. In this study, the contribution of several individual genes to the de novo acquisition of antibiotic resistance in Escherichia coli was investigated using mutants with deletions of genes known to be involved in antibiotic resistance. The results indicate that recA, vital for the SOS response, plays a crucial role in the development of antibiotic resistance. Likewise, deletion of global transcriptional regulators, such as gadE or soxS, involved in pH homeostasis and superoxide removal, respectively, can slow the acquisition of resistance to a degree depending on the antibiotic. Deletion of the transcriptional regulator soxS, involved in superoxide removal, slowed the acquisition of resistance to enrofloxacin. Acquisition of resistance occurred at a lower rate in the presence of a second stress factor, such as a lowered pH or increased salt concentration, than in the presence of optimal growth conditions. The overall outcome suggests that a central cellular mechanism is crucial for the development of resistance and that genes involved in the regulation of transcription play an essential role. The actual cellular response, however, depends on the class of antibiotic in combination with environmental conditions. A ntibiotic-resistant bacteria pose a serious threat to human health, as the costs of therapy of infections caused by such bacteria increase and the treatment outcome is negatively affected. Bacteria can become resistant de novo by genetic or phenotypic changes and also through the acquisition of resistance-conferring mobile genetic elements. Resistance to antibiotics is rapidly induced as a result of exposure to stepwise increasing sublethal drug concentrations (1). In less than 100 generations, bacterial cells developed genetic mutations and permanent transcriptional changes (2). On the one hand, these cellular modifications allow the population to grow in the presence of high antibiotic concentrations, but on the other hand, they may decrease fitness or cause a metabolic burden (1-3). This metabolic cost does not necessarily come in the form of an increased energy requirement. For example, the adaptation of Escherichia coli to amoxicillin was accompanied by a reduced ecological range, because resistant cells were less able to grow well under adverse external conditions (4).In order to devise measures to prevent or at least slow the development of antibiotic resistance, it is essential to understand the reaction of bacteria to drug exposure at the molecular level. Genes that were permanently differentially regulated in E. coli cells made resistant to amoxicillin, enrofloxacin, or tetracycline compared to their regulation in their sensitive ancestor (2) are likely to play a role in the development of resistance. For example, gadABC and hdeA, which confer resistance to acidic conditions, were differentially expressed in cells made permanently resistant to amoxicillin, enrofloxacin, or tetracycline by exposure to stepwise increasing antibiotic levels. The change in the expression of gadABC and hdeA was the ...