Gac-mediated downregulation of <i>fleQ</i> expression is independent of Vfr but dependent on AmrZ and AlgU.

<p>(<b>A</b>) Analysis of the swimming motility of F113 wild-type, mutants in the Gac-AlgU cascade, and complemented <i>amrZ</i> (p<i>amrZ</i>). Different letters indicate significant statistical difference (p<0.05). At 18 h, F113 wild-type strain swimming halo diameter is 11±0.55 (mm). (<b>B</b>) Western blot analysis of extracellular proteins from F113 wild-type strain and its isogenic mutants <i>amrZ</i><b><i>⁻</i></b> and <i>algU</i><b><i>⁻</i></b>, reacted with an anti-flagellin antiserum. Loading control corresponds to a Coomassie-stained gel portion. (<b>C</b>) qRT-PCR expression analysis of the <i>fleQ</i> gene (primers qfleQF-R) in F113 wild-type, <i>amrZ</i><b><i>⁻</i></b> and <i>algU</i><b><i>⁻</i></b>. <i>16S</i> gene expression (primers 16SF-R) was used for normalization. To control for DNA contamination, PCR of RNA was performed using the same primer pairs. Different letters indicate significant statistical differences (p<0.05).