The aims of this project were to study the glycoproteome in two mycobacteria; <i>M. marinum</i> and <i>M. smegmatis</i>, and to assess the role of protein glycosylation by knocking out the Pmt homologues. Due to technical difficulties in the construction of knock out strains in mycobacteria, the aims were changed to further elucidate the protein <i>O</i>-glycosylation pathway identified in <i>S. coelicolor. </i>Reverse transcriptase PCR was used to show that <i>pmt, ppm1 </i>and the putative <i>ppm2 </i>(SCO1014) are expressed throughout the complex life cycle of <i>S. coelicolor. </i>The putative <i>ppm2<sup>-</sup></i> strain AV301, which can not be complemented with a wild type copy of SCO1014, was shown to harbour a point mutation in<i> ppm1</i>. In Western blots, soluble Ppm1 localised to both the cytosolic and membrane fractions whereas Ppm2 was only seen in the membrane fraction. Two bands at different molecular masses for Ppm2 were seen suggesting that this enzyme might be processed in <i>Streptomyces. </i>Using the bacterial two hybrid system, it was shown that unlike in mycobacteria, Ppm1 does not interact with Ppm2 <i>in vivo</i>. Furthermore, unlike the yeast Pmt enzymes, <i>Streptomyces </i>Pmt does not dimerise <i>in vivo</i>, suggesting that bacterial Pmt homologues might have an alternative mode of action from the eukaryote enzymes. To study the role of GDP-Mannose (GDP-Man) in protein glycosylation, three putative GDP-Man synthases were identified and disrupted; disruption in SCO1388 caused no obvious phenotypes whereas the SCO3039 and SCO4238 disruption strains had an earlier onset of pigment production as a sign of stress. In attempts to disrupt all three GDP-Man synthases, it was discovered that the disruption of both SCO3039 and SCO4238 was lethal.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:521155 |
| Date | January 2010 |
| Creators | Anttonen, Katri Pauliina |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=92515 |
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