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 ).