Monitoring Bacterial Flagellar Secretion Dynamics

The luciferase assay was performed as described before [18 (link)]. The plasmid pRG19 (motA::luxCDABE) was used for monitoring the switch from early substrate secretion to late substrate secretion. The class 3 gene motA is expressed only upon HBB completion. Synchronization of flagellar gene expression was achieved by placing the AnTc inducible tetA promotor upstream of the flhDC gene [44 (link)]. Cells were grown in white clear-bottom 96-well plates (Greiner) and production of light and the OD₆₀₀ were measured over time using a Varioskan Flash multiplate reader (Thermo Fisher). After blank correction, relative light units (RLU) were calculated as:
$RLU=light\bullet \left(\frac{O{D}_{595}t=0}{O{D}_{595}t=n}\right).$