1887

Microbiology Society logo

Volume 150, Issue 1

Biosynthesis of the dichloroacetyl component of chloramphenicol in ISP5230: genes required for halogenation Free

Abstract

Five ORFs were detected in a fragment from the ISP5230 genomic DNA library by hybridization with a PCR product amplified from primers representing a consensus of known halogenase sequences. Sequencing and functional analyses demonstrated that ORFs 11 and 12 (but not ORFs 13–15) extended the partially characterized gene cluster for chloramphenicol (Cm) biosynthesis in the chromosome. Disruption of ORF11 () or ORF12 () and conjugal transfer of the insertionally inactivated genes to gave mutant strains VS1111 and VS1112, each producing a similar series of Cm analogues in which unhalogenated acyl groups replaced the dichloroacetyl substituent of Cm. H-NMR established that the principal metabolite in the disrupted strains was the --propionyl analogue. The sequence of CmlK implicated the protein in adenylation, and involvement in halogenation was inferred from biosynthesis of analogues by the -disrupted mutant. A role in generating the dichloroacetyl substituent was supported by partial restoration of Cm biosynthesis when a cloned copy of was introduced into VS1111. Complementation of the mutant also indicated that inactivation of rather than a polar effect of the disruption on expression had interfered with dichloroacetyl biosynthesis. The deduced CmlS sequence resembled sequences of FADH-dependent halogenases. Conjugal transfer of or into , a chlorination-deficient strain with a mutation mapped genetically to the Cm biosynthesis gene cluster, did not complement the lesion, suggesting that one or more genes in addition to and is needed to assemble the dichloroacetyl substituent. Insertional inactivation of ORF13 did not affect Cm production, and the products of ORF14 and ORF15 matched A3(2) proteins lacking plausible functions in Cm biosynthesis. Thus appears to mark the downstream end of the gene cluster.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): Cm, chloramphenicol , COSY, correlated spectroscopy , PAPA, p-aminophenylalanine and PAPS, p-aminophenylserine
SGM
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.26319-0
2004-01-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/1/mic1500085.html?itemId=/content/journal/micro/10.1099/mic.0.26319-0&mimeType=html&fmt=ahah

References

  1. Brown M. P., Aidoo K. A., Vining L. C. 1996; A role for pabAB, a p-aminobenzoate synthase gene of Streptomyces venezuelae ISP5230, in chloramphenicol biosynthesis. . Microbiology 142:1345–1355 [CrossRef]
     [Google Scholar]
  2. Chang Z., Sun Y., He J., Vining L. C. 2001; p-Aminobenzoic acid and chloramphenicol biosynthesis in Streptomyces venezuelae: gene sets for a key enzyme, 4-amino-4-deoxychorismate synthase. Microbiology 147:2113–2126
     [Google Scholar]
  3. Chen H. W., Walsh C. T. 2001; Coumarin formation in novobiocin biosynthesis: β-hydroxylation of the aminoacyl enzyme tyrosyl-S-NovH by a cytochrome P450 NovI. Chem Biol 8:1–12 [CrossRef]
     [Google Scholar]
  4. Conti E., Franks N. P., Brick P. 1996; Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes. Structure 4:287–298 [CrossRef]
     [Google Scholar]
  5. Conti E., Stachelhaus T., Marahiel M. A., Brick P. 1997; Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S. EMBO J 16:4174–4183 [CrossRef]
     [Google Scholar]
  6. Dairi T., Nakano T., Aisaka K., Katsumata R., Hasegawa M. 1995; Cloning and nucleotide sequence of the gene responsible for chlorination of tetracycline. Biosci Biotechnol Biochem 59:1099–1106 [CrossRef]
     [Google Scholar]
  7. DiMarco A. A., Averhoff B. A., Kim E. E., Ornston L. N. 1993; Evolutionary divergence of pobA, the structural gene encoding p-hydroxybenzoate hydroxylase in an Acinetobacter calcoaceticus strain well suited for genetic analysis. Gene 125:25–33 [CrossRef]
     [Google Scholar]
  8. Doull J. L., Ahmed Z., Stuttard C., Vining L. C. 1985; Isolation and characterization of Streptomyces venezuelae mutants blocked in chloramphenicol biosynthesis. J Gen Microbiol 131:97–104
     [Google Scholar]
  9. Doull J. L., Vats S., Chaliciopoulos M., Stuttard C., Wong K., Vining L. C. 1986; Conjugational fertility and location of chloramphenicol biosynthesis genes on the chromosomal linkage map of Streptomyces venezuelae. J Gen Microbiol 132:1327–1338
     [Google Scholar]
  10. Entsch B., Ballou D. P., Massey V. 1976; Flavin-oxygen derivatives involved in hydroxylation by p-hydroxybenzoate hydroxylase. J Biol Chem 251:2550–2563
     [Google Scholar]
  11. Facey S. J., Groß F., Vining L. C., Yang K., van Pee K.-H. 1996; Cloning, sequencing and disruption of a bromoperoxidase-catalase gene in Streptomyces venezuelae: evidence that it is not required for chlorination in chloramphenicol biosynthesis. Microbiology 142:657–665 [CrossRef]
     [Google Scholar]
  12. Gaitatzis N., Kunze B., Müller R. 2001; In vitro reconstitution of the myxochelin biosynthetic machinery of Stigmatella aurantiaca Sg a15: biochemical characterization of a reductive release mechanism from nonribosomal peptide synthetases. Proc Natl Acad Sci U S A 98:11136–11141 [CrossRef]
     [Google Scholar]
  13. Gatti D. L., Palfey B. A., Lah M. S., Entsch B., Massey V., Ballou D. P., Ludwig M. L. 1994; The mobile flavin of 4-OH benzoate hydroxylase. Science 266:110–114 [CrossRef]
     [Google Scholar]
  14. Groß F., Lewis E. A., Piraee M., van Pée K.-H., Vining L. C., White R. L. 2002; Isolation of 3′-O-acetylchloramphenicol: a possible intermediate in chloramphenicol biosynthesis. Bioorg Med Chem Lett 12:283–286 [CrossRef]
     [Google Scholar]
  15. He J., Magarvey N., Piraee M., Vining L. C. 2001; The gene cluster for chloramphenicol biosynthesis in Streptomyces venezuelae ISP5230 includes novel shikimate pathway homologues and a monomodular nonribosomal peptide synthetase gene. Microbiology 147:2817–2829
     [Google Scholar]
  16. Hohaus K., Altmann A., Burd W., Fischer I., Hammer P. E., Hill D. S., Ligon J. M., van Pee K.-H. 1997; NADH-dependent halogenases are more likely to be involved in halometabolite biosynthesis than haloperoxidases. Angew Chem Int Ed Engl 36:2012–2013 [CrossRef]
     [Google Scholar]
  17. Hopwood D. A., Bibb M. J., Chater K. F.7 other authors 1985 Genetic Manipulation of Streptomyces, a Laboratory Manual Norwich: John Innes Foundation;
  18. Ishikawa J., Hotta K. 1999; Frameplot: a new implementation of the frame analysis for predicting protein-coding regions in bacterial DNA with a high G+C content. FEMS Microbiol Lett 174:251–253 [CrossRef]
     [Google Scholar]
  19. Jez J. M., Bennett M. J., Schlegel B. P., Lewis M., Penning T. M. 1997; Comparative anatomy of the aldo–keto reductase superfamily. Biochem J 326:625–636
     [Google Scholar]
  20. Keller S., Wage T., Hohaus K., Hölzer M., Eichhorn E., van Pee K.-H. 2000; Purification and partial characterization of tryptophan 7-halogenase (PrnA) from Pseudomonas fluorescens. Angew Chem Int Ed Engl 39:2300–2302 [CrossRef]
     [Google Scholar]
  21. Kirner S., Hammer P. E., Hill D. S., Altmann A., Fischer I., Weislo L. J., Lanahan M., van Pée K.-H., Ligon J. M. 1998; Functions encoded by pyrrolnitrin biosynthetic genes from Pseudomonas fluorescens. J Bacteriol 180:1939–1943
     [Google Scholar]
  22. Konz D., Marahiel M. A. 1999; How do peptide synthetases generate structural diversity?. Chem Biol 6:R39–R48 [CrossRef]
     [Google Scholar]
  23. Lauer B., Russwurm R., Bormann C. 2000; Molecular characterization of two genes from Streptomyces tendae Tu901 required for the formation of the 4-formyl-4-imidazolin-2-one-containing nucleoside moiety of the peptidyl nucleoside antibiotic nikkomycin. Eur J Biochem 267:1698–1706 [CrossRef]
     [Google Scholar]
  24. Lewis E. A., Adamek T. L., Vining L. C., White R. L. 2003; Metabolites of a blocked chloramphenicol producer. J Nat Prod 66:62–66 [CrossRef]
     [Google Scholar]
  25. MacNeil D. J., Gewain K. M., Rudy C. L., Dezeny G., Gibbons P. H., MacNeil T. 1992; Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene 111:61–68 [CrossRef]
     [Google Scholar]
  26. Mazodier P., Petter R., Thompson C. 1989; Intergenic conjugation between Escherichia coli and Streptomyces species. J Bacteriol 171:3583–3585
     [Google Scholar]
  27. Mootz H. D., Marahiel M. A. 1997; The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains. J Bacteriol 179:6843–6850
     [Google Scholar]
  28. Mosher R. H., Ranade N. P., Schrempf H., Vining L. C. 1990; Chloramphenicol resistance in Streptomyces: cloning and characterization of a chloramphenicol hydrolase gene fromStreptomyces venezuelae. J Gen Microbiol 136:293–301 [CrossRef]
     [Google Scholar]
  29. Mosher R. H., Camp D. J., Yang K., Brown M. P., Shaw W. V., Vining L. C. 1995; Inactivation of chloramphenicol by O-phosphorylation. J Biol Chem 270:27000–27006 [CrossRef]
     [Google Scholar]
  30. Mosher R. H., Paradkar A. S., Anders C., Barton B., Jensen S. E. 1999; Genes specific for the biosynthesis of clavam metabolites antipodal to clavulanic acid are clustered with the gene for clavaminate synthase 1 in Streptomyces clavuligerus. Antimicrob Agents Chemother 43:1215–1224
     [Google Scholar]
  31. Nowak-Thompson B., Chaney N., Wing J. S., Gould S. J., Loper J. E. 1999; Characterization of the pyoluteorin biosynthetic gene cluster of Pseudomonas fluorescens Pf-5. J Bacteriol 181:2166–2174
     [Google Scholar]
  32. Page R. D. M. 1996; TREEVIEW: an application to display phylogenetic trees on personal computers. Comp Appl Biosci 12:357–358
     [Google Scholar]
  33. Paradkar A. S., Jensen S. E. 1995; Functional analysis of the gene encoding the clavaminate synthase 2 isoenzyme involved in clavulanic acid biosynthesis in Streptomyces clavuligerus. J Bacteriol 177:1307–1314
     [Google Scholar]
  34. Pelzer S., Sußmuth R., Heckmann D., Recktenwald J., Huber P., Jung G., Wohlleben W. 1999; Identification and analysis of the balhimycin biosynthetic gene cluster and its use for manipulating glycopeptide biosynthesis in Amycolatopsis mediterranei DSM5908. . Antimicrob Agents Chemother 43:1565–1573
     [Google Scholar]
  35. Piraee M., Vining L. C. 2002; Use of degenerate primers and touchdown PCR to amplify a halogenase gene fragment from Streptomyces venezuelae ISP5230. . J Ind Microbiol Biotechnol 29:1–5 [CrossRef]
     [Google Scholar]
  36. Pospiech A., Bietenhader J., Schupp T. 1996; Two multifunctional peptide synthetases and an O-methyltransferase are involved in the biosynthesis of the DNA-binding antibiotic and antitumour agent saframycin Mx1 from Myxococcus xanthus. Microbiology 142:741–746 [CrossRef]
     [Google Scholar]
  37. Puk O., Huber P., Bischoff D., Rechtenwald J., Jung G., Süßmuth R. D., van Pée K.-H., Wohlleben W., Pelzer S. 2002; Glycopeptide biosynthesis in Amycolatopsis mediterranei DSM5908: function of a halogenase and a haloperoxidase/perhydrolase. Chem Biol 9:225–235 [CrossRef]
     [Google Scholar]
  38. Quadri L. E. N., Keating T. A., Patel H. M., Walsh C. T. 1999; Assembly of the Pseudomonas aeruginosa nonribosomal peptide siderophore pyochelin: in vitro reconstitution of aryl-4,2-bisthiazoline synthetase activity from PchD, PchE, and PchF. Biochemistry 38:14941–14954 [CrossRef]
     [Google Scholar]
  39. Rowland B. M., Grossman T. H., Osburne M. S., Taber H. W. 1996; Sequence and genetic organization of a Bacillus subtilis operon encoding 2,3-dihydroxybenzoate biosynthetic enzymes. Gene 178:119–123 [CrossRef]
     [Google Scholar]
  40. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  41. Scrutton N. S., Berry A., Perham R. N. 1990; Redesign of the coenzyme specificity of a dehydrogenase by protein engineering. Nature 343:38–43 [CrossRef]
     [Google Scholar]
  42. Shirahata K., Hayashi T., Deguchi T., Suzuki T., Matsubara I. 1972; The structures of corynecins, chloramphenicol analogues produced by an n-paraffin-grown bacterium. Agric Biol Chem 36:2229–2232 [CrossRef]
     [Google Scholar]
  43. Simonsen J. N., Paramasigamani K., Vining L. C., McInnes A. G., Walter J. A., Wright J. L. C. 1978; Biosynthesis of chloramphenicol. Studies on the origin of the dichloroacetyl moiety. Can J Microbiol 24:136–142 [CrossRef]
     [Google Scholar]
  44. Strohl W. 1992; Compilation and analysis of DNA sequences associated with apparent Streptomyces promoters. Nucleic Acids Res 20:961–974 [CrossRef]
     [Google Scholar]
  45. Stuttard C. 1982; Temperate phages of Streptomyces venezuelae: lysogeny and host specificity shown by phages SV1 and SV2. J Gen Microbiol 128:115–121
     [Google Scholar]
  46. van Pée K.-H. 2001; Microbial biosynthesis of halometabolites. Arch Microbiol 175:250–258 [CrossRef]
     [Google Scholar]
  47. Vining L. C., Stuttard C. 1995; Chloramphenicol. In Genetics and Biochemistry of Antibiotic Production pp. 505–530Edited by Vining L. C., Stuttard C. Newton, MA: Butterworth-Heinemann;
     [Google Scholar]
  48. Wang Z.-X., Li S.-M., Heide L. 2000; Identification of the coumermycin A1 biosynthetic gene cluster of Streptomyces rishiriensis DSM 40489. Antimicrob Agents Chemother 44:3040–3048 [CrossRef]
     [Google Scholar]
  49. Wang L., White R. L., Vining L. C. 2002; Biosynthesis of the dideoxysugar component of jadomycin B: genes in the jad cluster of Streptomyces venezuelae ISP5230 for l-digitoxose assembly and transfer to the angucycline aglycone. Microbiology 148:1091–1103
     [Google Scholar]
  50. Wright F., Bibb M. J. 1992; Codon usage in the G+C rich Streptomyces genome. Gene 113:55–65 [CrossRef]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.26319-0
Loading
Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230: genes required for halogenation
Microbiology 150, 85 (2004); https://doi.org/10.1099/mic.0.26319-0
/content/journal/micro/10.1099/mic.0.26319-0
/content/journal/micro/10.1099/mic.0.26319-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

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