Project description: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.
Project description: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.
Project description:Three genes encoding AfsK, AfsR, AfsS homologues in Streptomyces pristinaespiralis were studied, respectively, to investigate regulatory role of AfsKRS system for pristinamycin biosynthesis. Transcription change and gene inactivation analysis indicated that these genes had active transcription and positive regulation for the improvement of pristinamycin production in S. pristinaespiralis. The analysis of AfsKRS-defective mutagenesis indicated that there might be a positive correlation between the product of afsK and pristinamycin I biosynthesis, and a negative correlation to pristinamycin II biosynthesis. However, both afsR and afsS might have negative correlation to pristinamycin I production and positive correlation to pristinamycin II production. The effects on pristinamycin production of AfsKRS disruptants by protein kinase inhibitor K252a indicated that AfsR, both not AfsK and AfsS, was the inhibition target of K252a in S. pristinaespiralis, and AfsR should serve as a pleiotropic regulator to have differential regulation on biosynthesis of pristinamycin I and II components. Based on above study, it might be deduced that different signal transduction patterns via AfsK, AfsR, AfsS of AfsKRS system should be involved in respective regulation for biosynthesis of pristinamycin I and II in S. pristinaespiralis. In conclusion, the investigation could give some valuable clues for exploring furtherly regulatory function of AfsKRS system in S. pristinaespiralis.
Project description:Pristinamycin biosynthesis in Streptomyces pristinaespiralis is governed by a complex hierarchical signaling cascade involving seven different transcriptional regulators (SpbR, PapR1, PapR2, PapR3, PapR4, PapR5, and PapR6). The signaling cascade is triggered by γ-butyrolactone (GBL)-like effector molecules, whereby the chemical structure of the effector, as well as its biosynthetic origin is unknown so far. Three of the pristinamycin transcriptional regulators (SpbR, PapR3, and PapR5) belong to the type of γ-butyrolactone receptor (GBLR). GBLRs are known to either act as "real" GBLRs, which bind GBLs as ligands or as "pseudo" GBLRs binding antibiotics or intermediates thereof as effector molecules. In this study, we performed electromobility shift assays (EMSAs) with SpbR, PapR3, and PapR5, respectively, in the presence of potential ligand samples. Thereby we could show that all three GBLRs bind synthetic 1,4-butyrolactone but not pristinamycin as ligand, suggesting that SpbR, PapR3, and PapR5 act as "real" GBLRs in S. pristinaespiralis. Furthermore, we identified a cytochrome P450 monooxygenase encoding gene snbU as potential biosynthesis gene for the GBLR-interacting ligand. Inactivation of snbU resulted in an increased pristinamycin production, which indicated that SnbU has a regulatory influence on pristinamycin production. EMSAs with culture extract samples from the snbU mutant did not influence the target binding ability of SpbR, PapR3, and PapR5 anymore, in contrast to culture supernatant samples from the S. pristinaespiralis wild-type or the pristinamycin deficient mutant papR2::apra, which demonstrates that SnbU is involved in the synthesis of the GBLR-interacting ligand.
Project description:Specialised metabolites made by the genus Streptomyces form the basis of 55% of clinically used antibiotics as well as cancer therapeutics, immunosuppressants and other anti-infectives. Streptomyces venezuelae is a model for the genus because it is fast-growing, sporulates in liquid culture within 24 hours and coordinates production of the antibiotic chloramphenicol with sporulation. In this work we demonstrate that the conserved orphan response regulator OrrA directly controls the production of the conserved WhiB-like protein WblA which in turn binds to 90 target gene promoters, including genes required for aerial hyphae production and sporulation. We provide evidence that WblA directly controls the production of AdpA and BldN, key developmental regulators that are required for entry into sporulation and show that the cellular levels of the key developmental regulators AdpA, BldM, BldN, RsbN, WhiA, WhiB and WhiD are significantly altered in a ∆wblA mutant. Proteins encoded by 32 of the 33 predicted specialised metabolite biosynthetic gene clusters in S. venezuelae are also significantly affected by loss of WblA and we show that the ∆wblA mutant over produces the antibiotic chloramphenicol. This is the first time the WblA regulon has been defined in the genus Streptomyces and is consistent with previous reports that deletion of wblA has pleiotropic effects on antibiotic production and sporulation in diverse Streptomyces species.
Project description:α-Glucuronidase from Streptomyces pristinaespiralis (SpGlcA115A) is composed of a single-chain peptide containing a catalytic domain belonging to glycosyl hydrolase family 115, a novel family of hemicellulolytic α-glucuronidases. The enzyme catalyzes the hydrolysis of α-linked 4-O-methylglucuronosyl and glucuronosyl residues from both polymeric xylans and oligosaccharides. SpGlcA115A was crystallized at 293 K using the sitting-drop vapour-diffusion method. The crystals belonged to space group R3 and diffracted to a resolution of 1.9 Å.