E. coli cells lacking LHR are sensitive to oxidative stress
Oxidative damage to DNA in E. coli cells includes deamination of cytosine to uracil and further oxidized uracil derivatives [18-21], triggering cytosine to adenine transversion mutations. We therefore assessed for a contribution from Lhr to DNA repair in E. colicells that is consistent with in vitro uracil-DNA glycosylase activity. The lhr gene was deleted in E. coli MG1655 (Δlhr ) by recombineering, and we removed the inactivating antibiotic resistance marker, verified by sequencing across the deletion site. We first tested Δlhr cells for sensitivity to azidothymidine (AZT), a previously reported phenotype for Lhr inE. coli cells [14]. In a viability plate assay after growing cells in broth (LB) containing a fixed 7.5 µg/mL AZT we observed 10-fold reduced viability of Δlhr cells compared with wild type cells (Figure 4A ), and similar moderate sensitivity of Δlhrcells across AZT concentrations (Figure 4B ), agreeing with the previous study [14]. We next measured survival of cells when grown in media containing hydrogen peroxide as a potent oxidizing agent. Hydrogen peroxide (12.5 mM) added to growth media after cells had reached OD600 of 0.3 resulted in significantly reduced growth of Δlhr cells in exponential phase compared with wild type cells (Figure 4C ). This agreed with 10-100-fold reduced cell viability compared with wild type cells when Δlhr cells grown in the same way, but without hydrogen peroxide, were then spotted onto LB agar containing increasing concentrations of hydrogen peroxide to count their viability (Figure 4D ).