Fig. 2

Characterization of Hfq3 Overexpression Toxicity. a Anomalous colony morphology of Hfq3 overexpression strain transformants as compared to Hfq2 and GFP overexpression strain transformants (non-inducing/air conditions). Cropped images reflect the same plate area in each case; scale bar 10 mm. b Western blot depicting level of Hfq3 whole cell expression vs. soluble expression level (probed with anti-Hfq3 antibody) in an overnight culture grown under inducing conditions. “Total” sample was prepared by lysing cells in 8 M urea, as compared to “soluble” sample, which was prepared by lysing cells by beadbeating in 1X PBS. Equal amounts of total cellular protein were loaded in each well, as verified by a parallel protein gel. Dotted arrow indicates the 50 kDa marker band. c Growth of B. anthracis strains patched to LB incubated at 37 °C in air or to NBY/bicarbonate incubated at 37 °C in 15% CO₂, as indicated. (i) Ames 35 parent strain, (ii) Hfq2 overexpression strain, (iii) Hfq3 overexpression strain, and (iv) GFP overexpression strain. d Expression of Hfq3 in the absence (LB) and presence (NBY) of inducing conditions. Equal amounts of total soluble protein from cell lysates were subjected to SDS-PAGE and western blotting with anti-Hfq3. Samples were analyzed under semi-native (-) and boiled (+) conditions. Due to the growth phenotype, log-phase OD₆₀₀ values for Hfq3 are difficult to attain, so Hfq3 levels depicted here for both LB and NBY are derived from a 10 mL culture grown to an OD₆₀₀ of 0.14–0.2. Dotted arrow represents the 50 kDa marker band. e Total cell lysate gel comparing overexpression levels of Hfq2, Hfq3, and GFP in B. anthracis grown under inducing conditions to an OD₆₀₀ of 0.7–1.0; the Hfq3 strain was prepared as described above in panel (d). Equal amounts of total soluble protein were subjected to SDS-PAGE. Samples were analyzed under semi-native (-) and boiled (+) conditions. Dotted arrow represents the 55 kDa marker band, solid arrow represents the 7 kDa marker band, and asterisks denote approximate locations of Hfq2, Hfq3, and GFP protein bands