Figure 4. Decreased expression of wall-teichoic acids during fermentative growth makes S. aureus more amenable to cleavage by AtlA.
Panel A; Schematic of wall-teichoic acid (WTA) biosynthesis in S. aureus. The diagram displays select proteins involved in WTA biosynthesis and is redrawn as initially presented by Campbell et al. (2012). The initial transformations in the pathway catalyzed by TarO and TarA are non-essential, while the latter steps are essential. Tunicamycin inhibits TarO, as well as the 2-epimerase MnaA, which modulates the substrate levels for TarO (Campbell et al., 2011; Mann et al., 2016). MnaA is not displayed. Panel B; Transcript levels corresponding to genes encoding for WTA biosynthesis proteins are decreased upon fermentative growth. Biofilms of the WT (JMB 1100) were cultured aerobically or fermentatively, mRNA was extracted, and the abundances of the tarO, tarA, tarB, and tarH transcripts were quantified. The data were normalized to 16S rRNA levels, and thereafter to levels observed aerobically. Panel C; AtlA-dependent cleavage of heat-killed cells at a decreased pH is modulated via wall-teichoic acids. Murein-hydrolase activity at pH of 5 for cell-wall associated proteins (CW-extracts) detached from a ΔatlA strain (KB 5000) carrying patlA and incubated with heat-killed cells of the WT cultured aerobically or fermentatively in the presence or absence of 100 ng/mL tunicamycin as substrates is displayed. Panel D; AtlA-dependent autolysis of intact whole cells at decreased pH is modulated via wall-teichoic acids. The WT and atlA::Tn (JMB 6625) strains were cultured aerobically or fermentatively in the presence or absence of 100 ng/mL tunicamycin. Autolysis was examined in intact cells resuspended in a buffer with pH of 5. Panels E; Heat-killed aerobic or fermenting WT bind similar amounts of AtlA. CW-extract detached from a ΔatlA strain (KB 5000) carrying patlA was incubated at pH of 5 with heat-killed WT, cultured aerobically or fermentatively in the presence or absence of 100 ng/mL tunicamycin, or in the absence of cells (control) for 8 min. The cells were separated by centrifugation and bacteriolytic activity in the resultant supernatant was assessed upon heat-killed M. luteus as a substrate is displayed. Data in Panel B represents the average value of triplicates. Statistical significance was calculated using a two-tail Student's t-test and * indicates p-value of <0.05. Data in Panels C-E represent the average value of technical duplicates from one set of substrate preparation or autolysis assays. The heat-killed substrates were prepared or autolysis assays were conducted on least three separate occasions and similar results were obtained. Error bars in all panels represent standard deviations. Error bars are displayed for all data, but might be too small to see on occasion.
Figure 4—figure supplement 1. AtlA- and AM-dependent cleavage of heat-killed cells is modulated via altered expression of wall-teichoic acids.
Figure 4—figure supplement 2. AtlA-dependent lysis rates of heat-killed tunicamycin treated cells are not altered upon alterations in the assay buffer pH.
