FIG 3.
ManA* results in alterations to specific cellular envelope components. (A) ManA regulates the interconversion of F6P and M6P. These metabolites are important precursors for the synthesis of cellular envelope components, including LPS, PG, and EPS. (B) LPS was extracted from wild-type, manA* (KD145), Tn5::wbqP (CJW1249), and Tn5::wbqP/Pxyl-wbqP (EK393) cells grown for 48 h in HIGG with 1 μM phosphate and 0.03% xylose and separated by Tris-Tricine SDS-PAGE. (C) Comparisons of muropeptides from wild-type and manA* cells grown in HIGG with 1 μM phosphate did not yield any gross changes in PG composition. (D) Wild-type and manA* cells were grown in HIGG with 1 μM phosphate for 48 h prior to pulse-chase labeling with HADA for 30 min to label regions of active PG synthesis. Both strains showed labeling at the base of the stalk. Scale bars, 5 μm. (E) The indicated strains were streaked onto HIGG plates supplemented with 3% sucrose to induce EPS production and mucoidy. Wild-type cells had a distinct mucoid appearance, whereas manA* was matte in appearance, signifying a decrease in EPS production. CB15 and NA1000 ΔMGE (EK717) were non-EPS-producing control strains. (F) The indicated strains were grown in HIGG with1 μM phosphate for 48 h, and stalk lengths were measured using ImageJ (error bars are SEM, ANOVA F(4,377) = 115.5, P < 0.0001; *, post hoc comparisons using Bonferroni test, P < 0.05). The Tn5::wbqP strain, which does not make O-antigen, had a partial effect on stalk elongation compared to the manA* strain. The effect of the wbqP transposon insertion was fully complemented by inducible expression of wbqP.