Figure 3. Amidase activity of LytA is required for capsule shedding.

(a) TIGR4 (WT) with empty vector, the lytA mutant (LytA⁻) with empty vector or vector containing a WT lytA allele (pLytA) or an allele with mutations in active-site residues (pLytA⁻) were subjected to capsule shedding assay. The supernatant fractions were then analysed by capsule blot. (b) Cultures from a were harvested, mechanically lysed, and lysates, normalized for total protein, were analysed by western blot using LytA antiserum (see full blot in Supplementary Fig. 6a). (c) Representative growth curve of strains from a grown in C+Y. At the arrow LL-37 was added to indicated cultures (open symbols) at 8 μg ml⁻¹. (d) The lytA mutant was subjected to a capsule-shedding assay, and recombinant LytA protein was added at the indicated concentration to the assay. LL-37 was added to the indicated cultures at 4 μg ml⁻¹. The supernatant fraction was then analysed by capsule blot.