Figure 5.
Multiple EF-G mutations confer kanamycin resistance which is increased by second site mutations. (A) Growth curves of the EF-G mutants in 8-kan and (B) plain LB (error bars represent standard deviation from eight replicates; legend from B is applicable throughout the figure). The stationary-phase OD600 for the isolates containing the FusAA608E alone, and in combination with gatC .-CC (cytosine insertions) mutation, is lower in 8-kan than for any of the other mutants. This suggests that the second site mutations in RpoD, CpxA, TopA and CyaA may improve FusAA608E strain's ability to grow at low kanamycin concentrations. Of note, the FusAP610T mutation isolated from 4-kan populations does not grow as well as the other fusA mutant strains in 8-kan. (C) Kanamycin MICs (n = 8; box plots as defined in description of Figure 2C) for the isolates with the mutations in EF-G (fusA). The FusAP610L mutation might provide higher resistance to kanamycin than the FusAP610T mutation (P < 10−5; two-sample independent t-test). However, the FusAA608E mutation alone or in combination with gatC .-CC (cytosine insertions) or TopAS180L confers resistance levels similar to the FusAP610T mutation (P = 0.289, 0.817 and 0.151, respectively). In combination with CyaAN600Y, CpxAF218Y,and RpoDL261Q, the FusAA608E mutation does better and has higher resistance than either FusAP610T or FusAA608E alone (P = 0.006, 0.0003 and <10−5, respectively). The emergence of some of these mutations in P1, although a resistant mutation is already present, may be explained by this observation.