1887

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.

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2004-01-01
2020-07-14
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