Previously, a protein O-mannosyl transferase (Pmt, SCO3154) and a polyprenol phosphate mannose synthase (Ppm1, SCO1423) were found to be required for the glycosylation of PstS, a phosphate binding protein, in the bacterium Streptomyces coelicolor. Bacteria in this genus are prolific producers of antibiotics and are often phenotypically resistant to multiple antibiotics. S. coelicolor pmt and ppm1 deficient mutants were hypersusceptible to cell-wall active antibiotics, suggesting that the protein modification could be required for cell wall and membrane homeostasis. The aim of this project was to investigate the S. coelicolor glycoproteome in order to better understand the physiological role of protein O-glycosylation in this model actinobacterium. Glycoproteins were detected in, and enriched from the membrane and culture filtrate proteomes of the S. coelicolor parent strain, J1929 and these were absent from the glycosylation deficient pmt (DT1025) and ppm1 (DT3017) mutants. Liquid chromatography coupled to mass spectrometry was used to characterise the membrane glycoproteome from the S. coelicolor parent strain, J1929 and 37 new glycoproteins were identified. Glycopeptides were modified on Ser/Thr residues with up to 3 hexoses; consistent with previous observations that the glycoprotein PstS is modified with a trihexose. The S. coelicolor glycoprotein glycans were shown to consist of Hex₂ and Hex₃ oligosaccharides. A carbohydrate linkage analysis led to the observation of 2-substituted, 4-substituted and terminal mannose residues, suggesting presence of (1->2) and (1->4) linkages in S. coelicolor glycoprotein glycans. The S. coelicolor glycoproteome comprises glycoproteins with various biological roles including solute binding, transport and cell wall biosynthesis. The genes encoding two S. coelicolor glycoproteins with putative roles in cell wall biosynthesis, an L, D transpeptidase (SCO4934) and a D-Ala-D-Ala, carboxypeptidase (SCO4847) were disrupted. Both mutants were hypersusceptible to β-lactam antibiotics, while the sco4847 mutant was hypersusceptible to lysozyme. These findings suggest that both proteins could be required for cell wall biosynthesis. As the phenotypes of the knockout mutants are reminiscent of the glycosylation deficient strains, we propose that glycosylation might be required for enzyme function
