Investigation of the phosphorylation of Bacillus subtilis LTA synthases by the serine/threonine kinase PrkC
Résumé
Bacillus subtilis possesses four lipoteichoic acid synthases LtaS, YfnI, YvgJ and YqgS involved in the synthesis of cell wall. The crystal structure of the extracellular domain of LtaS revealed a phosphorylated threonine and YfnI was identified in two independent phosphoproteome studies. Here, we show that the four LTA synthases can be phosphorylated in vitro by the Ser/Thr kinase PrkC. Phosphorylation neither affects the export/release of YfnI nor its substrate binding. However, we observed that a phosphomimetic form of YfnI was active whereas its phosphoablative form was inactive. The phenotypes of the strains deleted for prkC or prpC (coding for a phosphatase) are fairly similar to those of the strains producing the phosphoablative or phosphomimetic YfnI proteins. Clear evidence proving that PrkC phosphorylates YfnI in vivo is still missing but our data suggest that the activity of all LTA synthases may be regulated by phosphorylation. Nonetheless, their function is non-redundant in cell. Indeed, the deletion of either ltaS or yfnI gene could restore a normal growth and shape to a ΔyvcK mutant strain but this was not the case for yvgJ or yqgS. The synthesis of cell wall must then be highly regulated to guarantee correct morphogenesis whatever the growth conditions. The bacterial cell wall is a crucial structure that protects the cell from external stresses or damages and confers shape and resistance to osmotic pressure. Lipoteichoic acid (LTA) is an important component of the cell wall in Gram-positive bacteria, which is linked to the membrane by a lipid anchor. The polymer is composed of a polyglycerolphosphate (poly(GroP)) backbone chain tethered to the membrane by a diglucosyl-diacylglycerol glycolipid 1-3. LTA has been shown to play an important role in bacterial growth and physiology, cation homeo-stasis, morphogenesis and cell division 4-7. Unlike Staphylococcus aureus which only has one LTA synthase (LtaS) that produces LTA 8 , four LtaS paralogues LtaS, YfnI, YvgJ and YqgS are present in Bacillus subtilis. Three of them are authentic synthases whereas YvgJ acts as a primase that adds the first glycerolphosphate subunit to the lipid anchor 9. All enzymes are active in vitro. LtaS is described as the "house-keeping" enzyme necessary during veg-etative growth, YfnI is assumed to be the "stress" enzyme important during cell envelope stresses and YqgS is important during sporulation. They are predicted to have five N-terminal transmembrane helices followed by a large extracellular enzymatic domain that is cleaved just after a conserved AXA motif by type I signal pepti-dases and released in the culture medium 10,11. Proteomic studies showed that both extracellular domains of LtaS and YfnI (eLtaS and eYfnI) are found in B. subtilis late-exponential phase culture supernatants when grown in minimal medium 12. Interestingly, in the absence of LtaS, YfnI becomes more efficient in LTA production and synthesizes polymers with increased length suggesting that YfnI activity is modulated by LtaS 9. The four LTA paralogues, that possess a high percentage of sequence similarity, therefore seem to have interdependent activities and a partial functional redundancy in B. subtilis 9. In addition, YfnI and LtaS were found to be phosphorylated. YfnI has been identified as phosphorylated protein in two independent phosphoproteome studies on B. subtilis 13,14. Furthermore, in the eLtaS crystal structure the active site Thr297 was found to be modified by a phosphate group 5. The replacement of this Thr by an Ala residue abolished LtaS activity. The authors suggested that it rather mimics an intermediate state than a phos-phorylation by a kinase since this residue is located in a deep pocket but the donor for this phosphate group is 1
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