Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Apr;179(7):2247–2258. doi: 10.1128/jb.179.7.2247-2258.1997

Multiple loci of Pseudomonas syringae pv. syringae are involved in pathogenicity on bean: restoration of one lesion-deficient mutant requires two tRNA genes.

J J Rich 1, D K Willis 1
PMCID: PMC178961  PMID: 9079910

Abstract

A mutational analysis of lesion-forming ability was undertaken in Pseudomonas syringae pv. syringae B728a, causal agent of bacterial brown spot disease of bean. Following a screen of 6,401 Tn5-containing derivatives of B728a on bean pods, 26 strains that did not form disease lesions were identified. Nine of the mutant strains were defective in the ability to elicit the hypersensitive reaction (HR) and were shown to contain Tn5 insertions within the P. syringae pv. syringae hrp region. Ten HR+ mutants were defective in the production of the toxin syringomycin, and a region of the chromosome implicated in the biosynthesis of syringomycin was deleted in a subset of these mutants. The remaining seven lesion-defective mutants retained the ability to produce protease and syringomycin. Marker exchange mutagenesis confirmed that the Tn5 insertion was causal to the mutant phenotype in several lesion-defective, HR+ strains. KW239, a lesion- and syringomycin-deficient mutant, was characterized at the molecular level. Sequence analysis of the chromosomal region flanking the Tn5 within KW239 revealed strong similarities to a number of known Escherichia coli gene products and DNA sequences: the nusA operon, including the complete initiator tRNA(Met) gene, metY; a tRNA(Leu) gene; the tpiA gene product; and the MrsA protein. Removal of sequences containing the two potential tRNA genes prevented restoration of mutant KW239 in trans. The Tn5 insertions within the lesion-deficient strains examined, including KW239, were not closely linked to each other or to the lemA or gacA genes previously identified as involved in lesion formation by P. syringae pv. syringae.

Full Text

The Full Text of this article is available as a PDF (538.1 KB).

Selected References

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

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Barta T. M., Kinscherf T. G., Willis D. K. Regulation of tabtoxin production by the lemA gene in Pseudomonas syringae. J Bacteriol. 1992 May;174(9):3021–3029. doi: 10.1128/jb.174.9.3021-3029.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chandler P. M., Krishnapillai V. Characterization of Pseudomonas aeruginosa derepressed R-plasmids. J Bacteriol. 1977 May;130(2):596–603. doi: 10.1128/jb.130.2.596-603.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen K. S., Peters T. C., Walker J. R. A minor arginine tRNA mutant limits translation preferentially of a protein dependent on the cognate codon. J Bacteriol. 1990 May;172(5):2504–2510. doi: 10.1128/jb.172.5.2504-2510.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen M. X., Bouquin N., Norris V., Casarégola S., Séror S. J., Holland I. B. A single base change in the acceptor stem of tRNA(3Leu) confers resistance upon Escherichia coli to the calmodulin inhibitor, 48/80. EMBO J. 1991 Oct;10(10):3113–3122. doi: 10.1002/j.1460-2075.1991.tb07865.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Debenham P. G., Pongs O., Travers A. A. Formylmethionyl-tRNA alters RNA polymerase specificity. Proc Natl Acad Sci U S A. 1980 Feb;77(2):870–874. doi: 10.1073/pnas.77.2.870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  10. Friden P., Newman T., Freundlich M. Nucleotide sequence of the ilvB promoter-regulatory region: a biosynthetic operon controlled by attenuation and cyclic AMP. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6156–6160. doi: 10.1073/pnas.79.20.6156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gray J., Wang J., Gelvin S. B. Mutation of the miaA gene of Agrobacterium tumefaciens results in reduced vir gene expression. J Bacteriol. 1992 Feb;174(4):1086–1098. doi: 10.1128/jb.174.4.1086-1098.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gross D. C. Regulation of syringomycin synthesis in Pseudomonas syringae pv. syringae and defined conditions for its production. J Appl Bacteriol. 1985 Feb;58(2):167–174. doi: 10.1111/j.1365-2672.1985.tb01444.x. [DOI] [PubMed] [Google Scholar]
  13. Hickman M. J., Orser C. S., Willis D. K., Lindow S. E., Panopoulos N. J. Molecular cloning and biological characterization of the recA gene from Pseudomonas syringae. J Bacteriol. 1987 Jun;169(6):2906–2910. doi: 10.1128/jb.169.6.2906-2910.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hirano S. S., Upper C. D. Diel Variation in Population Size and Ice Nucleation Activity of Pseudomonas syringae on Snap Bean Leaflets. Appl Environ Microbiol. 1989 Mar;55(3):623–630. doi: 10.1128/aem.55.3.623-630.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hrabak E. M., Willis D. K. The lemA gene required for pathogenicity of Pseudomonas syringae pv. syringae on bean is a member of a family of two-component regulators. J Bacteriol. 1992 May;174(9):3011–3020. doi: 10.1128/jb.174.9.3011-3020.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hromockyj A. E., Tucker S. C., Maurelli A. T. Temperature regulation of Shigella virulence: identification of the repressor gene virR, an analogue of hns, and partial complementation by tyrosyl transfer RNA (tRNA1(Tyr)). Mol Microbiol. 1992 Aug;6(15):2113–2124. doi: 10.1111/j.1365-2958.1992.tb01385.x. [DOI] [PubMed] [Google Scholar]
  17. Huang H. C., Schuurink R., Denny T. P., Atkinson M. M., Baker C. J., Yucel I., Hutcheson S. W., Collmer A. Molecular cloning of a Pseudomonas syringae pv. syringae gene cluster that enables Pseudomonas fluorescens to elicit the hypersensitive response in tobacco plants. J Bacteriol. 1988 Oct;170(10):4748–4756. doi: 10.1128/jb.170.10.4748-4756.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jahn D., Verkamp E., Söll D. Glutamyl-transfer RNA: a precursor of heme and chlorophyll biosynthesis. Trends Biochem Sci. 1992 Jun;17(6):215–218. doi: 10.1016/0968-0004(92)90380-r. [DOI] [PubMed] [Google Scholar]
  19. Jones J. D., Gutterson N. An efficient mobilizable cosmid vector, pRK7813, and its use in a rapid method for marker exchange in Pseudomonas fluorescens strain HV37a. Gene. 1987;61(3):299–306. doi: 10.1016/0378-1119(87)90193-4. [DOI] [PubMed] [Google Scholar]
  20. KING E. O., WARD M. K., RANEY D. E. Two simple media for the demonstration of pyocyanin and fluorescin. J Lab Clin Med. 1954 Aug;44(2):301–307. [PubMed] [Google Scholar]
  21. Keen N. T., Tamaki S., Kobayashi D., Trollinger D. Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene. 1988 Oct 15;70(1):191–197. doi: 10.1016/0378-1119(88)90117-5. [DOI] [PubMed] [Google Scholar]
  22. Kinscherf T. G., Coleman R. H., Barta T. M., Willis D. K. Cloning and expression of the tabtoxin biosynthetic region from Pseudomonas syringae. J Bacteriol. 1991 Jul;173(13):4124–4132. doi: 10.1128/jb.173.13.4124-4132.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kitten T., Willis D. K. Suppression of a sensor kinase-dependent phenotype in Pseudomonas syringae by ribosomal proteins L35 and L20. J Bacteriol. 1996 Mar;178(6):1548–1555. doi: 10.1128/jb.178.6.1548-1555.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Laville J., Voisard C., Keel C., Maurhofer M., Défago G., Haas D. Global control in Pseudomonas fluorescens mediating antibiotic synthesis and suppression of black root rot of tobacco. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1562–1566. doi: 10.1073/pnas.89.5.1562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lawlor E. J., Baylis H. A., Chater K. F. Pleiotropic morphological and antibiotic deficiencies result from mutations in a gene encoding a tRNA-like product in Streptomyces coelicolor A3(2). Genes Dev. 1987 Dec;1(10):1305–1310. doi: 10.1101/gad.1.10.1305. [DOI] [PubMed] [Google Scholar]
  26. Leibowitz M. J., Soffer R. L. A soluble enzyme from Escherichia coli which catalyzes the transfer of leucine and phenylalanine from tRNA to acceptor proteins. Biochem Biophys Res Commun. 1969 Jul 7;36(1):47–53. doi: 10.1016/0006-291x(69)90647-0. [DOI] [PubMed] [Google Scholar]
  27. Leskiw B. K., Lawlor E. J., Fernandez-Abalos J. M., Chater K. F. TTA codons in some genes prevent their expression in a class of developmental, antibiotic-negative, Streptomyces mutants. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2461–2465. doi: 10.1073/pnas.88.6.2461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Manoil C. Analysis of protein localization by use of gene fusions with complementary properties. J Bacteriol. 1990 Feb;172(2):1035–1042. doi: 10.1128/jb.172.2.1035-1042.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mo Y. Y., Gross D. C. Plant signal molecules activate the syrB gene, which is required for syringomycin production by Pseudomonas syringae pv. syringae. J Bacteriol. 1991 Sep;173(18):5784–5792. doi: 10.1128/jb.173.18.5784-5792.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nomura T., Fujita N., Ishihama A. Promoter selectivity of Escherichia coli RNA polymerase: alteration by fMet-tRNAfMet. Nucleic Acids Res. 1986 Sep 11;14(17):6857–6870. doi: 10.1093/nar/14.17.6857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pongs O., Ulbrich N. Specific binding of formylated initiator-tRNA to Escherichia coli RNA polymerase. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3064–3067. doi: 10.1073/pnas.73.9.3064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Quigley N. B., Mo Y. Y., Gross D. C. SyrD is required for syringomycin production by Pseudomonas syringae pathovar syringae and is related to a family of ATP-binding secretion proteins. Mol Microbiol. 1993 Aug;9(4):787–801. doi: 10.1111/j.1365-2958.1993.tb01738.x. [DOI] [PubMed] [Google Scholar]
  33. Rich J. J., Hirano S. S., Willis D. K. Pathovar-specific requirement for the Pseudomonas syringae lemA gene in disease lesion formation. Appl Environ Microbiol. 1992 May;58(5):1440–1446. doi: 10.1128/aem.58.5.1440-1446.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rich J. J., Kinscherf T. G., Kitten T., Willis D. K. Genetic evidence that the gacA gene encodes the cognate response regulator for the lemA sensor in Pseudomonas syringae. J Bacteriol. 1994 Dec;176(24):7468–7475. doi: 10.1128/jb.176.24.7468-7475.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rich J. J., Willis D. K. A single oligonucleotide can be used to rapidly isolate DNA sequences flanking a transposon Tn5 insertion by the polymerase chain reaction. Nucleic Acids Res. 1990 Nov 25;18(22):6673–6676. doi: 10.1093/nar/18.22.6673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Staskawicz B., Dahlbeck D., Keen N., Napoli C. Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea. J Bacteriol. 1987 Dec;169(12):5789–5794. doi: 10.1128/jb.169.12.5789-5794.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stewart T. S., Roberts R. J., Strominger J. L. Novel species of tRNA. Nature. 1971 Mar 5;230(5288):36–38. doi: 10.1038/230036a0. [DOI] [PubMed] [Google Scholar]
  38. Tamura F., Nishimura S., Ohki M. The E. coli divE mutation, which differentially inhibits synthesis of certain proteins, is in tRNASer1. EMBO J. 1984 May;3(5):1103–1107. doi: 10.1002/j.1460-2075.1984.tb01936.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tobias J. W., Shrader T. E., Rocap G., Varshavsky A. The N-end rule in bacteria. Science. 1991 Nov 29;254(5036):1374–1377. doi: 10.1126/science.1962196. [DOI] [PubMed] [Google Scholar]
  40. Williamson R. M., Oxender D. L. Premature termination of in vivo transcription of a gene encoding a branched-chain amino acid transport protein in Escherichia coli. J Bacteriol. 1992 Mar;174(6):1777–1782. doi: 10.1128/jb.174.6.1777-1782.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Willis D. K., Rich J. J., Kinscherf T. G., Kitten T. Genetic regulation in plant pathogenic pseudomonads. Genet Eng (N Y) 1994;16:167–193. [PubMed] [Google Scholar]
  42. Xu G. W., Gross D. C. Evaluation of the Role of Syringomycin in Plant Pathogenesis by Using Tn5 Mutants of Pseudomonas syringae pv. syringae Defective in Syringomycin Production. Appl Environ Microbiol. 1988 Jun;54(6):1345–1353. doi: 10.1128/aem.54.6.1345-1353.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Xu G. W., Gross D. C. Physical and functional analyses of the syrA and syrB genes involved in syringomycin production by Pseudomonas syringae pv. syringae. J Bacteriol. 1988 Dec;170(12):5680–5688. doi: 10.1128/jb.170.12.5680-5688.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES