It was also tested if the see more growth of LVS and ΔmglA on solid medium was affected by the oxygen concentration. Approximately 100 bacteria were spread onto agar plates that were incubated in an aerobic or a microaerobic milieu. LVS formed colonies > two mm in size in both environments within 6 days but with delayed kinetics aerobically (Table 1). ΔmglA formed only few and small colonies on plates incubated aerobically. In the microaerobic milieu, however, it formed colonies JQ-EZ-05 of the same size as LVS, but with slightly delayed kinetics. Thus, regardless of growth medium used, ΔmglA appeared to

exhibit markedly impaired growth under aerobic conditions. Table 1 Size of colonies formed by LVS and ΔmglA on agar plates under aerobic or microaerobic conditions   Colony sizea Incubation time (days) Aerobic Microaerobic   LVS Δ mglA LVS Δ mglA

GSK1210151A research buy 2 0 0 1 0 3 1 0 2 1 6 3 MCb 3 3 a Colony size was graded as follows: 0 = Not visible, 1 = colonies <1 mm in diameter, 2 = 1.0 -2.0 mm. 3 = >2 mm in diameter b Mixed colonies, a few large colonies growing in close proximity to each other but most colonies were hardly visible Oxidized proteins in LVS and ΔmglA cultivated under aerobic or microaerobic conditions We hypothesized that the aberrant oxidative stress response of ΔmglA reported previously [8, 10] may lead to suboptimal handling of the effects of oxidation. We therefore attempted to quantify such effects at a more general level. To this end, we analyzed the presence of oxidized proteins using the OxyBlot method. Preparations from

ΔmglA cultivated under the aerobic conditions contained significantly more oxidized proteins than did those prepared from LVS (Figure 2). In contrast, the amounts of oxidized proteins were similar after cultivation in the microaerobic milieu. We noted some inter-experimental variation, but there were markedly increased amounts of oxidized proteins in the ΔmglA preparations under aerobic conditions in a majority of the experiments performed. FUU301 contained similar amounts of oxidized proteins as LVS regardless of growth condition (Figure 2). Figure 2 Analysis Tangeritin of oxidized proteins by the Oxyblot assay. Relative amounts of oxidized proteins in LVS, ΔmglA, or FUU301 during growth in an aerobic or microaerobic environment. Similar results were seen in two additional experiments. The first well of each preparation contained 2.5 ng of protein and the following wells two-fold dilutions thereof. Controls contain non-derivatized samples, and demonstrate the specificity of the antibodies used for detection of oxidative damage. In summary, the marked accumulation of oxidized proteins in ΔmglA during growth in the aerobic milieu strongly suggested that the mutant had an impaired response to oxidation. This may have been a reason for its delayed and lower maximal growth in the aerobic milieu.