A Complex Signaling Cascade Governs Pristinamycin Biosynthesis in Streptomyces pristinaespiralis.

Pristinamycin production in Streptomyces pristinaespiralis Pr11 is tightly regulated by an interplay between different repressors and activators. A γ-butyrolactone receptor gene (spbR), two TetR repressor genes (papR3 and papR5), three SARP (Streptomyces antibiotic regulatory protein) genes (papR1, papR2, and papR4), and a response regulator gene (papR6) are carried on the large 210-kb pristinamycin biosynthetic gene region of Streptomyces pristinaespiralis Pr11. A detailed investigation of all pristinamycin regulators revealed insight into a complex signaling cascade, which is responsible for the fine-tuned regulation of pristinamycin production in S. pristinaespiralis.

The bifunctional role of aconitase in Streptomyces viridochromogenes Tü494.

In many organisms, aconitases have dual functions; they serve as enzymes in the tricarboxylic acid cycle and as regulators of iron metabolism. In this study we defined the role of the aconitase AcnA in Streptomyces viridochromogenes Tü494, the producer of the herbicide phosphinothricyl-alanyl-alanine, also known as phosphinothricin tripeptide or bialaphos. A mutant in which the aconitase gene acnA was disrupted showed severe defects in morphology and physiology, as it was unable to form any aerial mycelium, spores nor phosphinothricin tripeptide. AcnA belongs to the iron regulatory proteins (IRPs). In addition to its catalytic function, AcnA plays a regulatory role by binding to iron responsive elements (IREs) located on the untranslated region of certain mRNAs. A mutation preventing the formation of the [4Fe-4S] cluster of AcnA eliminated its catalytic activity, but did not inhibit RNA-binding ability. In silico analysis of the S. viridochromogenes genome revealed several IRE-like structures. One structure is located upstream of recA, which is involved in the bacterial SOS response, and another one was identified upstream of ftsZ, which is required for the onset of sporulation in streptomycetes. The functionality of different IRE structures was proven with gel shift assays and specific IRE consensus sequences were defined. Furthermore, RecA was shown to be upregulated on post-transcriptional level under oxidative stress conditions in the wild-type strain but not in the acnA mutant, suggesting a regulatory role of AcnA in oxidative stress response.

Characterization of the 'pristinamycin supercluster' of Streptomyces pristinaespiralis.

Pristinamycin, produced by Streptomyces pristinaespiralis Pr11, is a streptogramin antibiotic consisting of two chemically unrelated compounds, pristinamycin I and pristinamycin II. The semi-synthetic derivatives of these compounds are used in human medicine as therapeutic agents against methicillin-resistant Staphylococcus aureus strains. Only the partial sequence of the pristinamycin biosynthetic gene cluster has been previously reported. To complete the sequence, overlapping cosmids were isolated from a S. pristinaespiralis Pr11 gene library and sequenced. The boundaries of the cluster were deduced, limiting the cluster size to approximately 210 kb. In the central region of the cluster, previously unknown pristinamycin biosynthetic genes were identified. Combining the current and previously identified sequence information, we propose that all essential pristinamycin biosynthetic genes are included in the 210 kb region. A pristinamycin biosynthetic pathway was established. Furthermore, the pristinamycin gene cluster was found to be interspersed by a cryptic secondary metabolite cluster, which probably codes for a glycosylated aromatic polyketide. Gene inactivation experiments revealed that this cluster has no influence on pristinamycin production. Overall, this work provides new insights into pristinamycin biosynthesis and the unique genetic organization of the pristinamycin gene region, which is the largest antibiotic 'supercluster' known so far.

Identification and functional characterization of phenylglycine biosynthetic genes involved in pristinamycin biosynthesis in Streptomyces pristinaespiralis.

Pristinamycin I (PI), a streptogramin type B antibiotic produced by Streptomyces pristinaespiralis, contains the aproteinogenic amino acid L-phenylglycine. Recent sequence analysis led to the identification of a set of putative phenylglycine biosynthetic genes. Successive inactivation of the individual genes resulted in a loss of PI production. Production was restored by supplementation with externally added L-phenylglycine, which demonstrates that these genes are involved in phenylglycine biosynthesis and thus probably disclosing the last essential pristinamycin biosynthetic genes. Finally, a putative pathway for phenylglycine synthesis is proposed.

Phosphinothricin-tripeptide biosynthesis: an original version of bacterial secondary metabolism?

Streptomyces viridochromogenes Tü494 produces the herbicide phosphinothricyl-alanyl-alanine (phosphinothricin-tripeptide=PTT; bialaphos). Its bioactive moiety phosphinothricin competitively inhibits bacterial and plant glutamine synthetases. The biosynthesis of PTT includes the synthesis of the unusual amino acid N-acetyl-demethyl-phosphinothricin and a three step condensation via non-ribosomal peptide synthetases. Two characteristics within the PTT biosynthesis make it suitable to study the evolution of secondary metabolism biosynthesis. First, PTT biosynthesis represents the only known system where all peptide synthetase modules are located on separate proteins. This 'single enzyme system' might be an archetype of the multimodular and multienzymatic non-ribosomal peptide synthetases in evolutionary terms. The second interesting feature of PTT biosynthesis is the pathway-specific aconitase Pmi that is involved in the supply of N-acetyl-demethyl-phosphinothricin. Pmi is highly similar to the tricarboxylic acid aconitase AcnA. They share 64% identity at the DNA level and both belong to the Iron-Regulatory-Protein/AcnA family. Despite their high sequence similarity, AcnA and Pmi catalyze different reactions and are not able to substitute for each other. Thus, the enzyme pair AcnA/Pmi presents an example of the evolution of a secondary metabolite-specific enzyme from a primary metabolism enzyme.