, 2008). Kawai et al. (2011) recently suggested that LCP proteins transfer WTAs and other anionic polymers from the lipid carrier to the cell wall AZD2281 datasheet peptidoglycan in B. subtilis. Comparative growth of LCP mutants on bacitracin gradient plates showed that the LCP triple mutant was highly susceptible (Fig. 3a). The bacitracin MIC of the triple mutant was 4 μg mL−1 compared to 32 μg mL−1 for wild type and all LCP single

and double mutants. The hyper susceptibility of the LCP triple mutant to bacitracin could therefore be due to an additional shortage of the lipid carriers caused by the lack of the putative WTA ligase function of LCP proteins. In line with the proposed function of LCP proteins, previous studies showed a decrease in the WTA content of LCP mutants in different species (Hübscher et al., 2008; Kawai et al., 2011). Therefore, we analysed the WTA content of LCP single mutants and the triple mutant in S. aureus, via detection of the cell wall phosphorus BMS-907351 mouse content (Ames & Dubin, 1960). The previously described decrease in the WTA content of the ΔmrsR mutant (Hübscher et al., 2009) could be confirmed here, and the WTA contents of the Δsa0908 and Δsa2103 mutants were decreased to 62% and 95% of the wild type level, respectively (Fig. 3b). An almost complete depletion of

WTA was observed in the triple LCP mutant, with cell wall phosphorus content down to 2% of the wild type. Re-introduction of single LCP genes into the triple mutant restored WTA levels to 94%, 81% and 69% of wild type levels for sa2103, msrR and sa0908, respectively. The capacity of all LCP proteins to restore the WTA content to

a certain degree confirmed a partial functional redundancy that has been shown for other phenotypes such as growth defects, beta-lactam resistance, biofilm formation and self-agglutination (Over et al., 2011). The very low WTA content of the LCP triple mutant confirmed that LCP proteins in S. aureus have an essential function in WTA loading of the cell wall. The three LCP genes in B. subitlis are conditionally essential, meaning that an LCP triple mutant in B. subtilis is only viable when tagO (tarO) is also deleted, thereby preventing the flux of precursors into the WTA synthesis pathway (Kawai et al. 2011). The effect of TarO (TagO) inhibition on the LCP triple mutant was tested to Dichloromethane dehalogenase detect a possible connection between LCP proteins with WTA synthesis or assembly in S. aureus, as found for B. subtilis (Kawai et al., 2011). Subinhibitory concentrations of tunicamycin, which are known to inhibit TarO (TagO; Campbell et al., 2011), could partially complement the growth defect of the LCP triple mutant (Fig. 4a). The minimal doubling time of the triple mutant decreased from 49 ± 2 to 34 ± 2 min upon tunicamycin treatment. Inhibition of TarO in the wild type did not significantly affect the minimal doubling time of 25 ± 0.