Fig. 3

Characterization of Hfq3-associated Toxicity in Liquid Media. Growth curves were performed at 37 °C, 225 rpm, and 15% CO₂ in NBY/bicarbonate media. a Growth comparison of Ames 35 WT (A35; blue) and Ames 35-Hfq3 overexpression strain (Hfq3; black). Data is expressed as the mean of three biological replicates for each strain +/- the standard error of the mean (SEM). b Growth comparison of recovered colonies #1–6 to the parent Hfq3 overexpression strain. Error bars reflect standard error of the mean (SEM) of three biological replicates for the parent Hfq3 strain. The doubling time during log phase averaged across the six recovered colonies was 41 ± 1 min. and the doubling time over this time course for the Ames-35 Hfq3 cells was ≈ 90+ min. In contrast, the doubling time for wild-type Ames 35 grown under these conditions (but no antibiotic) in parallel experiments is ≈ 33–35 min. c Expression levels of Hfq3 from recovered colonies as compared to the parent Hfq3 overexpression strain. Equal amounts of total soluble protein from cell lysates (as verified by protein gels) were subjected to SDS-PAGE and western blotting with anti-Hfq3. Samples were analyzed under semi-native (-) and boiled (+) conditions. Dotted arrow represents the 50 kDa marker band. Based on ImageJ analysis, Hfq3 expression levels were reduced between 6 and 17-fold as compared to the Hfq3 overexpression parent strain in recovered isolates with detectable protein expression. No protein expression was detectable in isolates carrying the nonsense mutations described in Results. d Growth of Ames 35 Δhfq1hfq2hfq3 and Ames 35 Δhfq1hfq2hfq3 overexpressing Hfq3 patched to LB incubated at 37 °C in air or to NBY/bicarbonate incubated at 37 °C in 15% CO₂, as indicated. As described in Results, the severity of the phenotype of Hfq3-overexpressing strains in air is variable across independent experiments