M., Knoll, A., Boehringer, D., Hey, T., Krauss, G. & Krausch, G. ( 2002 ). UV light-damaged DNA and its interaction with human replication protein A: an atomic force microscopy study. Nucleic Acids Res 30, 2686–2691.[CrossRef]
    [Google Scholar]
  27. Lyubchenko, Y., Shlyakhtenko, L., Harrington, R., Oden, P. & Lindsay, S. ( 1993 ). Atomic force microscopy of long DNA: imaging in air and under water. Proc Natl Acad Sci U S A 90, 2137–2140.[CrossRef]
    [Google Scholar]
  28. Merkel, R., Nassoy, P., Leung, A., Ritchie, K. & Evans, E. ( 1999 ). Energy landscapes of receptor-ligand bonds explored with dynamic force spectroscopy. Nature 397, 50–53.[CrossRef]
    [Google Scholar]
  29. Miller, P. F. & Sulavik, M. C. ( 1996 ). Overlaps and parallels in the regulation of intrinsic multiple-antibiotic resistance in Escherichia coli. Mol Microbiol 21, 441–448.[CrossRef]
    [Google Scholar]
  30. Oke, V. & Long, S. R. ( 1999 ). Bacteroid formation in the Rhizobium-legume symbiosis. Curr Opin Microbiol 2, 641–646.[CrossRef]
    [Google Scholar]
  31. Oscarsson, J., Mizunoe, Y., Uhlin, B. E. & Haydon, D. J. ( 1996 ). Induction of haemolytic activity in Escherichia coli by the slyA gene product. Mol Microbiol 20, 191–199.[CrossRef]
    [Google Scholar]
  32. Pellock, B. J., Teplitski, M., Boinay, R. P., Bauer, W. D. & Walker, G. C. ( 2002 ). A LuxR homolog controls production of symbiotically active extracellular polysaccharide II by Sinorhizobium meliloti. J Bacteriol 184, 5067–5076.[CrossRef]
    [Google Scholar]
  33. Perez-Rueda, E. & Collado-Vides, J. ( 2001 ). Common history at the origin of the position-function correlation in transcriptional regulators in archaea and bacteria. J Mol Evol 53, 172–179.[CrossRef]
    [Google Scholar]
  34. Popp, R., Kohl, T., Patz, P., Trautwein, G. & Gerischer, U. ( 2002 ). Differential DNA binding of transcriptional regulator PcaU from Acinetobacter sp. strain ADP1. J Bacteriol 184, 1988–1997.[CrossRef]
    [Google Scholar]
  35. Rüberg, S., Pühler, A. & Becker, A. ( 1999 ). Biosynthesis of the exopolysaccharide galactoglucan in Sinorhizobium meliloti is subject to a complex control by the phosphate-dependent regulator PhoB and the proteins ExpG and MucR. Microbiology 145, 603–611.[CrossRef]
    [Google Scholar]
  36. Sauer, R. T., Yocum, R. R., Doolittle, R. F., Lewis, M. & Pabo, C. O. ( 1982 ). Homology among DNA-binding proteins suggests use of a conserved super-secondary structure. Nature 298, 447–451.[CrossRef]
    [Google Scholar]
  37. Schell, M. A. ( 1993 ). Molecular biology of the LysR family of transcriptional regulators. Annu Rev Microbiol 47, 597–626.[CrossRef]
    [Google Scholar]
  38. Schwesinger, F., Ros, R., Strunz, T., Anselmetti, D., Guntherodt, H. J., Honegger, A., Jermutus, L., Tiefenauer, L. & Pluckthun, A. ( 2000 ). Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates. Proc Natl Acad Sci U S A 97, 9972–9977.[CrossRef]
    [Google Scholar]
  39. Spaink, H. P. ( 2000 ). Root nodulation and infection factors produced by rhizobial bacteria. Annu Rev Microbiol 54, 257–288.[CrossRef]
    [Google Scholar]
  40. Strunz, T., Oroszlan, K., Schafer, R. & Guntherodt, H. J. ( 1999 ). Dynamic force spectroscopy of single DNA molecules. Proc Natl Acad Sci U S A 96, 11277–11282.[CrossRef]
    [Google Scholar]
  41. Sulavik, M. C., Gambino, L. F. & Miller, P. F. ( 1995 ). The MarR repressor of the multiple antibiotic resistance (mar) operon in Escherichia coli: prototypic member of a family of bacterial regulatory proteins involved in sensing phenolic compounds. Mol Med 1, 436–446.
    [Google Scholar]
  42. Wang, L. X., Wang, Y., Pellock, B. & Walker, G. C. ( 1999 ). Structural characterization of the symbiotically important low-molecular-weight succinoglycan of Sinorhizobium meliloti. J Bacteriol 181, 6788–6796.
    [Google Scholar]
  43. 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]
    [Google Scholar]
  44. Young, R. A. & Davis, R. W. ( 1983 ). Yeast RNA polymerase II genes: isolation with antibody probes. Science 222, 778–782.[CrossRef]
    [Google Scholar]
  45. Zhan, H. J., Levery, S. B., Lee, C. C. & Leigh, J. A. ( 1989 ). A second exopolysaccharide of Rhizobium meliloti strain SU47 that can function in root nodule invasion. Proc Natl Acad Sci U S A 86, 3055–3059.[CrossRef]
    [Google Scholar]
  46. Zhan, H. J., Lee, C. C. & Leigh, J. A. ( 1991 ). Induction of the second exopolysaccharide (EPSb) in Rhizobium meliloti SU47 by low phosphate concentrations. J Bacteriol 173, 7391–7394.
    [Google Scholar]
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