MinD-like ATPase FlhG effects location and number of bacterial flagella during C-ring assembly.

The number and location of flagella, bacterial organelles of locomotion, are species specific and appear in regular patterns that represent one of the earliest taxonomic criteria in microbiology. However, the mechanisms that reproducibly establish these patterns during each round of cell division are poorly understood. FlhG (previously YlxH) is a major determinant for a variety of flagellation patterns. Here, we show that FlhG is a structural homolog of the ATPase MinD, which serves in cell-division site determination. Like MinD, FlhG forms homodimers that are dependent on ATP and lipids. It interacts with a complex of the flagellar C-ring proteins FliM and FliY (also FliN) in the Gram-positive, peritrichous-flagellated Bacillus subtilis and the Gram-negative, polar-flagellated Shewanella putrefaciens. FlhG interacts with FliM/FliY in a nucleotide-independent manner and activates FliM/FliY to assemble with the C-ring protein FliG in vitro. FlhG-driven assembly of the FliM/FliY/FliG complex is strongly enhanced by ATP and lipids. The protein shows a highly dynamic subcellular distribution between cytoplasm and flagellar basal bodies, suggesting that FlhG effects flagellar location and number during assembly of the C-ring. We describe the molecular evolution of a MinD-like ATPase into a flagellation pattern effector and suggest that the underappreciated structural diversity of the C-ring proteins might contribute to the formation of different flagellation patterns.

FlhG employs diverse intrinsic domains and influences FlhF GTPase activity to numerically regulate polar flagellar biogenesis in Campylobacter jejuni.

Flagellation in polar flagellates is one of the rare biosynthetic processes known to be numerically regulated in bacteria. Polar flagellates must spatially and numerically regulate flagellar biogenesis to create flagellation patterns for each species that are ideal for motility. FlhG ATPases numerically regulate polar flagellar biogenesis, yet FlhG orthologs are diverse in motif composition. We discovered that Campylobacter jejuni FlhG is at the center of a multipartite mechanism that likely influences a flagellar biosynthetic step to control flagellar number for amphitrichous flagellation, rather than suppressing activators of flagellar gene transcription as in Vibrio and Pseudomonas species. Unlike other FlhG orthologs, the FlhG ATPase domain was not required to regulate flagellar number in C. jejuni. Instead, two regions of C. jejuni FlhG that are absent or significantly altered in FlhG orthologs are involved in numerical regulation of flagellar biogenesis. Additionally, we found that C. jejuni FlhG influences FlhF GTPase activity, which may mechanistically contribute to flagellar number regulation. Our work suggests that FlhG ATPases divergently evolved in each polarly flagellated species to employ different intrinsic domains and extrinsic effectors to ultimately mediate a common output - precise numerical control of polar flagellar biogenesis required to create species-specific flagellation patterns optimal for motility.

The role of FlhF and HubP as polar landmark proteins in Shewanella putrefaciens†CN-32.

Spatiotemporal regulation of cell polarity plays a role in many fundamental processes in bacteria and often relies on 'landmark' proteins which recruit the corresponding clients to their designated position. Here, we explored the localization of two multi-protein complexes, the polar flagellar motor and the chemotaxis array, in Shewanella putrefaciens CN-32. We demonstrate that polar positioning of the flagellar system, but not of the chemotaxis system, depends on the GTPase FlhF. In contrast, the chemotaxis array is recruited by a transmembrane protein which we identified as the functional ortholog of Vibrio cholerae HubP. Mediated by its periplasmic N-terminal LysM domain, SpHubP exhibits an FlhF-independent localization pattern during cell cycle similar to its Vibrio counterpart and also has a role in proper chromosome segregation. In addition, while not affecting flagellar positioning, SpHubP is crucial for normal flagellar function and is involved in type IV pili-mediated twitching motility. We hypothesize that a group of HubP/FimV homologs, characterized by a rather conserved N-terminal periplasmic section required for polar targeting and a highly variable acidic cytoplasmic part, primarily mediating recruitment of client proteins, serves as polar markers in various bacterial species with respect to different cellular functions.