Characterization and cellular roles of a bacterial O-GlcNAc transferase in Synechococcus elongatus PCC 7942

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Characterization and cellular roles of a bacterial O-GlcNAc transferase in Synechococcus elongatus PCC 7942

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The post-translational addition of a single O-linked 2-N-acetylglucosamine (O-GlcNAc) to serine or threonine residues is an important element in an ever-increasing array of metazoan cellular and regulatory processes. The enzyme responsible for this modification, O-GlcNAc transferase (OGT), is conserved among a wide variety of organisms and is critical for viability in many eukaryotes, including humans, mice, and Drosophila. Although OGTs with domain structure similar to eukaryotic OGTs are predicted for numerous bacteria species, their cellular roles remain unknown. I have identified an OGT in the cyanobacterium Synechococcus elongatus PCC 7942 that has active site homology and similar domain structure to eukaryotic OGTs. An OGT deletion mutant was created ([delta]ogt), which is viable and has no defect in growth rate, but has several phenotypes of interest. Without agitation, [delta]ogt cells aggregate, and settle out of media. Compared to wild type cells, the [delta]ogt cells also have higher free cellular phosphate levels, wider thylakoid lumen, and differential organization of electron dense inclusion bodies. These phenotypes are rescued by re-introduction 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 E. coli hydrolyzes the donor sugar, UDP-GlcNAc, while mutant OGTs that do not fully rescue the deletion mutant have reduced or no activity. These results suggest that the eukaryotic-like OGTs of bacteria affect multiple processes. Although the substrates for the SeOGT remain elusive, I have uncovered a relationship between the SeOGT and pili through a mutation that suppresses the [delta]ogt settling phenotype. A single amino acid substitution (alanine 107 to aspartic acid) in the protein PilA suppresses two mutant phenotypes, settling and loss of pili. PilA is a subunit of type four pili (fimbriae), which are frequently found on the surfaces of gram-negative bacteria. Fimbriae are involved in virulence, DNA uptake, twitching motility, and adhesion. Although pilus assembly is a complex process, pili are primarily homopolymers of PilA, which is added or subtracted via ATPases to either extend or retract the pilus. No pili are detectable in [delta]ogt cells, while pili are restored to [delta]ogt cells by the pilA(A107D) mutation. When present together, mutant and wild type pilA genes prevent the assembly of pili, suggesting that there is a toxic interaction between mutant and wild type proteins which results in inability to assemble pili. This occurs regardless of the presence of OGT. Glycosylation of extra-cellular appendages have been widely reported. Pili are known substrates of glycosylation and glycosylation states have been reported to be important in the function of pili. The suppression of [delta]ogt phenotypes by mutant PilA suggests that PilA is either a substrate of the OGT or that another protein involved in pilus synthesis is modified by OGT.


University of Minnesota Ph.D. dissertation. May 2015. Major: Molecular, Cellular, Developmental Biology and Genetics. Advisor: Neil Olszewski. 1 computer file (PDF); 136 pages.

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Sokol, Kerry. (2015). Characterization and cellular roles of a bacterial O-GlcNAc transferase in Synechococcus elongatus PCC 7942. Retrieved from the University Digital Conservancy,

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