σ28-dependent sRNAs are primarily expressed in log phase.

A Overview of the flagellar regulon. The early genes initiate the transcription of the middle genes, including fliA which encodes σ28. In turn, σ28 initiates the transcription of the late genes and enhances the transcription of some of the middle genes. For the middle and late genes, only selected operons are shown. The sRNAs analyzed in this study are colored in blue. This model was inspired by (Kalir et al., 2005).

B Browser images showing levels of UhpU, MotR, FliX and FlgO sRNAs in total RNA (black) and Hfq co-immunoprecipitation (gray) libraries. Normalized read count ranges are shown in the upper right of each frame. Data analyzed is from (RIL-seq experiment 1, (Melamed et al., 2020).

C Northern blot analysis of total RNA from WT (GSO983) or ΔfliA cells grown to the indicated time points. The same membrane was probed for all four σ28-dependent sRNAs. A full-length transcript (∼260 nt) and several processed transcripts, of which one is predominant (UhpU-S, ∼60 nt), are detected for UhpU, one prominent band (∼95 nt) is detected for MotR, one prominent band (∼200 nt) is detected for FliX, and two close bands close in size (∼75 nt) are detected for FlgO.

D WT (GSO983) cells were grown to OD600 ∼0.6 and ∼1.0. RNA was extracted from total lysates as well as samples from co-immunoprecipitation with Hfq, separated on an acrylamide gel, transferred to a membrane, and probed for σ28-dependent sRNAs. A ∼100 nt FliX band (FliX-S) was revealed immunoprecipitating with Hfq.

In (C) and (D), RNAs were probed sequentially on the same membrane and 5S RNA served as a loading control.

Overexpression of the σ28-dependent sRNAs leads to differences in flagella number and motility.

A Moderate increase in flagella number with UhpU overexpression based on electron microscopy analysis for WT cells carrying an empty vector or overexpressing UhpU.

B Increased motility with UhpU overexpression based on motility in 0.3% agar for WT (crl+) cells carrying an empty vector or overexpressing UhpU.

C Increase in flagella number with MotR overexpression based on electron microscopy analysis for WT cells carrying an empty vector or overexpressing MotR.

D Slight increase in motility with MotR overexpression based on motility in 0.3% agar for WT (crl+) cells carrying an empty vector or overexpressing MotR.

E Reduction in flagella number with FliX overexpression based on electron microscopy analysis for WT cells carrying an empty vector or overexpressing FliX.

F Reduced motility with FliX overexpression based on motility in 0.3% agar for WT (crl-) cells carrying an empty vector or overexpressing FliX.

G No change in flagella number with FlgO overexpression based on electron microscopy analysis for WT cells carrying an empty vector or overexpressing FlgO.

H No change in motility with FlgO overexpression based on motility in 0.3% agar for WT (crl-) cells carrying an empty vector or overexpressing FlgO.

Cells in (A) and (B) were induced with 1 mM IPTG. Quantification for all the assays is shown on the right. For (A), (C), (E) and (G) quantification of the number of flagella per cell was done by counting the flagella for 20 cells (black dots), and a one-way ANOVA comparison was performed to calculate the significance of the change in flagella number (ns = not significant, ** = P<0.01, **** = P<0.0001). Each experiment was repeated three times and one representative experiment is shown. The bottom and top of the box are the 25th and 75th percentiles, the line inside the box is the median, the lower and the upper whiskers represent the minimum and the maximum values of the dataset, respectively. The experiments presented in (C) and (E) were carried out on same day, and the same pZE sample is shown. Graphs for (B), (D), (F) and (H) show the average of nine biological repeats. Error bars represent one SD and a one-way ANOVA comparison was performed to calculate the significance of the change in motility (ns = not significant, * = P<0.05, **** = P<0.0001).

Multiple sRNAs repress LrhA synthesis.

A Browser image showing chimeras (in red) for UhpU, ArcZ, RprA and McaS, at the 5’ UTR region of lrhA. Blue highlighting indicates position of sRNA-lrhA base pairing. Data analyzed is from (Melamed et al., 2020).

B Base-pairing between lrhA and UhpU with sequences of mutants assayed. Seed sequence predicted by (Melamed et al., 2016) is underlined. Numbering is from AUG of lrhA mRNA and +1 of UhpU sRNA.

C UhpU represses lrhA-lacZ fusion. β-galactosidase assay detecting the levels of lrhA-lacZ and lrhA-M1-lacZ translational fusions in response to UhpU and UhpU-M1 overexpression.

D UhpU does not affect motility when LrhA is absent, based on motility in 0.3% agar for WT (crl+) cells or ΔlrhA cells carrying an empty vector or overexpressing UhpU. Graph shows the average of three biological repeats, and error bars represent one SD. One-way ANOVA comparison was performed to calculate the significance of the change in motility (ns = not significant, **** = P<0.0001).

E Predicted base-pairing between lrhA and ArcZ, RprA or McaS. Numbering is from AUG of lrhA mRNA and +1 of indicated sRNAs.

F Down regulation of lrhA by ArcZ and RprA but not McaS. β-galactosidase assay detecting the levels of lrhA-lacZ translational fusions in response to ArcZ, RprA and McaS overexpression.

For (C) and (F), graphs show the average of three biological repeats, and error bars represent one SD. One-way ANOVA comparison was performed to calculate the significance of the change in β-galactosidase activity (ns = not significant, **** = P<0.0001).

Multiple sRNAs regulate flagellin synthesis.

A Browser image showing chimeras (red and blue) for UhpU, MotR, and FliX at the fliCX region. Data analyzed is from (RIL-seq experiment 1, (Melamed et al., 2020). Red and blue lines indicate the RNA in the region is first or second RNA in the chimera, respectively. Blue highlighting indicates position of sRNA-fliC base pairing

B Immunoblot analysis showing UhpU and MotR overexpression leads to increased flagellin levels and FliX overexpression leads to reduced flagellin levels. Flagellin levels were determined by immunoblot analysis using α-FliC antibody. A sample from a ΔfliC strain was included as a control given the detection of a cross-reacting band slightly larger than flagellin. The Ponceau S-stained membrane serves as a loading control.

C Northern blot analysis showing UhpU and MotR overexpression increases fliC mRNA levels and FliX overexpression reduces fliC levels. 5S RNA served as a loading control.

D Predicted base-pairing between fliC and UhpU, MotR or FliX. Seed sequences predicted by (Melamed et al., 2016) or by this study are underlined. Numbering is from AUG of fliC mRNA and +1 of indicated sRNAs.

MotR and FliX base pair with the S10 mRNA leading to upregulation and downregulation, respectively.

A Browser image showing MotR chimeras (in red) in S10 leader and rpsJ region. Data analyzed is from (RIL-seq experiment 1, (Melamed et al., 2020)). Coverage of the region in total RNA-seq libraries is shown for empty vector (pZE) and for pZE-MotR* overexpression (Table S2). The Hfq and MotR binding sites as detected in Fig S8A and S8B are highlighted in light blue.

B Base-pairing between rpsJ and MotR with sequences of mutants assayed. Predicted MotR seed sequence is underlined. Numbering is from +1 of rpsJ mRNA and MotR sRNA.

C MotR induces rpsJ-gfp reporter fusion. Test of rpsJ-MotR interaction with reporter assays of rpsJ-gfp expressed from pXG10-SF with MotR or MotR* expressed from pZE.

D MotR increases FLAG tagged S10 levels. 3xFLAG-S10 was expressed from pBAD33 and MotR or MotR* was expressed from pZE. A mutation in MotR eliminates this regulation. 3xFLAG-S10 levels were determined by immunoblot analysis using α-FLAG antibody. The Ponceau S-stained membrane serves as a loading control.

E RNase III-mediated cleavage of rpsJ directed by MotR in region of base pairing. 32P-labeled rpsJ and rpsJ-M1 were treated with lead for 10 min with or without MotR and MotR -M1 and separated on a sequencing gel. Numbering is from +1 of rpsJ mRNA.

F Browser image showing FliX chimeras (in red) in the S10 operon. Highlighted in light blue are the base pairing regions between FliX and the S10 operon mRNA. Data analyzed is from (RIL-seq experiment 1, (Melamed et al., 2020)).

G Base pairing between rplC, rpsS, rpsQ and FliX with sequences of mutants assayed. FliX seed sequence predicted by (Melamed et al., 2016) is underlined. Numbering is from AUG of indicated CDS and +1 of FliX sRNA.

H Test of FliX interactions with reporter assays of rplC-gfp, rpsS-rplV-gfp and rpsQ-gfp expressed from pXG10-SF or pXG30-SF with FliX or FliX-S expressed from pZE. I RNase III-mediated cleavage of rpsS directed by FliX-S in region of base pairing. 32P-labeled rpsS and rpsS-M1 were treated with RNase III for 1.5 min with or without FliX-S and FliX-S -M1 and separated on a sequencing gel. Numbering is from AUG of rpsS CDS.

For (C) and (H), the average of three independent measurements is shown. Error bars represent one SD. One-way ANOVA comparison was performed to calculate the significance of the change in GFP signal (ns = not significant, * = P<0.05, ** = P<0.01, **** = P<0.0001).

MotR overexpression leads to a nusB-dependent increase in expression from flagellar operons.

A MotR* and S10Δloop overexpression increase the number of flagella. The number of flagella per cell detected by electron microscopy were counted for WT cells harboring the indicated plasmids.

B MotR effect is eliminated in ΔnusB background. The number of flagella per cell detected by electron microscopy were counted for WT or ΔnusB cells harboring the indicated plasmids.

C MotR induces mRNA levels throughout the flagellar operons in WT background but not in ΔnusB background. MotR was expressed from pZE plasmid and the levels of motB, cheW, tar, cheZ, ssrA and cadB were monitored in comparison to their levels in the pZE control vector by RT-qPCR. cadB served as a non-flagellar gene control and ssrA served as a reference gene; the same cadB data is shown in both plots. Experiments were done in 4 biological replicates and one-way ANOVA comparison was performed to calculate the significance of the change in mRNA levels (ns = not significant, **** = P<0.0001).

For (A) and (B), flagella were counted for 20 cells (black dots), and a one-way ANOVA comparison was performed to calculate the significance of the change in flagella number (ns = not significant, **** = P<0.0001). Box plot and error bars descriptions as in Fig 2. Each experiment was repeated three times and one representative experiment is shown.

MotR and FliX overproduction leads to increased and decreased expression of flagellar genes, respectively.

A MotR* induces flagellar genes. Green symbols represent flagellar regulon genes as indicated on the graph.

B FliX reduces flagellar genes. Red symbols represent flagellar regulon genes as indicated on the graph. In (A) and (B) Differential expression analysis was conducted with DESeq2 and threshold for differentially expressed transcripts was set to adjusted value of p < 0.05.

C MotR overexpression increases the activity of GFP fusions to PflgB and PfliL. The activity of the promoters was monitored for 330 min by measuring the GFP signal and dividing it with the culture OD600nm in the presence of MotR expressed from pZE plasmid.

D FliX overexpression decreases the activity of GFP fusions to PflgB and PfliL. The activity of the promoters was monitored for 330 min by measuring the GFP signal and dividing it with the culture OD600nm in the presence of FliX expressed from pZE plasmid. For (A) and (B), WT harboring the control vector pZE or the MotR* or the FliX expression plasmid were grown to OD600 ∼ 0.2; total RNA was extracted and used for the construction of cDNA libraries, which were analyzed as described in Materials and Methods.

For (C) and (D), three biological repeats are shown in the graph. One-way ANOVA comparison was performed to calculate the significance of the change in GFP signal (ns = not significant, * = P<0.05, ** = P<0.01, *** = P<0.001, **** = P<0.0001). The experiments presented in 7C and S13B, and in 7D and S13A, were carried out on same day, respectively, and the same pZE samples are shown.

Complex regulatory network of sRNAs controlling flagella synthesis.

A Reduction in flagella number in motR-M1 mutant.

B Increase in flagella number in fliX-M1 mutant.

C Reduced motility in motR-M1 mutant (GSO1087) based on a competition assay with its corresponding WT (GSO1088).

D No difference in motility in fliX-M1 mutant (GSO1076) based on a competition assay with its corresponding WT (GSO1088).

E Reduction in PflgB-gfp and PfliL-gfp expression in motR-M1 mutant (GSO1087) background compared to WT background.

F No difference in PflgB-gfp and PfliL-gfp expression in fliX-M1 mutant (GSO1076) background compared to WT background.

G σ28-dependent sRNAs control flagella synthesis at different levels. UhpU activates the flagellar regulon by repressing a regulator of flhDC. MotR and FliX, respectively, activate and repress middle and the late gene expression (dotted line indicates exact mechanism is not known, though we document base pairing with the fliC mRNA). MotR and FliX also connect ribosome and flagella synthesis by regulating genes in the S10 operon (solid line indicates documented base pairing with this mRNA).

In (A) and (B), the number of flagella per cell detected by electron microscopy were counted for for 40 cells (black dots) for the WT, motR-M1 and fliX-M1 strains at three points in growth (OD600 ∼ 0.2, OD600 ∼ 0.6, and OD600 ∼ 2.0). A one-way ANOVA comparison was performed to calculate the significance of the change in flagella number (ns = not significant, ** = P<0.01, *** = P<0.001, **** = P<0.0001). Each experiment was repeated three times and one representative experiment is shown. Box plot and error bars descriptions as in Fig 2. For (C) and (D), WT or the corresponding mutant, expressed either green fluorescent signal or red fluorescent signal by carrying pCON1.proC-GFP or pCON1.proC-mCherry plasmid, respectively. In the left images, WT cells expressing GFP were mixed with mutant cells expressing mCherry; in the middle images, WT cells expressing mCherry were mixed with mutant cells expressing GFP; in the right images, WT cells expressing GFP were mixed with WT cells expressing mCherry. The indicated mixed cultures were spotted on a soft agar (0.3%) plate, incubated at 30°C, and imaged after 18 h. For (E) and (F), three biological repeats are shown in the graph (except for PfliL-gfp in fliX-M1, for which two repeats are shown). One-way ANOVA comparison was performed to calculate the significance of the change in GFP signal (ns = not significant, ** = P<0.01, *** = P<0.001, **** = P<0.0001).