The bacterial predator Bdellovibrio bacteriovorus is considered to be obligatorily prey (host)-dependent (H-D), and thus unable to form biofilms. However, spontaneous host-independent (H-I) variants grow axenically and can form robust biofilms. A screen of 350 H-I mutants revealed that single mutations in stator genes fliL or motA were sufficient to generate flagellar motility-defective H-I strains able to adhere to surfaces but unable to develop biofilms. The variants showed large transcriptional shifts in genes related to flagella, prey-invasion, and cyclic-di-GMP (CdG), as well as large changes in CdG cellular concentration relative to the H-D parent. The introduction of the parental fliL allele resulted in a full reversion to the H-D phenotype, but we propose that specific interactions between stator proteins prevented functional complementation by fliL paralogs. In contrast, specific mutations in a pilus-associated protein (Bd0108) mutant background were necessary for biofilm formation, including secretion of extracellular DNA (eDNA), proteins, and polysaccharides matrix components. Remarkably, fliL disruption strongly reduced biofilm development. All H-I variants grew similarly without prey, showed a strain-specific reduction in predatory ability in prey suspensions, but maintained similar high efficiency in prey biofilms. Population-wide allele sequencing suggested additional routes to host independence. Thus, stator and invasion pole-dependent signaling control the H-D and the H-I biofilm-forming phenotypes, with single mutations overriding prey requirements, and enabling shifts from obligate to facultative predation, with potential consequences on community dynamics. Our findings on the facility and variety of changes leading to facultative predation also challenge the concept of Bdellovibrio and like organisms being obligate predators.
OBJECTIVE: The ability of bacteria to form biofilms is a central research theme in biology, medicine, and the environment. We show that cultures of the obligate (host-dependent) \"solitary\" predatory bacterium Bdellovibrio bacteriovorus, which cannot replicate without prey, can use various genetic routes to spontaneously yield host-independent (H-I) variants that grow axenically (as a single species, in the absence of prey) and exhibit various surface attachment phenotypes, including biofilm formation. These routes include single mutations in flagellar stator genes that affect biofilm formation, provoke motor instability and large motility defects, and disrupt cyclic-di-GMP intracellular signaling. H-I strains also exhibit reduced predatory efficiency in suspension but high efficiency in prey biofilms. These changes override the requirements for prey, enabling a shift from obligate to facultative predation, with potential consequences on community dynamics.

Bacteria swim using membrane-spanning, electrochemical gradient-powered motors that rotate semi-rigid helical filaments. This primer provides a brief overview of the basic synthesis, structure and operation of these nanomachines. Details and variations on the basic system can be found in suggested further reading.

The flagellar motor of marine Vibrio is driven by the sodium-motive force across the inner membrane. The stator complex, consisting of two membrane proteins PomA and PomB, is responsible for energy conversion in the motor. To understand the coupling of the Na+ flux with torque generation, it is essential to clearly identify the Na+-binding sites and the Na+ flux pathway through the stator channel. Although residues essential for Na+ flux have been identified by using mutational analysis, it has been difficult to observe Na+ binding to the PomAB stator complex. Here we describe a method to monitor the binding of Na+ to purified PomAB stator complex using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. This method demonstrates that Na+-binding sites are formed by critical aspartic acid and threonine residues located in the transmembrane segments of PomAB.

The bacterial flagellum employs a rotary motor embedded on the cell surface. The motor consists of the stator and rotor elements and is driven by ion influx (typically H+ or Na+) through an ion channel of the stator. Ion influx induces conformational changes in the stator, followed by changes in the interactions between the stator and rotor. The driving force to rotate the flagellum is thought to be generated by changing the stator-rotor interactions. In this chapter, we describe two methods for investigating the interactions between the stator and rotor: site-directed in vivo photo-crosslinking and site-directed in vivo cysteine disulfide crosslinking.

Many motile bacteria swim and swarm toward favorable environments using the flagellum, which is rotated by a motor embedded in the inner membrane. The motor is composed of the rotor and the stator, and the motor torque is generated by the change of the interaction between the rotor and the stator induced by the ion flow through the stator. A stator unit consists of two types of membrane proteins termed A and B. Recent cryo-EM studies on the stators from mesophiles revealed that the stator consists of five A and two B subunits, whereas the low-resolution EM analysis showed that purified hyperthermophilic MotA forms a tetramer. To clarify the assembly formation and factors enhancing thermostability of the hyperthermophilic stator, we determined the cryo-EM structure of MotA from Aquifex aeolicus (Aa-MotA), a hyperthermophilic bacterium, at 3.42 Å resolution. Aa-MotA forms a pentamer with pseudo C5 symmetry. A simulated model of the Aa-MotA5MotB2 stator complex resembles the structures of mesophilic stator complexes, suggesting that Aa-MotA can assemble into a pentamer equivalent to the stator complex without MotB. The distribution of hydrophobic residues of MotA pentamers suggests that the extremely hydrophobic nature in the subunit boundary and the transmembrane region is a key factor to stabilize hyperthermophilic Aa-MotA.

Vibrio has a polar flagellum driven by sodium ions for swimming. The force-generating stator unit consists of PomA and PomB. PomA contains four transmembrane regions and a cytoplasmic domain of approximately 100 residues, which interacts with the rotor protein, FliG, to be important for the force generation of rotation. The 3D structure of the stator shows that the cytosolic interface (CI) helix of PomA is located parallel to the inner membrane. In this study, we investigated the function of CI helix and its role as stator. Systematic proline mutagenesis showed that residues K64, F66 and M67 were important for this function. The mutant stators did not assemble around the rotor. Moreover, the growth defect caused by PomB plug deletion was suppressed by these mutations. We speculate that the mutations affect the structure of the helices extending from TM3 and TM4 and reduce the structural stability of the stator complex. This study suggests that the helices parallel to the inner membrane play important roles in various processes, such as the hoop-like function in securing the stability of the stator complex and the ion conduction pathway, which may lead to the elucidation of the ion permeation and assembly mechanism of the stator.

3D printing has evolved into an invaluable tool for rapid and cost-effective production of intricate parts. In this paper we describe 3D printing and other rapid prototyping methods to fabricate 3.2 mm stators and drive caps for use in magic angle spinning (MAS) NMR experiments. These components can be fabricated with the assistance of computer-aided design (CAD) software and at a fraction of the cost of commercial parts. Additionally, we show that the performance of these 3D printed stators and drive caps is comparable to commercially available systems and that they have significant advantages over their machined counterparts.

We developed a robust bead assay for studying flagellar motor behavior of Pseudomonas aeruginosa. Using this assay, we studied the dynamics of the two stator systems in the flagellar motor. We found that the two sets of stators function differently, with MotAB stators providing higher total torque and MotCD stators ensuring more stable motor speed. The motors in wild-type cells adjust the stator compositions according to the environment, resulting in an optimal performance in environmental exploration compared to that of mutants with one set of stators. The bead assay we developed in this investigation can be further used to study P. aeruginosa chemotaxis at the level of a single cell using the motor behavior as the chemotaxis output. IMPORTANCE Cells of Pseudomonas aeruginosa possess a single polar flagellum, driven by a rotatory motor powered by two sets of torque-generating units (stators). We developed a robust bead assay for studying the behavior of the flagellar motor in P. aeruginosa, by attaching a microsphere to shortened flagellar filament and using it as an indicator of motor rotation. Using this assay, we revealed the dynamics of the two stator systems in the flagellar motor and found that the motors in wild-type cells adjust the stator compositions according to the environment, resulting in an optimal performance in environmental exploration compared to that of mutants with one set of stators.

The bacterial flagellar motor (BFM) is a protein complex that confers motility to cells and contributes to survival and virulence. The BFM consists of stators that are ion-selective membrane protein complexes and a rotor that directly connects to a large filament, acting as a propeller. The stator complexes couple ion transit across the membrane to torque that drives rotation of the motor. The most common ion gradients that drive BFM rotation are protons (H+) and sodium ions (Na+). The sodium-powered stators, like those in the PomA/PomB stator complex of Vibrio spp., can be inhibited by sodium channel inhibitors, in particular, by phenamil, a potent and widely used inhibitor. However, relatively few new sodium motility inhibitors have been described since the discovery of phenamil. In this study, we characterized two possible motility inhibitors, HM2-16F and BB2-50F, from a small library of previously reported amiloride derivatives. We used three approaches: effect on rotation of tethered cells, effect on free-swimming bacteria, and effect on rotation of marker beads. We showed that both HM2-16F and BB2-50F stopped rotation of tethered cells driven by Na+ motors comparable to phenamil at matching concentrations and could also stop rotation of tethered cells driven by H+ motors. Bead measurements in the presence and absence of stators confirmed that the compounds did not inhibit rotation via direct association with the stator, in contrast to the established mode of action of phenamil. Overall, HM2-16F and BB2-50F stopped swimming in both Na+ and H+ stator types and in pathogenic and nonpathogenic strains. IMPORTANCE Here, we characterized two novel amiloride derivatives in the search for antimicrobial compounds that target bacterial motility. These compounds were shown to inhibit flagellar motility at 10 μM across multiple strains: from nonpathogenic Escherichia coli with flagellar rotation driven by proton or chimeric sodium-powered stators, to proton-powered pathogenic E. coli (enterohemorrhagic E. coli or uropathogenic E. coli [EHEC or UPEC, respectively]), and finally, sodium-powered Vibrio alginolyticus. Broad antimotility compounds such as these are important tools in our efforts to control virulence of pathogens in health and agricultural settings.

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.