Dysregulation of a nucleotidyltransferase induces division and surface glycan defects in <i>Escherichia coli</i> by altering related metabolite levels

The cell surface of gram-negative <i>Escherichia coli</i> is rich in glycoconjugates, including O-antigen (O-Ag) and enterobacterial common antigen polysaccharides, which mediate interactions with the environment. Enzymes called nucleotidyltransferases produce nucleoside diphosphate sugars (NDP-sugars), the precursors to these and many other cellular glycans. Across bacteria, glucose-1-phosphate thymidylyltransferases (G1P-Ts) couple deoxythymidine triphosphate (dTTP) and glucose-1-phosphate to produce a key secondary metabolite, deoxythymidine diphosphate-glucose (dTDP-Glc). Flux through many glycan biosynthetic pathways is regulated through feedback inhibition at an allosteric site of the conserved bacterial G1P-T RmlA. Here, we sought to address the cellular consequences of G1P-T dysregulation on metabolic flux and <i>E. coli</i> physiology. Expression of a hyperactive RmlA variant in <i>E. coli</i> cells lacking native G1P-Ts abrogated growth, induced morphological defects, and increased sensitivity toward cell division inhibitors. These defects were suppressed through nucleotide supplementation, leading to the hypothesis that growth and division defects result from observed depletion of nucleotide pools required for DNA synthesis. Inspection of nucleotide metabolite compositions in <i>E. coli</i> lysates carrying mutant G1P-T, however, also indicated increased cellular concentrations of the primary metabolite uridine diphosphate glucose (UDP-Glc), which is known to block cell division. We found that high intracellular levels of UDP-Glc also unexpectedly halt the expression of O-Ag, which reveals that this metabolite is key for coordinating surface glycan biosynthesis and DNA precursor production prior to cell division. Overall, this work highlights the importance of G1P-T regulation and uncovers additional roles of UDP-Glc as a molecular sensor that mediates diverse bacterial biosynthetic processes.