Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Mar;179(5):1646–1654. doi: 10.1128/jb.179.5.1646-1654.1997

Functional analysis of exsC and exsB in regulation of exoenzyme S production by Pseudomonas aeruginosa.

J Goranson 1, A K Hovey 1, D W Frank 1
PMCID: PMC178878  PMID: 9045825

Abstract

Expression of ExsC, ExsB, and ExsA (the exoenzyme S trans-regulatory locus) of Pseudomonas aeruginosa was analyzed by using complementation, RNase protection, translational fusion, and T7-directed protein expression analyses. T7 expression analyses in E. coli hosts demonstrated that ExsC, ExsA, and a truncated form of ExsD (a partial open reading frame located 3' of ExsA) were translated; however, a product corresponding to ExsB was undetectable. T7-mediated transcription and translation of the antisense strand resulted in production of a 18.5-kDa product, termed ExsB', which overlapped the predicted ExsB product. In complementation experiments, deletion of the region encoding ExsB and most of ExsB' severely reduced exoenzyme S production. Site-specific mutagenesis of the start codons for ExsB and ExsB', however, did not affect exoenzyme S production. RNase protection studies were initiated to examine the hypothesis that RNA encoded within the ExsB/ExsB' region exerted a regulatory effect. RNA encoding ExsB' was not detectable from chromosomal genes or complementation constructs, indicating that ExsB' was not expressed in P. aeruginosa. To determine the pattern of translation, a chloramphenicol acetyltransferase gene (cat) reporter was fused in frame with ExsB and with ExsA in the context of the entire locus or in the absence of the exsB region. These experiments indicated that exsB was not translated but that deletion of the exsB region affected the translation of ExsA-CAT. RNase protection assays further suggested that deletion of exsB resulted in a processing of ExsA mRNA. Our data indicate that the untranslated exsB region of the trans-regulatory locus mRNA mediates either the stability or the translation of exsA. Complementation analysis further suggests that ExsC may play a role in the translation or stability of ExoS.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

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

  1. Allaoui A., Scheen R., Lambert de Rouvroit C., Cornelis G. R. VirG, a Yersinia enterocolitica lipoprotein involved in Ca2+ dependency, is related to exsB of Pseudomonas aeruginosa. J Bacteriol. 1995 Aug;177(15):4230–4237. doi: 10.1128/jb.177.15.4230-4237.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bodey G. P., Bolivar R., Fainstein V., Jadeja L. Infections caused by Pseudomonas aeruginosa. Rev Infect Dis. 1983 Mar-Apr;5(2):279–313. doi: 10.1093/clinids/5.2.279. [DOI] [PubMed] [Google Scholar]
  3. Bula C., Wilcox K. W. Negative effect of sequential serine codons on expression of foreign genes in Escherichia coli. Protein Expr Purif. 1996 Feb;7(1):92–103. doi: 10.1006/prep.1996.0013. [DOI] [PubMed] [Google Scholar]
  4. Coburn J., Dillon S. T., Iglewski B. H., Gill D. M. Exoenzyme S of Pseudomonas aeruginosa ADP-ribosylates the intermediate filament protein vimentin. Infect Immun. 1989 Mar;57(3):996–998. doi: 10.1128/iai.57.3.996-998.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Coburn J., Gill D. M. ADP-ribosylation of p21ras and related proteins by Pseudomonas aeruginosa exoenzyme S. Infect Immun. 1991 Nov;59(11):4259–4262. doi: 10.1128/iai.59.11.4259-4262.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Coburn J., Kane A. V., Feig L., Gill D. M. Pseudomonas aeruginosa exoenzyme S requires a eukaryotic protein for ADP-ribosyltransferase activity. J Biol Chem. 1991 Apr 5;266(10):6438–6446. [PubMed] [Google Scholar]
  7. Coburn J. Pseudomonas aeruginosa exoenzyme S. Curr Top Microbiol Immunol. 1992;175:133–143. doi: 10.1007/978-3-642-76966-5_7. [DOI] [PubMed] [Google Scholar]
  8. Cornelis G., Sluiters C., de Rouvroit C. L., Michiels T. Homology between virF, the transcriptional activator of the Yersinia virulence regulon, and AraC, the Escherichia coli arabinose operon regulator. J Bacteriol. 1989 Jan;171(1):254–262. doi: 10.1128/jb.171.1.254-262.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Frank D. W., Iglewski B. H. Cloning and sequence analysis of a trans-regulatory locus required for exoenzyme S synthesis in Pseudomonas aeruginosa. J Bacteriol. 1991 Oct;173(20):6460–6468. doi: 10.1128/jb.173.20.6460-6468.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Frank D. W., Nair G., Schweizer H. P. Construction and characterization of chromosomal insertional mutations of the Pseudomonas aeruginosa exoenzyme S trans-regulatory locus. Infect Immun. 1994 Feb;62(2):554–563. doi: 10.1128/iai.62.2.554-563.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Friedman A. M., Long S. R., Brown S. E., Buikema W. J., Ausubel F. M. Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene. 1982 Jun;18(3):289–296. doi: 10.1016/0378-1119(82)90167-6. [DOI] [PubMed] [Google Scholar]
  13. Fu H., Coburn J., Collier R. J. The eukaryotic host factor that activates exoenzyme S of Pseudomonas aeruginosa is a member of the 14-3-3 protein family. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2320–2324. doi: 10.1073/pnas.90.6.2320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Green P. J., Pines O., Inouye M. The role of antisense RNA in gene regulation. Annu Rev Biochem. 1986;55:569–597. doi: 10.1146/annurev.bi.55.070186.003033. [DOI] [PubMed] [Google Scholar]
  15. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  16. Hoe N. P., Goguen J. D. Temperature sensing in Yersinia pestis: translation of the LcrF activator protein is thermally regulated. J Bacteriol. 1993 Dec;175(24):7901–7909. doi: 10.1128/jb.175.24.7901-7909.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hoe N. P., Minion F. C., Goguen J. D. Temperature sensing in Yersinia pestis: regulation of yopE transcription by lcrF. J Bacteriol. 1992 Jul;174(13):4275–4286. doi: 10.1128/jb.174.13.4275-4286.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Hovey A. K., Frank D. W. Analyses of the DNA-binding and transcriptional activation properties of ExsA, the transcriptional activator of the Pseudomonas aeruginosa exoenzyme S regulon. J Bacteriol. 1995 Aug;177(15):4427–4436. doi: 10.1128/jb.177.15.4427-4436.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Iglewski B. H., Sadoff J., Bjorn M. J., Maxwell E. S. Pseudomonas aeruginosa exoenzyme S: an adenosine diphosphate ribosyltransferase distinct from toxin A. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3211–3215. doi: 10.1073/pnas.75.7.3211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kamath-Loeb A. S., Gross C. A. Translational regulation of sigma 32 synthesis: requirement for an internal control element. J Bacteriol. 1991 Jun;173(12):3904–3906. doi: 10.1128/jb.173.12.3904-3906.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kulich S. M., Frank D. W., Barbieri J. T. Purification and characterization of exoenzyme S from Pseudomonas aeruginosa 388. Infect Immun. 1993 Jan;61(1):307–313. doi: 10.1128/iai.61.1.307-313.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kulich S. M., Yahr T. L., Mende-Mueller L. M., Barbieri J. T., Frank D. W. Cloning the structural gene for the 49-kDa form of exoenzyme S (exoS) from Pseudomonas aeruginosa strain 388. J Biol Chem. 1994 Apr 8;269(14):10431–10437. [PubMed] [Google Scholar]
  24. 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]
  25. Lambert de Rouvroit C., Sluiters C., Cornelis G. R. Role of the transcriptional activator, VirF, and temperature in the expression of the pYV plasmid genes of Yersinia enterocolitica. Mol Microbiol. 1992 Feb;6(3):395–409. [PubMed] [Google Scholar]
  26. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  27. McNally L. M., McNally M. T. SR protein splicing factors interact with the Rous sarcoma virus negative regulator of splicing element. J Virol. 1996 Feb;70(2):1163–1172. doi: 10.1128/jvi.70.2.1163-1172.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McNally M. T., Gontarek R. R., Beemon K. Characterization of Rous sarcoma virus intronic sequences that negatively regulate splicing. Virology. 1991 Nov;185(1):99–108. doi: 10.1016/0042-6822(91)90758-4. [DOI] [PubMed] [Google Scholar]
  29. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nagai H., Yuzawa H., Yura T. Interplay of two cis-acting mRNA regions in translational control of sigma 32 synthesis during the heat shock response of Escherichia coli. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10515–10519. doi: 10.1073/pnas.88.23.10515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nicas T. I., Bradley J., Lochner J. E., Iglewski B. H. The role of exoenzyme S in infections with Pseudomonas aeruginosa. J Infect Dis. 1985 Oct;152(4):716–721. doi: 10.1093/infdis/152.4.716. [DOI] [PubMed] [Google Scholar]
  32. Nicas T. I., Iglewski B. H. Contribution of exoenzyme S to the virulence of Pseudomonas aeruginosa. Antibiot Chemother (1971) 1985;36:40–48. doi: 10.1159/000410470. [DOI] [PubMed] [Google Scholar]
  33. Nicas T. I., Iglewski B. H. Isolation and characterization of transposon-induced mutants of Pseudomonas aeruginosa deficient in production of exoenzyme S. Infect Immun. 1984 Aug;45(2):470–474. doi: 10.1128/iai.45.2.470-474.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nomura M., Gourse R., Baughman G. Regulation of the synthesis of ribosomes and ribosomal components. Annu Rev Biochem. 1984;53:75–117. doi: 10.1146/annurev.bi.53.070184.000451. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Stout V., Torres-Cabassa A., Maurizi M. R., Gutnick D., Gottesman S. RcsA, an unstable positive regulator of capsular polysaccharide synthesis. J Bacteriol. 1991 Mar;173(5):1738–1747. doi: 10.1128/jb.173.5.1738-1747.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
  40. 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]
  41. Yahr T. L., Barbieri J. T., Frank D. W. Genetic relationship between the 53- and 49-kilodalton forms of exoenzyme S from Pseudomonas aeruginosa. J Bacteriol. 1996 Mar;178(5):1412–1419. doi: 10.1128/jb.178.5.1412-1419.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yahr T. L., Frank D. W. Transcriptional organization of the trans-regulatory locus which controls exoenzyme S synthesis in Pseudomonas aeruginosa. J Bacteriol. 1994 Jul;176(13):3832–3838. doi: 10.1128/jb.176.13.3832-3838.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yahr T. L., Goranson J., Frank D. W. Exoenzyme S of Pseudomonas aeruginosa is secreted by a type III pathway. Mol Microbiol. 1996 Dec;22(5):991–1003. doi: 10.1046/j.1365-2958.1996.01554.x. [DOI] [PubMed] [Google Scholar]
  44. Yahr T. L., Hovey A. K., Kulich S. M., Frank D. W. Transcriptional analysis of the Pseudomonas aeruginosa exoenzyme S structural gene. J Bacteriol. 1995 Mar;177(5):1169–1178. doi: 10.1128/jb.177.5.1169-1178.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. von Gabain A., Belasco J. G., Schottel J. L., Chang A. C., Cohen S. N. Decay of mRNA in Escherichia coli: investigation of the fate of specific segments of transcripts. Proc Natl Acad Sci U S A. 1983 Feb;80(3):653–657. doi: 10.1073/pnas.80.3.653. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES