Regulation of the biosynthesis of the macrolide antibiotic spiramycin in Streptomyces ambofaciens.

Streptomyces ambofaciens synthesizes the macrolide antibiotic spiramycin. The biosynthetic gene cluster for spiramycin has been characterized for S. ambofaciens. In addition to the regulatory gene srmR (srm22), previously identified (M. Geistlich et al., Mol. Microbiol. 6:2019-2029, 1992), three putative regulatory genes had been identified by sequence analysis. Gene expression analysis and gene inactivation experiments showed that only one of these three genes, srm40, plays a major role in the regulation of spiramycin biosynthesis. The disruption of srm22 or srm40 eliminated spiramycin production while their overexpression increased spiramycin production. Expression analysis was performed by reverse transcription-PCR (RT-PCR) for all the genes of the cluster in the wild-type strain and in the srm22 (srmR) and srm40 deletion mutants. The results from the expression analysis, together with the ones from the complementation experiments, indicated that Srm22 is required for srm40 expression, Srm40 being a pathway-specific activator that controls most, if not all, of the spiramycin biosynthetic genes.

Glycosylation steps during spiramycin biosynthesis in Streptomyces ambofaciens: involvement of three glycosyltransferases and their interplay with two auxiliary proteins.

Streptomyces ambofaciens synthesizes spiramycin, a 16-membered macrolide antibiotic used in human medicine. The spiramycin molecule consists of a polyketide lactone ring (platenolide) synthesized by a type I polyketide synthase, to which three deoxyhexoses (mycaminose, forosamine, and mycarose) are attached successively in this order. These sugars are essential to the antibacterial activity of spiramycin. We previously identified four genes in the spiramycin biosynthetic gene cluster predicted to encode glycosyltransferases. We individually deleted each of these four genes and showed that three of them were required for spiramycin biosynthesis. The role of each of the three glycosyltransferases in spiramycin biosynthesis was determined by identifying the biosynthetic intermediates accumulated by the corresponding mutant strains. This led to the identification of the glycosyltransferase responsible for the attachment of each of the three sugars. Moreover, two genes encoding putative glycosyltransferase auxiliary proteins were also identified in the spiramycin biosynthetic gene cluster. When these two genes were deleted, one of them was found to be dispensable for spiramycin biosynthesis. However, analysis of the biosynthetic intermediates accumulated by mutant strains devoid of each of the auxiliary proteins (or of both of them), together with complementation experiments, revealed the interplay of glycosyltransferases with the auxiliary proteins. One of the auxiliary proteins interacted efficiently with the two glycosyltransferases transferring mycaminose and forosamine while the other auxiliary protein interacted only with the mycaminosyltransferase.

Organization of the biosynthetic gene cluster for the macrolide antibiotic spiramycin in Streptomyces ambofaciens.

Spiramycin, a 16-membered macrolide antibiotic used in human medicine, is produced by Streptomyces ambofaciens; it comprises a polyketide lactone, platenolide, to which three deoxyhexose sugars are attached. In order to characterize the gene cluster governing the biosynthesis of spiramycin, several overlapping cosmids were isolated from an S. ambofaciens gene library, by hybridization with various probes (spiramycin resistance or biosynthetic genes, tylosin biosynthetic genes), and the sequences of their inserts were determined. Sequence analysis showed that the spiramycin biosynthetic gene cluster spanned a region of over 85 kb of contiguous DNA. In addition to the five previously described genes that encode the type I polyketide synthase involved in platenolide biosynthesis, 45 other genes have been identified. It was possible to propose a function for most of the inferred proteins in spiramycin biosynthesis, in its regulation, in resistance to the produced antibiotic or in the provision of extender units for the polyketide synthase. Two of these genes, predicted to be involved in deoxysugar biosynthesis, were inactivated by gene replacement, and the resulting mutants were unable to produce spiramycin, thus confirming their involvement in spiramycin biosynthesis. This work reveals the main features of spiramycin biosynthesis and constitutes a first step towards a detailed molecular analysis of the production of this medically important antibiotic.

Conjugal immunity of Streptomyces strains carrying the integrative element pSAM2 is due to the pif gene (pSAM2 immunity factor).

Mechanisms of conjugal immunity preventing redundant exchange between two cells harbouring the same conjugative element have been reported in diverse bacteria. Such a system does exist for pSAM2, a conjugative and integrative element of Streptomyces. The apparition of the conjugative free form of pSAM2 in the donor strain during mating can be considered as the initial step of transfer. We analysed the genes involved in transfer inhibition by mating donors harbouring pSAM2 with recipient strains containing different regions of pSAM2. The conjugal immunity was previously thought to be mediated by the transcriptional repressor KorSA. Although the transfer efficiency is reduced by its presence in the recipient, the initiation of the transfer process is not affected. In contrast, the presence in the recipient strain of a single pSAM2 gene, pif (pSAM2 immunity factor), was sufficient to abolish both transfer and initiation of transfer. Thus, the clustered genes korSA and pif act complementarily to maintain pSAM2 in a 'prophage' state under non-conjugal conditions. KorSA is involved in intracellular signalling, whereas Pif participates in intercellular signalling. The Pif nudix motif is essential for its activity. This is the first protein of the nudix family shown to be involved in bacterial conjugation.

Disruption of sblA in Streptomyces lividans permits expression of a heterologous alpha-amylase gene in the presence of glucose.

In a transposition mutant of Streptomyces lividans TK24, the usually glucose-repressible expression of a heterologous alpha-amylase gene (aml) became resistant to glucose repression. The transposon had inserted into an ORF called sblA which encodes a 274 aa product sharing significant sequence similarities with various phosphatases that act on small phosphorylated substrates. sblA was transcribed as a monocistronic mRNA and its transcription was enhanced at the transition phase. Because its transcriptional and putative translational start points coincide, sblA is likely to be translated in the absence of a conventional RBS. The sblA-disrupted mutant is characterized by early growth arrest in glucose-grown cultures and by partial relief of glucose repression of aml expression.