Project name: Gene expression in a Streptomyces coelicolor strain defective in sco1423 encoding a polyprenol phosphate mannose synthase (Ppm1) compared to the complemented mutant and the wild type parent

Description: Polyprenol phosphate mannose (PPM) is a lipid linked sugar donor used by extra-cytoplasmic glycosyl tranferases in bacteria. PPM is synthesised by polyprenol phosphate mannose synthase, Ppm1, and in most Actinobacteria is used as the sugar donor for protein O-mannosyl transferase, Pmt, in protein glycosylation. Ppm1 and Pmt have homologues in yeasts and humans, where they are required for protein O-mannosylation. Actinobacteria also use PPM for lipoglycan biosynthesis. Here we show that ppm1 mutants of Streptomyces coelicolor have increased susceptibility to a number of antibiotics that target cell wall biosynthesis. The pmt mutants also have mildly increased antibiotic susceptibilities, in particular to -lactams and vancomycin. Despite normal induction of the vancomycin gene cluster, vanSRJKHAX, the pmt and ppm1 mutants remained highly vancomycin sensitive indicating that the mechanism of resistance is blocked post-transcriptionally. Differential RNA expression analysis indicated that catabolic pathways were downregulated and anabolic ones upregulated in the ppm1 mutant compared to the parent or complemented strains. Of note was the increase in expression of fatty acid biosynthetic genes in the ppm1- mutant. A change in lipid composition was confirmed using Raman spectroscopy, which showed that the ppm1- mutant had a greater relative proportion of unsaturated fatty acids compared to the parent or the complemented mutant. Taken together these data suggest that an inability to synthesise PPM (ppm1-) and loss of the glycoproteome (pmt- mutant) can detrimentally affect membrane or cell envelope functions leading to loss of intrinsic and, in the case of vancomycin, acquired antibiotic resistance.Overall design: Three strains of Streptomyces coelicolor were used; J1929 (the parent strain), DT3017 (a ppm1- mutant derivative of J1929) and DT3017pDT16 (DT3017 containing a plasmid that expressed the wild type allele of ppm1). All three strains were grown in a fermenter and mRNA extracted for RNA sequencing. The biomass from three biological replicates(r1, r2 and r3) of each strain grown in a fermenter was harvested at 6 hours post-inoculation and therefore during rapid vegetative growth.

Data type: Transcriptome or Gene expression

Sample scope: Multiisolate

Relevance: Environmental

Last updated: 2017-12-12