1887

Abstract

The function of the PrpR protein of serovar Typhimurium LT2 was studied and . The PrpR protein is a sensor of 2-methylcitrate (2-MC), an intermediate of the 2-methylcitric acid cycle used by this bacterium to convert propionate to pyruvate. PrpR was unresponsive to citrate (a close structural analogue of 2-MC) and to propionate, suggesting that 2-MC, not propionate, is the metabolite that signals the presence of propionate in the environment to . alleles encoding mutant proteins with various levels of 2-MC-independent activity were isolated. All lesions causing constitutive PrpR activity were mapped to the N-terminal domain of the protein. Removal of the entire sensing domain resulted in a protein (PrpR) with the highest 2-MC-independent activity. Residue A162 is critical to 2-MC sensing, since the mutant PrpR protein PrpR was as active as the PrpR protein in the absence of 2-MC. DNA footprinting studies identified the site in the region between and the operon to which the PrpR protein binds. Analysis of the binding-site sequence revealed two sites with dyad symmetry. Results from DNase I footprinting assays suggested that the PrpR protein may have higher affinity for the site proximal to the P promoter.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27299-0
2004-11-01
2019-10-15
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/11/mic1503877.html?itemId=/content/journal/micro/10.1099/mic.0.27299-0&mimeType=html&fmt=ahah

References

  1. Austin, S. & Dixon, R. ( 1992; ). The prokaryotic enhancer binding protein NTRC has an ATPase activity which is phosphorylation and DNA dependent. EMBO J 11, 2219–2228.
    [Google Scholar]
  2. Ausubel, F. A., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. ( 1989; ). Current Protocols in Molecular Biology. New York, NY: Greene Publishing Associates & Wiley Interscience.
  3. Bargo, F., Muller, L. D., Varga, G. A., Delahoy, J. E. & Cassidy, T. W. ( 2002; ). Ruminal digestion and fermentation of high-producing dairy cows with three different feeding systems combining pasture and total mixed rations. J Dairy Sci 85, 2964–2973.[CrossRef]
    [Google Scholar]
  4. Barnes, E. M., Impey, C. S. & Stevens, B. J. ( 1979; ). Factors affecting the incidence and anti-salmonella activity of the anaerobic caecal flora of the young chick. J Hyg (Lond) 82, 263–283.[CrossRef]
    [Google Scholar]
  5. Berger, S. L. ( 1984; ). The use of Cerenkov radiation for monitoring reactions performed in minute volumes: examples from recombinant DNA technology. Anal Biochem 136, 515–519.[CrossRef]
    [Google Scholar]
  6. Berkowitz, D., Hushon, J. M., Whitfield, H. J., Roth, J. & Ames, B. N. ( 1968; ). Procedure for identifying nonsense mutations. J Bacteriol 96, 215–220.
    [Google Scholar]
  7. Brock, M., Fischer, R., Linder, D. & Buckel, W. ( 2000; ). Methylcitrate synthase from Aspergillus nidulans: implications for propionate as an antifungal agent. Mol Microbiol 35, 961–973.[CrossRef]
    [Google Scholar]
  8. Bryant, M. ( 1997; ). Introduction to gastrointestinal microbial ecology. In Gastrointestinal Microbiology, pp. 3–10. Edited by R. Mackie, B. White & R. Isaacson. New York: Chapman and Hall.
  9. Buckel, W. ( 1999; ). Anaerobic energy metabolism. In Biology of the Procaryotes, pp. 278–326. Edited by J. W. Lengler, G. Drews & H. G. Chlegel. Stuttgart, Germany: Thieme.
  10. Busch, M., Stein, G., Poppitz, W., Hein, G. & Muller, A. ( 2002; ). Validated capillary gas chromatographic-mass spectrometric assay to determine 2-methylcitric acid I and II levels in human serum by using a pulsed splitless injection procedure. J Chromatogr B Analyt Technol Biomed Life Sci 775, 215–223.[CrossRef]
    [Google Scholar]
  11. Byrne, B. M. & Dankert, J. ( 1979; ). Volatile fatty acids and aerobic flora in the gastrointestinal tract of mice under various conditions. Infect Immun 23, 559–563.
    [Google Scholar]
  12. Castilho, B. A., Olfson, P. & Casadaban, M. J. ( 1984; ). Plasmid insertion mutagenesis and lac gene fusions with mini-Mu bacteriophage transposons. J Bacteriol 158, 488–495.
    [Google Scholar]
  13. Chaney, M. & Buck, M. ( 1999; ). The sigma 54 DNA-binding domain includes a determinant of enhancer responsiveness. Mol Microbiol 33, 1200–1209.
    [Google Scholar]
  14. Collier, L. S., Gaines, G. L., 3rd & Neidle, E. L. ( 1998; ). Regulation of benzoate degradation in Acinetobacter sp. strain ADP1 by BenM, a LysR-type transcriptional activator. J Bacteriol 180, 2493–2501.
    [Google Scholar]
  15. Cummings, J. H. ( 1995; ). Short chain fatty acids. In Human Colonic Bacteria: Role in Nutrition, Physiology and Pathology, pp. 101–105. Edited by G. Gibson & G. Macfarlane. London: CRC Press.
  16. Escalante-Semerena, J. C. & Roth, J. R. ( 1987; ). Regulation of cobalamin biosynthetic operons in Salmonella typhimurium. J Bacteriol 169, 2251–2258.
    [Google Scholar]
  17. Fernandez, S., de Lorenzo, V. & Perez-Martin, J. ( 1995; ). Activation of the transcriptional regulator XylR of Pseudomonas putida by release of repression between functional domains. Mol Microbiol 16, 205–213.[CrossRef]
    [Google Scholar]
  18. Garmendia, J. & de Lorenzo, V. ( 2000; ). The role of the interdomain B linker in the activation of the XylR protein of Pseudomonas putida. Mol Microbiol 38, 401–410.[CrossRef]
    [Google Scholar]
  19. Horswill, A. R. & Escalante-Semerena, J. C. ( 1997; ). Propionate catabolism in Salmonella typhimurium LT2: two divergently transcribed units comprise the prp locus at 8·5 centisomes, prpR encodes a member of the sigma-54 family of activators, and the prpBCDE genes constitute an operon. J Bacteriol 179, 928–940.
    [Google Scholar]
  20. Horswill, A. R. & Escalante-Semerena, J. C. ( 2001; ). In vitro conversion of propionate to pyruvate by Salmonella enterica enzymes: 2-methylcitrate dehydratase (PrpD) and aconitase enzymes catalyze the conversion of 2-methylcitrate to 2-methylisocitrate. Biochemistry 40, 4703–4713.[CrossRef]
    [Google Scholar]
  21. Horswill, A. R., Dudding, A. R. & Escalante-Semerena, J. C. ( 2001; ). Studies of propionate toxicity in Salmonella enterica identify 2-methylcitrate as a potent inhibitor of cell growth. J Biol Chem 276, 19094–19101.[CrossRef]
    [Google Scholar]
  22. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef]
    [Google Scholar]
  23. Lee, J. H., Scholl, D., Nixon, B. T. & Hoover, T. R. ( 1994; ). Constitutive ATP hydrolysis and transcription activation by a stable, truncated form of Rhizobium meliloti DCTD, a sigma 54-dependent transcriptional activator. J Biol Chem 269, 20401–20409.
    [Google Scholar]
  24. Lee, S. Y., De La Torre, A., Yan, D., Kustu, S., Nixon, B. T. & Wemmer, D. E. ( 2003; ). Regulation of the transcriptional activator NtrC1: structural studies of the regulatory and AAA+ ATPase domains. Genes Dev 17, 2552–2563.[CrossRef]
    [Google Scholar]
  25. Lupas, A., Van Dyke, M. & Stock, J. ( 1991; ). Predicting coiled coils from protein sequences. Science 252, 1162–1164.[CrossRef]
    [Google Scholar]
  26. Man, W.-J., Li, Y., Connor, C. D. & Wilton, D. C. ( 1995; ). The binding of propionyl-CoA and carboxymethyl-CoA to Escherichia coli citrate synthase. Biochim Biophys Acta 1250, 69–75.[CrossRef]
    [Google Scholar]
  27. Maruyama, K. & Kitamura, H. ( 1985; ). Mechanisms of growth inhibition by propionate and restoration of the growth by sodium bicarbonate or acetate in Rhodopseudomonas sphaeroides S. J Biochem (Tokyo) 98, 819–824.
    [Google Scholar]
  28. Morett, E. & Segovia, L. ( 1993; ). The σ 54 bacterial enhancer-binding protein family: mechanism of action and phylogenetic relationship of their functional domains. J Bacteriol 175, 6067–6074.
    [Google Scholar]
  29. O'Neill, E., Ng, L. C., Sze, C. C. & Shingler, V. ( 1998; ). Aromatic ligand binding and intramolecular signalling of the phenol-responsive sigma54-dependent regulator DmpR. Mol Microbiol 28, 131–141.
    [Google Scholar]
  30. O'Neill, E., Wikstrom, P. & Shingler, V. ( 2001; ). An active role for a structured B-linker in effector control of the sigma54-dependent regulator DmpR. EMBO J 20, 819–827.[CrossRef]
    [Google Scholar]
  31. Palacios, S. & Escalante-Semerena, J. C. ( 2000; ). prpR, ntrA, and ihf functions are required for expression of the prpBCDE operon, encoding enzymes that catabolize propionate in Salmonella enterica serovar typhimurium LT2. J Bacteriol 182, 905–910.[CrossRef]
    [Google Scholar]
  32. Perez-Martin, J. & De Lorenzo, V. ( 1995; ). The amino-terminal domain of the prokaryotic enhancer-binding protein XylR is a specific intramolecular repressor. Proc Natl Acad Sci U S A 92, 9392–9396.[CrossRef]
    [Google Scholar]
  33. Sasse, J. ( 1991; ). Detection of proteins. In Current Protocols in Molecular Biology, pp. 10.16.11–10.16.18. Edited by F. A. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith & K. Struhl. New York: Wiley Interscience.
  34. Schneider, D. A., Gaal, T. & Gourse, R. L. ( 2002; ). NTP-sensing by rRNA promoters in Escherichia coli is direct. Proc Natl Acad Sci U S A 99, 8602–8607.[CrossRef]
    [Google Scholar]
  35. Tang, X., Nakata, Y., Li, H.-O., Zhang, M., Gao, H., Fujita, A., Sakatsume, O., Ohta, T. & Yokoyama, K. ( 1994; ). The optimization of preparations of competent cells for transformation of E. coli. Nucleic Acid Res 22, 2857–2858.[CrossRef]
    [Google Scholar]
  36. Tsai, S. P., Hartin, R. J. & Ryu, J. ( 1989; ). Transformation in restriction-deficient Salmonella typhimurium LT2. J Gen Microbiol 135, 2561–2567.
    [Google Scholar]
  37. Tsang, A. W., Horswill, A. R. & Escalante-Semerena, J. C. ( 1998; ). Studies of regulation of expression of the propionate (prpBCDE) operon provide insights into how Salmonella typhimurium LT2 integrates its 1,2-propanediol and propionate catabolic pathways. J Bacteriol 180, 6511–6518.
    [Google Scholar]
  38. Vale, R. D. ( 2000; ). AAA proteins. Lords of the ring. J Cell Biol 150, F13–19.[CrossRef]
    [Google Scholar]
  39. Weiss, D. S., Batut, J., Klose, K. E., Keener, J. & Kustu, S. ( 1991; ). The phosphorylated form of the enhancer-binding protein NTRC has an ATPase activity that is essential for activation of transcription. Cell 67, 155–167.[CrossRef]
    [Google Scholar]
  40. Wyman, C., Rombel, I., North, A. K., Bustamante, C. & Kustu, S. ( 1997; ). Unusual oligomerization required for activity of NtrC, a bacterial enhancer-binding protein. Science 275, 1658–1661.[CrossRef]
    [Google Scholar]
  41. Xu, H. & Hoover, T. R. ( 2001; ). Transcriptional regulation at a distance in bacteria. Curr Opin Microbiol 4, 138–144.[CrossRef]
    [Google Scholar]
  42. Zhang, X., Chaney, M., Wigneshweraraj, S. R., Schumacher, J., Bordes, P., Cannon, W. & Buck, M. ( 2002; ). Mechanochemical ATPases and transcriptional activation. Mol Microbiol 45, 895–903.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27299-0
Loading
/content/journal/micro/10.1099/mic.0.27299-0
Loading

Data & Media loading...

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