Two antagonistic response regulators control Pseudomonas aeruginosa polarization during mechanotaxis.

The opportunistic pathogen Pseudomonas aeruginosa adapts to solid surfaces to enhance virulence and infect its host. Type IV pili (T4P), long and thin filaments that power surface-specific twitching motility, allow single cells to sense surfaces and control their direction of movement. T4P distribution is polarized to the sensing pole by the chemotaxis-like Chp system via a local positive feedback loop. However, how the initial spatially resolved mechanical signal is translated into T4P polarity is incompletely understood. Here, we demonstrate that the two Chp response regulators PilG and PilH enable dynamic cell polarization by antagonistically regulating T4P extension. By precisely quantifying the localization of fluorescent protein fusions, we show that phosphorylation of PilG by the histidine kinase ChpA controls PilG polarization. Although PilH is not strictly required for twitching reversals, it becomes activated upon phosphorylation and breaks the local positive feedback mechanism established by PilG, allowing forward-twitching cells to reverse. Chp thus uses a main output response regulator, PilG, to resolve mechanical signals in space and employs a second regulator, PilH, to break and respond when the signal changes. By identifying the molecular functions of two response regulators that dynamically control cell polarization, our work provides a rationale for the diversity of architectures often found in non-canonical chemotaxis systems.

Wrapped Up: The Motility of Polarly Flagellated Bacteria.

A huge number of bacterial species are motile by flagella, which allow them to actively move toward favorable environments and away from hazardous areas and to conquer new habitats. The general perception of flagellum-mediated movement and chemotaxis is dominated by the Escherichia coli paradigm, with its peritrichous flagellation and its famous run-and-tumble navigation pattern, which has shaped the view on how bacteria swim and navigate in chemical gradients. However, a significant amount-more likely the majority-of bacterial species exhibit a (bi)polar flagellar localization pattern instead of lateral flagella. Accordingly, these species have evolved very different mechanisms for navigation and chemotaxis. Here, we review the earlier and recent findings on the various modes of motility mediated by polar flagella.

Mechanotaxis directs Pseudomonas aeruginosa twitching motility.

The opportunistic pathogen Pseudomonas aeruginosa explores surfaces using twitching motility powered by retractile extracellular filaments called type IV pili (T4P). Single cells twitch by sequential T4P extension, attachment, and retraction. How single cells coordinate T4P to efficiently navigate surfaces remains unclear. We demonstrate that P. aeruginosa actively directs twitching in the direction of mechanical input from T4P in a process called mechanotaxis. The Chp chemotaxis-like system controls the balance of forward and reverse twitching migration of single cells in response to the mechanical signal. Collisions between twitching cells stimulate reversals, but Chp mutants either always or never reverse. As a result, while wild-type cells colonize surfaces uniformly, collision-blind Chp mutants jam, demonstrating a function for mechanosensing in regulating group behavior. On surfaces, Chp senses T4P attachment at one pole, thereby sensing a spatially resolved signal. As a result, the Chp response regulators PilG and PilH control the polarization of the extension motor PilB. PilG stimulates polarization favoring forward migration, while PilH inhibits polarization, inducing reversal. Subcellular segregation of PilG and PilH efficiently orchestrates their antagonistic functions, ultimately enabling rapid reversals upon perturbations. The distinct localization of response regulators establishes a signaling landscape known as local excitation-global inhibition in higher-order organisms, identifying a conserved strategy to transduce spatially resolved signals.

Polar flagellar wrapping and lateral flagella jointly contribute to Shewanella putrefaciens environmental spreading.

Flagella enable bacteria to actively spread within the environment. A number of species possess two separate flagellar systems, where in most cases a primary polar flagellar system is supported by distinct secondary lateral flagella under appropriate conditions. Using functional fluorescence tagging on one of these species, Shewanella putrefaciens, as a model system, we explored how two different flagellar systems can exhibit efficient joint function. The S. putrefaciens secondary flagellar filaments are composed as a mixture of two highly homologous non-glycosylated flagellins, FlaA2 and FlaB2 . Both are solely sufficient to form a functional filament, however, full spreading motility through soft agar requires both flagellins. During swimming, lateral flagella emerge from the cell surface at angles between 30° and 50°, and only filaments located close to the cell pole may form a bundle. Upon a directional shift from forward to backward swimming initiated by the main polar flagellum, the secondary filaments flip over and thus support propulsion into either direction. Lateral flagella do not inhibit the wrapping of the polar flagellum around the cell body at high load. Accordingly, screw thread-like motility mediated by the primary flagellum and activity of lateral flagella cumulatively supports spreading through constricted environments such as polysaccharide matrices.

Roadmap on emerging concepts in the physical biology of bacterial biofilms: from surface sensing to community formation.

Bacterial biofilms are communities of bacteria that exist as aggregates that can adhere to surfaces or be free-standing. This complex, social mode of cellular organization is fundamental to the physiology of microbes and often exhibits surprising behavior. Bacterial biofilms are more than the sum of their parts: single-cell behavior has a complex relation to collective community behavior, in a manner perhaps cognate to the complex relation between atomic physics and condensed matter physics. Biofilm microbiology is a relatively young field by biology standards, but it has already attracted intense attention from physicists. Sometimes, this attention takes the form of seeing biofilms as inspiration for new physics. In this roadmap, we highlight the work of those who have taken the opposite strategy: we highlight the work of physicists and physical scientists who use physics to engage fundamental concepts in bacterial biofilm microbiology, including adhesion, sensing, motility, signaling, memory, energy flow, community formation and cooperativity. These contributions are juxtaposed with microbiologists who have made recent important discoveries on bacterial biofilms using state-of-the-art physical methods. The contributions to this roadmap exemplify how well physics and biology can be combined to achieve a new synthesis, rather than just a division of labor.

Bacteria exploit a polymorphic instability of the flagellar filament to escape from traps.

Many bacterial species swim by rotating single polar helical flagella. Depending on the direction of rotation, they can swim forward or backward and change directions to move along chemical gradients but also to navigate their obstructed natural environment in soils, sediments, or mucus. When they get stuck, they naturally try to back out, but they can also resort to a radically different flagellar mode, which we discovered here. Using high-speed microscopy, we monitored the swimming behavior of the monopolarly flagellated species Shewanella putrefaciens with fluorescently labeled flagellar filaments at an agarose-glass interface. We show that, when a cell gets stuck, the polar flagellar filament executes a polymorphic change into a spiral-like form that wraps around the cell body in a spiral-like fashion and enables the cell to escape by a screw-like backward motion. Microscopy and modeling suggest that this propagation mode is triggered by an instability of the flagellum under reversal of the rotation and the applied torque. The switch is reversible and bacteria that have escaped the trap can return to their normal swimming mode by another reversal of motor direction. The screw-type flagellar arrangement enables a unique mode of propagation and, given the large number of polarly flagellated bacteria, we expect it to be a common and widespread escape or motility mode in complex and structured environments.

Spatial control of sensory adaptation modulates mechanosensing in Pseudomonas aeruginosa.

Sensory signaling pathways use adaptation to dynamically respond to changes in their environment. Here, we report the mechanism of sensory adaptation in the Pil-Chp mechanosensory system, which the important human pathogen Pseudomonas aeruginosa uses to sense mechanical stimuli during surface exploration. Using biochemistry, genetics, and cell biology, we discovered that the enzymes responsible for adaptation, a methyltransferase and a methylesterase, are segregated to opposing cell poles as P. aeruginosa explore surfaces. By coordinating the localization of both enzymes, we found that the Pil-Chp response regulators influence local receptor methylation, the molecular basis of bacterial sensory adaptation. We propose a model in which adaptation during mechanosensing spatially resets local receptor methylation, and thus Pil-Chp signaling, to modulate the pathway outputs, which are involved in P. aeruginosa virulence. Despite decades of bacterial sensory adaptation studies, our work has uncovered an unrecognized mechanism that bacteria use to achieve adaptation to sensory stimuli.

Spatial arrangement of several flagellins within bacterial flagella improves motility in different environments.

Bacterial flagella are helical proteinaceous fibers, composed of the protein flagellin, that confer motility to many bacterial species. The genomes of about half of all flagellated species include more than one flagellin gene, for reasons mostly unknown. Here we show that two flagellins (FlaA and FlaB) are spatially arranged in the polar flagellum of Shewanella putrefaciens, with FlaA being more abundant close to the motor and FlaB in the remainder of the flagellar filament. Observations of swimming trajectories and numerical simulations demonstrate that this segmentation improves motility in a range of environmental conditions, compared to mutants with single-flagellin filaments. In particular, it facilitates screw-like motility, which enhances cellular spreading through obstructed environments. Similar mechanisms may apply to other bacterial species and may explain the maintenance of multiple flagellins to form the flagellar filament.

A polar bundle of flagella can drive bacterial swimming by pushing, pulling, or coiling around the cell body.

Bacteria swim in sequences of straight runs that are interrupted by turning events. They drive their swimming locomotion with the help of rotating helical flagella. Depending on the number of flagella and their arrangement across the cell body, different run-and-turn patterns can be observed. Here, we present fluorescence microscopy recordings showing that cells of the soil bacterium Pseudomonas putida that are decorated with a polar tuft of helical flagella, can alternate between two distinct swimming patterns. On the one hand, they can undergo a classical push-pull-push cycle that is well known from monopolarly flagellated bacteria but has not been reported for species with a polar bundle of multiple flagella. Alternatively, upon leaving the pulling mode, they can enter a third slow swimming phase, where they propel themselves with their helical bundle wrapped around the cell body. A theoretical estimate based on a random-walk model shows that the spreading of a population of swimmers is strongly enhanced when cycling through a sequence of pushing, pulling, and wrapped flagellar configurations as compared to the simple push-pull-push pattern.