Purification and Cryo-Electron Microscopy Analysis of Bacterial Appendages

The cryo-EM structure of ETB-Gi complex, obtained using the Fusion-G system, provides insights into the distinct activation mechanism and G-protein promiscuity of ETB, aiding in the design of ETB agonists for therapeutic applications.

The bacterial flagellar filament controls the swimming mode of bacteria by its polymorphic structural change. We investigated dynamics of the flagellar filament by massive molecular dynamic simulation and found that three types of inter-molecular interactions, permanent, sliding and switch interactions, play essential roles in the structural change. The polymorphic structure of the flagellar filament is thought to be due to energy frustration between intra- and inter-molecular interactions.

The evolution of archaeal flagellar filaments

Cryo-EM structure and functional analyses show that human iron exporter ferroportin (Fpn) can bind and transport calcium ions and the transport activity is reduced when the concentration of ferrous ions is high.

Motility allows microbes to explore and maximize success in their environment; however, many laboratory bacterial strains have a reduced or altered capacity for motility. Swimming motility in Bacillus subtilis depends on peritrichous flagella and is carried out individually as cells move by biased random walks toward attractants. Previously, we adapted Bacillus subtilis strain 3610 to the laboratory for 300 generations in lysogeny broth (LB) batch culture and isolated lab-adapted strains. Strain SH2 is motility-defective and in broth culture forms large, frequently spherical aggregates of cells. A single point mutation in the flagellin gene hag that causes amino acid 259 to switch from A to T is necessary and sufficient to cause these social cell aggregates, and aggregation occurs between flagellated cells bearing this point mutation regardless of the strain background. Cells associate when bearing this mutation, but flagellar rotation is needed to pull associating cells into spherical aggregates. Using electron microscopy, we are able to show that the SH2 flagellar filament has limited polymorphism when compared to other flagellar structures. This limited polymorphism hinders the flagellum's ability to function as a motility apparatus but appears to alter its function to that of cell aggregation/adhesion. We speculate that the genotype-specific aggregation of cells producing HagA259T flagella could have increased representation in a batch-culture experiment by allowing similar cells to go through a transfer together and also that this mutation could serve as an early step to evolve sociality in the natural world.IMPORTANCEThe first life forms on this planet were prokaryotic, and the earliest environments were aquatic, and from these relatively simple starting conditions, complex communities of microbes and ultimately multicellular organisms were able to evolve. Usually, motile cells in aqueous environments swim as individuals but become social by giving up motility and secreting extracellular substances to become a biofilm. Here, we identify a single point mutation in the flagellum that is sufficient to allow cells containing this mutation to specifically form large, suspended groups of cells. The specific change in the flagellar filament protein subunits causes a unique change in the flagellar structure. This could represent a distinct way for closely related cells to associate as an early precursor to sociality.

The bacterial flagellum is an elaborate nanomachine that powers motility in a variety of environments. While recent cryo-electron tomography studies have revealed great complexity as well as diversity in flagellar motor structures, less is known about the components that constitute the auxiliary structures observed in the periplasm for several species. One example is the E-ring, which was first observed in 1979 in Caulobacter crescentus but whose composition has only recently been shown to be a single protein, FlgY and its homologs. Multiple FlgY dimers form a conserved ring-spoke structure encircling the MS-ring, although the impact of the E-ring on motility seems to differ across bacterial phyla. Remarkably, the E-ring is widely present in flagellated species in the Bacteria domain except β- and γ-proteobacteria, suggesting an ancient origin that likely traces back to the last bacterial common ancestor. Future investigation is required to determine the exact role of this conserved structure in motor function, which may reveal mechanisms distinct from the current working model based on Escherichia coli and Salmonella enterica, which lack the E-ring, and also shed light on the architecture and function of the ancestral motor.

Purified and crude flagellar isolates from cells of Bacillus pumilus NRS 236 were treated with acid, alcohol, acid-alcohol, or heat, and were examined electron microscopically in negatively stained and shadow-cast preparations. Under certain conditions, each of these agents causes the flagella to break between the proximal hooks and the spiral filaments. In such preparations, filaments are seen in various stages of disintegration, whereas hooks of fairly constant length retain their integrity and morphological identity. When crude isolates of flagella are treated under these conditions, the hooks remain attached to membrane fragments or bear basal material. These findings substantiate previous structural observations that led to the view that the proximal hook is a distinct part of the bacterial flagellum and further confirm that the hook is tightly associated with basal material and the cytoplasmic membrane. It appears that the hook is a polarly oriented structure, and that the interactions between the hook and the basal material or the cytoplasmic membrane are different from those between the hook and the filamentous portion of the organelle. Moreover, both types of interaction apparently differ still from those by which the flagellin subunits are held together in the flagellar filament. Hooks were isolated by exploiting the differences in relative stability shown by the various morphological regions of the bacterial flagellum.

Bacterial flagella are the best-known rotational organelles in the biological world. The spiral-shaped flagellar filaments that extend from the cell surface rotate like a screw to create a propulsive force. At the base of the flagellar filament lies a protein motor that consists of a stator and a rotor embedded in the membrane. The stator is composed of two types of membrane subunits, PomA (similar to MotA in Escherichia coli) and PomB (similar to MotB in E. coli), which are energy converters that assemble around the rotor to couple rotation with the ion flow. Recently, stator structures, where two MotB molecules are inserted into the center of a ring made of five MotA molecules, were reported. This structure inspired a model in which the MotA ring rotates around the MotB dimer in response to ion influx. Here, we focus on the Vibrio PomB plug region, which is involved in flagellar motor activation. We investigated the plug region using site-directed photo-cross-linking and disulfide cross-linking experiments. Our results demonstrated that the plug interacts with the extracellular short loop region of PomA, which is located between transmembrane helices 3 and 4. Although the motor stopped rotating after cross-linking, its function recovered after treatment with a reducing reagent that disrupted the disulfide bond. Our results support the hypothesis, which has been inferred from the stator structure, that the plug region terminates the ion influx by blocking the rotation of the rotor as a spanner. IMPORTANCE The biological flagellar motor resembles a mechanical motor. It is composed of a stator and a rotor. The force is transmitted to the rotor by the gear-like stator movements. It has been proposed that the pentamer of MotA subunits revolves around the axis of the B subunit dimer in response to ion flow. The plug region of the B subunit regulates the ion flow. Here, we demonstrated that the ion flow was terminated by cross-linking the plug region of PomB with PomA. These findings support the rotation hypothesis and explain the role of the plug region in blocking the rotation of the stator unit.

Convergent evolution in the supercoiling of prokaryotic flagellar filaments

Structural Biology is a scientific field that seeks to understand the biological function of macromolecules by analyzing their threedimensional structure and organization.Insights from structural biology can inform the development of novel therapeutics for disease research and lead to a better understanding of fundamental biological processes.Cryogenic electron microscopy (cryo-EM) is one of the latest tools for structural biology research.Due to numerous advantages inherent to cryo-EM technology for structural biology research, the 2017 Nobel Prize in Chemistry was awarded for the development of cryo-EM for high-resolution structure determination of biomolecules in solution [1].However, cryo-EM data collection is still a labor-intensive process that often requires lengthy and iterative sample screening for a given protein sample as experimental conditions for data collection are optimized [2].Sample screening, wherein the user attempts to determine the best quality images for data collection, requires significant operator time; furthermore, it is usually difficult to determine which areas will yield the highest resolution reconstructions [3].The challenge of automating sample screening by machine learning is an opportunity to improve the throughput of drug development with cryo-EM.CryoFAST (Fast Acquisition for Single-particle and Tomography) is a machine-learning platform for automated Cryo-EM data acquisition.It works by analyzing images at multiple levels of magnification to optimally select targets.As targets are prioritized, CryoFAST automatically queues image acquisition and processing, providing real-time quality feedback, and updating target priorities with Bayesian optimization.CryoFAST is integrated with SerialEM and Gatan GMS/Latitude.Last year, we presented our early development and product roadmap for CryoFAST.This year, we will report the results of our machine learning development.We present the results of simulation studies as well as early alpha testing of the performance of our algorithm and microscope control automation in collaboration with the Pacific Northwest CryoEM center and the Lyumkis lab at Salk Institute.

Electron energy filtering significantly improves amplitude contrast of frozen-hydrated protein at 300 kV

The transcription factor FlbD regulates the temporal and spatial transcription of flagellar genes in the bacterium Caulobacter crescentus. Activation of FlbD requires cell cycle progression and the assembly of an early (class II) flagellum structure. In this report, we identify 20 independent gain-of-function mutations in flbD that relieve regulation by flagellar assembly. One of these, flbD-1204, contained a mutation in the receiver domain (V17M) and another, flbD-1231, in the DNA binding domain (V451G). Both of these mutations resulted in an aberrant pattern of cell cycle transcription. The presence of the FlbD-1204 allele also resulted in a loss of swarmer-pole-specific transcription. These results indicate that temporal and spatial transcription is influenced by the assembly of the nascent flagellar structure. The trans-acting positive and negative regulatory factor, FliX, couples flagellar assembly to the activation of FlbD and, as we show here, also influences temporal transcription. Furthermore, we show that FliX can suppress the activity of FlbD mutants that cannot be phosphorylated, and that FliX is required for FlbD stability, and vice versa. These results indicate that FliX may interact directly with FlbD to regulate its activity.

Archaeal flagella are similar functionally to bacterial flagella, but structurally they are completely different. Helical archaeal flagellar filaments are formed of protein subunits called flagellins (archaellins). Notwithstanding progress in studies of archaeal flagella achieved in recent years, many problems in this area are still unsolved. In this review, we analyze the formation of these supramolecular structures by the example of flagellar filaments of halophilic archaea. Recent data on the structure of the flagellar filaments demonstrate that their supramolecular organization differs considerably in different haloarchaeal species.

Helical Packing of Needles from Functionally Altered Shigella Type III Secretion Systems

Many bacteria use rotating helical flagella in swimming motility. In the search for food or migration towards a new habitat, bacteria occasionally unbundle their flagellar filaments and tumble, leading to an abrupt change in direction. Flexible flagella can also be easily deformed by external shear flow, leading to complex bacterial trajectories. Here, we examine the effects of flagella flexibility on the navigation of bacteria in two fundamental shear flows: planar shear and Poiseuille flow realized in long channels. On the basis of slender body elastodynamics and numerical analysis, we discovered a variety of non-trivial effects stemming from the interplay of self-propulsion, elasticity and shear-induced flagellar bending. We show that in planar shear flow the bacteria execute periodic motion, whereas in Poiseuille flow, they migrate towards the centre of the channel or converge towards a limit cycle. We also find that even a small amount of random reorientation can induce a strong response of bacteria, leading to overall non-periodic trajectories. Our findings exemplify the sensitive role of flagellar flexibility and shed new light on the navigation of bacteria in complex shear flows.