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. 2023 Sep 14;12:e85690. doi: 10.7554/eLife.85690

Figure 3. Aromatic substitutions at position 12 retain SsrB pH sensitivity.

(a) Normalized PsifA-lacZ activity in the presence of SsrB or H12 mutants of SsrB grown in magnesium minimal medium (MGM) pH 7.4 (blue bars) and pH 5.6 (red bars) after 3 hr of 0.1% (w/v) arabinose induction. The H12K, H12Y, and H12F substitutions showed a decrease in activity at pH 7.4 compared to SsrB (34 ± 4%, 30 ± 4%, and 24 ± 8%). (b) SsrB H12K only showed a moderate increase in activity at pH 5.6 (2.7-fold); however, the SsrB H12Y and H12F substitutions retained an acid-stimulated increase in the activity (6.9- and 6.7-fold, respectively) comparable to the wild-type (5-fold). (c) PsifA-lacZ activity measured from strains recovered at 8 hr post HeLa infection. The activity of the SsrB H12Q and SsrB H12Y-expressing strains was 62 ± 12% and 58 ± 3%, respectively, relative to the SsrB-expressing strain (p<0.001, n = 3). (d) Intracellular survival of strains in HeLa cells at 16 hr post infection (hpi). During HeLa cell infection, the c.f.u./ml of the SsrB-expressing strain increased 19-fold at 16 hpi. For SsrB H12Q and SsrB H12Y-expressing strains, the c.f.u./ml only increased 6.4- and 7.6-fold, respectively (p<0.0001, n = 3).

Figure 3—source data 1. PsifA-lacZ activities and fold replication values for individual experiments used to create plots for Figure 3.

Figure 3.

Figure 3—figure supplement 1. DW637 strains expressing SsrB, SsrBc, H12Q, or H12Y infecting HeLa cells at 16 hr post infection (hpi).

Figure 3—figure supplement 1.

Salmonellae were stained with anti-LPS antibody (green), HeLa cells were stained using phalloidin (red) and DAPI (blue). Fewer intracellular bacteria were present in the HeLa cells infected with SsrBc and H12 mutants.
Figure 3—figure supplement 2. Intracellular survival of 14028s strains expressing SsrB wild-type or H12Q or lacking SsrB during infection of RAW 264.7 macrophages.

Figure 3—figure supplement 2.

As compared to the wild-type, which exhibits a 14.2-fold increase in replication at 16 hr post infection (hpi) vs. 2 hpi, Salmonellae expressing H12Q only showed a twofold increase in replication, similar to that of a ΔssrB strain (twofold) (**p<0.01, n.s. = p>0.05, n = 3).
Figure 3—figure supplement 2—source data 1. Fold replication values for individual experiments used to create plots for Figure 3—figure supplement 2.
Figure 3—figure supplement 3. Immunoblotting of strains expressing various SsrB constructs grown in magnesium minimal medium (MGM) pH 7.4.

Figure 3—figure supplement 3.

An anti-6X-HisTag monoclonal antibody was used to detect SsrB, and an anti-DnaK antibody was used as a loading control. Lane 1 contains purified SsrB (6.9 μg) as a positive control. Lanes 2–5 contain cell-free extracts from strains expressing the empty vector (lane 2), SsrB (lane 3), H12Q (lane 4), and H12Y (lane 5).
Figure 3—figure supplement 3—source data 1. Original uncropped images of immunoblot used to generate Figure 3—figure supplement 2.