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Title: Protein glycosylation in actinomycetes
Author: Anttonen, Katri Pauliina
ISNI:       0000 0004 2689 7346
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 2010
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The aims of this project were to study the glycoproteome in two mycobacteria; M. marinum and M. smegmatis, 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 O-glycosylation pathway identified in S. coelicolor.  Reverse transcriptase PCR was used to show that pmt, ppm1 and the putative ppm2 (SCO1014) are expressed throughout the complex life cycle of S. coelicolor.  The putative ppm2- strain AV301, which can not be complemented with a wild type copy of SCO1014, was shown to harbour a point mutation in ppm1.  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 Streptomyces.  Using the bacterial two hybrid system, it was shown that unlike in mycobacteria, Ppm1 does  not interact with Ppm2 in vivo.  Furthermore, unlike the yeast Pmt enzymes, Streptomyces Pmt does not dimerise in vivo, 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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Streptomyces