Figure 3. Temperature-sensitive growth of CMR300, CMR301, and CMR302 on LB broth.

Cells were grown on LB broth with 100 µg/mL ampicillin and 1 mM IPTG from A₆₀₀ 0.02 to 0.2 at 30 °C; then half of each culture was shifted to 37 or 42 °C, as indicated. Cultures were diluted 10-fold whenever A₆₀₀ reached 0.3–0.4. The cumulative A₆₀₀ was corrected for the dilutions. Panels A, B and C show the temperature shifts of CMR300, CMR301 and CMR302, respectively, from 30 to 37 °C. CMR300 nearly matches the W3110(pWMsbA) control at both temperatures. Reduced growth rates, albeit with good viability, were observed for CMR301 and CMR302 at both 30 and 37 °C versus their matched vector control strains. Panels D, E and F show the temperature shifts of exponentially growing CMR300, CMR301 and CMR302, respectively, from 30 to 42 °C in LB broth, demonstrating the rapid growth cessation of all three kdtA deletion constructs.