Abstract
In Bacillus subtilis, Listeria monocytogenes and in two Mycobacteria, it was previously shown that yvcK is a gene required for normal cell shape, for optimal carbon source utilization and for virulence of pathogenic bacteria. Here we report that the B. subtilis protein YvcK binds to Uridine diphosphate-sugars like Uridine diphosphate-Glucose (UDP-Glc) and Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) in vitro. Using the crystal structure of Bacillus halodurans YvcK, we identified residues involved in this interaction. We tested the effect of point mutations affecting the ability of YvcK to bind UDP-sugars on B. subtilis physiology and on cell size. Indeed, it was shown that UDP-Glc serves as a metabolic signal to regulate B. subtilis cell size. Interestingly, we observed that, whereas a yvcK deletion results in the formation of unusually large cells, inactivation of YvcK UDP-sugar binding site does not affect cell length. However, these point mutations result in an increased sensitivity to bacitracin, an antibiotic which targets peptidoglycan synthesis. We thus propose that UDP-GlcNAc, a precursor of peptidoglycan, could be a good physiological ligand candidate of YvcK.
Highlights
To cite this version: Elodie Foulquier, Anne Galinier
We have undertaken the biochemical characterization of YvcK from B. subtilis and we have shown that this protein is able to bind UDP-sugars like UDP-GlcNAc and Uridine diphosphate-Glucose (UDP-Glc) in vitro
Since a yvcK mutant has an increased sensitivity to bacitracin[12], we tested the binding of YvcK with two UDP-sugars, UDP-Glc and UDP-GlcNAc, that are cell wall precursor components
Summary
To test whether the effect of a yvcK deletion associated with a deletion of a gene encoding another enzyme of the glycolipid biosynthesis pathway (in conditions where UDP-Glc synthesis is altered), we constructed a gtaB yvcK double mutant and analyzed its cell size in LB rich conditions (Fig. 3D and E). Strains expressing the YvcK-T14A or YvcK-N218A protein, whose ability to bind UDP-sugars is more weakly affected (Fig. 4D and E), have IC50 values that tend towards that of the WT strain (about 282 μg/ml and 261 μg/ml for strains producing YvcK-T14A or YvcK-N218A protein respectively and 368 μg/ml for the WT strain) These data show a correlation between the increase of bacitracin sensitivity and the alteration of the corresponding mutant YvcK protein to bind UDP-GlcNAc and demonstrate a physiological role for the residues involved in UDP-sugars binding. In B. subtilis, we can imagine that the phosphorylation of Thr[304], that is located near the Arg[301] in the sequence and in the structure, modifies YvcK ability to bind UDP-sugars and the bacitracin sensitivity of Bacillus cells
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