1887

Abstract

The human opportunistic pathogen is the major cause of morbidity and mortality of cystic fibrosis patients and is responsible for a variety of infections in compromised hosts. Using PCR-based signature-tagged mutagenesis, we identified a STM5437 mutant with an insertion into the PA5437 gene (called for putative pyruvate carboxylase regulator). PycR inactivation results in 100 000-fold attenuation of virulence in the rat lung . PycR has the signature of a transcriptional regulator with a predicted helix–turn–helix motif binding to a typical LysR DNA binding site in the PA5436 ()–PA5437 () intercistronic region. Two pyruvate carboxylase subunits ( and ) are divergently transcribed upstream of . Transcriptional start sites of and are located at −127 and −88 bp upstream of their initiation codons with Shine–Dalgarno and putative promoter sequences containing −10 and −35 sequences. The DNA binding of PycR was confirmed by DNA mobility shift assay. Genome-wide transcriptional profiling and quantitative real-time PCR (qRT-PCR) indicated that the genes differentially regulated by PycR include two pyruvate carboxylase genes and genes necessary for lipid metabolism, lipolytic activity, anaerobic respiration and biofilm formation. PycR is a regulator with pleiotropic effects on virulence factors, such as lipase and esterase expression and biofilm formation, which are important for maintenance of in chronic lung infection.

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2008-07-01
2019-11-16
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References

  1. Arai, H., Mizutani, M. & Igarashi, Y. ( 2003; ). Transcriptional regulation of the nos genes for nitrous oxide reductase in Pseudomonas aeruginosa. Microbiology 149, 29–36.[CrossRef]
    [Google Scholar]
  2. Baek, S. H., Rajashekara, G., Splitter, G. A. & Shapleigh, J. P. ( 2004; ). Denitrification genes regulate Brucella virulence in mice. J Bacteriol 186, 6025–6031.[CrossRef]
    [Google Scholar]
  3. Bochner, B. R., Gadzinski, P. & Panomitros, E. ( 2001; ). Phenotype microarrays for high-throughput phenotypic testing and assay of gene function. Genome Res 11, 1246–1255.[CrossRef]
    [Google Scholar]
  4. Bowtell, D. & Sambrook, J. ( 2003; ). DNA Microarrays: a Molecular Cloning Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  5. Branson, J. P., Nezic, M., Wallace, J. C. & Attwood, P. V. ( 2002; ). Kinetic characterization of yeast pyruvate carboxylase isozyme Pyc1. Biochemistry 41, 4459–4466.[CrossRef]
    [Google Scholar]
  6. Carty, N. L., Rumbaugh, K. P. & Hamood, A. N. ( 2003; ). Regulation of toxA by PtxR in Pseudomonas aeruginosa PA103. Can J Microbiol 49, 450–464.[CrossRef]
    [Google Scholar]
  7. Cash, H. A., Woods, D. E., McCullough, B., Johanson, W. G., Jr & Bass, J. A. ( 1979; ). A rat model of chronic respiratory infection with Pseudomonas aeruginosa. Am Rev Respir Dis 119, 453–459.
    [Google Scholar]
  8. deHaseth, P. L., Zupancic, M. L. & Record, M. T., Jr ( 1998; ). RNA polymerase–promoter interactions: the comings and goings of RNA polymerase. J Bacteriol 180, 3019–3025.
    [Google Scholar]
  9. Delic-Attree, I., Toussaint, B., Garin, J. & Vignais, P. M. ( 1997; ). Cloning, sequence and mutagenesis of the structural gene of Pseudomonas aeruginosa CysB, which can activate algD transcription. Mol Microbiol 24, 1275–1284.[CrossRef]
    [Google Scholar]
  10. Filiatrault, M. J., Wagner, V. E., Bushnell, D., Haidaris, C. G., Iglewski, B. H. & Passador, L. ( 2005; ). Effect of anaerobiosis and nitrate on gene expression in Pseudomonas aeruginosa. Infect Immun 73, 3764–3772.[CrossRef]
    [Google Scholar]
  11. Firoved, A. M., Wood, S. R., Ornatowski, W., Deretic, V. & Timmins, G. S. ( 2004; ). Microarray analysis and functional characterization of the nitrosative stress response in nonmucoid and mucoid Pseudomonas aeruginosa. J Bacteriol 186, 4046–4050.[CrossRef]
    [Google Scholar]
  12. Hoang, T. T., Karkhoff-Schweizer, R. R., Kutchma, A. J. & Schweizer, H. P. ( 1998; ). A broad-host-range Flp–FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212, 77–86.[CrossRef]
    [Google Scholar]
  13. Holloway, B. W., Krishnapillai, V. & Morgan, A. F. ( 1979; ). Chromosomal genetics of Pseudomonas. Microbiol Rev 43, 73–102.
    [Google Scholar]
  14. Jaeger, K. E., Dijkstra, B. W. & Reetz, M. T. ( 1999; ). Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annu Rev Microbiol 53, 315–351.[CrossRef]
    [Google Scholar]
  15. Jitrapakdee, S. & Wallace, J. C. ( 1999; ). Structure, function and regulation of pyruvate carboxylase. Biochem J 340, 1–16.[CrossRef]
    [Google Scholar]
  16. Kolenbrander, P. E. & Andersen, R. N. ( 1989; ). Inhibition of coaggregation between Fusobacterium nucleatum and Porphyromonas (Bacteroides) gingivalis by lactose and related sugars. Infect Immun 57, 3204–3209.
    [Google Scholar]
  17. Kovacikova, G., Lin, W. & Skorupski, K. ( 2005; ). Dual regulation of genes involved in acetoin biosynthesis and motility/biofilm formation by the virulence activator AphA and the acetate-responsive LysR-type regulator AlsR in Vibrio cholerae. Mol Microbiol 57, 420–433.[CrossRef]
    [Google Scholar]
  18. Lai, H., Kraszewski, J. L., Purwantini, E. & Mukhopadhyay, B. ( 2006; ). Identification of pyruvate carboxylase genes in Pseudomonas aeruginosa PAO1 and development of a P. aeruginosa-based overexpression system for α4- and α4β4-type pyruvate carboxylases. Appl Environ Microbiol 72, 7785–7792.[CrossRef]
    [Google Scholar]
  19. Lehoux, D. E., Sanschagrin, F. & Levesque, R. C. ( 2002; ). Identification of in vivo essential genes from Pseudomonas aeruginosa by PCR-based signature-tagged mutagenesis. FEMS Microbiol Lett 210, 73–80.[CrossRef]
    [Google Scholar]
  20. Lehoux, D. E., Sanschagrin, F., Kukavica-Ibrulj, I., Potvin, E. & Levesque, R. C. ( 2004; ). Identification of novel pathogenicity genes by PCR signature-tagged mutagenesis and related technologies. Methods Mol Biol 266, 289–304.
    [Google Scholar]
  21. Lim, F., Morris, C. P., Occhiodoro, F. & Wallace, J. C. ( 1988; ). Sequence and domain structure of yeast pyruvate carboxylase. J Biol Chem 263, 11493–11497.
    [Google Scholar]
  22. Lizewski, S. E., Schurr, J. R., Jackson, D. W., Frisk, A., Carterson, A. J. & Schurr, M. J. ( 2004; ). Identification of AlgR-regulated genes in Pseudomonas aeruginosa by use of microarray analysis. J Bacteriol 186, 5672–5684.[CrossRef]
    [Google Scholar]
  23. Mukhopadhyay, B., Stoddard, S. F. & Wolfe, R. S. ( 1998; ). Purification, regulation, and molecular and biochemical characterization of pyruvate carboxylase from Methanobacterium thermoautotrophicum strain ΔH. J Biol Chem 273, 5155–5166.[CrossRef]
    [Google Scholar]
  24. O'Toole, G. A. & Kolter, R. ( 1998a; ). Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol Microbiol 28, 449–461.[CrossRef]
    [Google Scholar]
  25. O'Toole, G. A. & Kolter, R. ( 1998b; ). Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol 30, 295–304.[CrossRef]
    [Google Scholar]
  26. Phibbs, P. V., Jr, Feary, T. W. & Blevins, W. T. ( 1974; ). Pyruvate carboxylase deficiency in pleiotropic carbohydrate-negative mutant strains of Pseudomonas aeruginosa. J Bacteriol 118, 999–1009.
    [Google Scholar]
  27. Philippot, L. ( 2005; ). Denitrification in pathogenic bacteria: for better or worst? Trends Microbiol 13, 191–192.[CrossRef]
    [Google Scholar]
  28. Potvin, E., Lehoux, D. E., Kukavica-Ibrulj, I., Richard, K. L., Sanschagrin, F., Lau, G. W. & Levesque, R. C. ( 2003; ). In vivo functional genomics of Pseudomonas aeruginosa for high-throughput screening of new virulence factors and antibacterial targets. Environ Microbiol 5, 1294–1308.[CrossRef]
    [Google Scholar]
  29. Rosenau, F. & Jaeger, K. ( 2000; ). Bacterial lipases from Pseudomonas: regulation of gene expression and mechanisms of secretion. Biochimie 82, 1023–1032.[CrossRef]
    [Google Scholar]
  30. Rozen, S. & Skaletsky, H. ( 2000; ). Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132, 365–386.
    [Google Scholar]
  31. Rukholm, G., Mugabe, C., Azghani, A. O. & Omri, A. ( 2006; ). Antibacterial activity of liposomal gentamicin against Pseudomonas aeruginosa: a time-kill study. Int J Antimicrob Agents 27, 247–252.[CrossRef]
    [Google Scholar]
  32. Sambrook, J. & Russell, D. W. ( 2001; ). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  33. Samols, D., Thornton, C. G., Murtif, V. L., Kumar, G. K., Haase, F. C. & Wood, H. G. ( 1988; ). Evolutionary conservation among biotin enzymes. J Biol Chem 263, 6461–6464.
    [Google Scholar]
  34. Schell, M. A. ( 1993; ). Molecular biology of the LysR family of transcriptional regulators. Annu Rev Microbiol 47, 597–626.[CrossRef]
    [Google Scholar]
  35. Schuster, M., Lostroh, C. P., Ogi, T. & Greenberg, E. P. ( 2003; ). Identification, timing, and signal specificity of Pseudomonas aeruginosa quorum-controlled genes: a transcriptome analysis. J Bacteriol 185, 2066–2079.[CrossRef]
    [Google Scholar]
  36. Schuster, M., Urbanowski, M. L. & Greenberg, E. P. ( 2004; ). Promoter specificity in Pseudomonas aeruginosa quorum sensing revealed by DNA binding of purified LasR. Proc Natl Acad Sci U S A 101, 15833–15839.[CrossRef]
    [Google Scholar]
  37. Segura, D. & Espin, G. ( 2004; ). Inactivation of pycA, encoding pyruvate carboxylase activity, increases poly-β-hydroxybutyrate accumulation in Azotobacter vinelandii on solid medium. Appl Microbiol Biotechnol 65, 414–418.[CrossRef]
    [Google Scholar]
  38. Shan, Z., Xu, H., Shi, X., Yu, Y., Yao, H., Zhang, X., Bai, Y., Gao, C., Saris, P. E. & Qiao, M. ( 2004; ). Identification of two new genes involved in twitching motility in Pseudomonas aeruginosa. Microbiology 150, 2653–2661.[CrossRef]
    [Google Scholar]
  39. Simon, R., Priefer, U. & Pühler, A. ( 1983; ). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Bio/technology (NY) 1, 784–791.[CrossRef]
    [Google Scholar]
  40. Stehr, F., Kretschmar, M., Kröger, C., Hube, B. & Schäfer, W. ( 2003; ). Microbial lipases as virulence factors. J Mol Catal, B Enzym 22, 347–355.[CrossRef]
    [Google Scholar]
  41. Vallet, I., Olson, J. W., Lory, S., Lazdunski, A. & Filloux, A. ( 2001; ). The chaperone/usher pathways of Pseudomonas aeruginosa: identification of fimbrial gene clusters (cup) and their involvement in biofilm formation. Proc Natl Acad Sci U S A 98, 6911–6916.[CrossRef]
    [Google Scholar]
  42. van Heeckeren, A. M. & Schluchter, M. D. ( 2002; ). Murine models of chronic Pseudomonas aeruginosa lung infection. Lab Anim 36, 291–312.[CrossRef]
    [Google Scholar]
  43. Wade, D. S., Calfee, M. W., Rocha, E. R., Ling, E. A., Engstrom, E., Coleman, J. P. & Pesci, E. C. ( 2005; ). Regulation of Pseudomonas quinolone signal synthesis in Pseudomonas aeruginosa. J Bacteriol 187, 4372–4380.[CrossRef]
    [Google Scholar]
  44. Waite, R. D., Papakonstantinopoulou, A., Littler, E. & Curtis, M. A. ( 2005; ). Transcriptome analysis of Pseudomonas aeruginosa growth: comparison of gene expression in planktonic cultures and developing and mature biofilms. J Bacteriol 187, 6571–6576.[CrossRef]
    [Google Scholar]
  45. West, S. E., Schweizer, H. P., Dall, C., Sample, A. K. & Runyen-Janecky, L. J. ( 1994; ). Construction of improved Escherichia–Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 148, 81–86.[CrossRef]
    [Google Scholar]
  46. Wilhelm, S., Tommassen, J. & Jaeger, K. E. ( 1999; ). A novel lipolytic enzyme located in the outer membrane of Pseudomonas aeruginosa. J Bacteriol 181, 6977–6986.
    [Google Scholar]
  47. Yoon, S. S., Hennigan, R. F., Hilliard, G. M., Ochsner, U. A., Parvatiyar, K., Kamani, M. C., Allen, H. L., DeKievit, T. R., Gardner, P. R. & other authors ( 2002; ). Pseudomonas aeruginosa anaerobic respiration in biofilms: relationships to cystic fibrosis pathogenesis. Dev Cell 3, 593–603.[CrossRef]
    [Google Scholar]
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