FIG 4.

Exogenous expression of slyA rescues the Congo red binding deficiency in a virB mutant. (A) Qualitative analysis of Congo red binding. S. flexneri 2457T wild-type and virB::Tn5 strains harboring pBAD, pBAD-virB, or pBAD-slyA were serially diluted (40-fold dilutions) and spotted (approximately 6 μl) onto TSA Congo red plates supplemented with 0.2% (wt/vol) d-glucose (noninduced) or 0.2% (wt/vol) l-arabinose (induced). (B) Quantitative analysis of Congo red binding. Two culture spots (shown in panel A) were scraped off the agar plate and resuspended in 0.75 ml 25% ethanol to remove the Congo red bound to cells. The OD₆₀₀ was determined to normalize samples against cell number. Cell suspensions were centrifuged to pellet cells, and the OD₄₉₈ of the supernatant was determined. Relative Congo red binding was calculated as [(OD₄₉₈/OD₆₀₀)/(average (OD₄₉₈/OD₆₀₀)_{2457T pBAD})] × 100. Data shown are representative of three independent trials. A two-tailed Student's t test (assuming equal variance) was used for statistical analysis. *, P < 0.01; **, P < 0.001.