Mechanistic Insights into the Antimicrobial Effect of Benzodioxane-Benzamides Against Escherichia coli

<b>Background/Objectives</b>: The bacterial cell division machinery is emerging as an attractive target for antimicrobial compounds. FtsZ, a highly conserved essential division protein, is the target for a number of small molecules such as benzamides. Recent studies show that benzodioxane-benzamides (BDOBs) are among the most potent inhibitors of FtsZ function in Gram-positive bacteria, although their ability to inhibit Gram-negative FtsZ, in particular <i>Escherichia coli</i> FtsZ, has been more controversial. <b>Methods</b>: Here, we use genetic and cytological methods to demonstrate that FtsZ of efflux pump-disabled <i>E. coli</i> can be efficiently targeted by BDOBs. <b>Results</b>: We show that engineered mutants and spontaneous variants map in or near the interdomain cleft (IDC) of FtsZ that confers resistance to BDOBs, similar to previous results with Gram-positive FtsZs. We also uncover spontaneous extragenic mutants that can confer high levels of resistance to at least one potent BDOB, including a mutant that encodes a novel hyperfission variant of the essential cell division protein FtsW. <b>Conclusions</b>: Our evidence indicates that as with Gram-positive bacteria, the IDC of Gram-negative bacterial FtsZ is directly targeted by BDOBs, provided efflux pumps are disabled. We also conclude that FtsZ-independent factors can influence the effect of BDOBs on <i>E. coli</i> cell division, including activation of division septum synthesis.