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.

Suggested EC number for PcyX: 1.3.7.6 DATABASES: The PcyX X-ray structure was deposited in the PDB with the accession code 5OWG.

We have studied the localization and dynamics of bacterial Ffh, part of the SRP complex, its receptor FtsY, and of ribosomes in the Gamma-proteobacterium Shewanella putrefaciens. Using structured illumination microscopy, we show that ribosomes show a pronounced accumulation at the cell poles, whereas SRP and FtsY are distributed at distinct sites along the cell membrane, but they are not accumulated at the poles. Single molecule dynamics can be explained by assuming that all three proteins/complexes move as three distinguishable mobility fractions: a low mobility/static fraction may be engaged in translation, medium-fast diffusing fractions may be transition states, and high mobility populations likely represent freely diffusing molecules/complexes. Diffusion constants suggest that SRP and FtsY move together with slow-mobile ribosomes. Inhibition of transcription leads to loss of static molecules and reduction of medium-mobile fractions, in favor of freely diffusing subunits, while inhibition of translation appears to stall the medium mobile fractions. Depletion of FtsY leads to aggregation of Ffh, but not to loss of the medium mobile fraction, indicating that Ffh/SRP can bind to ribosomes independently from FtsY. Heat maps visualizing the three distinct diffusive populations show that while static molecules are mostly clustered at the cell membrane, diffusive molecules are localized throughout the cytosol. The medium fast populations show an intermediate pattern of preferential localization, suggesting that SRP/FtsY/ribosome transition states may form within the cytosol to finally find a translocon.

A number of bacterial species control the function of the flagellar motor in response to the levels of the secondary messenger c-di-GMP, which is often mediated by c-di-GMP-binding proteins that act as molecular brakes or clutches to slow the motor rotation. The gammaproteobacterium Shewanella putrefaciens possesses two distinct flagellar systems, the primary single polar flagellum and a secondary system with one to five lateral flagellar filaments. Here, we identified a protein, MotL, which specifically regulates the activity of the lateral, but not the polar, flagellar motors in response to the c-di-GMP levels. MotL only consists of a single PilZ domain binding c-di-GMP, which is crucial for its function. Deletion and overproduction analyses revealed that MotL slows down the lateral flagella at elevated levels of c-di-GMP, and may speed up the lateral flagellar-mediated movement at low c-di-GMP concentrations. In vitro interaction studies hint at an interaction of MotL with the C-ring of the lateral flagellar motors. This study shows a differential c-di-GMP-dependent regulation of the two flagellar systems in a single species, and implicates that PilZ domain-only proteins can also act as molecular regulators to control the flagella-mediated motility in bacteria.

Many bacterial species are able to synthesize flagella, long proteinaceous helical fibres that extend from the cell's surface. Rotation of the flagellar helix, mediated by a membrane-embedded motor, enables efficient active movement of the cell through liquid environments (swimming) or across surfaces (swarming) (Kearns, 2010;Thormann et al., 2022;Wadhwa & Berg, 2022). In addition, the flagellum can serve as an adhesive or pathogenicity factor or as a sensor the bacteria use to determine environmental conditions such as viscosity or surface wetness (Chaban et al., 2015;Laventie & Jenal, 2020). The flagellum is an intricate nanomachine, which -in its general design -is well-conserved between different bacterial species. It consists of the helical filament, the cell envelope-embedded basal body housing the rotary motor and a type III export apparatus, and a universal joint structure, the hook, which connects the motor and filament (Figure 1a) (Johnson et al., 2021;Tan et al., 2021). The whole structure is formed from about 20 different protein building blocks with different stoichiometries ranging from one to nine copies, e.g. for parts of the flagellar type III export system (fT3SS), to many thousands of the main filament building block, the flagellin