TisB protein is the single molecular determinant underlying multiple downstream effects of ofloxacin in Escherichia coli.

Bactericidal antibiotics can cause metabolic perturbations that contribute to antibiotic-induced lethality. The molecular mechanism underlying these downstream effects remains unknown. Here, we show that ofloxacin, a fluoroquinolone that poisons DNA gyrase, induces a cascade of metabolic changes that are dependent on an active SOS response. We identified the SOS-regulated TisB protein as the unique molecular determinant responsible for cytoplasmic condensation, proton motive force dissipation, loss of pH homeostasis, and H2O2 accumulation in Escherichia coli cells treated with high doses of ofloxacin. However, TisB is not required for high doses of ofloxacin to interfere with the function of DNA gyrase or the resulting rapid inhibition of DNA replication and lethal DNA damage. Overall, the study sheds light on the molecular mechanisms by which ofloxacin affects bacterial cells and highlights the role of the TisB protein in mediating these effects.

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli.

Antibiotic persistence refers to the capacity of small bacterial subpopulations to transiently tolerate high doses of bactericidal antibiotics. Upon bactericidal antibiotic treatment, the bulk of the bacterial population is rapidly killed. This first rapid phase of killing is followed by a substantial decrease in the rate of killing as the persister cells remain viable. Classically, persistence is determined at the population level by time/kill assays performed with high doses of antibiotics and for defined exposure times. While this method provides information about the level of persister cells and the killing kinetics, it fails to reflect the intrinsic cell-to-cell heterogeneity underlying the persistence phenomenon. The protocol described here combines classical time/kill assays with single-cell analysis using real-time fluorescence microscopy. By using appropriate fluorescent reporters, the microscopy imaging of live cells can provide information regarding the effects of the antibiotic on cellular processes, such as chromosome replication and segregation, cell elongation, and cell division. Combining population and single-cell analysis allows for the molecular and cellular characterization of the persistence phenotype.