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...

Diameter-limit harvesting has long been suspected as a dysgenic forestry practice, but a conclusive, practical demonstration of the effects of this selection technique on residual tree performance is lacking. To determine the effects of repeated diameter-limit harvesting on the phenotypes of residual trees, we compared radial growth patterns of residual red spruce trees (with ages greater than 100 years) after diameter-limit harvests with those of residual trees in stands subjected to positive selection harvesting. After nearly 50 years of repeated harvesting, residual trees in the diameter-limit stands were nearly 40% smaller and had grown 32% slower than residual trees in positive selection stands. Furthermore, diameter-limit residuals were initially smaller and remained significantly smaller than positive selection residuals throughout their lifespan, despite major release events. After release, the diameter-limit trees responded with increased growth rates, but the increase was relatively small. Growth rates were consistently and significantly lower for diameter-limit residuals until the final 20 yr when growth rates in each treatment converged. Our results indicate that red spruce stands subjected to repeated diameter-limit harvesting will develop progressively less valuable growing stock with limited growth potential.

The opportunity to trace the evolution of a triplet repeat is rare, especially for seed-plant lineages with a well-defined fossil record. Microsatellite PtTX2133 sequences from 18 species in 2 conifer genera were used to calibrate the birth of a CAGn repeat, from its protomicrosatellite origins to its repeat expansion. Birth occurred in the hard-pine genome ~ 136 million years ago, or 14 million generations ago, then expanded as a polymorphic triplet repeat 136-100 million years before a major North American vicariance event. Calibration of the triplet-repeat birth and expansion is supported by the shared allelic lineages among Old and New World hard pines and the shared alleles solely among North American diploxylon or hard pines. Five CAGn repeat units appeared to be the expansion threshold for Old and New World diploxylon pines. Haploxylon pine species worldwide did not undergo birth and repeat expansion, remaining monomorphic, with a single imperfect 198-bp allele. A sister genus, Picea, had only a region of cryptic simplicity, preceding a proto-microsatellite region. The polymorphic triplet repeat in hard pines is older than some long-lived microsatellites reported for reptiles, yet younger than those reported for insects. Some cautionary points are raised about phylogenetic applications for this long-lived microsatellite.

O‐GlcNAc modification, the posttranslational addition of a single O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) to a serine and/or threonine, regulates many processes in plants and animals. Although O‐GlcNAc transferases (OGTs) are predicted to exist in many bacteria species, the function of these OGTs is largely unknown. When the OGT of the photosynthetic cyanobacterium Synechococcus elongatus PCC 7942 was deleted the resulting mutant was viable and had no growth rate defects. The mutant however did have several defects, including cell aggregation that led to the cell settling out of culture, higher free cellular phosphate levels and altered organization of both thylakoids and polyphosphate bodies. These phenotypes were rescued by re‐introduction of the wild‐type OGT, but were not fully rescued by OGTs with mutations affecting the predicted catalytic domain indicating the phenotypes are due to deletion of the OGT. During the course of this research a mutant that was suppressed for the settling phenotype arose spontaneously. Whole genome sequencing has identified a single new mutation in this strain and experiments are underway to determine if this mutation is responsible for suppression of the settling phenotype and to determine if it affects the other phenotypes associated with deletion of the OGT.