Figure 3

Effects of the absence of RpoS and YcgG2 on IHE3034 bacteria. For the citrate utilization assays, all conditions, i.e., days (d), temperature (°C) and percentage of glucose added (%), are stated in the upper corner of the subfigures. (a) YcgG2 levels of IHE3034 bacteria cultured in LB (sample 2), AUM (sample 4) and LB supplemented with 0.5 M NaCl (sample 6). Positive control – in vitro-produced YcgG2. ∆ycgG2 bacterial extract, used as a negative control, showed very faint bands due to cross-reactivity of the antibody (samples 3 and 5). (b) SfaA levels of IHE3034 and ∆ycgG2 bacteria cultured in LB, AUM and LB supplemented with 0.5 M NaCl. (c) SfaA levels of IHE3034 and ∆ycgG2 bacteria with restored RpoS (strains 3 and 4, respectively) activity cultured in AUM compared to the levels of the empty-vector strains (strains 1 and 2). (d) Citrate utilization by IHE3034 cells. Box I: Lack of bacterial growth when citrate was provided as a single carbon and energy source. Box II: Citrate utilization by IHE3034 bacteria when glucose was provided as a co-substrate. Representatives of the rest of the strains utilizing only glucose, i.e., MC4100 and RH90 (MC4100∆rpoS), were used as negative controls. (e) Citrate utilization by IHE3034, ∆ycgG2 and ∆sfaY bacteria on Simmon’s plates supplemented with 0.1% glucose and incubated at 37 °C (Box I) and at 22 °C (Box II), and with 0.2% glucose at 22 °C (Box III). (f) Citrate utilization by IHE3034, ∆ycgG2 and ∆sfaY bacteria with restored RpoS activity compared to that of their empty-vector derivatives. (g) Schematic illustration of the effects of RpoS activity on S-fimbrial expression.