FIG. 2.
Affinity of HlpA for S. gallolyticus in exponential and stationary growth phases. In-cell Western analysis of S. gallolyticus bacteria in exponential and stationary growth phases exposed to HlpA-His. Approximately 1 × 109 exponential- or stationary-phase-grown bacteria were coated onto a 96-well plate. Detection of HlpA and HlpA-His was established by rabbit anti-HlpA antibodies and secondary Alexa Fluor 680-labeled anti-rabbit antibody. The bars represent the fluorescence intensities of extracytoplasmic recombinant HlpA-His and endogenous HlpA of bacteria. Note that nonstripped (top row, −) bacteria have, in addition to HlpA-His, endogenous HlpA at their surfaces, whereas trypsin-stripped (top row, +) bacteria have reduced endogenous levels of HlpA on the bacterial cell wall. Exponential- or stationary-phase bacteria were incubated either with (bottom row, +) or without (bottom row, −) HlpA-His. The fluorescence intensity was corrected for background measurement of the incubation medium. Error bars indicate the standard errors from two replicate experiments. *, P < 0.05; **, P < 0.01; ns, not significant (Student's t test).