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Volume 147, Issue 8

Quorum-sensing-dependent regulation of biosynthesis of the polyketide antibiotic mupirocin in NCIMB 10586

The GenBank accession numbers for the sequences determined in this work are AF318063 (), AF318064 () and AF318065 ().

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Abstract

Mupirocin (pseudomonic acid) is a polyketide antibiotic, targeting isoleucyl-tRNA synthase, and produced by NCIMB 10586. It is used clinically as a topical treatment for staphylococcal infections, particularly in contexts where there is a problem with methicillin-resistant (MRSA). In studying the mupirocin biosynthetic cluster the authors identified two putative regulatory genes, and , whose predicted amino acid sequences showed significant identity to proteins involved in quorum-sensing-dependent regulatory systems such as LasR/LuxR (transcriptional activators) and LasI/LuxI (synthases for acylhomoserine lactones – AHLs – that activate LasR/LuxR). Inactivation by deletion mutations using a suicide vector strategy confirmed the requirement for both genes in mupirocin biosynthesis. Cross-feeding experiments between bacterial strains as well as solvent extraction showed that, as predicted, wild-type NCIMB 10586 produces a diffusible substance that overcomes the defect of a mutant. Use of biosensor strains showed that the MupI product can activate the system and that produces one or more compounds that can replace the MupI product. Insertion of a reporter gene into , the first ORF of the mupirocin biosynthetic operon, showed that together / control expression of the operon in such a way that the cluster is switched on late in exponential phase and in stationary phase.

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Keyword(s): 3-OH-C10-HSL, N-(3 hydroxydecanoyl)-l-homoserine lactone , 3-OH-C14:1-HSL, N-(3-hydroxytetradecenoyl)-l-homoserine lactone , 3-OH-C6-HSL, N-(3-hydroxyhexanoyl)-l-homoserine lactone , 3-OH-C8-HSL, N-(3 hydroxyoctanoyl)-l-homoserine lactone , 3-oxo-C12-HSL, N-(3-oxododecanoyl)-l-homoserine lactone , 3-oxo-C6-HSL, N-(3-oxohexanoyl)-l-homoserine lactone , AHL, N-acylhomoserine lactone , C10-HSL, N-decanoyl-l-homoserine lactone , C4-HSL, N-butyryl-l-homoserine lactone , C6-HSL, N-hexanoyl-l-homoserine lactone , diffusible activator , genetic manipulation , N-acylhomoserine lactone , reporter genes and Staphylococcus aureus
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2001-08-01
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References

  1. Bever R. A., Iglewski B. H. 1988; Molecular characterization and nucleotide sequence of the Pseudomonas aeruginosa elastase structural gene. J Bacteriol 170:4309–4314
     [Google Scholar]
  2. Birnboim H. C., Doly J. 1979; A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523 [CrossRef]
     [Google Scholar]
  3. Choi S. H., Greenberg E. P. 1991; The C-terminal region of the Vibrio fischeri LuxR contains an inducer-dependent lux gene activating domain. Proc Natl Acad Sci USA 88:11115–11119 [CrossRef]
     [Google Scholar]
  4. Cohen S. N., Chang A. C. Y., Hsu H. 1972; Non-chromosomal antibiotic resistance in bacteria; genetic transformation of Escherichia coli by R factor DNA. Proc Natl Acad Sci USA 69:2110–2114 [CrossRef]
     [Google Scholar]
  5. Cohn D. H., Mileham A. J., Simon M. I., Nealson K. H., Rausch S. K., Bonam D., Baldwin T. O. 1985; Nucleotide sequence of the luxA gene of Vibrio harveyi and the complete amino acid sequence of the α subunit of bacterial luciferase. J Biol Chem 260:6139–6146
     [Google Scholar]
  6. DeKievit T., Seed P. C., Nezezon J., Passador L., Iglewski B. H. 1999; RsaL, a novel repressor of virulence gene expression in Pseudomonas aeruginosa . J Bacteriol 181:2175–2184
     [Google Scholar]
  7. Devine J. H., Countryman C., Baldwin T. O. 1988; Nucleotide sequence of the luxR and luxI genes and structure of the primary regulatory region of the lux regulon of Vibrio fischeri ATCC7744. Biochemistry 27:837–842 [CrossRef]
     [Google Scholar]
  8. Devine J. H., Shadel G. S., Baldwin T. O. 1989; Identification of the operator of the lux regulon from Vibrio fischeri ATCC7744. Proc Natl Acad Sci USA 86:5688–5692 [CrossRef]
     [Google Scholar]
  9. Devereux J., Haeberli P., Smithies O. 1984; A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395 [CrossRef]
     [Google Scholar]
  10. Dunn D. K., Michaliszyn G. A., Bogaki I. B., Meighen E. A. 1973; Conversion of aldehyde to acid into the bacterial bioluminescent reaction. Biochemistry 12:4911–4918 [CrossRef]
     [Google Scholar]
  11. Flavier A. B., Ganova-Raeva L. M., Schell M. A., Denny T. P. 1997; Hierarchical autoinduction in Ralstonia solanacearum : control of acyl-homoserine lactone production by a novel autoregulatory system responsive to 3-hydroxypalmitic acid methyl ester. J Bacteriol 179:7089–7097
     [Google Scholar]
  12. Fuqua W. C., Winans S. C. 1994; A LuxR–LuxI type regulatory system activates Agrobacterium Ti plasmid conjugal transfer in the presence of a plant tumour metabolite. J Bacteriol 176:2796–2806
     [Google Scholar]
  13. Fuqua W. C., Winans S. C., Greenberg E. P. 1994; Quorum-sensing in bacteria: the LuxR–LuxI family of cell density-responsive transcriptional regulators. J Bacteriol 176:269–275
     [Google Scholar]
  14. Fuqua W. C., Winans S. C., Greenberg E. P. 1996; Census and consensus in bacterial ecosystems: the LuxR–LuxI family of quorum-sensing transcriptional regulators. Annu Rev Microbiol 50:727–751 [CrossRef]
     [Google Scholar]
  15. Gambello M. J., Kaye S., Iglewski B. H. 1991; Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression. J Bacteriol 173:3000–3009
     [Google Scholar]
  16. Gambello M. J., Kaye S., Iglewski B. H. 1993; LasR of Pseudomonas aeruginosa is a transcriptional activator of the alkaline protease gene ( apr ) and an enhancer of exotoxin A expression. Infect Immum 61:1180–1184
     [Google Scholar]
  17. Gay P., Le Coq D., Steinmetz M., Berkelman T., Hado C. I. 1985; Positive selection procedure for the entrapment of insertion sequence elements in Gram-negative bacteria. J Bacteriol 164:918–921
     [Google Scholar]
  18. Gibson T. J. 1984; Studies on the Epstein–Barr virus genome. PhD thesisCambridge University, UK
     [Google Scholar]
  19. Gornall A. G., Bardawill C. J., David M. M. 1949; Determination of serum proteins by means of the biuret reaction. J Biol Chem 177:751–766
     [Google Scholar]
  20. Gray K. M., Greenberg E. P. 1992; Sequence analysis of luxR and luxI , the luminescence regulatory genes from the squid light organ symbiont Vibrio fischeri ES114. Mol Mar Biol Biotechnol 1:414–419
     [Google Scholar]
  21. Greenberg E. P. 2000; Acyl-homoserine lactone quorum sensing in bacteria. J Microbiol 38:117–121
     [Google Scholar]
  22. Hanzelka B. L., Greenberg E. P. 1995; Evidence that the N-terminal region of the Vibrio fischeri LuxR constitutes an autoinducer-binding domain. J Bacteriol 177:815–817
     [Google Scholar]
  23. Hughes J., Mellows G. 1978; Inhibition of isoleucyl-transfer ribonucleic acid synthase in Escherichia coli by pseudomonic acid. Biochem J 176:305–318
     [Google Scholar]
  24. Hughes J., Mellows G. 1980; Interaction of pseudomonic acid A with Escherichia coli B isoleucyl-tRNA synthase. Biochem J 191:209–219
     [Google Scholar]
  25. Hwang I., Li P.-L., Zhang L., Piper K. R., Cook D. M., Tate M. E., Farrand S. K. 1994; TraI, a LuxI homologue, is responsible for production of conjugation factor, the Ti plasmid N- acylhomoserine lactone autoinducer. Proc Natl Acad Sci USA 91:4639–4643 [CrossRef]
     [Google Scholar]
  26. Jagura-Burdzy G., Kostellidou K., Pole J., Khare D., Jones A. C., Williams D. R., Thomas C. M. 1999; IncC of broad-host-range plasmid RK2 modulates KorB transcriptional repressor activity in vivo and operator binding in vitro . J Bacteriol 181:2807–2815
     [Google Scholar]
  27. Johnston T. C., Thompson R. B., Baldwin T. O. 1986; Nucleotide sequence of luxB gene of Vibrio harveyi and the complete amino acid sequence of the β subunit of the bacterial luciferase. J Biol Chem 261:4805–4811
     [Google Scholar]
  28. Kahn M., Kolter R., Thomas C. M., Figurski D. H., Meyer R., Remeut E., Helinski D. R. 1979; Plasmid cloning vehicles derived from plasmids ColE1 and RK2. Methods Enzymol 68:268–280
     [Google Scholar]
  29. Kaiser D., Losick R. 1993; How and why bacteria talk to each other. Cell 73:873–885 [CrossRef]
     [Google Scholar]
  30. Kaplan H. B., Greenberg E. P. 1985; Diffusion of autoinducer is involved in regulation of Vibrio fischeri luminescence system. J Bacteriol 163:1210–1214
     [Google Scholar]
  31. Kuo A., Blough N. V., Dunlap P. V. 1994; Multiple N- acyl-l-homoserine lactone autoinducers of luminescence in the marine symbiotic bacterium Vibrio fischeri . J Bacteriol 176:7558–7565
     [Google Scholar]
  32. Latifi A., Winson M. K., Foglino M., Bycroft B. W., Stewart G. S. A. B., Lazdunski A., Williams P. 1995; Multiple homologues of LuxR and LuxI control expression of virulence determinants and secondary metabolites through quorum sensing in Pseudomonas aeruginosa PAO1. Mol Microbiol 17:333–343 [CrossRef]
     [Google Scholar]
  33. Latifi A., Foglino M., Tanaka K., Williams P., Lazdunski A. 1996; A hierarchical quorum-sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhlR (VsmR) to expression of the stationary-phase sigma factor RpoS. Mol Microbiol 21:1137–1146 [CrossRef]
     [Google Scholar]
  34. Laue B. E., Jiang Y., Chhabra S. R., Jacob S., Stewart G. S. A. B., Hardman A., Downie J. A., O’Gara F., Williams P. 2000; The biocontrol strain Pseudomonas fluorescens F113 produces the Rhizobium small bacteriocin, N -(3-hydroxy-7- cis -tetradecanoyl)homoserine lactone, via HdtS, a putative novel N- acylhomoserine lactone synthase. Microbiology 146:2469–2480
     [Google Scholar]
  35. Lewenza S., Conway B., Greenberg E. P., Sokol P. A. 1999; Quorum sensing in Burkholderia cepacia : identification of the LuxRI homologue CepRI. J Bacteriol 181:748–756
     [Google Scholar]
  36. Macartney P. 1996; Molecular and genetic analyses of the central control regions of broad host range bacterial plasmids RK2 and R75 1. PhD thesisUniversity of Birmingham
     [Google Scholar]
  37. McClean K. H., Winson M. K., Fish L. 9 other authors 1997; Quorum sensing and Chromobacterium violaceum : exploitation of violacein production and inhibition for the detection of N- acylhomoserine lactones. Microbiology 143:3703–3711 [CrossRef]
     [Google Scholar]
  38. Martin M., Showalter R., Silverman M. 1989; Identification of the luminescence genes in Vibrio harveyi . J Bacteriol 171:2406–2414
     [Google Scholar]
  39. Meighen E. A. 1988; Enzymes and genes from the lux operon of bioluminescent bacteria. Annu Rev Microbiol 42:151–176 [CrossRef]
     [Google Scholar]
  40. Moir A., Lafferty E., Smith D. A. 1979; Genetic analysis of spore germination mutants of Bacillus subtilis 168: the correlation of phenotype with map location. J Gen Microbiol 111:165–168 [CrossRef]
     [Google Scholar]
  41. Mullis K., Faloona F., Scharf S., Saiki R., Horn G., Erlich H. 1986; Specific enzymic amplification of DNA in vitro : the polymerase chain reaction. Cold Spring Harbor Symp Quant Biol 51:263–273 [CrossRef]
     [Google Scholar]
  42. Nealson K. H., Platt T., Hastings J. W. 1970; Cellular control of the synthesis and activity of the bacterial luminescence system. J Bacteriol 104:313–322
     [Google Scholar]
  43. Ochsner U. S., Koch A. K., Reiser J. 1994; Isolation and characterization of regulatory gene affecting rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa . J Bacteriol 176:2044–2054
     [Google Scholar]
  44. Parsek M. R., Schaefer A. L., Greenberg E. P. 1997; Analysis of random and site-directed mutations in rhlI , a Pseudomonas aeruginosa gene encoding an acylhomoserine lactone synthase. Mol Microbiol 26:301–310 [CrossRef]
     [Google Scholar]
  45. Passador L., Cook J. M., Gambello M. J., Rust L., Iglewski B. H. 1993; Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science 260:1127–1130 [CrossRef]
     [Google Scholar]
  46. Pearson J. P., Gray K., Passador L., Tucker K. D., Eberhard A., Iglewski B. H., Greenberg E. P. 1994; Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes. Proc Natl Acad Sci USA 91:197–201 [CrossRef]
     [Google Scholar]
  47. Pearson J. P., Pesci E. C., Iglewski B. H. 1997; Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in the control of elastase and rhamnolipid biosynthesis genes. J Bacteriol 179:5756–5767
     [Google Scholar]
  48. Pierson L. S., Keppenne V. D., Wood D. W. 1994; Phenazine antibiotic biosynthesis in Pseudomonas aureofaciens 30-84 is regulated by PhzR in response to cell density. J Bacteriol 176:3966–3974
     [Google Scholar]
  49. Pierson L. S., Gaffney T., Lam S., Gong F. 1995; Molecular analysis of genes encoding phenazine biosynthesis in the biological control bacterium Pseudomonas aureofaciens 30-84. FEMS Microbiol Lett 134:299–307
     [Google Scholar]
  50. Rode H., deWet P. M., Millar A. J. W., Cywes S. 1991; Mupirocin resistance. Lancet 338:578
     [Google Scholar]
  51. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  52. Sanger F., Nicklen S., Coulson A. R. 1977; DNA-sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:4563–4567
     [Google Scholar]
  53. Schweizer H. P., Hoang T. T. 1995; An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa . Gene 158:15–22 [CrossRef]
     [Google Scholar]
  54. Seed P. C., Passador L., Iglewski B. H. 1995; Activation of Pseudomonas aeruginosa lasI gene by LasR and Pseudomonas autoinducer PAI: an autoinduction regulatory hierarchy. J Bacteriol 177:654–659
     [Google Scholar]
  55. Shaw P. D., Ping G., Daly S. L., Cha C., Cronan J. E., Rinehart K. L., Farrand S. K. 1997; Detecting and characterizing N- acyl-homoserine lactone signal molecules by thin-layer chromatography. Proc Natl Acad Sci USA 94:6036–6042 [CrossRef]
     [Google Scholar]
  56. Simon R., Priefer U., Puhler A. 1983; A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Biotechnology 1:784–791 [CrossRef]
     [Google Scholar]
  57. Smith C. A., Thomas C. M. 1983; Deletion mapping of kil and kor functions in the trfA and trbB regions of broad-host-range plasmid RK2. Mol Gen Genet 190:245–254 [CrossRef]
     [Google Scholar]
  58. Steinmetz M., Le Coq D., Aymerich S., Gonzy-Treboul G., Gay P. 1985; The DNA sequence of the gene for the secreted Bacillus subtilis enzyme levansucrase and its genetic control sites. Mol Gen Genet 200:220–228 [CrossRef]
     [Google Scholar]
  59. Swift S., Karlyshev A. V., Fish L., Durant E. L., Winson M. K., Chhabra S. R., Williams P., MacIntyre S., Stewart G. S. A. B. 1997; Quorum sensing in Aeromonas hydrophila and Aeromonas salmonicida : identification of the LuxRI homologues AhyRI and AsaRI and their cognate N- acylhomoserine lactone signal molecules. J Bacteriol 179:5271–5281
     [Google Scholar]
  60. Szittner R., Meighen E. 1990; Nucleotide sequence, expression and properties of luciferase coded by lux genes from a terrestrial bacterium. J Biol Chem 265:16581–16587
     [Google Scholar]
  61. Thomas C. M., Hussain A. A. K., Smith C. A. 1982; Maintenance of broad host range plasmid RK2 replicons in Pseudomonas aeruginosa . Nature 298:674–676 [CrossRef]
     [Google Scholar]
  62. Warne S. 1986; Studies of gene expression signals suitable for the regulation of foreign proteins in Escherichia coli and Pseudomonas species. PhD thesisDepartment of Biological Sciences, University of Birmingham, UK
     [Google Scholar]
  63. Whatling C. 1993; Molecular analysis of the genes involved in the biosynthesis of pseudomonic acid, a polyketide antibiotic produced by Pseudomonas fluorescens NCIMB10586. PhD thesisDepartment of Biological Sciences, University of Birmingham, UK
     [Google Scholar]
  64. Whatling C. A., Hodgson J. E., Burnham M. K., Clarke N. J., Franklin F. C. H., Thomas C. M. 1995; Identification of a 60 kb region of the chromosome of Pseudomonas fluorescens 10586 required for the biosynthesis of pseudomonic acid (mupirocin). Microbiology 141:973–982 [CrossRef]
     [Google Scholar]
  65. Winson M. K., Camara M., Latifi A. 10 other authors 1995; Multiple N- acyl-l-homserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa. Proc Natl Acad Sci USA 92:9427–9431 [CrossRef]
     [Google Scholar]
  66. Winson M. K., Swift S., Fish L., Throup J. P., Jorgensen F., Chhabra S. R., Bycroft B. W., Williams P., Stewart G. S. A. B. 1998; Construction and analysis of luxCDABE -based plasmid sensors for investigating N- acyl homoserine lactone-mediated quorum sensing. FEMS Microbiol Lett 163:185–192 [CrossRef]
     [Google Scholar]
  67. Wood D. W., Pierson L. S. 1996; The phzI gene of Pseudomonas aureofaciens 30-84 is responsible for the production of a diffusible signal required for phenazine antibiotic production. Gene 168:49–53 [CrossRef]
     [Google Scholar]
  68. Yanisch-Perron C., Viera J., Messing J. 1985; Improved M13 phage cloning vectors and host strains: nucleotide sequence of M13mp18 and pUC19 vectors. Gene 33:103–119 [CrossRef]
     [Google Scholar]
  69. Zukowski M. M., Gaffney D. F., Speck D., Kauffman M., Findelli A., Wisecup A., Lecocq J. P. 1983; Chromogenic identification of genetic regulatory signals in Bacillus subtilis based on expression of a cloned Pseudomonas gene. Proc Natl Acad Sci USA 80:1101–1105 [CrossRef]
     [Google Scholar]
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Quorum-sensing-dependent regulation of biosynthesis of the polyketide antibiotic mupirocin in Pseudomonas fluorescens NCIMB 10586The GenBank accession numbers for the sequences determined in this work are AF318063 (mupA), AF318064 (mupR) and AF318065 (mupI).
Microbiology 147, 2127 (2001); https://doi.org/10.1099/00221287-147-8-2127
/content/journal/micro/10.1099/00221287-147-8-2127
/content/journal/micro/10.1099/00221287-147-8-2127
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