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Functional Angucycline-Like Antibiotic Gene Cluster in the Terminal Inverted Repeats of the Streptomyces ambofaciens Linear Chromosome
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Edité par CCSD ; American Society for Microbiology -
International audience. Streptomyces ambofaciens has an 8-Mb linear chromosome ending in 200-kb terminal inverted repeats.Analysis of the F6 cosmid overlapping the terminal inverted repeats revealed a locus similar to type IIpolyketide synthase (PKS) gene clusters. Sequence analysis identified 26 open reading frames, including genesencoding the -ketoacyl synthase (KS), chain length factor (CLF), and acyl carrier protein (ACP) that makeup the minimal PKS. These KS, CLF, and ACP subunits are highly homologous to minimal PKS subunitsinvolved in the biosynthesis of angucycline antibiotics. The genes encoding the KS and ACP subunits aretranscribed constitutively but show a remarkable increase in expression after entering transition phase. Fivegenes, including those encoding the minimal PKS, were replaced by resistance markers to generate single anddouble mutants (replacement in one and both terminal inverted repeats). Double mutants were unable toproduce either diffusible orange pigment or antibacterial activity against Bacillus subtilis. Single mutantsshowed an intermediate phenotype, suggesting that each copy of the cluster was functional. Transformation ofdouble mutants with a conjugative and integrative form of F6 partially restored both phenotypes. Thepigmented and antibacterial compounds were shown to be two distinct molecules produced from the samebiosynthetic pathway. High-pressure liquid chromatography analysis of culture extracts from wild-type anddouble mutants revealed a peak with an associated bioactivity that was absent from the mutants. Twoadditional genes encoding KS and CLF were present in the cluster. However, disruption of the second KS genehad no effect on either pigment or antibiotic production.
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