Functional Angucycline-Like Antibiotic Gene Cluster in the Terminal Inverted Repeats of the Streptomyces ambofaciens Linear Chromosome

Abstract : 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 II polyketide synthase (PKS) gene clusters. Sequence analysis identified 26 open reading frames, including genes encoding the-ketoacyl synthase (KS), chain length factor (CLF), and acyl carrier protein (ACP) that make up the minimal PKS. These KS, CLF, and ACP subunits are highly homologous to minimal PKS subunits involved in the biosynthesis of angucycline antibiotics. The genes encoding the KS and ACP subunits are transcribed constitutively but show a remarkable increase in expression after entering transition phase. Five genes, including those encoding the minimal PKS, were replaced by resistance markers to generate single and double mutants (replacement in one and both terminal inverted repeats). Double mutants were unable to produce either diffusible orange pigment or antibacterial activity against Bacillus subtilis. Single mutants showed an intermediate phenotype, suggesting that each copy of the cluster was functional. Transformation of double mutants with a conjugative and integrative form of F6 partially restored both phenotypes. The pigmented and antibacterial compounds were shown to be two distinct molecules produced from the same biosynthetic pathway. High-pressure liquid chromatography analysis of culture extracts from wild-type and double mutants revealed a peak with an associated bioactivity that was absent from the mutants. Two additional genes encoding KS and CLF were present in the cluster. However, disruption of the second KS gene had no effect on either pigment or antibiotic production. Streptomyces spp. are gram-positive soil-inhabiting filamen-tous bacteria which undergo a complex process of morphological differentiation. One of the most striking traits of these microorganisms is their ability to produce a vast array of secondary metabolites, many of which possess antibiotic or other pharmacologically useful activities (9). Polyketide synthases (PKSs) are involved in the production of a large number of these antibiotics and are usually classified into three types. Modular (type I) and aromatic (type II) PKSs are the most common in streptomycetes, although type III PKSs, members of the chalcone synthase superfamily of condensing enzymes previously characterized in plants, have also been described (36). In type I PKSs, the catalytic sites for the various biosynthetic steps are present as domains along the length of large multifunctional proteins. In contrast, type II PKSs comprise several generally monofunctional proteins that possess one enzymatic activity which is used reiteratively. The core component of type II PKSs is called the minimal PKS. This is responsible for assembling the polyketide chain and is composed of a-ketoacyl synthase (KS), a chain length factor, and an acyl carrier protein (22). Additional PKS sub-units, usually cyclase/dehydrase and ketoreductase enzymes, are responsible for modification of the nascent chain into a specific cyclic polyketide compound, while open reading frames (ORF) encoding enzymes such as oxygenases or meth-ylases tailor the polyketide backbone into the final product. With the advent of whole-genome sequencing programs, previously uncharacterized gene clusters deduced to be responsible for secondary metabolite biosynthesis have been found in Streptomyces. In the avermectin-producing species Streptomyces avermitilis, 30 secondary metabolite gene clusters, including 12 type I and II PKS clusters, were identified by searching for homologues to polypeptides with a defined role in secondary metabolism (24, 38). Streptomyces coelicolor A3(2), the model Streptomyces strain, has long been known to produce four chemically distinct antibiotics (23), but on completion of its genome sequence, new type I and type II PKS gene clusters were revealed among a total of 18 additional clusters that code for enzymes characteristic of secondary metabolism (6). The Streptomyces linear chromosome is composed of a central core comprising about half the chromosome and a pair of chromosome arms, and it appears that the distribution of these clusters shows some preponderance in the arms (6, 38). Following our studies on genetic instability in Streptomyces ambofaciens, a phenomenon that affects morphological and
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Antimicrobial Agents and Chemotherapy, American Society for Microbiology, 2004, 48 (2), pp.575 - 588. 〈10.1128/AAC.48.2.575–588.2004〉
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Xiuhua Pang, Bertrand Aigle, Jean-Michel Girardet, Sophie Mangenot, Jean-Luc Pernodet, et al.. Functional Angucycline-Like Antibiotic Gene Cluster in the Terminal Inverted Repeats of the Streptomyces ambofaciens Linear Chromosome. Antimicrobial Agents and Chemotherapy, American Society for Microbiology, 2004, 48 (2), pp.575 - 588. 〈10.1128/AAC.48.2.575–588.2004〉. 〈hal-01659031〉



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