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. 1993 Oct;175(20):6695–6703. doi: 10.1128/jb.175.20.6695-6703.1993

Isolation and analysis of eight exe genes and their involvement in extracellular protein secretion and outer membrane assembly in Aeromonas hydrophila.

S P Howard 1, J Critch 1, A Bedi 1
PMCID: PMC206782  PMID: 8407845

Abstract

The exeE gene of Aeromonas hydrophila has been shown to be required for the secretion of most if not all of the extracellular proteins produced by this bacterium. In addition, an exeE::Tn5-751 insertion mutant of A. hydrophila was found to be deficient in the amounts of a number of its major outer membrane proteins (B. Jiang and S. P. Howard, J. Bacteriol. 173:1241-1249, 1991). The exeE gene and the exeF gene were previously isolated as part of a fragment which complemented this mutant. In this study, we have isolated and sequenced a further eight exe genes, exeG through exeN, which constitute the 3' end of the exe operon. These genes have a high degree of similarity with the extracellular secretion operons of a number of different gram-negative bacteria. Marker exchange mutagenesis was used to insert kanamycin resistance cassettes into three different regions of the exe operon. The phenotypes of these mutants showed that in A. hydrophila this operon is required not only for extracellular protein secretion but also for normal assembly of the outer membrane, in particular with respect to the quantities of the major porins. Five of the Exe proteins contain type IV prepilin signal sequences, although the prepilin peptidase gene does not appear to form part of the exe operon. Limited processing of the ExeG protein was observed when it was expressed in Escherichia coli, and this processing was greatly accelerated in the presence of the prepilin peptidase of Pseudomonas 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. Bally M., Ball G., Badere A., Lazdunski A. Protein secretion in Pseudomonas aeruginosa: the xcpA gene encodes an integral inner membrane protein homologous to Klebsiella pneumoniae secretion function protein PulO. J Bacteriol. 1991 Jan;173(2):479–486. doi: 10.1128/jb.173.2.479-486.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bally M., Filloux A., Akrim M., Ball G., Lazdunski A., Tommassen J. Protein secretion in Pseudomonas aeruginosa: characterization of seven xcp genes and processing of secretory apparatus components by prepilin peptidase. Mol Microbiol. 1992 May;6(9):1121–1131. doi: 10.1111/j.1365-2958.1992.tb01550.x. [DOI] [PubMed] [Google Scholar]
  3. Bo J. N., Howard S. P. Mutagenesis and isolation of Aeromonas hydrophila genes which are required for extracellular secretion. J Bacteriol. 1991 Feb;173(3):1241–1249. doi: 10.1128/jb.173.3.1241-1249.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brendel V., Trifonov E. N. A computer algorithm for testing potential prokaryotic terminators. Nucleic Acids Res. 1984 May 25;12(10):4411–4427. doi: 10.1093/nar/12.10.4411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chakraborty T., Huhle B., Hof H., Bergbauer H., Goebel W. Marker exchange mutagenesis of the aerolysin determinant in Aeromonas hydrophila demonstrates the role of aerolysin in A. hydrophila-associated systemic infections. Infect Immun. 1987 Sep;55(9):2274–2280. doi: 10.1128/iai.55.9.2274-2280.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dalbey R. E., Von Heijne G. Signal peptidases in prokaryotes and eukaryotes--a new protease family. Trends Biochem Sci. 1992 Nov;17(11):474–478. doi: 10.1016/0968-0004(92)90492-r. [DOI] [PubMed] [Google Scholar]
  7. Dupuy B., Taha M. K., Possot O., Marchal C., Pugsley A. P. PulO, a component of the pullulanase secretion pathway of Klebsiella oxytoca, correctly and efficiently processes gonococcal type IV prepilin in Escherichia coli. Mol Microbiol. 1992 Jul;6(14):1887–1894. doi: 10.1111/j.1365-2958.1992.tb01361.x. [DOI] [PubMed] [Google Scholar]
  8. Eisenberg D., Weiss R. M., Terwilliger T. C. The hydrophobic moment detects periodicity in protein hydrophobicity. Proc Natl Acad Sci U S A. 1984 Jan;81(1):140–144. doi: 10.1073/pnas.81.1.140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Engelman D. M., Steitz T. A., Goldman A. Identifying nonpolar transbilayer helices in amino acid sequences of membrane proteins. Annu Rev Biophys Biophys Chem. 1986;15:321–353. doi: 10.1146/annurev.bb.15.060186.001541. [DOI] [PubMed] [Google Scholar]
  10. Filip C., Fletcher G., Wulff J. L., Earhart C. F. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. J Bacteriol. 1973 Sep;115(3):717–722. doi: 10.1128/jb.115.3.717-722.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  13. Hokama A., Iwanaga M. Purification and characterization of Aeromonas sobria pili, a possible colonization factor. Infect Immun. 1991 Oct;59(10):3478–3483. doi: 10.1128/iai.59.10.3478-3483.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Holland I. B., Blight M. A., Kenny B. The mechanism of secretion of hemolysin and other polypeptides from gram-negative bacteria. J Bioenerg Biomembr. 1990 Jun;22(3):473–491. doi: 10.1007/BF00763178. [DOI] [PubMed] [Google Scholar]
  15. Howard S. P., Buckley J. T. Activation of the hole-forming toxin aerolysin by extracellular processing. J Bacteriol. 1985 Jul;163(1):336–340. doi: 10.1128/jb.163.1.336-340.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Howard S. P., Buckley J. T. Intracellular accumulation of extracellular proteins by pleiotropic export mutants of Aeromonas hydrophila. J Bacteriol. 1983 Apr;154(1):413–418. doi: 10.1128/jb.154.1.413-418.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Howard S. P., Buckley J. T. Molecular cloning and expression in Escherichia coli of the structural gene for the hemolytic toxin aerolysin from Aeromonas hydrophila. Mol Gen Genet. 1986 Aug;204(2):289–295. doi: 10.1007/BF00425512. [DOI] [PubMed] [Google Scholar]
  18. Howard S. P., Buckley J. T. Protein export by a gram-negative bacterium: production of aerolysin by Aeromonas hydrophila. J Bacteriol. 1985 Mar;161(3):1118–1124. doi: 10.1128/jb.161.3.1118-1124.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Iwanaga M., Hokama A. Characterization of Aeromonas sobria TAP13 pili: a possible new colonization factor. J Gen Microbiol. 1992 Sep;138(9):1913–1919. doi: 10.1099/00221287-138-9-1913. [DOI] [PubMed] [Google Scholar]
  20. Jeanteur D., Gletsu N., Pattus F., Buckley J. T. Purification of Aeromonas hydrophila major outer-membrane proteins: N-terminal sequence analysis and channel-forming properties. Mol Microbiol. 1992 Nov;6(22):3355–3363. doi: 10.1111/j.1365-2958.1992.tb02203.x. [DOI] [PubMed] [Google Scholar]
  21. Jiang B., Howard S. P. The Aeromonas hydrophila exeE gene, required both for protein secretion and normal outer membrane biogenesis, is a member of a general secretion pathway. Mol Microbiol. 1992 May;6(10):1351–1361. doi: 10.1111/j.1365-2958.1992.tb00856.x. [DOI] [PubMed] [Google Scholar]
  22. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Leung K. Y., Stevenson R. M. Tn5-induced protease-deficient strains of Aeromonas hydrophila with reduced virulence for fish. Infect Immun. 1988 Oct;56(10):2639–2644. doi: 10.1128/iai.56.10.2639-2644.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lindeberg M., Collmer A. Analysis of eight out genes in a cluster required for pectic enzyme secretion by Erwinia chrysanthemi: sequence comparison with secretion genes from other gram-negative bacteria. J Bacteriol. 1992 Nov;174(22):7385–7397. doi: 10.1128/jb.174.22.7385-7397.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nunn D. N., Lory S. Product of the Pseudomonas aeruginosa gene pilD is a prepilin leader peptidase. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3281–3285. doi: 10.1073/pnas.88.8.3281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nunn D., Bergman S., Lory S. Products of three accessory genes, pilB, pilC, and pilD, are required for biogenesis of Pseudomonas aeruginosa pili. J Bacteriol. 1990 Jun;172(6):2911–2919. doi: 10.1128/jb.172.6.2911-2919.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Parker L. L., Hall B. G. Characterization and nucleotide sequence of the cryptic cel operon of Escherichia coli K12. Genetics. 1990 Mar;124(3):455–471. doi: 10.1093/genetics/124.3.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pasloske B. L., Paranchych W. The expression of mutant pilins in Pseudomonas aeruginosa: fifth position glutamate affects pilin methylation. Mol Microbiol. 1988 Jul;2(4):489–495. doi: 10.1111/j.1365-2958.1988.tb00055.x. [DOI] [PubMed] [Google Scholar]
  30. Peek J. A., Taylor R. K. Characterization of a periplasmic thiol:disulfide interchange protein required for the functional maturation of secreted virulence factors of Vibrio cholerae. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6210–6214. doi: 10.1073/pnas.89.13.6210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pugsley A. P. A mutation in the dsbA gene coding for periplasmic disulfide oxidoreductase reduces transcription of the Escherichia coli ompF gene. Mol Gen Genet. 1993 Mar;237(3):407–411. doi: 10.1007/BF00279445. [DOI] [PubMed] [Google Scholar]
  32. Pugsley A. P., Dupuy B. An enzyme with type IV prepilin peptidase activity is required to process components of the general extracellular protein secretion pathway of Klebsiella oxytoca. Mol Microbiol. 1992 Mar;6(6):751–760. doi: 10.1111/j.1365-2958.1992.tb01525.x. [DOI] [PubMed] [Google Scholar]
  33. Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pugsley A. P. Translocation of a folded protein across the outer membrane in Escherichia coli. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):12058–12062. doi: 10.1073/pnas.89.24.12058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pugsley A. P., d'Enfert C., Reyss I., Kornacker M. G. Genetics of extracellular protein secretion by gram-negative bacteria. Annu Rev Genet. 1990;24:67–90. doi: 10.1146/annurev.ge.24.120190.000435. [DOI] [PubMed] [Google Scholar]
  36. Sandhu G. S., Precup J. W., Kline B. C. Rapid one-step characterization of recombinant vectors by direct analysis of transformed Escherichia coli colonies. Biotechniques. 1989 Jul-Aug;7(7):689–690. [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. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  39. Strom M. S., Lory S. Amino acid substitutions in pilin of Pseudomonas aeruginosa. Effect on leader peptide cleavage, amino-terminal methylation, and pilus assembly. J Biol Chem. 1991 Jan 25;266(3):1656–1664. [PubMed] [Google Scholar]
  40. Strom M. S., Lory S. Kinetics and sequence specificity of processing of prepilin by PilD, the type IV leader peptidase of Pseudomonas aeruginosa. J Bacteriol. 1992 Nov;174(22):7345–7351. doi: 10.1128/jb.174.22.7345-7351.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  42. Twining S. S. Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes. Anal Biochem. 1984 Nov 15;143(1):30–34. doi: 10.1016/0003-2697(84)90553-0. [DOI] [PubMed] [Google Scholar]
  43. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  44. Whitchurch C. B., Hobbs M., Livingston S. P., Krishnapillai V., Mattick J. S. Characterisation of a Pseudomonas aeruginosa twitching motility gene and evidence for a specialised protein export system widespread in eubacteria. Gene. 1991 May 15;101(1):33–44. doi: 10.1016/0378-1119(91)90221-v. [DOI] [PubMed] [Google Scholar]
  45. Wong K. R., Buckley J. T. Proton motive force involved in protein transport across the outer membrane of Aeromonas salmonicida. Science. 1989 Nov 3;246(4930):654–656. doi: 10.1126/science.2814486. [DOI] [PubMed] [Google Scholar]
  46. Yu J., Webb H., Hirst T. R. A homologue of the Escherichia coli DsbA protein involved in disulphide bond formation is required for enterotoxin biogenesis in Vibrio cholerae. Mol Microbiol. 1992 Jul;6(14):1949–1958. doi: 10.1111/j.1365-2958.1992.tb01368.x. [DOI] [PubMed] [Google Scholar]
  47. Zubay G. The isolation and properties of CAP, the catabolite gene activator. Methods Enzymol. 1980;65(1):856–877. doi: 10.1016/s0076-6879(80)65079-4. [DOI] [PubMed] [Google Scholar]
  48. d'Enfert C., Ryter A., Pugsley A. P. Cloning and expression in Escherichia coli of the Klebsiella pneumoniae genes for production, surface localization and secretion of the lipoprotein pullulanase. EMBO J. 1987 Nov;6(11):3531–3538. doi: 10.1002/j.1460-2075.1987.tb02679.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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