An ATP-dependent partner switch links flagellar C-ring assembly with gene expression

Abstract

Bacterial flagella differ in their number and spatial arrangement. In many species, the MinD-type ATPase FlhG (also YlxH/FleN) is central to the numerical control of bacterial flagella, and its deletion in polarly flagellated bacteria typically leads to hyperflagellation. The molecular mechanism underlying this numerical control, however, remains enigmatic. Using the model species Shewanella putrefaciens, we show that FlhG links assembly of the flagellar C ring with the action of the master transcriptional regulator FlrA (named FleQ in other species). While FlrA and the flagellar C-ring protein FliM have an overlapping binding site on FlhG, their binding depends on the ATP-dependent dimerization state of FlhG. FliM interacts with FlhG independent of nucleotide binding, while FlrA exclusively interacts with the ATP-dependent FlhG dimer and stimulates FlhG ATPase activity. Our in vivo analysis of FlhG partner switching between FliM and FlrA reveals its mechanism in the numerical restriction of flagella, in which the transcriptional activity of FlrA is down-regulated through a negative feedback loop. Our study demonstrates another level of regulatory complexity underlying the spationumerical regulation of flagellar biogenesis and implies that flagellar assembly transcriptionally regulates the production of more initial building blocks.

Keywords: ATPase; flagellum; nanomachine; regulation; spatiotemporal.

Fig. 1.

FliM and FlhG in the context of the C-ring assembly. (A) Cryo-ET images showing 100 × 100 nm slices through symmetrized subtomogram averages of the S. putrefaciens wt (Left) and ΔflhG motor (Right). (B) Fluorescence and DIC microscopy images depicting FliM-mCherry/ΔflhG/FlhG-GFP localization (Upper) and FliM-ΔN-mCherry/ΔflhG/FlhG-GFP (Lower). (Scale bar, 5 µm.)

Fig. 2.

Dissection of the FlhG/FlrA interaction. (A) Y2H experiments show that FlhG interacts with FliM, FlhF, and FlrA, but not with FliG and FliN. The growth of cells, coexpressing the FlhG bait protein and the indicated prey proteins, was assessed on -LT, -HLT (HIS3 reporter), and -ALT (ADE2 reporter) plates. (B) Domain architecture of FlrA (Upper) and structural details of the FlrA linker region and HTH domain (Lower); SpFlrA model based on 5m7n (NtrX from Brucella abortus), created with SWISS-MODEL. (C) Pulldown assay probing the interaction of FlrA truncations with FlhG in the presence of 1 mM ATP. (D) Pulldown assay probing the interaction of the FlrA-HTH domain and its preceding linker region with FlhG in the presence of 1 mM ATP. (E) Interaction of FlhG with the FlrA-HTH domain probed by MST in the presence of 0.25 mM ATP (Upper) and in its absence (Lower). Data represent mean ± SD of n = 3 technical replicates. (F) Velocity/substrate characteristic of FlhG ATPase activity in the absence (gray curve) or presence (black curve) of equimolar FlrA-HTH. Data represent mean ± SD of n = 3 technical replicates.

Fig. 3.

FliM and FlrA share a binding site at FlhG. (A) Peptides exhibiting reduced HDX in the FlhG_D58A/FlrA-HTH complex are mapped onto a structural model of FlhG (generated with SWISS-MODEL based on the structure of homodimeric FlhG from Geobacillus thermodenitrificans; PDB ID code 4RZ3, ref. 8). The different coloring of the peptides denotes the presumed reason for the observed differences in HDX based on their implication in establishing the homodimeric interface of FlhG (green), interface establishment and/or FlrA binding (cyan), or FlrA binding (blue). (B) Peptides exhibiting reduced (blue) or increased (red) HDX in the FlhG/FliN complex are mapped onto a structural model of FlhG (generated with SWISS-MODEL based on the structure of monomeric FlhG from Geobacillus thermodenitrificans; PDB ID code 4RZ2, ref. 8). (C) Key residues residing in helices α6 and α7 of FlhG involved in the interaction with FliM and FlrA-HTH. (D) GST pulldown with an immobilized FlrA-HTH domain against FlhG single mutants (in the presence of 1 mM ATP). Mutants prevent a binding interaction. (E) GST pulldown with an immobilized FliM-N against FlhG single mutants (absence of ATP). Mutants prevent a binding interaction. (F) Fluorescence microscopy (Alexa Fluor 488 maleimide staining) and DIC microscopy images of FlhG wt and mutants, showing the change in flagellation pattern and location. FlhG_D58A leads to loss of flagella in most cells. (Scale bar, 5 µm.)

Fig. 4.

FlrA–FlhG interaction affects transcriptional and spationumerical control of flagella. (A) Fluorescence microscopy images (Alexa Fluor 488 maleimide) with stained filaments and DIC images depicting FlrA mutants in the FlhG-binding interface with additional controls. (Scale bar, 5 µm.) (B) Quantification of the number of hooks per cell in Shewanella putrefaciens wt and hyperflagellation mutants (see A for corresponding fluorescence images). (C) qPCR data highlighting the close alignment of FlrA_L400E and flhG deletion phenotypes, in comparison with a FlhG overexpression control. (D) Quantification of FLAG-tagged FlrA, FliM, and FlhG by Western blot highlights the threefold excess of FlhG in S. putrefaciens wt. (Scale bar, 5 µm.) (E) Fluorescence microscopy images (Alexa Fluor 488 maleimide) with stained filaments and DIC images depicting overexpression of FlhG in FlrA mutants in the FlhG-binding interface with additional controls. (F) Western blots depicting the increase of FlhG and FlhF protein levels in FlrA_L400E and FlrA_Δ389–409 strains.

Fig. 5.

ATP-dependent partner switch links flagellar C-ring assembly with gene expression. The color code is given in the legend. Abbreviations are OM, outer membrane; PG, peptidoglycan; IM, inner membrane; and fT3SS, flagellar type III secretion system. Further explanations for the steps indicated by "A" to "I" are given in Discussion.