(B) As a comparison the Rd KW20 was grown in BHI (■) and CDM (■) and the adhC mutant was grown in BHI (▲) and CDM (▲ with dotted lines). (C) Rd KW20 (■) and adhC mutant (♦) were then grown with high oxygen until 24 hr when the oxygen tension was changed to low oxygen. To assess whether AdhC was being expressed under these aerobic conditions in the wild type cells we Captisol solubility dmso firstly monitored AdhC activity during the growth cycle. The cells were assayed for AdhC activity (by assay of GSNO reductase activity), at different time points through the growth cycle. Figure 2A shows that AdhC activity increases during exponential phase, and then decreases in late exponential and stationary
phase. RNA was also extracted from H. influenzae wild-type strain at early, mid and late log phase and RT-PCR was performed using 16 S and adhC-estD
primers (Figure 2B). We also investigated the effect of differences in oxygen tension on AdhC expression by growing cultures in low, medium and high oxygen levels; Figure 2C shows that AdhC activity was highest in cells grown at highest oxygen tension and activity decreased as oxygen tension in the culture decreased. Taken together these results indicated that adhC expression in H. influenzae is highest under aerobic conditions and this is associated with glucose metabolism. Figure 2 Change in AdhC specific activity during growth of H. influenzae . (A) Samples were Metalloexopeptidase taken and assayed for AdhC enzyme activity from early log phase (3 hr), mid-log phase (4.5 h), Doramapimod research buy log phase (5.5 h) late log phase (8 h) and stationary phase (18 h). (B) RT-PCR for the 16SrDNA (lanes 1–4) and adhC-estD (lanes 5–8) using RNA from the time points 3 h (lanes 1 and 6), 5.5 h (lanes 2 and 7) and 8 h (lanes 3 and 8). Lanes 4 and 5 are representative negative controls. Lane 9 is the ladder. (C) At time points throughout the H. influenzae growth phase AdhC specific activity was measured from cells grown with different oxygen tensions (low tension are the black
bar and high oxygen tension are the grey bars). The enzyme activity is presented as change in NADH consumed per minute per mg total protein. Y-error bars indicate +/− 1 standard deviation of the mean. Units are μmol NADH oxidized min-1 mg protein-1. The growth KPT-330 in vitro curves are indicated by the OD600 of cells grown at low oxygen levels (black circle) and high oxygen levels (gray box). (*P < 0.001, **P < 0.002, ***P < 0.0005). AdhC is required for defense against reactive aldehydes To determine whether AdhC had a role in protection against the reactive aldehydes known to be relevant and toxic during aerobic growth, we grew wild-type (Rd KW20) and its isogenic adhC mutant in the presence of some of these compounds and measured the end point of growth (OD600), growth of any culture did not continue beyond the 18 hr point.