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. 1994 Dec;176(24):7532–7542. doi: 10.1128/jb.176.24.7532-7542.1994

The vfr gene product, required for Pseudomonas aeruginosa exotoxin A and protease production, belongs to the cyclic AMP receptor protein family.

S E West 1, A K Sample 1, L J Runyen-Janecky 1
PMCID: PMC197210  PMID: 8002577

Abstract

The synthesis of exotoxin A (ETA) by Pseudomonas aeruginosa is a complex, regulated event. Several ETA putative regulatory mutants of P. aeruginosa PA103 have previously been characterized (S. E. H. West, S. A. Kaye, A. N. Hamood, and B. H. Iglewski, Infect. Immun. 62:897-903, 1994). In addition to ETA production, these mutants, PA103-15, PA103-16, and PA103-19, were also deficient in the production of protease and in regA P1 promoter activity. RegA is a positive regulator of ETA transcription. We cloned a gene, designated vfr for virulence factor regulator, that restored ETA and protease production to parental levels in these mutants. In addition, transcription from the regA P1 promoter was restored. In Escherichia coli, when vfr was overexpressed from a phage T7 promoter, a protein with an apparent molecular mass of 28.5 kDa was produced. Analysis of the deduced amino acid sequence of vfr revealed that the expected protein is 67% identical and 91% similar over a 202-amino-acid overlap to the E. coli cyclic AMP receptor protein (CAP or Crp). The cloned vfr gene complemented the beta-galactosidase- and tryptophanase-deficient phenotypes of E. coli RZ1331, a crp deletion mutant. However, the E. coli crp gene under the control of the tac promoter did not complement the ETA-deficient or protease-deficient phenotype of PA103-15 or PA103-16. The ability of vfr to restore both ETA and protease production to these mutants suggests that vfr is a global regulator of virulence factor expression in P. aeruginosa.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allen L. N., Hanson R. S. Construction of broad-host-range cosmid cloning vectors: identification of genes necessary for growth of Methylobacterium organophilum on methanol. J Bacteriol. 1985 Mar;161(3):955–962. doi: 10.1128/jb.161.3.955-962.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bilezikian J. P., Kaempfer R. O., Magasanik B. Mechanism of tryptophanase induction in Escherichia coli. J Mol Biol. 1967 Aug 14;27(3):495–506. doi: 10.1016/0022-2836(67)90054-x. [DOI] [PubMed] [Google Scholar]
  3. Blake M. S., Johnston K. H., Russell-Jones G. J., Gotschlich E. C. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem. 1984 Jan;136(1):175–179. doi: 10.1016/0003-2697(84)90320-8. [DOI] [PubMed] [Google Scholar]
  4. Blumentals I. I., Kelly R. M., Gorziglia M., Kaufman J. B., Shiloach J. Development of a defined medium and two-step culturing method for improved exotoxin A yields from Pseudomonas aeruginosa. Appl Environ Microbiol. 1987 Sep;53(9):2013–2020. doi: 10.1128/aem.53.9.2013-2020.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Botsford J. L., Harman J. G. Cyclic AMP in prokaryotes. Microbiol Rev. 1992 Mar;56(1):100–122. doi: 10.1128/mr.56.1.100-122.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  7. Chung D. W., Collier R. J. Enzymatically active peptide from the adenosine diphosphate-ribosylating toxin of Pseudomonas aeruginosa. Infect Immun. 1977 Jun;16(3):832–841. doi: 10.1128/iai.16.3.832-841.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. DeVault J. D., Hendrickson W., Kato J., Chakrabarty A. M. Environmentally regulated algD promoter is responsive to the cAMP receptor protein in Escherichia coli. Mol Microbiol. 1991 Oct;5(10):2503–2509. doi: 10.1111/j.1365-2958.1991.tb02096.x. [DOI] [PubMed] [Google Scholar]
  9. Ebright R. H., Cossart P., Gicquel-Sanzey B., Beckwith J. Molecular basis of DNA sequence recognition by the catabolite gene activator protein: detailed inferences from three mutations that alter DNA sequence specificity. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7274–7278. doi: 10.1073/pnas.81.23.7274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ebright R. H., Kolb A., Buc H., Kunkel T. A., Krakow J. S., Beckwith J. Role of glutamic acid-181 in DNA-sequence recognition by the catabolite gene activator protein (CAP) of Escherichia coli: altered DNA-sequence-recognition properties of [Val181]CAP and [Leu181]CAP. Proc Natl Acad Sci U S A. 1987 Sep;84(17):6083–6087. doi: 10.1073/pnas.84.17.6083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Eschenlauer A. C., Reznikoff W. S. Escherichia coli catabolite gene activator protein mutants defective in positive control of lac operon transcription. J Bacteriol. 1991 Aug;173(16):5024–5029. doi: 10.1128/jb.173.16.5024-5029.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Essar D. W., Eberly L., Han C. Y., Crawford I. P. DNA sequences and characterization of four early genes of the tryptophan pathway in Pseudomonas aeruginosa. J Bacteriol. 1990 Feb;172(2):853–866. doi: 10.1128/jb.172.2.853-866.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Farinha M. A., Kropinski A. M. Construction of broad-host-range vectors for general cloning and promoter selection in Pseudomonas and Escherichia coli. Gene. 1989 Apr 30;77(2):205–210. doi: 10.1016/0378-1119(89)90068-1. [DOI] [PubMed] [Google Scholar]
  14. Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Frank D. W., Iglewski B. H. Kinetics of toxA and regA mRNA accumulation in Pseudomonas aeruginosa. J Bacteriol. 1988 Oct;170(10):4477–4483. doi: 10.1128/jb.170.10.4477-4483.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Frank D. W., Storey D. G., Hindahl M. S., Iglewski B. H. Differential regulation by iron of regA and toxA transcript accumulation in Pseudomonas aeruginosa. J Bacteriol. 1989 Oct;171(10):5304–5313. doi: 10.1128/jb.171.10.5304-5313.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fürste J. P., Pansegrau W., Frank R., Blöcker H., Scholz P., Bagdasarian M., Lanka E. Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene. 1986;48(1):119–131. doi: 10.1016/0378-1119(86)90358-6. [DOI] [PubMed] [Google Scholar]
  18. Gambello M. J., Iglewski B. H. Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression. J Bacteriol. 1991 May;173(9):3000–3009. doi: 10.1128/jb.173.9.3000-3009.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gambello M. J., Kaye S., Iglewski B. H. LasR of Pseudomonas aeruginosa is a transcriptional activator of the alkaline protease gene (apr) and an enhancer of exotoxin A expression. Infect Immun. 1993 Apr;61(4):1180–1184. doi: 10.1128/iai.61.4.1180-1184.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ghosaini L. R., Brown A. M., Sturtevant J. M. Scanning calorimetric study of the thermal unfolding of catabolite activator protein from Escherichia coli in the absence and presence of cyclic mononucleotides. Biochemistry. 1988 Jul 12;27(14):5257–5261. doi: 10.1021/bi00414a046. [DOI] [PubMed] [Google Scholar]
  21. Gray G. L., Vasil M. L. Isolation and genetic characterization of toxin-deficient mutants of Pseudomonas aeruginosa PAO. J Bacteriol. 1981 Aug;147(2):275–281. doi: 10.1128/jb.147.2.275-281.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gronenborn A. M., Sandulache R., Gärtner S., Clore G. M. Mutations in the cyclic AMP binding site of the cyclic AMP receptor protein of Escherichia coli. Biochem J. 1988 Aug 1;253(3):801–807. doi: 10.1042/bj2530801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hamood A. N., Olson J. C., Vincent T. S., Iglewski B. H. Regions of toxin A involved in toxin A excretion in Pseudomonas aeruginosa. J Bacteriol. 1989 Apr;171(4):1817–1824. doi: 10.1128/jb.171.4.1817-1824.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hedstrom R. C., Funk C. R., Kaper J. B., Pavlovskis O. R., Galloway D. R. Cloning of a gene involved in regulation of exotoxin A expression in Pseudomonas aeruginosa. Infect Immun. 1986 Jan;51(1):37–42. doi: 10.1128/iai.51.1.37-42.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Higgins D. E., DiRita V. J. Transcriptional control of toxT, a regulatory gene in the ToxR regulon of Vibrio cholerae. Mol Microbiol. 1994 Oct;14(1):17–29. doi: 10.1111/j.1365-2958.1994.tb01263.x. [DOI] [PubMed] [Google Scholar]
  26. Holloway B. W., Krishnapillai V., Morgan A. F. Chromosomal genetics of Pseudomonas. Microbiol Rev. 1979 Mar;43(1):73–102. doi: 10.1128/mr.43.1.73-102.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hull R. A., Gill R. E., Hsu P., Minshew B. H., Falkow S. Construction and expression of recombinant plasmids encoding type 1 or D-mannose-resistant pili from a urinary tract infection Escherichia coli isolate. Infect Immun. 1981 Sep;33(3):933–938. doi: 10.1128/iai.33.3.933-938.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kolb A., Busby S., Buc H., Garges S., Adhya S. Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem. 1993;62:749–795. doi: 10.1146/annurev.bi.62.070193.003533. [DOI] [PubMed] [Google Scholar]
  29. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  30. Liu P. V. Exotoxins of Pseudomonas aeruginosa. I. Factors that influence the production of exotoxin A. J Infect Dis. 1973 Oct;128(4):506–513. doi: 10.1093/infdis/128.4.506. [DOI] [PubMed] [Google Scholar]
  31. Lory S. Effect of iron on accumulation of exotoxin A-specific mRNA in Pseudomonas aeruginosa. J Bacteriol. 1986 Dec;168(3):1451–1456. doi: 10.1128/jb.168.3.1451-1456.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. MacGregor C. H., Wolff J. A., Arora S. K., Phibbs P. V., Jr Cloning of a catabolite repression control (crc) gene from Pseudomonas aeruginosa, expression of the gene in Escherichia coli, and identification of the gene product in Pseudomonas aeruginosa. J Bacteriol. 1991 Nov;173(22):7204–7212. doi: 10.1128/jb.173.22.7204-7212.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ohman D. E., Sadoff J. C., Iglewski B. H. Toxin A-deficient mutants of Pseudomonas aeruginosa PA103: isolation and characterization. Infect Immun. 1980 Jun;28(3):899–908. doi: 10.1128/iai.28.3.899-908.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Phillips A. T., Mulfinger L. M. Cyclic adenosine 3',5'-monophosphate levels in Pseudomonas putida and Pseudomonas aeruginosa during induction and carbon catabolite repression of histidase synthesis. J Bacteriol. 1981 Mar;145(3):1286–1292. doi: 10.1128/jb.145.3.1286-1292.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Prince R. W., Cox C. D., Vasil M. L. Coordinate regulation of siderophore and exotoxin A production: molecular cloning and sequencing of the Pseudomonas aeruginosa fur gene. J Bacteriol. 1993 May;175(9):2589–2598. doi: 10.1128/jb.175.9.2589-2598.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ratnaningsih E., Dharmsthiti S., Krishnapillai V., Morgan A., Sinclair M., Holloway B. W. A combined physical and genetic map of Pseudomonas aeruginosa PAO. J Gen Microbiol. 1990 Dec;136(12):2351–2357. doi: 10.1099/00221287-136-12-2351. [DOI] [PubMed] [Google Scholar]
  37. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schweizer H. P. Escherichia-Pseudomonas shuttle vectors derived from pUC18/19. Gene. 1991 Jan 2;97(1):109–121. doi: 10.1016/0378-1119(91)90016-5. [DOI] [PubMed] [Google Scholar]
  39. Shadel G. S., Devine J. H., Baldwin T. O. Control of the lux regulon of Vibrio fischeri. J Biolumin Chemilumin. 1990 Apr-Jun;5(2):99–106. doi: 10.1002/bio.1170050205. [DOI] [PubMed] [Google Scholar]
  40. Shortridge V. D., Pato M. L., Vasil A. I., Vasil M. L. Physical mapping of virulence-associated genes in Pseudomonas aeruginosa by transverse alternating-field electrophoresis. Infect Immun. 1991 Oct;59(10):3596–3603. doi: 10.1128/iai.59.10.3596-3603.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Siegel L. S., Hylemon P. B., Phibbs P. V., Jr Cyclic adenosine 3',5'-monophosphate levels and activities of adenylate cyclase and cyclic adenosine 3',5'-monophosphate phosphodiesterase in Pseudomonas and Bacteroides. J Bacteriol. 1977 Jan;129(1):87–96. doi: 10.1128/jb.129.1.87-96.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Smith A. W., Iglewski B. H. Transformation of Pseudomonas aeruginosa by electroporation. Nucleic Acids Res. 1989 Dec 25;17(24):10509–10509. doi: 10.1093/nar/17.24.10509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Smith G. E., Summers M. D. The bidirectional transfer of DNA and RNA to nitrocellulose or diazobenzyloxymethyl-paper. Anal Biochem. 1980 Nov 15;109(1):123–129. doi: 10.1016/0003-2697(80)90019-6. [DOI] [PubMed] [Google Scholar]
  44. Storey D. G., Frank D. W., Farinha M. A., Kropinski A. M., Iglewski B. H. Multiple promoters control the regulation of the Pseudomonas aeruginosa regA gene. Mol Microbiol. 1990 Mar;4(3):499–503. doi: 10.1111/j.1365-2958.1990.tb00616.x. [DOI] [PubMed] [Google Scholar]
  45. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Weber I. T., Steitz T. A. Structure of a complex of catabolite gene activator protein and cyclic AMP refined at 2.5 A resolution. J Mol Biol. 1987 Nov 20;198(2):311–326. doi: 10.1016/0022-2836(87)90315-9. [DOI] [PubMed] [Google Scholar]
  48. West S. E., Iglewski B. H. Codon usage in Pseudomonas aeruginosa. Nucleic Acids Res. 1988 Oct 11;16(19):9323–9335. doi: 10.1093/nar/16.19.9323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. West S. E., Kaye S. A., Hamood A. N., Iglewski B. H. Characterization of Pseudomonas aeruginosa mutants that are deficient in exotoxin A synthesis and are altered in expression of regA, a positive regulator of exotoxin A. Infect Immun. 1994 Mar;62(3):897–903. doi: 10.1128/iai.62.3.897-903.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wick M. J., Frank D. W., Storey D. G., Iglewski B. H. Identification of regB, a gene required for optimal exotoxin A yields in Pseudomonas aeruginosa. Mol Microbiol. 1990 Mar;4(3):489–497. doi: 10.1111/j.1365-2958.1990.tb00615.x. [DOI] [PubMed] [Google Scholar]
  51. Williams R., Bell A., Sims G., Busby S. The role of two surface exposed loops in transcription activation by the Escherichia coli CRP and FNR proteins. Nucleic Acids Res. 1991 Dec 25;19(24):6705–6712. doi: 10.1093/nar/19.24.6705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wolff J. A., MacGregor C. H., Eisenberg R. C., Phibbs P. V., Jr Isolation and characterization of catabolite repression control mutants of Pseudomonas aeruginosa PAO. J Bacteriol. 1991 Aug;173(15):4700–4706. doi: 10.1128/jb.173.15.4700-4706.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wretlind B., Pavlovskis O. R. Genetic mapping and characterization of Pseudomonas aeruginosa mutants defective in the formation of extracellular proteins. J Bacteriol. 1984 Jun;158(3):801–808. doi: 10.1128/jb.158.3.801-808.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zhang X. P., Ebright R. H. Identification of a contact between arginine-180 of the catabolite gene activator protein (CAP) and base pair 5 of the DNA site in the CAP-DNA complex. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4717–4721. doi: 10.1073/pnas.87.12.4717. [DOI] [PMC free article] [PubMed] [Google Scholar]

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