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, peritrichousflagellated 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.flagellum | FlhG | C-ring | Bacillus | Shewanella

SummarySpatiotemporal 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 pilimediated 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.

As numerous bacterial species, Shewanella putrefaciens CN-32 possesses a complete secondary flagellar system. A significant subpopulation of CN-32 cells induces expression of the secondary system under planktonic conditions, resulting in formation of one, sometimes two, filaments at lateral positions in addition to the primary polar flagellum. Mutant analysis revealed that the single chemotaxis system primarily or even exclusively addresses the main polar flagellar system. Cells with secondary filaments outperformed their monopolarly flagellated counterparts in spreading on soft-agar plates and through medium-filled channels despite having lower swimming speed. While mutant cells with only polar flagella navigate by a "run-reverse-flick" mechanism resulting in effective cell realignments of about 90°, wild-type cells with secondary filaments exhibited a range of realignment angles with an average value of smaller than 90°. Mathematical modeling and computer simulations demonstrated that the smaller realignment angle of wild-type cells results in the higher directional persistence, increasing spreading efficiency both with and without a chemical gradient. Taken together, we propose that in S. putrefaciens CN-32, cell propulsion and directional switches are mainly mediated by the polar flagellar system, while the secondary filament increases the directional persistence of swimming and thus of spreading in the environment.bacterial motility | cell reorientation | CheY | lateral flagella T he ability to actively explore and exploit the environment provides a major advantage for all kinds of organisms, including bacteria (1, 2). Among bacteria, flagella are common and efficient organelles of locomotion that consist of long, helical, proteinaceous filaments extending from the cell's surface and are rotated by a membrane-embedded motor to which they are attached by the flexible hook structure. The majority of flagellar motors function in a bidirectional fashion and can rotate either counterclockwise (CCW) or clockwise (CW) (3, 4). Most bacterial species navigate using a random walk that originates from an alternation of straight runs and cell reorientations. In the absence of gradients, such random walk results in a uniform spreading in the environment. In gradients of environmental stimuli, bacterial random walk becomes biased, whereby cells use temporal comparisons of the stimulus strength to suppress reorientations while swimming in a favorable direction. This behavior is controlled by one or more chemotaxis systems, which transduce environmental stimuli to control flagellar motors (5). Signals perceived by an array of sensor proteins are converted into the phosphorylation state of a soluble signal-transmitting protein, CheY. Phosphorylated CheY can directly interact with the flagellar motor and induce a switch in rotation or a motor break. In peritrichously flagellated bacteria with several filaments, such as the paradigm system of Escherichia coli, CCW rotation leads to formation of a flagellar bundle that drives ...