1887

Abstract

The exopolyphosphatase (Ppx) of is encoded by the PA5241 gene (). Ppx catalyses the hydrolysis of inorganic polyphosphates to orthophosphate (P). In the present work, we identified and characterized the promoter region of and its regulation under environmental stress conditions. The role of Ppx in the production of several virulence factors was demonstrated through studies performed on a null mutant. We found that is under the control of two interspaced promoters, dually regulated by nitrogen and phosphate limitation. Under nitrogen-limiting conditions, its expression was controlled from a σ-dependent promoter activated by the response regulator NtrC. However, under P limitation, the expression was controlled from a σ promoter, activated by PhoB. Results obtained from the null mutant demonstrated that Ppx is involved in the production of virulence factors associated with both acute infection (e.g. motility-promoting factors, blue/green pigment production, C6–C12 quorum-sensing homoserine lactones) and chronic infection (e.g. rhamnolipids, biofilm formation). Molecular and physiological approaches used in this study indicated that maintains consistently proper levels of Ppx regardless of environmental conditions. The precise control of expression appeared to be essential for the survival of and the occurrence of either acute or chronic infection in the host.

Funding
This study was supported by the:
  • SECYT-UNRC
  • MinCyT-Cba
  • CONICET, Argentina
  • DGAPA
  • UNAM
  • CONACyT, México
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2014-02-01
2021-10-26
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References

  1. Achbergerová L., Nahálka J. ( 2011). Polyphosphate – an ancient energy source and active metabolic regulator. Microb Cell Fact 10:63 [View Article][PubMed]
    [Google Scholar]
  2. Akiyama M., Crooke E., Kornberg A. ( 1993). An exopolyphosphatase of Escherichia coli. The enzyme and its ppx gene in a polyphosphate operon. J Biol Chem 268:633–639[PubMed]
    [Google Scholar]
  3. Ault-Riché D., Fraley C. D., Tzeng C. M., Kornberg A. ( 1998). Novel assay reveals multiple pathways regulating stress-induced accumulations of inorganic polyphosphate in Escherichia coli . J Bacteriol 180:1841–1847[PubMed]
    [Google Scholar]
  4. Barrios H., Valderrama B., Morett E. ( 1999). Compilation and analysis of σ54-dependent promoter sequences. Nucleic Acids Res 27:4305–4313 [View Article][PubMed]
    [Google Scholar]
  5. Beassoni P. R., Otero L. H., Lisa A. T., Domenech C. E. ( 2008). Using a molecular model and kinetic experiments in the presence of divalent cations to study the active site and catalysis of Pseudomonas aeruginosa phosphorylcholine phosphatase. Biochim Biophys Acta 1784:2038–2044 [View Article][PubMed]
    [Google Scholar]
  6. Blanco A. G., Canals A., Bernués J., Solà M., Coll M. ( 2011). The structure of a transcription activation subcomplex reveals how σ70 is recruited to PhoB promoters. EMBO J 30:3776–3785 [View Article][PubMed]
    [Google Scholar]
  7. Caiazza N. C., Shanks R. M., O’Toole G. A. ( 2005). Rhamnolipids modulate swarming motility patterns of Pseudomonas aeruginosa . J Bacteriol 187:7351–7361 [View Article][PubMed]
    [Google Scholar]
  8. Cha C. E., Gao P., Chen Y. C., Shaw P. D., Farrand S. K. ( 1998). Production of acyl-homoserine lactone quorum-sensing signals by Gram-negative plant-associated bacteria. Mol Plant Microbe Interact 11:1119–1129 [View Article][PubMed]
    [Google Scholar]
  9. Choi K. H., Schweizer H. P. ( 2005). An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants. BMC Microbiol 5:30 [View Article][PubMed]
    [Google Scholar]
  10. Choi K. H., Schweizer H. P. ( 2006). mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa . Nat Protocols 1:153–161 [CrossRef]
    [Google Scholar]
  11. Choi K. H., Kumar A., Schweizer H. P. ( 2006). A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J Microbiol Methods 64:391–397 [View Article][PubMed]
    [Google Scholar]
  12. Dacheux D., Epaulard O., de Groot A., Guery B., Leberre R., Attree I., Polack B., Toussaint B. ( 2002). Activation of the Pseudomonas aeruginosa type III secretion system requires an intact pyruvate dehydrogenase aceAB operon. Infect Immun 70:3973–3977 [View Article][PubMed]
    [Google Scholar]
  13. Del Peso-Santos T., Landfors M., Skärfstad E., Ryden P., Shingler V. ( 2012). Pr is a member of a restricted class of σ70-dependent promoters that lack a recognizable –10 element. Nucleic Acids Res 40:11308–11320 [View Article][PubMed]
    [Google Scholar]
  14. Delic-Attree I., Toussaint B., Froger A., Willison J. C., Vignais P. M. ( 1996). Isolation of an IHF-deficient mutant of a Pseudomonas aeruginosa mucoid isolate and evaluation of the role of IHF in algD gene expression. Microbiology 142:2785–2793 [View Article][PubMed]
    [Google Scholar]
  15. Dubois M., Gilles K. A., Hamilton J. K., Rebers P. A., Smith F. ( 1956). Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356 [View Article]
    [Google Scholar]
  16. Essar D. W., Eberly L., Hadero A., Crawford I. P. ( 1990). Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications. J Bacteriol 172:884–900[PubMed]
    [Google Scholar]
  17. Geissdörfer W., Ratajczak A., Hillen W. ( 1998). Transcription of ppk from Acinetobacter sp. strain ADP1, encoding a putative polyphosphate kinase, is induced by phosphate starvation. Appl Environ Microbiol 64:896–901[PubMed]
    [Google Scholar]
  18. Hervás A. B., Canosa I., Santero E. ( 2008). Transcriptome analysis of Pseudomonas putida in response to nitrogen availability. J Bacteriol 190:416–420 [View Article][PubMed]
    [Google Scholar]
  19. Heurlier K., Dénervaud V., Pessi G., Reimmann C., Haas D. ( 2003). Negative control of quorum sensing by RpoN (σ54) in Pseudomonas aeruginosa PAO1. J Bacteriol 185:2227–2235 [View Article][PubMed]
    [Google Scholar]
  20. Ishige K., Zhang H., Kornberg A. ( 2002). Polyphosphate kinase (PPK2), a potent, polyphosphate-driven generator of GTP. Proc Natl Acad Sci U S A 99:16684–16688 [View Article][PubMed]
    [Google Scholar]
  21. Jimenez P. N., Koch G., Thompson J. A., Xavier K. B., Cool R. H., Quax W. J. ( 2012). The multiple signaling systems regulating virulence in Pseudomonas aeruginosa . Microbiol Mol Biol Rev 76:46–65 [View Article][PubMed]
    [Google Scholar]
  22. Jishage M., Kvint K., Shingler V., Nyström T. ( 2002). Regulation of sigma factor competition by the alarmone ppGpp. Genes Dev 16:1260–1270 [View Article][PubMed]
    [Google Scholar]
  23. Kato J., Yamamoto T., Yamada K., Ohtake H. ( 1993). Cloning, sequence and characterization of the polyphosphate kinase-encoding gene (ppk) of Klebsiella aerogenes . Gene 137:237–242 [View Article][PubMed]
    [Google Scholar]
  24. Kornberg A., Rao N. N., Ault-Riché D. ( 1999). Inorganic polyphosphate: a molecule of many functions. Annu Rev Biochem 68:89–125 [View Article][PubMed]
    [Google Scholar]
  25. Lee S.-J., Lee Y.-S., Lee Y.-C., Choi Y.-L. ( 2006). Molecular characterization of polyphosphate (PolyP) operon from Serratia marcescens . J Basic Microbiol 46:108–115 [View Article][PubMed]
    [Google Scholar]
  26. Leech A. J., Sprinkle A., Wood L., Wozniak D. J., Ohman D. E. ( 2008). The NtrC family regulator AlgB, which controls alginate biosynthesis in mucoid Pseudomonas aeruginosa, binds directly to the algD promoter. J Bacteriol 190:581–589 [View Article][PubMed]
    [Google Scholar]
  27. Li W., Lu C. D. ( 2007). Regulation of carbon and nitrogen utilization by CbrAB and NtrBC two-component systems in Pseudomonas aeruginosa . J Bacteriol 189:5413–5420 [View Article][PubMed]
    [Google Scholar]
  28. Lisa T. A., Lucchesi G. I., Domenech C. E. ( 1994). Pathogenicity of Pseudomonas aeruginosa and its relationship to the choline metabolism through the action of cholinesterase, acid phosphatase, and phospholipase C. Curr Microbiol 29:193–199 [View Article]
    [Google Scholar]
  29. Lisa A. T., Beassoni P. R., Masssimelli M. J., Otero L. H., Domenech C. E. ( 2007). A glance on Pseudomonas aeruginosa phosphorylcholine phosphatase, an enzyme whose synthesis depends on the presence of choline in its environment. Communicating Current Research and Educational Topics and Trends in Applied Microbiology vol. 1255–262 Méndez-Vilas A. Badajoz: Formatex Press;
    [Google Scholar]
  30. Long J., Zaborina O., Holbrook C., Zaborin A., Alverdy J. ( 2008). Depletion of intestinal phosphate after operative injury activates the virulence of P aeruginosa causing lethal gut-derived sepsis. Surgery 144:189–197 [View Article][PubMed]
    [Google Scholar]
  31. Lucchesi G. I., Lisa T. A., Domenech C. E. ( 1989). Choline and betaine as inducer agents of Pseudomonas aeruginosa phospholipase C activity in high phosphate medium. FEMS Microbiol Lett 57:335–338 [View Article][PubMed]
    [Google Scholar]
  32. Makino K., Amemura M., Kawamoto T., Kimura S., Shinagawa H., Nakata A., Suzuki M. ( 1996). DNA binding of PhoB and its interaction with RNA polymerase. J Mol Biol 259:15–26 [View Article][PubMed]
    [Google Scholar]
  33. Massimelli M. J., Sánchez D. G., Buchieri M. V., Olvera L., Beassoni P. R., Schweizer H. P., Morett E., Lisa A. T. ( 2011). Choline catabolism, σ54 factor and NtrC are required for the full expression of the Pseudomonas aeruginosa phosphorylcholine phosphatase gene. Microbiol Res 166:380–390 [View Article][PubMed]
    [Google Scholar]
  34. Mendoza-Vargas A., Olvera L., Olvera M., Grande R., Vega-Alvarado L., Taboada B., Jimenez-Jacinto V., Salgado H., Juárez K. & other authors ( 2009). Genome-wide identification of transcription start sites, promoters and transcription factor binding sites in E. coli . PLoS ONE 4:e7526 [View Article][PubMed]
    [Google Scholar]
  35. Miller J. H. ( 1972). Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press;
    [Google Scholar]
  36. Monds R. D., Newell P. D., Schwartzman J. A., O’Toole G. A. ( 2006). Conservation of the Pho regulon in Pseudomonas fluorescens Pf0-1. Appl Environ Microbiol 72:1910–1924 [View Article][PubMed]
    [Google Scholar]
  37. Morett E., Segovia L. ( 1993). The sigma 54 bacterial enhancer-binding protein family: mechanism of action and phylogenetic relationship of their functional domains. J Bacteriol 175:6067–6074[PubMed]
    [Google Scholar]
  38. Münch R., Hiller K., Barg H., Heldt D., Linz S., Wingender E., Jahn D. ( 2003). PRODORIC: prokaryotic database of gene regulation. Nucleic Acids Res 31:266–269 [View Article][PubMed]
    [Google Scholar]
  39. Nievas F., Bogino P., Sorroche F., Giordano W. ( 2012). Detection, characterization, and biological effect of quorum-sensing signaling molecules in peanut-nodulating bradyrhizobia. Sensors (Basel) 12:2851–2873 [View Article][PubMed]
    [Google Scholar]
  40. Österberg S., del Peso-Santos T., Shingler V. ( 2011). Regulation of alternative sigma factor use. Annu Rev Microbiol 65:37–55 [View Article][PubMed]
    [Google Scholar]
  41. Rao N. N., Liu S., Kornberg A. ( 1998). Inorganic polyphosphate in Escherichia coli: the phosphate regulon and the stringent response. J Bacteriol 180:2186–2193[PubMed]
    [Google Scholar]
  42. Rao N. N., Gómez-García M. R., Kornberg A. ( 2009). Inorganic polyphosphate: essential for growth and survival. Annu Rev Biochem 78:605–647 [View Article][PubMed]
    [Google Scholar]
  43. Rashid M. H., Kornberg A. ( 2000). Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa . Proc Natl Acad Sci U S A 97:4885–4890 [View Article][PubMed]
    [Google Scholar]
  44. Rashid M. H., Rao N. N., Kornberg A. ( 2000a). Inorganic polyphosphate is required for motility of bacterial pathogens. J Bacteriol 182:225–227 [View Article][PubMed]
    [Google Scholar]
  45. Rashid M. H., Rumbaugh K., Passador L., Davies D. G., Hamood A. N., Iglewski B. H., Kornberg A. ( 2000b). Polyphosphate kinase is essential for biofilm development, quorum sensing, and virulence of Pseudomonas aeruginosa . Proc Natl Acad Sci U S A 97:9636–9641 [View Article][PubMed]
    [Google Scholar]
  46. Sambrook J., Russell D. W. ( 2001). Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press;
    [Google Scholar]
  47. Sánchez D. G., Otero L. H., Hernández C. M., Serra A. L., Encarnación S., Domenech C. E., Lisa A. T. ( 2012). A Pseudomonas aeruginosa PAO1 acetylcholinesterase is encoded by the PA4921 gene and belongs to the SGNH hydrolase family. Microbiol Res 167:317–325 [View Article][PubMed]
    [Google Scholar]
  48. Shi X., Rao N. N., Kornberg A. ( 2004). Inorganic polyphosphate in Bacillus cereus: motility, biofilm formation, and sporulation. Proc Natl Acad Sci U S A 101:17061–17065 [View Article][PubMed]
    [Google Scholar]
  49. Shinagawa H., Makino K., Amemura M., Nakata A. ( 1987). Structure and function of the regulatory genes for the phosphate regulon in Escherichia coli . Phosphate Metabolism and Cellular Regulation in Microorganisms20–25 Torriani-Gorini A., Rothman F. G., Silver S., Wright A., Yagil E. Washington, DC: American Society for Microbiology Press;
    [Google Scholar]
  50. Silby M. W., Nicoll J. S., Levy S. B. ( 2009). Requirement of polyphosphate by Pseudomonas fluorescens Pf0-1 for competitive fitness and heat tolerance in laboratory media and sterile soil. Appl Environ Microbiol 75:3872–3881 [View Article][PubMed]
    [Google Scholar]
  51. Silva S. N., Farías C. B., Rufino R. D., Luna J. M., Sarubbo L. A. ( 2010). Glycerol as substrate for the production of biosurfactant by Pseudomonas aeruginosa UCP0992. Colloids Surf B Biointerfaces 79:174–183 [View Article][PubMed]
    [Google Scholar]
  52. Son M. S., Matthews W. J. Jr, Kang Y., Nguyen D. T., Hoang T. T. ( 2007). In vivo evidence of Pseudomonas aeruginosa nutrient acquisition and pathogenesis in the lungs of cystic fibrosis patients. Infect Immun 75:5313–5324 [View Article][PubMed]
    [Google Scholar]
  53. Sonnleitner E., Abdou L., Haas D. ( 2009). Small RNA as global regulator of carbon catabolite repression in Pseudomonas aeruginosa . Proc Natl Acad Sci U S A 106:21866–21871 [View Article][PubMed]
    [Google Scholar]
  54. Studholme D. J., Dixon R. ( 2003). Domain architectures of σ54-dependent transcriptional activators. J Bacteriol 185:1757–1767 [View Article][PubMed]
    [Google Scholar]
  55. Thayil S. M., Morrison N., Schechter N., Rubin H., Karakousis P. C. ( 2011). The role of the novel exopolyphosphatase MT0516 in Mycobacterium tuberculosis drug tolerance and persistence. PLoS ONE 6:e28076 [View Article][PubMed]
    [Google Scholar]
  56. Wang L., Gralla J. D. ( 1998). Multiple in vivo roles for the –12-region elements of sigma 54 promoters. J Bacteriol 180:5626–5631[PubMed]
    [Google Scholar]
  57. Yeom J., Park W. ( 2012). Pleiotropic effects of the mioC mutation on the physiology of Pseudomonas aeruginosa PAO1. FEMS Microbiol Lett 335:47–57 [View Article][PubMed]
    [Google Scholar]
  58. Zaborin A., Romanowski K., Gerdes S., Holbrook C., Lepine F., Long J., Poroyko V., Diggle S. P., Wilke A. & other authors ( 2009). Red death in Caenorhabditis elegans caused by Pseudomonas aeruginosa PAO1. Proc Natl Acad Sci U S A 106:6327–6332 [View Article][PubMed]
    [Google Scholar]
  59. Zago A., Chugani S., Chakrabarty A. M. ( 1999). Cloning and characterization of polyphosphate kinase and exopolyphosphatase genes from Pseudomonas aeruginosa 8830. J Bacteriol 182:6687–6693
    [Google Scholar]
  60. Zhang Q., Li Y., Tang C. M. ( 2010). The role of the exopolyphosphatase PPX in avoidance by Neisseria meningitidis of complement-mediated killing. J Biol Chem 285:34259–34268 [View Article][PubMed]
    [Google Scholar]
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