PMC

Panels A-B) Quantitative Western blot analysis of FliY and FliG. Error bars indicate the standard deviation of 3 biological replicates and the raw values can be found in . The following strains were used to generate these panels: WT (DK1042), efp (DK2050), efp fliY^soe (DK5518), and efp nusG^soe (DK5955). Panel C) β-galactosidase activity reported in Miller Units (MU) of a translational fusion of lacZ to fliY. Error bars indicate the standard deviation of 3 biological replicates and the raw values can be found in . The following strains were used to generate this panel: WT fliY-lacZ (DK5185), efp fliY-lacZ (DK5186), WT fliY^soe-lacZ (DK5168), and efp fliY^soe-lacZ (DK5169).

Fold change in expression detected by RNA sequencing of genes in the σ^D regulon relative to wild type. The levels of expression in wild type are indicated by a dashed line. Error bars represent the standard deviation of 3 biological replicates. The following strains were used to generate this figure: WT (DK1042), efp (DK2050), efp fliY^soe (DK5518), and efp nusG^soe (DK5955).

Panel A) Schematic depicting the genetic hierarchy of B. subtilis flagellar biosynthesis. Open arrows represent genes and the position of fliY is indicated in red. Bent arrows indicate promoters. Closed arrows indicate activation and T bars indicate inhibition. Panel B) Schematic depicting the putative structure of the B. subtilis flagellum. The predicted locations of relevant flagellar components are labeled. PG indicates peptidoglycan and PM indicates plasma membrane. Panel C) Sequence logo of 282 FliY homologs. The accession numbers that were included can be found in .

Panels A, C, E, G) Average ribosome profiling pause scores of each codon within the FliY open reading frame. The position of the SPP motif is indicated by a red asterisk on the X-axis. Panels B, D, F, H) Average pause scores for FliY codons 145–175. The box indicates the location of the SPP motif. Error bars indicate the standard deviation of 3 biological replicates. The following strains were used to generate this figure: WT (DK1042), efp (DK2050), efp fliY^soe (DK5518), and efp nusG^soe (DK5955).

β-galactosidase activity reported in Miller Units (MU) of a transcriptional fusion of lacZ to the σ^D-dependent flagellin promoter P_hag. The levels of expression in WT and the efp mutant are indicated by dashed lines for comparison. Data represent the average of three biological replicates, error bars indicate the standard deviation, and the raw values can be found in . The following strains were used to generate this panel: WT (DK5457), efp (DK5458), swrA (DK7032), efp swrA (DK7033), flgM (DK7074), efp flgM (DK7078), swrA flgM (DK7076), and efp swrA flgM (DK7077), efp fliY^soe (DK7049), and efp nusG^soe (DK7050).

Panels A-B) Weighted sequence logos of amino acid sequences in which the P-site codon had a pause score of 10 or greater in the ribosome profiling datasets from WT (DK1042) or efp (DK2050). Panel C) Average pause scores of tripeptides centered around the P-site (EPA, grey circles) or E site (-2EP, black circles) of the ribosome in WT (DK1042) and efp mutant (DK2050). In both cases, only the pause score for the P-site codon was used to determine the average. The dashed line indicates the distribution expected for equal scores in both strains. The 10 tripeptides with the highest pause score in the efp mutant are listed and the average pause scores for all 8,000 tripeptides at both positions can be found in .

Panel A) Top views of centrally inoculated swarm plates incubated overnight at 37°C and imaged against a black background. Zones of colonization appear light grey. The following strains were used to generate the panel: WT (DK1042), efp (DK2050), efp fliY^soe (DK5518), efp nusG^soe (DK5955), and efp nusG^soe fliY^soe (DK6640). Panels B-H) Quantitative swarm expansion assays in which mid-log phase cultures were concentrated and used to inoculate swarm agar plates. Swarm expansion was monitored along the same axis every 30 min for 6.5 hrs. Each data point represents the average of three replicates and the raw values can be found in . The following strains were used as the inoculum: B) WT (DK1042) and efp (DK2050). C) efp P_IPTG-yeeI^T19K (DK2779) and efp P_IPTG-yeeI (DK2777). D) efp yacO rae1 (DK5415), efp yacO (DK5413), and efp rae1 (DK5414). E) efp ydiF (DK4093). F) efp rpsC^R142H (DK6656), efp rpsJ^M88R (DK6657), and efp rpsC^H99L (DK6656). G) efp fliY^soe (DK5518) and fliY (DK1481). H) efp nusG^soe fliY^soe (DK6640), efp nusG^soe (DK5955), and efp P_IPTG-nusG (DK5412).

Panel A) Fluorescence micrographs of the flagellar filament (Hag^T209C), hook (FlgE^T123C), or basal body (FliM-GFP) in the indicated genetic background. Membranes were stained with FM4-64 and false colored red. Filaments and hooks were stained with Alexa Fluor 488 C5 maleimide and false colored green. FliM-GFP was false colored green. Scale bar corresponds to 5 μm. The following strains were used to generate this panel: WT hag^T209C (DS8521), efp hag^T209C (DK1053), swrA hag^T209C (DS8600), fliY hag^T209C (DK2155), WT flgE^T123C (DS7673), efp flgE^T123C (DK1054), swrA flgE^T123C (DK480), fliY flgE^T123C (DK1563), WT fliM-GFP (DS1919), efp fliM-GFP (DK1055), swrA fliM-GFP (DS9515), and fliY fliM-GFP (DS5628). Panels B-C) 3-D SIM microscopy and Imaris software were used to quantify the number of basal body puncta (panel B) or hook puncta (panel C) per cell relative to cell length on 30 individual cells per strain. The averages and standard deviations for each strain are provided in the panel legends and the raw values can be found in . The following strains were used to generate panel B: WT (DS8521), efp (DK1053), and swrA (DS8600). The following strains were used to generate panel C: WT (DS7673), efp (DK1054), and swrA (DK480).