Mupirocin, a polyketide antibiotic active against Gram-positive bacteria is used clinically for treatment of bacterial skin infections, to clear Stapylococcus aureus ftom nasal passages and as a surgical scrub to inhibit bacterial growth, particularly that of MRSA. Mupirocin is synthesised by polyketide synthases (PKS) in a series of reactions involving many enzymes encoded by genes from the mupirocin cluster. The mupirocin cluster consists of six larger ORFs (mmpA-F. ) encoding multifunctional proteins, and twenty nine individual genes (mupA-X and macpA-E) all of which have been shown to be required for normal mupirocin biosynthesis and presumably create a biosynthetic assembly line. Sub-groups of mutants produce identical novel metabolites implying the presence of multi-protein complexes. To determine the interactions between proteins of the Mup assembly line Bacterial and Yeast Two Hybrid Systems were used but no evidence has been provided that tested proteins are partners. Although no interaction has been revealed, complementation studies suggest that MupE protein requires coexpression of MupD protein to be functional. Furthermore, inactivation of MupD by amino acid substitution suggests that MupD is not essential on its own for any step in mupirocin biosynthesis except for the proper function of MupE protein. BPLC analysis of wild type P. fluorescens overexpressing mupOlmacpElUIVICIF in trans showed that this did not abolish PA-B production as had been hypothesised but did increase production of total antibiotic (PA-A and PA-B) up to more than two-fold indicating a need to modify our model for the production of PA-B. Results using fluorescence microscopy demonstrate that small Mup proteins are not localized within the bacterial cell and that they are spread evenly in the cells of P. fluorescens.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:510237 |
| Date | January 2009 |
| Creators | Macioszek, Malgorzata |
| Publisher | University of Birmingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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