cThere are up to seven regulatory genes in the pristinamycin biosynthetic gene cluster of Streptomyces pristinaespiralis, which infers a complicated regulation mechanism for pristinamycin production. In this study, we revealed that PapR6, a putative atypical response regulator, acts as a pathway-specific activator of pristinamycin II (PII) biosynthesis. Deletion of the papR6 gene resulted in significantly reduced PII production, and its overexpression led to increased PII formation, compared to that of the parental strain HCCB 10218. However, either papR6 deletion or overexpression had very little effect on pristinamycin I (PI) biosynthesis. Electrophoretic mobility shift assays (EMSAs) demonstrated that PapR6 bound specifically to the upstream region of snaF, the first gene of the snaFE1E2GHIJK operon, which is likely responsible for providing the precursor isobutyryl-coenzyme A (isobutyryl-CoA) and the intermediate C 11 ␣␤-unsaturated thioester for PII biosynthesis. A signature PapR6-binding motif comprising two 4-nucleotide (nt) inverted repeat sequences (5=-GAGG-4 nt-CCTC-3=) was identified. Transcriptional analysis showed that inactivation of the papR6 gene led to markedly decreased expression of snaFE1E2GHIJK. Furthermore, we found that a mutant (snaFmu) with base substitutions in the identified PapR6-binding sequence in the genome exhibited the same phenotype as that of the ⌬papR6 strain. Therefore, it may be concluded that pathway-specific regulation of PapR6 in PII biosynthesis is possibly exerted via controlling the provision of isobutyryl-CoA as well as the intermediate C 11 ␣␤-unsaturated thioester. Streptomyces pristinaespiralis produces pristinamycin, which is a streptogramin-like antibiotic and a mixture of two structurally different components: pristinamycin I (PI), a branched cyclic hexadepsipeptide, and pristinamycin II (PII), a polyunsaturated cyclopeptide macrolactone (1). PI and PII are cosynthesized at a ratio of 30:70 and show strong synergistic activity. The combined application of both components yields an antibacterial activity up to 100 times higher than that of each single component (2). Pristinamycin is attracting more and more interest for its activity against many multiple-drug-resistant Gram-positive bacteria, such as methicillin-resistant staphylococci and vancomycin-resistant Enterococcus faecium (2).The gene cluster responsible for the biosynthesis of PI and PII has been well characterized for S. pristinaespiralis (3-9); all essential PI/PII biosynthetic genes are proposed to be included in a 210-kb "supercluster," in which the pristinamycin gene cluster is interspersed with a cryptic type II polyketide biosynthetic gene cluster (10). Interestingly, there are seven regulatory genes in the pristinamycin biosynthetic gene cluster, including papR1 to papR6 and spbR, which infers a complicated regulatory mechanism for pristinamycin production. It has long been suggested that manipulation of the regulatory network governing antibiotic biosynthesis in Streptomyces may represent an ef...

No abstract

No abstract

How to sample a seizure plant:the role of visualization spatial distribution analysis of Lophophora williamsii as an exampleAbstract: Natural compounds in plants are often unevenly distributed, and determining the best sampling locations to obtain the most representative results is technically challenging. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can provide the basis for formulating sampling guideline. For a succulent plant sample, ensuring the authenticity and in situ nature of the spatial distribution analysis results during MSI analysis also needs to be thoroughly considered. In this study, we developed a well-established and reliable MALDI-MSI method based on preservation methods, slice conditions, auxiliary matrices, and MALDI parameters to detect and visualize the spatial distribution of mescaline in situ in L. williamsii. The MALDI-MSI results were validated using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Low-temperature storage at −80°C and drying of ‘bookmarks’ were the appropriate storage methods for succulent plant samples and their flower samples, and cutting into 40μm thick sections at −20°C using gelatin as the embedding medium is the appropriate sectioning method. The use of DCTB (trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile) as an auxiliary matrix and a laser intensity of 45 are favorable MALDI parameter conditions for mescaline analysis. The region of interest (ROI) semi-quantitative analysis revealed that mescaline is concentrated in the epidermal tissues of L. williamsii as well as in the meristematic tissues of the crown. The study findings not only help to provide a basis for determining the best sampling locations for mescaline in L. williamsii, but they also provide a reference for the optimization of storage and preparation conditions for raw plant organs before MALDI detection.

No abstract