The Putative Eukaryote-LikeO-GlcNAc Transferase of the Cyanobacterium Synechococcus elongatus PCC 7942 Hydrolyzes UDP-GlcNAc and Is Involved in Multiple Cellular Processes

Abstract

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...

A Putative O-Linked β- N -Acetylglucosamine Transferase Is Essential for Hormogonium Development and Motility in the Filamentous Cyanobacterium Nostoc punctiforme

“…These data support a conserved role for O-GlcNAc protein modification in the generation of motile filaments in both hormogonium-forming and non-hormogonium-forming filamentous cyanobacteria. Although we do not provide biochemical evidence for O-GlcNAcylation activity of the putative OGT encoded by ogtA, such evidence has been reported for the OGT of Synechococcus elongatus PCC 7942 (seOGT) (23). Given that the catalytic GT41 domain of N. punctiforme OgtA (amino acids 374 to 729 of OgtA) shares 29% sequence identity with seOGT (NCBI BLASTP) and maintains conserved histidine and lysine residues (H280 and K445 of seOGT, corresponding to H386 and K554 of N. punctiforme OgtA) essential for seOGT function (23), it seems likely that ogtA encodes a functional OGT.…”

“…While structurally similar putative OGTs are also widespread among prokaryotes, their function is largely unknown (20)(21)(22). A recent study found that inactivation of a gene encoding a putative OGT in the cyanobacterium Synechococcus elongatus PCC 7942 resulted in a pleiotropic phenotype, and it provided biochemical evidence of OGT enzymatic activity for this gene product (23). Here, we provide a detailed characterization of the phenotype for an N. punctiforme ΔogtA mutant strain, the results of which implicate a role for O-GlcNAc protein modification in hormogonium development.…”

Genomic Signatures of Honey Bee Association in an Acetic Acid Symbiont

“…To date, only a handful of prokaryotic OGTs have been identified, and the targets of these OGTs remain unclear ( Shen et al. 2006 ; Sokol and Olszewski 2015 ). Given the role OGTs play in eukaryotic posttranslational modification and the fact that many bacterial effector proteins show homology to eukaryotic proteins ( Galan 2009 ), it is possible that the presence of OGT in B. apis represents a pathway for host–microbe interaction and symbiont-mediated protein modification.…”

Evidence for a Functional O-Linked N-Acetylglucosamine (O-GlcNAc) System in the Thermophilic Bacterium Thermobaculum terrenum

“…Several are secreted pathogenicity factors, like the NagJ from Clostridium perfringens ( 39 ) (hereafter Cp OGA), precluding a role in modulating intracellular O -GlcNAc signaling. A noteworthy exception is the recently identified OGT homologue found in the cyanobacterium Synechococcus elongates that appears to be involved in phosphorus retention within the cell, and genetic disruption causes the cells to aggregate ( 40 ). The underlying biological mechanisms of these phenotypes are not understood, and the organism lacks a predicted OGA homologue, precluding the existence of a dynamic O -GlcNAc proteome in this organism.…”