Fig. 3.
UgtP is subject to nutrient-dependent, post-translational regulation by the Clp proteases. Semi-quantitative immunoblot of UgtP-His expressed from a xylose-inducible promoter (Pxyl-ugtP-his) in the absence of (a) 5 proteases, YluC, CptA, ClpP, YvjB, and Lon (PL2022, PL2028, PL2102, PL2032, and PL2033, BH10 as WT) or (b) single/combinatorial deletions of the Clp chaperones (ClpC, ClpE, and/or ClpX) (BH127, BH128, BH130, PL2102, BH135, BH136, BH137, BH138). Cells were cultured in either LB + 0.5% xylose or minimal sorbitol + 0.5% xylose, and immunoblotted against His and FtsZ. Samples shown in (a) and also for (b) were run on the same blot, but cropped during image processing. (c) Fold change in UgtP-His levels for Pxyl-ugtP-his (BH10) and Pxyl-ugtP-his + ∆clpP (BH129) after adding spectinomycin to inhibit translation. Cells were cultured in minimal sorbitol + 0.5% xylose, sampled every 30 min after spectinomycin addition, and subjected to immunoblotting against His antibody. Values are mean ± SD (n = 3). A two-way ANOVA was used to assess differences in UgtP-His levels ± clpP over time (P < 0.0001) and a Bonferroni multiple comparisons test (shown) was used to determine significance between the two strains at specific time intervals (***p < 0.001, **p < 0.01). (d) qRT-PCR measurements of clpC, clpE, clpX, and clpP expression in minimal sorbitol versus LB. Values are mean ± SD (n = 4). An unpaired T-test was applied to the ∆Ct values to access significance (***p < 0.001, **p < 0.01, ns p > 0.05)