Protein-O-Mannosylation By Non-Sec/Tat Secretion Translocons In Actinobacteria

Protein-O-mannosylation (POM) is a form of O-glycosylation that is ubiquitous throughout all domains of life and has been extensively characterized in eukaryotic systems. However, in prokaryotes this process has only been investigated in terms of pathogenicity (in Mycobacterium tuberculosis) even though there are many non-pathogenic bacteria that are known to regularly carry out POM. To date, there is no consensus on what benefit POM imparts to the non-pathogenic bacteria that can perform it. Though the generation of a POM deficient mutant of Corynebacterium glutamicum - a widely utilized and known mannosylating actinobacteria - this work shows that even closely related actinobacterial GT-39s (the enzymes responsible for the initiation of POM) can potentially have different activities and substrate specificities for targets of POM. Moreover, presented here is evidence that POM does not only occur in a SEC-dependent manner; POM also occurs with TAT and non-SEC secreted substrates in a specific and likely tightly regulated manner. Together these results highlight the need for further biochemical characterization of POM in these and other bacterial species to help elucidate the true nature of its biological functions. ImportanceBoth the mechanism and overall cellular function of protein-O-mannosylation, a ubiquitous subset of O-glycosylation, is poorly understood in bacterial systems. In Mycobacterium tuberculosis and other pathogenic actinobacteria, numerous secreted virulence factors were identified as mannoproteins, with protein-O-mannosylation deficient mutants displaying a less virulent phenotype due to these proteins lacking the modification. However, these findings do not offer any explanations as to why non-pathogenic strains of actinobacteria also perform this modification as in these organisms it is seemingly dispensable. Corynebacterium glutamicum is a widely utilized, industrially relevant actinobacteria that also performs protein-O-mannosylation. This manuscript describes the utilization of C. glutamicum as a Gram-positive recombinant host for the in vivo study of actinobacterial protein-O-mannosylation and demonstrates the distinct lack of first-hand biochemical data of the process in prokaryotes.