FliC phosphorylation affects T2SS secretion efficiency.

<p>(A) Immunoblot of extracellular LasB (top panel), intracellular LasB (middle panel) and intracellular RNA polymerase (RNA Pol) α-subunit (bottom panel) at 13 h for PAO1 WT, Δ<i>fliC</i>, Δ<i>fliC</i>-FL T27A, Δ<i>fliC</i>-FL and Δ<i>fliC</i>-FL S28A strains. Proteins were loaded based on equal number of cells as shown by RNA Pol α-subunit levels (bottom panel). (B) Quantification of extracellular LasB levels at 13 h in Δ<i>fliC</i>-FL T27A vs. Δ<i>fliC</i>-FL and Δ<i>fliC</i>-FL S28A vs. Δ<i>fliC</i>–FL. Error bars represent mean ±SD calculated from five biological replicates. Student’s t-test p-values < 0.05 for Δ<i>fliC</i>-FL T27A vs. Δ<i>fliC</i>–FL and Δ<i>fliC</i>-FL S28A vs. Δ<i>fliC</i>-FL. (C) Immunoblot of membrane proteins XcpY, XcpP, XcpQ in PAO1 WT, Δ<i>fliC</i>, Δ<i>fliC</i>-FL T27A, Δ<i>fliC</i>-FL and Δ<i>fliC</i>-FL S28A strains at 13 h. The proteins were loaded from equal number of bacterial cells as shown by immunoblotting of RNA Pol α-subunit levels.