(a) WTA was extracted from both cells and supernatant of the wild type and tcaA mutant of S. aureus and visualised on and SDS-PAGE gel stained with 1 mg/ml Alcian blue. The tcaA mutant had significantly less WTA in the cell wall but more in the supernatant. (b) The WTA extractions were performed on the wild type and tcaA mutant in triplicate the relative density of the WTA quantified by densitometry. (c) The phosphate content of the cell wall WTA extracts was quantified, which verified that the tcaA mutant has significantly less WTA. (d) The inactivation of the lcpA gene increases the sensitivity of S. aureus to killing by serum. (e) The wild type strain JE2 was grown in broth supplemented (at 10%) with supernatant of either itself (JE2), with that from a tcaA mutant, or with that from a lcpA mutant. These supernatants had no effect on the growth of JE2 in the absence of arachidonic acid. In the presence of arachidonic acid JE2 was unable to grow when supplemented with its own supernatant, however, when supplemented with the supernatant of the two mutants, which both contain soluble WTA, JE2 was able to grow. The addition of purified WTA extract (at 2%) from another S. aureus strain (LAC) also neutralised arachidonic acid, however an equivalent extract from an isogenic WTA mutant (LAC ΔtarO) did not. (f) The charge across the cell wall of the wild type and tcaA mutant was compared using cytochrome c, where the mutant was found to be less negatively charged. An mprF mutant has been included as a control. The dots represent individual data points (n=5 for (d), n=3 for remaining assays), the bars the mean value, and the error bars the standard deviation. Significance was determined as *<0.05, **<0.01, ***<0.001, ****<0.0001.