Enzymatic activities and functional interdependencies of Bacillus subtilis lipoteichoic acid synthesis enzymes

Abstract

Lipoteichoic acid (LTA) is an important cell wall polymer in Gram-positive bacteria. The enzyme responsible for polyglycerolphosphate LTA synthesis is LtaS, first described in Staphylococcus aureus. Four LtaS orthologues, LtaSBS, YfnI, YqgS and YvgJ, are present in Bacillus subtilis. Using an in vitro enzyme assay, we determined that all four proteins are Mn2+-dependent metal enzymes that use phosphatidylglycerol as a substrate. We show that LtaSBS, YfnI and YqgS can produce polymers, suggesting that these three proteins are bona-fide LTA synthases while YvgJ functions as an LTA primase, as indicated by the accumulation of a GroP-Glc2-DAG glycolipid. Western blot analysis of LTA produced by ltaSBS, yfnI, yqgS and yvgJ single, triple and the quadruple mutant, showed that LTA production was only abolished in the quadruple and the YvgJ-only expressing mutant. B. subtilis strains expressing YfnI in the absence of LtaSBS produced LTA of retarded mobility, presumably caused by an increase in chain length as suggested by a structural analysis of purified LTA. Taken together, the presented results indicate that the mere presence or absence of LTA cannot account for cell division and sporulation defects observed in the absence of individual enzymes and revealed an unexpected enzymatic interdependency of LtaS-type proteins in B. subtilis.

The <i>Bacillus subtilis</i> essential gene <i>dgkB</i> is dispensable in mutants with defective lipoteichoic acid synthesis

“…Although yfnI cannot fully substitute for ltaS in S. aureus (Gründling and Schneewind, 2007), the suppression by the yfnI single disruption (Fig. 2a) suggests that YfnI produces DG and thus synthesizes LTA in B. subtilis cells as recently shown by Wörmann et al (2011).…”

“…In the yfnI-disrupted strain, the ectopic expression of yflE from P xyl promoter did not, while ectopic yfnI expression did return the growth to IPTG-dependent, indicating that there was no cross-complementation in the opposite direction either. The results suggest that the two gene products, YflE and YfnI, are functionally different, although the two enzymes have the same predicted membrane topology and domain structure (Wörmann et al, 2011).…”

“…However, Schirner et al (2009) have reported that a quadruple mutant defective in all the homologues could be constructed, although it grew slowly and had a cell division Edited by Fujio Kawamuraand separation defect. Recently, Wörmann et al (2011) have shown that LTA is absent in the quadruple mutant. Therefore, the essentiality of the B. subtilis pgsA gene cannot be ascribed to its indispensability for LTA synthesis.…”

Small molecule inhibitor of lipoteichoic acid synthesis is an antibiotic for Gram-positive bacteria

“…Growth of S. aureus, B. anthracis, L. monocytogenes, or B. subitilis cannot occur without polyglycerol-phosphate LTA synthesis and ltaS expression (14,16,17,40). LtaS, the catalyst of LTA synthesis, harbors five transmembrane domains and a C-terminal catalytic domain that is found in bacteria but not in eukaryotes (14).…”

Abnormal morphology of Bacillus subtilis ugtP mutant cells lacking glucolipids

“…Therefore, there might conceivably be a relationship between the phenotype of the ugtP mutant and LTA. B. subtilis has four LTA synthases (the products of yflE, yfnI, yqgS, and yvgJ), and two enzymes (the products of yflE and yfnI) are believed to be the major LTA synthases (Wörmann et al, 2011). The cells of a strain with a combination of ugtP and yflE yfnI double mutations exhibit a shape similar to that of the ugtP mutant (data not shown).…”