Streptomyces coelicolor strains lacking polyprenol phosphate mannose synthase and protein O-mannosyl transferase are hyper-susceptible to multiple antibiotics

Polyprenol phosphate mannose (PPM) is a lipid-linked sugar donor used by extra-cytoplasmic glycosyl tranferases in bacteria. PPM is synthesiszed 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 β-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 synthesize 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

Investigation of Streptomyces coelicolor A3(2) glycosylation mutants

This project has established that glycosylation is important for the normal, vegetative growth of S. coelicolor.  Three genes SCO3154/pmt, SCO1423/ppm1 and a putative SCO1014/ppm2 have been identified to be part of the pathway that carries out glycosylation in general, as well as for glycosylation of a phage (πC31cδ25) receptor. The three genes encode protein mannosyltransferase, Pmt and polyprenol phosphate mannose synthases, Ppm1, and Ppm2.  Of the three, Ppm2 was previously uncharacterised.  Radiolabelling experiments have shown that Ppm1 and Ppm2 are essential for the synthesis of C45 polyprenol phosphate mannose (Ppm) in S. coelicolor. In collaboration with researchers at Imperial College, London, we have characterised the endogenous polyprenol in S. coelicolor, shown that it consists of nine polyprenol units (C45) and provided evidence for the transfer of mannose from GDP-mannose to C45 polyprenol phosphate.  The transfer of mannose to polyprenol phosphates can be inhibited by the addition of amphomycin.  The pmt mutant has been shown to contain C45-Ppm which implies that the role of Pmt is further down the glycosylation pathway.  Glycosylation mutants in ppm1, ppm2 and pmt were hypersensitive to rifampicin and cell wall acting drugs, bacitracin and tunicamycin.  The ppm1 and ppm2 mutants were hypersensitive to vancomycin.  The MIC of vancomycin for a putative ppm2 mutant was between 10-20 μg ml-1 whereas the WT was resistant to vancomycin at >200 μg ml-1.  Phage resistance and small colony phenotype can be complemented; antibiotic sensitivities were partially complemented.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Infrared Spectroscopic Study on the Modified Mechanism of Aluminum-Impregnated Bone Charcoal

Fluoride contamination in drinking water is a prominent and widespread problem in many parts of the world. Excessive ingestion of fluoride through water can lead to the high risk of fluorosis in human body. Bone charcoal, with the principal active component of hydroxyapatite, is a frequently used adsorbent for fluoride removal. Many laboratory experiments suggest that the aluminum-impregnated bone charcoal is an effective adsorbent in defluoridation. However, the mechanisms underlying this modification process are still not well understood, which in turn greatly impedes the further studies on other different modified adsorbents. To address this issue, we used the infrared spectroscopy to examine the bone charcoal and the aluminum-impregnated bone charcoal, respectively. The comparative results show that the −OH peak of infrared spectroscopy has been intensified after modification. This significant change helped speculate the modified mechanism of the aluminum-impregnated bone charcoal. In addition, it is found that the hydroxide ion dissociates from hydroxyapatite in the modification process. Such finding implies that the tetrahydroxoaluminate can be combined with the hydroxyapatite and the aluminum ion can be impregnated onto the bone char surface