1887

Abstract

In the soil bacterium M114, extracellular proteolytic activity and fluorescent siderophore (pseudobactin M114) production were previously shown to be co-ordinately negatively regulated in response to environmental iron levels. An iron-starvation extracytoplasmic function sigma factor, PbrA, required for the transcription of siderophore biosynthetic genes, was also implicated in M114 protease regulation. The current study centred on the characterization and genetic regulation of the gene(s) responsible for protease production in M114. A serralysin-type metalloprotease gene, , was identified and found to encode the major, if not only, extracellular protease produced by this strain. The expression of and its protein product were found to be subject to complex regulation. Transcription analysis confirmed that PbrA was required for full transcription under low iron conditions, while the ferric uptake regulator, Fur, was implicated in repression under high iron conditions. Interestingly, the iron regulation of AprA was dependent on culture conditions, with PbrA-independent AprA-mediated proteolytic activity observed on skim milk agar supplemented with yeast extract, when supplied with iron or purified pseudobactin M114. These effects were not observed on skim milk agar without yeast extract. PbrA-independent expression was also observed from a truncated transcriptional fusion when grown in sucrose asparagine tryptone broth supplied with iron or purified pseudobactin M114. Thus, experimental evidence suggested that iron mediated its effects via transcriptional activation by PbrA under low iron conditions, while an as-yet-unidentified sigma factor(s) may be required for the PbrA-independent expression and AprA proteolytic activity induced by siderophore and iron.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.28379-0
2006-01-01
2020-04-02
Loading full text...

Full text loading...

/deliver/fulltext/micro/152/1/29.html?itemId=/content/journal/micro/10.1099/mic.0.28379-0&mimeType=html&fmt=ahah

References

  1. Adams C, Dowling D. N, O'Sullivan D. J, O'Gara F. 1994; Isolation of a gene (pbsC) required for siderophore biosynthesis in fluorescent Pseudomonas sp. strain M114. Mol Gen Genet243:515–524[CrossRef]
    [Google Scholar]
  2. Ahn J. H, Pan J. G, Rhee J. S. 1999; Identification of the tliDEF ABC transporter specific for lipase in Pseudomonas fluorescens SIK W1. J Bacteriol181:1847–1852
    [Google Scholar]
  3. Altschul S. F, Madden T. L, Schaffer A. A, Zhang J, Zhang Z, Miller W, Lipman D. L. 1997; Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res25:3389–3402[CrossRef]
    [Google Scholar]
  4. Baysse C, Budzikiewicz H, Fernández D. U, Cornelis P. 2002; Impaired maturation of the siderophore pyoverdine chromophore in Pseudomonas fluorescens ATCC 17400 deficient for the cytochrome c biogenesis protein CcmC. FEBS Lett523:23–28[CrossRef]
    [Google Scholar]
  5. Beare P. A, For R. J, Martin L. W, Lamont I. L. 2003; Siderophore-mediated cell signalling in Pseudomonas aeruginosa : divergent pathways regulate virulence factor production and siderophore receptor synthesis. Mol Microbiol47:195–207
    [Google Scholar]
  6. Boyer H. W, Roulland-Dussoix D. 1969; A complementation analysis of the restriction and modification of DNA in Escherichia coli . J Mol Biol41:459–472[CrossRef]
    [Google Scholar]
  7. Burger M, Woods R. G, McCarthy C, Beacham I. R. 2000; Temperature regulation of protease in Pseudomonas fluorescens LS107d2 by an ECF sigma factor and a transmembrane activator. Microbiology146:3149–3155
    [Google Scholar]
  8. Callanan M, Sexton R, Dowling D. N, O'Gara F. 1996; Regulation of the iron uptake genes in Pseudomonas fluorescens M114 by pseudobactin M114: the pbrA sigma factor does not mediate the siderophore regulatory response. FEMS Microbiol Lett144:61–66[CrossRef]
    [Google Scholar]
  9. Chabeaud P, Bitter W, Tommassen J, Heulin T, Achouak W, de Groot A. 2001; Phase-variable expression of an operon encoding extracellular alkaline protease, a serine protease homolog, and lipase in Pseudomonas brassicacearum . J Bacteriol183:2117–2120[CrossRef]
    [Google Scholar]
  10. Chen W. P, Kuo T. T. 1993; A simple and rapid method for the preparation of gram-negative bacterial genomic DNA. Nucleic Acids Res21:2260[CrossRef]
    [Google Scholar]
  11. Chessa J. P, Petrescu I, Bentahir M, Gerday C, van Beeumen J. 2000; Purification, physico-chemical characterization and sequence of a heat labile alkaline metalloprotease isolated from a psychrophilic Pseudomonas species. Biochim Biophys Acta 1479;265–274[CrossRef]
    [Google Scholar]
  12. Duong F, Lazdunski A, Cami B, Murgier M. 1992; Sequence of a cluster of genes controlling synthesis and secretion of alkaline protease in Pseudomonas aeruginosa : relationships to other secretory pathways. Gene121:47–54[CrossRef]
    [Google Scholar]
  13. Escolar L, Pérez-Martín J, de Lorenzo V. 1999; Opening the iron box: transcriptional metalloregulation by the Fur protein. J Bacteriol181:6223–6229
    [Google Scholar]
  14. Figurski D. H, Helinski D. R. 1979; Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans . Proc Natl Acad Sci U S A76:1648–1652[CrossRef]
    [Google Scholar]
  15. Geels F. P, Schippers B. 1983; Reduction in yield depressions in high frequency potato cropping soil after seed tuber treatments with antagonistic fluorescent Pseudomonas spp. Phytopathol Z108:207–214[CrossRef]
    [Google Scholar]
  16. Helmann J. D. 2002; The extracytoplasmic function (ECF) sigma factors. Adv Microb Physiol46:47–110
    [Google Scholar]
  17. Kawai E, Idei A, Kumura H, Shimazaki K, Akatsuka H, Omori K. 1999; The ABC-exporter genes involved in the lipase secretion are clustered with the genes for lipase, alkaline protease, and serine protease homologues in Pseudomonas fluorescens no. 33. Biochim Biophys Acta1446:377–382[CrossRef]
    [Google Scholar]
  18. Kovach M. E, Phillips R. W, Elzer P. H, Roop R. M. I, Peterson K. M. 1994; pBBR1MCS: a broad-host-range cloning vector. Biotechniques16:800–802
    [Google Scholar]
  19. Kumeta H, Hoshino T, Goda T, Okayama T, Shimada T, Ohgiya S, Matsuyama H, Ishizaki K. 1999; Identification of a member of the serralysin family isolated from the psychrotrophic bacterium, Pseudomonas fluorescens 114. Biosci Biotechnol Biochem63:1165–1170[CrossRef]
    [Google Scholar]
  20. Lamont I. L, Beare P. A, Ochsner U, Vasil A. I, Vasil M. L. 2002; Siderophore-mediated signaling regulates virulence factor production in Pseudomonas aeruginosa . Proc Natl Acad Sci U S A99:7072–7077[CrossRef]
    [Google Scholar]
  21. Liao C. H, McCallus D. E. 1998; Biochemical and genetic characterisation of an extracellular protease from Pseudomonas fluorescens CY091. Appl Environ Microbiol64:914–921
    [Google Scholar]
  22. Marits R, Tshuikina M, Pirhonen M, Laasik E, Mäe A. 2002; Regulation of the expression of prtW  : :  gusA fusions in Erwinia carotovora subsp. carotovora . Microbiology148:835–842
    [Google Scholar]
  23. Meyer J. M, Abdallah M. A. 1978; The fluorescent pigment of Pseudomonas fluorescens : biosynthesis, purification and physicochemical properties. J Gen Microbiol107:319–328[CrossRef]
    [Google Scholar]
  24. Miller J. H. 1972; Experiments in Molecular Biology Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  25. Moënne-Loccoz Y, Tichy H. V, O'Donnell A, Simon R, O'Gara F. 2001; Impact of 2,4-diacetylphloroglucinol-producing biocontrol strain Pseudomonas fluorescens F113 on intraspecific diversity of resident culturable fluorescent pseudomonads associated with the roots of field-grown sugar beet seedlings. Appl Environ Microbiol67:3418–3425[CrossRef]
    [Google Scholar]
  26. Moores J. C, Magazin M, Ditta G. S, Leong J. 1984; Cloning of genes involved in biosynthesis of pseudobactin, a high affinity iron transport agent of a plant growth-promoting Pseudomonas strain. J Bacteriol157:53–58
    [Google Scholar]
  27. Morris J, O'Sullivan D. J, Koster M, Leong J, Weisbeek P. J, O'Gara F. 1992; Characterization of fluorescent siderophore-mediated iron uptake in Pseudomonas sp. strain M114: evidence for the existence of an additional ferric siderophore receptor. Appl Environ Microbiol58:630–635
    [Google Scholar]
  28. Ochsner U. A, Wilderman P. J, Vasil A. I, Vasil M. L. 2002; GeneChip expression analysis of the iron starvation response in Pseudomonas aeruginosa : identification of novel pyoverdine biosynthesis genes. Mol Microbiol45:1277–1287[CrossRef]
    [Google Scholar]
  29. O'Gara F, Treacy P, O'Sullivan D, O'Sullivan M, Higgins P. 1986; Biological control of phytopathogens by Pseudomonas spp. genetic aspects of siderophore production and root colonization. In Iron, Siderophores and Plant Diseases pp 331–339 Edited by Swinburne T. R.. New York: Plenum;
    [Google Scholar]
  30. Pujic P, Dervyn R, Sorokin A, Ehrlich S. D. 1998; The kdgRKAT operon of Bacillus subtilis : detection of the transcript and regulation by the kdgR and ccpA genes. Microbiology144:3111–3118[CrossRef]
    [Google Scholar]
  31. Rédly G. A, Poole K. 2003; Pyoverdine-mediated regulation of FpvA synthesis in Pseudomonas aeruginosa : involvement of a probable extracytoplasmic-function sigma factor, FpvI. J Bacteriol185:1261–1265[CrossRef]
    [Google Scholar]
  32. Rombel I. T, McMorran B. J, Lamont I. L. 1995; Identification of a DNA sequence motif required for expression of iron-regulated genes in pseudomonads. Mol Gen Genet246:519–528[CrossRef]
    [Google Scholar]
  33. Ruangviriyachai C. 1991; Liquid chromatographic and complexation analysis of natural and synthetic chelating agents. PhD thesis University College Cork; Cork, Ireland:
    [Google Scholar]
  34. Sambrook J, Fritsch E. F, Maniatis T. 1989; Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  35. Sarath G, Wagner F. W, de la Motte R. 1989; Protease assay methods. In Proteolytic Enzymes: a Practical Approach pp 25–55 Edited by Beynon R. J., Bond J. S.. Oxford: Oxford University Press;
    [Google Scholar]
  36. Schafer A, Tauch A, Jager W, Kalinowski J, Puhler A. 1994; Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum . Gene145:69–73[CrossRef]
    [Google Scholar]
  37. Scher F. M, Baker R. 1982; Effects of Pseudomonas putida and a synthetic iron chelator on induction of soil suppressiveness to Fusarium wilt pathogens. Phytopathology72:1567–1573[CrossRef]
    [Google Scholar]
  38. Sexton J. R. 1995; Cloning and characterisation of transcriptional activator(s) required for iron regulated gene expression in Pseudomonas fluorescens M114 PhD thesis University College Cork; Cork, Ireland:
    [Google Scholar]
  39. Sexton R, O'Sullivan D. J, Dowling D. N, O'Gara F, Gill P. R., Jr, Callanan M. J. 1995; Iron responsive gene expression in Pseudomonas fluorescens M114: cloning and characterization of a transcription-activating factor, PbrA. Mol Microbiol15:297–306[CrossRef]
    [Google Scholar]
  40. Sexton R, O'Gara F, Gill P. R., Jr, Dowling D. N. 1996; Transcriptional regulation of the iron-responsive sigma factor gene pbrA . Mol Gen Genet250:50–58
    [Google Scholar]
  41. Shanahan P, O'Sullivan D. J, Simpson P, Glennon J. D, O'Gara F. 1992; Isolation of 2,4-diacetylphloroglucinol from a fluorescent pseudomonad and investigation of physiological parameters influencing its production. Appl Environ Microbiol58:353–358
    [Google Scholar]
  42. Shen J, Meldrum A, Poole K. 2002; FpvA receptor involvement in pyoverdine biosynthesis in Pseudomonas aeruginosa . J Bacteriol184:3268–3275[CrossRef]
    [Google Scholar]
  43. Shigematsu T, Fukushima J, Oyama M, Tsuda M, Kawamoto S, Okuda K. 2001; Iron-mediated regulation of alkaline proteinase production in Pseudomonas aeruginosa . Microbiol Immunol45:579–590[CrossRef]
    [Google Scholar]
  44. Spaink H. P, Okker R. J. H, Wijffelman C. A, Pees E, Lugtenberg B. J. J. 1987; Promoters in the nodulation region of the Rhizobium leguminosarum Sym plasmid pRL1JI. Plant Mol Biol9:27–39[CrossRef]
    [Google Scholar]
  45. Vasil M. L, Ochsner U. A. 1999; The response of Pseudomonas aeruginosa to iron: genetics, biochemistry and virulence. Mol Microbiol34:399–413[CrossRef]
    [Google Scholar]
  46. Visca P, Leoni L, Wilson M. J, Lamont I. L. 2002; Iron transport and regulation, cell signalling and genomics: lessons from Escherichia coli and Pseudomonas . Mol Microbiol45:1177–1190[CrossRef]
    [Google Scholar]
  47. Voisard C, Bull C, Keel C, Laville J, Maurhofer M, Schnider U, Défago G, Haas D. 1994; Biocontrol of root diseases by Pseudomonas fluorescens CHA0: current concepts and experimental approaches. In Molecular Ecology of Rhizosphere Microorganisms pp 67–89 Edited by O'Gara F., Dowling D. N., Boesten B.. Weinheim, Germany: VCH Publishers;
    [Google Scholar]
  48. Wilderman P. J, Vasil A. I, Johnson Z, Wilson M. J, Cunliffe H. E, Lamont I. L, Vasil M. L. 2001; Characterization of an endoprotease (PrpL) encoded by a PvdS-regulated gene in Pseudomonas aeruginosa . Infect Immun69:5385–5394[CrossRef]
    [Google Scholar]
  49. Wilson M. J, McMorran B. J, Lamont I. L. 2001; Analysis of promoters recognized by PvdS, an extracytoplasmic-function sigma factor protein from Pseudomonas aeruginosa . J Bacteriol183:2151–2155[CrossRef]
    [Google Scholar]
  50. Woods R. G, Burger M, Beven C. A, Beacham I. R. 2001; The aprX–lipA operon of Pseudomonas fluorescens B52: a molecular analysis of metalloprotease and lipase production. Microbiology147:345–354
    [Google Scholar]
  51. Yarwood J. M, Volper E. M, Greenberg E. P. 2005; Delays in Pseudomonas aeruginosa quorum-sensing are conditional. Proc Natl Acad Sci U S A102:9008–9013[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.28379-0
Loading
/content/journal/micro/10.1099/mic.0.28379-0
Loading

Data & Media loading...

Most cited this month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error