1887

Abstract

Some strains produce a catechol-type siderophore named vanchrobactin, whose biosynthetic pathway has not been completely elucidated. In addition to the previously described genes , , , , and , in the present study we have identified the genes encoding a DAHP (3-deoxy--arabino-heptulosonate-7-phosphate) synthetase (), a phosphopantheteinyl transferase (), a LysR-family transcriptional regulator () and a putative siderophore receptor (). A deletion affecting or greatly reduced growth under iron-limiting conditions, whereas deletion of did not have significant effects. Vanchrobactin production was abolished in the mutant, whereas the mutant retained a residual vanchrobactin production ability. Reverse transcriptase-mediated PCR indicated that this 11-gene cluster is organized into six iron-regulated transcriptional units. Transcriptional fusions demonstrated that the ferric uptake regulator (Fur) protein is the main iron-responsive regulator of these genes. Interestingly, the gene was strongly iron-repressed, but Fur was not essential for this repression. In addition, the maximal expression from the promoter was achieved only in the presence of an intact copy of . Analysis of the -galactosidase activities of a  : :  fusion in a mutant and in the presence of added vanchrobactin suggested that a ferric-vanchrobactin-dependent activator plays a positive regulatory role in transcription of the operon. This possibility is reinforced by the presence of a predicted AraC box upstream of . We propose that vanchrobactin biosynthesis is subjected to a complex regulatory circuitry aimed at adjusting vanchrobactin production for the maintenance of iron homeostasis in .

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2008/016618-0
2008-05-01
2019-10-15
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/5/1400.html?itemId=/content/journal/micro/10.1099/mic.0.2008/016618-0&mimeType=html&fmt=ahah

References

  1. Alice, A. F., Lopez, C. S. & Crosa, J. H. ( 2005; ). Plasmid- and chromosome-encoded redundant and specific functions are involved in biosynthesis of the siderophore anguibactin in Vibrio anguillarum 775: a case of chance and necessity? J Bacteriol 187, 2209–2214.[CrossRef]
    [Google Scholar]
  2. Anderson, M. T. & Armstrong, S. K. ( 2004; ). The BfeR regulator mediates enterobactin-inducible expression of Bordetella enterobactin utilization genes. J Bacteriol 186, 7302–7311.[CrossRef]
    [Google Scholar]
  3. Arnow, L. E. ( 1937; ). Colorimetric determination of the components of 3,4 dihydroxyphenyl-alanine-tyrosine mixtures. J Biol Chem 118, 531–537.
    [Google Scholar]
  4. Balado, M., Osorio, C. R. & Lemos, M. L. ( 2006; ). A gene cluster involved in the biosynthesis of vanchrobactin, a chromosome-encoded siderophore produced by Vibrio anguillarum. Microbiology 152, 3517–3528.[CrossRef]
    [Google Scholar]
  5. Bateman, A., Coin, L., Durbin, R., Finn, R. D., Hollich, V., Griffiths-Jones, S., Khanna, A., Marshall, M., Moxon, S. & other authors ( 2004; ). The Pfam protein families database. Nucleic Acids Res 32, D138–D141.[CrossRef]
    [Google Scholar]
  6. Braun, V. & Killmann, H. ( 1999; ). Bacterial solutions to the iron-supply problem. Trends Biochem Sci 24, 104–109.[CrossRef]
    [Google Scholar]
  7. Braun, V., Hantke, K. & Koster, W. ( 1998; ). Bacterial iron transport: mechanisms, genetics, and regulation. Met Ions Biol Syst 35, 67–145.
    [Google Scholar]
  8. Brickman, T. J., Kang, H. Y. & Armstrong, S. K. ( 2001; ). Transcriptional activation of Bordetella alcaligin siderophore genes requires the AlcR regulator with alcaligin as inducer. J Bacteriol 183, 483–489.[CrossRef]
    [Google Scholar]
  9. Butterton, J. R., Stoebner, J. A., Payne, S. M. & Calderwood, S. B. ( 1992; ). Cloning, sequencing, and transcriptional regulation of viuA, the gene encoding the ferric vibriobactin receptor of Vibrio cholerae. J Bacteriol 174, 3729–3738.
    [Google Scholar]
  10. Challis, G. L. ( 2005; ). A widely distributed bacterial pathway for siderophore biosynthesis independent of nonribosomal peptide synthetases. ChemBioChem 6, 601–611.[CrossRef]
    [Google Scholar]
  11. Chen, Q. & Crosa, J. H. ( 1996; ). Antisense RNA, Fur, iron, and the regulation of iron transport genes in Vibrio anguillarum. J Biol Chem 271, 18885–18891.[CrossRef]
    [Google Scholar]
  12. Chen, Q., Actis, L. A., Tolmasky, M. E. & Crosa, J. H. ( 1994; ). Chromosome-mediated 2,3-dihydroxybenzoic acid is a precursor in the biosynthesis of the plasmid-mediated siderophore anguibactin in Vibrio anguillarum. J Bacteriol 176, 4226–4234.
    [Google Scholar]
  13. Conchas, R. F., Lemos, M. L., Barja, J. L. & Toranzo, A. E. ( 1991; ). Distribution of plasmid- and chromosome-mediated iron uptake systems in Vibrio anguillarum strains of different origins. Appl Environ Microbiol 57, 2956–2962.
    [Google Scholar]
  14. Crosa, J. H. ( 1989; ). Genetics and molecular biology of siderophore-mediated iron transport in bacteria. Microbiol Rev 53, 517–530.
    [Google Scholar]
  15. Crosa, J. H. & Walsh, C. T. ( 2002; ). Genetics and assembly line enzymology of siderophore biosynthesis in bacteria. Microbiol Mol Biol Rev 66, 223–249.[CrossRef]
    [Google Scholar]
  16. de Lorenzo, V., Wee, S., Herrero, M. & Neilands, J. B. ( 1987; ). Operator sequences of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulation (fur) repressor. J Bacteriol 169, 2624–2630.
    [Google Scholar]
  17. Di Lorenzo, M., Stork, M., Tolmasky, M. E., Actis, L. A., Farrell, D., Welch, T. J., Crosa, L. M., Wertheimer, A. M., Chen, Q. & other authors ( 2003; ). Complete sequence of virulence plasmid pJM1 from the marine fish pathogen Vibrio anguillarum strain 775. J Bacteriol 185, 5822–5830.[CrossRef]
    [Google Scholar]
  18. Escolar, L., Perez-Martin, J. & de Lorenzo, V. ( 1999; ). Opening the iron box: transcriptional metalloregulation by the Fur protein. J Bacteriol 181, 6223–6229.
    [Google Scholar]
  19. Fetherston, J. D., Bearden, S. W. & Perry, R. D. ( 1996; ). YbtA, an AraC-type regulator of the Yersinia pestis pesticin/yersiniabactin receptor. Mol Microbiol 22, 315–325.[CrossRef]
    [Google Scholar]
  20. Gallegos, M. T., Schleif, R., Bairoch, A., Hofmann, K. & Ramos, J. L. ( 1997; ). AraC/XylS family of transcriptional regulators. Microbiol Mol Biol Rev 61, 393–410.
    [Google Scholar]
  21. Hall, T. A. ( 1999; ). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41, 95–98.
    [Google Scholar]
  22. Hantke, K. ( 1987; ). Selection procedure for deregulated iron transport mutants (fur) in Escherichia coli K 12: fur not only affects iron metabolism. Mol Gen Genet 210, 135–139.[CrossRef]
    [Google Scholar]
  23. Herrero, M., de Lorenzo, V. & Timmis, K. N. ( 1990; ). Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in Gram-negative bacteria. J Bacteriol 172, 6557–6567.
    [Google Scholar]
  24. Hodgson, D. A. ( 2000; ). Primary metabolism and its control in streptomycetes: a most unusual group of bacteria. Adv Microb Physiol 42, 47–238.
    [Google Scholar]
  25. Kloosterman, H., Hessels, G. I., Vrijbloed, J. W., Euverink, G. J. & Dijkhuizen, L. ( 2003; ). (De)regulation of key enzyme steps in the shikimate pathway and phenylalanine-specific pathway of the actinomycete Amycolatopsis methanolica. Microbiology 149, 3321–3330.[CrossRef]
    [Google Scholar]
  26. Lemos, M. L., Salinas, P., Toranzo, A. E., Barja, J. L. & Crosa, J. H. ( 1988; ). Chromosome-mediated iron uptake system in pathogenic strains of Vibrio anguillarum. J Bacteriol 170, 1920–1925.
    [Google Scholar]
  27. Litwin, C. M. & Quackenbush, J. ( 2001; ). Characterization of a Vibrio vulnificus LysR homologue, HupR, which regulates expression of the haem uptake outer membrane protein, HupA. Microb Pathog 31, 295–307.[CrossRef]
    [Google Scholar]
  28. Liu, Q., Ma, Y., Zhou, L. & Zhang, Y. ( 2005; ). Gene cloning, expression and functional characterization of a phosphopantetheinyl transferase from Vibrio anguillarum serotype O1. Arch Microbiol 183, 37–44.[CrossRef]
    [Google Scholar]
  29. Masse, E. & Gottesman, S. ( 2002; ). A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli. Proc Natl Acad Sci U S A 99, 4620–4625.[CrossRef]
    [Google Scholar]
  30. Michel, L., Gonzalez, N., Jagdeep, S., Nguyen-Ngoc, T. & Reimmann, C. ( 2005; ). PchR-box recognition by the AraC-type regulator PchR of Pseudomonas aeruginosa requires the siderophore pyochelin as an effector. Mol Microbiol 58, 495–509.[CrossRef]
    [Google Scholar]
  31. Miethke, M., Westers, H., Blom, E. J., Kuipers, O. P. & Marahiel, M. A. ( 2006; ). Iron starvation triggers the stringent response and induces amino acid biosynthesis for bacillibactin production in Bacillus subtilis. J Bacteriol 188, 8655–8657.[CrossRef]
    [Google Scholar]
  32. Miller, J. H. ( 1992; ). A Short Course in Bacterial Genetics. Plainview, NY: Cold Spring Harbor Laboratory.
  33. Mouriño, S., Osorio, C. R. & Lemos, M. L. ( 2004; ). Characterization of heme uptake cluster genes in the fish pathogen Vibrio anguillarum. J Bacteriol 186, 6159–6167.[CrossRef]
    [Google Scholar]
  34. Osorio, C. R., Juiz-Rio, S. & Lemos, M. L. ( 2006; ). A siderophore biosynthesis gene cluster from the fish pathogen Photobacterium damselae subsp. piscicida is structurally and functionally related to the Yersinia high-pathogenicity island. Microbiology 152, 3327–3341.[CrossRef]
    [Google Scholar]
  35. Panina, E. M., Vitreschak, A. G., Mironov, A. A. & Gelfand, M. S. ( 2001; ). Regulation of aromatic amino acid biosynthesis in gamma-proteobacteria. J Mol Microbiol Biotechnol 3, 529–543.
    [Google Scholar]
  36. Panina, E. M., Vitreschak, A. G., Mironov, A. A. & Gelfand, M. S. ( 2003; ). Regulation of biosynthesis and transport of aromatic amino acids in low-GC Gram-positive bacteria. FEMS Microbiol Lett 222, 211–220.[CrossRef]
    [Google Scholar]
  37. Parales, R. E. & Harwood, C. S. ( 1993; ). Construction and use of a new broad-host-range lacZ transcriptional fusion vector, pHRP309, for Gram bacteria. Gene 133, 23–30.[CrossRef]
    [Google Scholar]
  38. Pelludat, C., Rakin, A., Jacobi, C. A., Schubert, S. & Heesemann, J. ( 1998; ). The yersiniabactin biosynthetic gene cluster of Yersinia enterocolitica: organization and siderophore-dependent regulation. J Bacteriol 180, 538–546.
    [Google Scholar]
  39. Pittard, J., Camakaris, H. & Yang, J. ( 2005; ). The TyrR regulon. Mol Microbiol 55, 16–26.
    [Google Scholar]
  40. Ratledge, C. & Dover, L. G. ( 2000; ). Iron metabolism in pathogenic bacteria. Annu Rev Microbiol 54, 881–941.[CrossRef]
    [Google Scholar]
  41. Sambrook, J. & Russell, D. W. ( 2001; ). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  42. Schell, M. A. ( 1993; ). Molecular biology of the LysR family of transcriptional regulators. Annu Rev Microbiol 47, 597–626.[CrossRef]
    [Google Scholar]
  43. Schwarzer, D., Finking, R. & Marahiel, M. A. ( 2003; ). Nonribosomal peptides: from genes to products. Nat Prod Rep 20, 275–287.[CrossRef]
    [Google Scholar]
  44. Schwyn, B. & Neilands, J. B. ( 1987; ). Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160, 47–56.[CrossRef]
    [Google Scholar]
  45. Soengas, R. G., Anta, C., Espada, A., Paz, V., Ares, I. R., Balado, M., Rodriguez, J., Lemos, M. L. & Jiménez, C. ( 2006; ). Structural characterization of vanchrobactin, a new catechol siderophore produced by the fish pathogen Vibrio anguillarum serotype O2. Tetrahedron Lett 47, 7113–7116.[CrossRef]
    [Google Scholar]
  46. Stojiljkovic, I., Baumler, A. J. & Hantke, K. ( 1994; ). Fur regulon in Gram-negative bacteria. Identification and characterization of new iron-regulated Escherichia coli genes by a Fur titration assay. J Mol Biol 236, 531–545.[CrossRef]
    [Google Scholar]
  47. Stork, M., Di Lorenzo, M., Welch, T. J., Crosa, L. M. & Crosa, J. H. ( 2002; ). Plasmid-mediated iron uptake and virulence in Vibrio anguillarum. Plasmid 48, 222–228.[CrossRef]
    [Google Scholar]
  48. Stork, M., Di Lorenzo, M., Welch, T. J. & Crosa, J. H. ( 2007; ). Transcription termination within the iron transport-biosynthesis operon of Vibrio anguillarum requires an antisense RNA. J Bacteriol 189, 3479–3488.[CrossRef]
    [Google Scholar]
  49. Tabor, S. & Richardson, C. C. ( 1985; ). A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A 82, 1074–1078.[CrossRef]
    [Google Scholar]
  50. Tolmasky, M. E. & Crosa, J. H. ( 1991; ). Regulation of plasmid-mediated iron transport and virulence in Vibrio anguillarum. Biol Met 4, 33–35.[CrossRef]
    [Google Scholar]
  51. Tolmasky, M. E., Actis, L. A. & Crosa, J. H. ( 1993; ). A single amino acid change in AngR, a protein encoded by pJM1-like virulence plasmids, results in hyperproduction of anguibactin. Infect Immun 61, 3228–3233.
    [Google Scholar]
  52. Tolmasky, M. E., Wertheimer, A. M., Actis, L. A. & Crosa, J. H. ( 1994; ). Characterization of the Vibrio anguillarum fur gene: role in regulation of expression of the FatA outer membrane protein and catechols. J Bacteriol 176, 213–220.
    [Google Scholar]
  53. Toranzo, A. E. & Barja, J. L. ( 1990; ). A review of the taxonomy and seroepizootiology of Vibrio anguillarum, with special reference to aquaculture in the northwest of Spain. Dis Aquat Organ 9, 73–82.[CrossRef]
    [Google Scholar]
  54. Touati, D. ( 2000; ). Iron and oxidative stress in bacteria. Arch Biochem Biophys 373, 1–6.[CrossRef]
    [Google Scholar]
  55. Vasil, M. L., Ochsner, U. A., Johnson, Z., Colmer, J. A. & Hamood, A. N. ( 1998; ). The fur-regulated gene encoding the alternative sigma factor PvdS is required for iron-dependent expression of the LysR-type regulator ptxR in Pseudomonas aeruginosa. J Bacteriol 180, 6784–6788.
    [Google Scholar]
  56. Wandersman, C. & Delepelaire, P. ( 2004; ). Bacterial iron sources: from siderophores to hemophores. Annu Rev Microbiol 58, 611–647.[CrossRef]
    [Google Scholar]
  57. Wang, R. F. & Kushner, S. R. ( 1991; ). Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene 100, 195–199.[CrossRef]
    [Google Scholar]
  58. Watnick, P. I., Butterton, J. R. & Calderwood, S. B. ( 1998; ). The interaction of the Vibrio cholerae transcription factors, Fur and IrgB, with the overlapping promoters of two virulence genes, irgA and irgB. Gene 209, 65–70.[CrossRef]
    [Google Scholar]
  59. Webster, A. C. & Litwin, C. M. ( 2000; ). Cloning and characterization of vuuA, a gene encoding the Vibrio vulnificus ferric vulnibactin receptor. Infect Immun 68, 526–534.[CrossRef]
    [Google Scholar]
  60. Welch, T. J., Chai, S. & Crosa, J. H. ( 2000; ). The overlapping angB and angG genes are encoded within the trans-acting factor region of the virulence plasmid in Vibrio anguillarum: essential role in siderophore biosynthesis. J Bacteriol 182, 6762–6773.[CrossRef]
    [Google Scholar]
  61. Wertheimer, A. M., Verweij, W., Chen, Q., Crosa, L. M., Nagasawa, M., Tolmasky, M. E., Actis, L. A. & Crosa, J. H. ( 1999; ). Characterization of the angR gene of Vibrio anguillarum: essential role in virulence. Infect Immun 67, 6496–6509.
    [Google Scholar]
  62. Wolf, M. K. & Crosa, J. H. ( 1986; ). Evidence for the role of a siderophore in promoting Vibrio anguillarum infections. J Gen Microbiol 132, 2949–2952.
    [Google Scholar]
  63. Yanofsky, C. ( 2000; ). Transcription attenuation: once viewed as a novel regulatory strategy. J Bacteriol 182, 1–8.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2008/016618-0
Loading
/content/journal/micro/10.1099/mic.0.2008/016618-0
Loading

Data & Media loading...

vol. , part 5, pp. 1400 - 1413

Oligonucleotides used in this study [PDF file](44 KB)



PDF
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