Small RNAs OmrA and OmrB promote class III flagellar gene expression by inhibiting the synthesis of anti-Sigma factor FlgM

Abstract

Bacteria can move by a variety of mechanisms, the best understood being flagella-mediated motility. Flagellar genes are organized in a three-tiered cascade allowing for temporally regulated expression that involves both transcriptional and post-transcriptional control. The class I operon encodes the master regulator FlhDC that drives class II gene transcription. Class II genes include fliA and flgM, which encode the Sigma factor σ²⁸, required for class III transcription, and the anti-Sigma factor FlgM, which inhibits σ²⁸ activity, respectively. The flhDC mRNA is regulated by several small regulatory RNAs (sRNAs). Two of these, the sequence-related OmrA and OmrB RNAs, inhibit FlhD synthesis. Here, we report on a second layer of sRNA-mediated control downstream of FhlDC in the flagella pathway. By mutational analysis, we confirm that a predicted interaction between the conserved 5' seed sequences of OmrA/B and the early coding sequence in flgM mRNA reduces FlgM expression. Regulation is dependent on the global RNA-binding protein Hfq. In vitro experiments support a canonical mechanism: binding of OmrA/B prevents ribosome loading and decreases FlgM protein synthesis. Simultaneous inhibition of both FlhD and FlgM synthesis by OmrA/B complicated an assessment of how regulation of FlgM alone impacts class III gene transcription. Using a combinatorial mutation strategy, we were able to uncouple these two targets and demonstrate that OmrA/B-dependent inhibition of FlgM synthesis liberates σ²⁸ to ultimately promote higher expression of the class III flagellin gene fliC.

Keywords: FlgM; Small RNAs; flagella; motility; post-transcriptional control; translational regulation.

Figure 1.

Effects of OmrA and OmrB in the regulatory networks for motile and sessile lifestyles in E. coli.

Figure 2.

OmrA and OmrB cause direct translational inhibition of flgM mRNA in vivo.

Figure 3.

Binding of OmrA and OmrB inhibit translation of flgM mRNA.

Figure 4.

In-frame deletion of flgM affects class III (fliC) but not class II (fliE) flagellar gene expression.

Figure 5.

Uncoupled regulation of flhD and flgM_M1 by mutant versions of OmrA.

Figure 6.

OmrA-mediated regulation of flgM promotes class III flagellar gene expression.