The posttranslational addition of a single O-linked -N-acetylglucosamine (O-GlcNAc) to serine or threonine residues regulates numerous metazoan cellular processes. The enzyme responsible for this modification, O-GlcNAc transferase (OGT), is conserved among a wide variety of organisms and is critical for the viability of many eukaryotes. Although OGTs with domain structures similar to those of eukaryotic OGTs are predicted for many bacterial species, the cellular roles of these OGTs are unknown. We have identified a putative OGT in the cyanobacterium Synechococcus elongatus PCC 7942 that shows active-site homology and similar domain structure to eukaryotic OGTs. An OGT deletion mutant was created and found to exhibit several phenotypes. Without agitation, mutant cells aggregate and settle out of the medium. The mutant cells have higher free inorganic phosphate levels, wider thylakoid lumen, and differential accumulation of electron-dense inclusion bodies. These phenotypes are rescued by reintroduction of the wild-type OGT but are not fully rescued by OGTs with single amino acid substitutions corresponding to mutations that reduce eukaryotic OGT activity. S. elongatus OGT purified from Escherichia coli hydrolyzed the sugar donor, UDP-GlcNAc, while the mutant OGTs that did not fully rescue the deletion mutant phenotypes had reduced or no activity. These results suggest that bacterial eukaryote-like OGTs, like their eukaryotic counterparts, influence multiple processes.A lthough long believed to be solely attributes of eukaryotes, many glycosylation pathways are now evident in prokaryotes. Bacterial proteins can be modified with a variety of N-linked (1) and O-linked glycans (2). Posttranslational modification of serine or threonine residues with single O-linked -N-acetylglucosamine (GlcNAc) by O-GlcNAc transferases (OGTs) is common in eukaryotes. All eukaryotic OGTs share a domain structure consisting of tetratricopeptide repeats (TPRs) that are involved in protein-protein interactions (3) followed by the glycosyltransferase catalytic region (Fig. 1A). The catalytic region is composed of two highly conserved domains (4), which are linked by a variable-length insertion. OGT homologs occur across prokaryotic phyla (4), and a number of these OGTs are eukaryote-like (Fig. 1B); this is best illustrated by the similarities in the crystal structures of the Xanthomonas campestris (5, 6) and human (7,8) OGTs. In addition to sharing similar domain structures, key amino acids in and around the active site are conserved in the eukaryotic and bacterial enzymes (see Fig. S1 in the supplemental material).Unlike other eukaryotic glycosylations, O-GlcNAcylation occurs in the nucleus and cytosol and is dynamically added and removed from proteins. O-GlcNAcylation can regulate a cellular process directly or by acting in competition with phosphorylation (9, 10). Notably, and unlike phosphorylation, which employs a plethora of site-specific kinases and phosphatases, the cycling of O-GlcNAcylation relies on two highly conserved enzyme...