Previous work indicated that hha ydgT mutants failed to swim on motility plates but the contribution of the individual genes to this phenotype was not known and the ability of these strains to make surface flagella was not tested [16]. To test the contribution of individual genes to this non-motile phenotype, we used a standard soft agar motility assay and confirmed that hha ydgT mutants were non-motile in accordance with previous data (Figure 2A). This phenotype required deletion of both

hha and ydgT as single Δhha or ΔydgT mutants remained motile (Figure 2B). To determine if the motility defect observed in Δhha ΔydgT was due to a defect in flagellar rotation or a lack of flagellar production we stained IWR-1 in vivo bacteria and examined them using transmission Screening Library ic50 electron microscopy to visualize surface flagella. We found that while wild type bacteria were highly flagellated, Δhha ΔydgT bacteria did not assemble flagella on their surface (Figure 2C). Figure 2 Repression of flagellar biosynthesis and motility is dependent on the loss of Hha and YdgT. (A) Wild type, Δhha, ΔydgT and Δhha ΔydgT were assessed for flagellar-based motility using a 0.25% soft agar motility assay BGB324 chemical structure in which

2 μL of overnight culture was inoculated into semi-solid agar and incubated at 37°C for 6 h. (B) The radius of the motility halo region was quantified after 6 h and is shown as means with standard errors. (C) Bacteria and surface flagella were negatively stained using a 0.1% uranyl acetate solution and visualized using scanning transmission electron microscopy. Data represents three independent experiments. Transcriptional activity of class

II/III and III promoters is decreased in a hha ydgT mutant Flagellar biosynthesis is organized into a transcriptional hierarchy of three distinct classes. To understand the non-flagellated phenotype in greater detail, we measured the activity of transcriptional reporters corresponding to each of the three promoter classes driving the expression of green fluorescent protein (GFP). While the transcriptional activity in single hha or ydgT mutants was not Rho significantly different when compared to wild type, transcriptional reporters for the hybrid class II/III promoter (fliA) [23, 24] and class III promoter (fliC) were significantly reduced in the hha ydgT double mutant compared to wild type cells (Figure 3A). Since flhDC promoter activity did not differ between wild type and the hha ydgT mutant, we tested whether the inhibition of class II/III and class III gene expression in Δhha ΔydgT involved an effect downstream of FlhD-FlhC protein production, since the FlhD4C2 complex is known to activate class II transcription. Using Western blot analysis with FlhC and FlhD-specific antisera, we observed a decrease in the levels of FlhC and FlhD in hha ydgT mutants compared to wild type (Figure 3B), which was consistent with the observed decrease in activity for FlhD4C2 target promoters.