The Effect of Media and Filtration in Inducing the Oxidative Stress Response in <i>Escherichia coli</i>

Abstract

Escherichia coli grown in complex medium (LB), but not defined medium (DGC), arrest DNA replication when collected on membrane filters and resuspended in fresh media. The arrest is similar to that observed when cells are challenged with hydrogen peroxide. Yet, the reason behind this arrest is unknown. I hypothesized that the arrest in replication in complex medium after filtering might be due to oxidative shock, and therefore cells grown in complex medium and filtered should induce an oxidative stress response similar to cells treated with hydrogen peroxide. Utilizing the indicator dye H2DCFDA, which fluoresces in response to reactive oxygen species, I established an assay to measure levels of oxidative stress in E.coli cultures treated with hydrogen peroxide and compared them to cells grown in complex medium and filtered. Using a deletion mutant in oxyR, which is sensitive to oxidative stress and unable to induce a cellular response to hydrogen peroxide, I assessed the fraction of fluorescent-positive oxyR cells grown in complex or defined media and filtered, to oxyR cells treated with hydrogen peroxide. Contrary to what I hypothesized, I found that filtration of cells grown in complex medium does not significantly induce oxidative stress. I discuss alternative reasons as to what may be causing replication to arrest, such as the lack of divalent metals like iron and manganese, osmotic stress and dehydration, the sensitivity of H2DCFDA, and the difference in medium makeup.