Characterisation of the Streptomyces coelicolor glycoproteome reveals glycoproteins important for cell wall biogenesis

In prokaryotes, the role of protein glycosylation is poorly understood due to our limited understanding of their glycoproteomes. In some Actinobacteria, defects in protein O-glycosylation have been shown to retard growth and result in hypersensitivity to cell wall-targeting antibiotics, suggesting that this modification is important for maintaining cell wall structure. Here, we have characterized the glycoproteome in Streptomyces coelicolor and shown that glycoproteins have diverse roles, including those related to solute binding, ABC transporters, and cell wall biosynthesis. We have generated mutants encoding two putative cell wall-active glycoproteins and shown them to be hypersensitive to cell wall-targeting antibiotics. These findings strongly suggest that both glycoproteins are required for maintaining cell wall integrity and that glycosylation affects enzyme function.The physiological role of protein O-glycosylation in prokaryotes is poorly understood due to our limited knowledge of the extent of their glycoproteomes. In Actinobacteria, defects in protein O-mannosyl transferase (Pmt)-mediated protein O-glycosylation have been shown to significantly retard growth (Mycobacterium tuberculosis and Corynebacterium glutamicum) or result in increased sensitivities to cell wall-targeting antibiotics (Streptomyces coelicolor), suggesting that protein O-glycosylation has an important role in cell physiology. Only a single glycoprotein (SCO4142, or PstS) has been identified to date in S. coelicolor. Combining biochemical and mass spectrometry-based approaches, we have isolated and characterized the membrane glycoproteome in S. coelicolor. A total of ninety-five high-confidence glycopeptides were identified which mapped to thirty-seven new S. coelicolor glycoproteins and a deeper understanding of glycosylation sites in PstS. Glycosylation sites were found to be modified with up to three hexose residues, consistent with what has been observed previously in other Actinobacteria. S. coelicolor glycoproteins have diverse roles and functions, including solute binding, polysaccharide hydrolases, ABC transporters, and cell wall biosynthesis, the latter being of potential relevance to the antibiotic-sensitive phenotype of pmt mutants. Null mutants in genes encoding a putative d-Ala-d-Ala carboxypeptidase (SCO4847) and an l,d-transpeptidase (SCO4934) were hypersensitive to cell wall-targeting antibiotics. Additionally, the sco4847 mutants displayed an increased susceptibility to lysozyme treatment. These findings strongly suggest that both glycoproteins are required for maintaining cell wall integrity and that glycosylation could be affecting enzyme function

Cross-resistance is modular in bacteria-phage interactions

Phages shape the structure of natural bacterial communities and can be effective therapeutic agents. Bacterial resistance to phage infection, however, limits the usefulness of phage therapies and could destabilize community structures, especially if individual resistance mutations provide cross-resistance against multiple phages. We currently understand very little about the evolution of cross-resistance in bacteria-phage interactions. Here we show that the network structure of cross-resistance among spontaneous resistance mutants of Pseudomonas aeruginosa evolved against each of 27 phages is highly modular. The cross-resistance network contained both symmetric (reciprocal) and asymmetric (non-reciprocal) cross-resistance, forming two cross-resistance modules defined by high within- but low between-module cross-resistance. Mutations conferring cross-resistance within-modules targeted either lipopolysaccharide or type-IV pilus biosynthesis, suggesting that the modularity of cross-resistance was structured by distinct phage receptors. In contrast, between-module cross-resistance was provided by mutations affecting the alternative sigma factor, RpoN, which controls many lifestyle-associated functions, including motility, biofilm formation and quorum sensing. Broader cross-resistance range was not associated with higher fitness costs or weaker resistance against the focal phage used to select resistance. However, mutations in rpoN, providing between-module cross-resistance, were associated with higher fitness costs than mutations associated with within-module cross-resistance, that is, in genes encoding either lipopolysaccharide or type-IV pilus biosynthesis. The observed structure of cross-resistance predicted both the frequency of resistance mutations and the ability of phage combinations to suppress bacterial growth. These findings suggest that the evolution of cross-resistance is common, is likely to play an important role in the dynamic structure of bacteria-phage communities, and could inform the design principles for phage therapy treatments