Visualization of functional rotor proteins of the bacterial flagellar motor in the cell membrane

The bacterial flagellar motor is a rotary motor driven by the electrochemical potentials of specific ions across the cell membrane. Direct interactions between the rotor protein FliG and the stator protein MotA are thought to generate the rotational torque. Here, we used total internal reflection fluorescent microscopy to observe the localization of green fluorescent protein (GFP)-fused FliG in Escherichia coli cells. We identified three types of fluorescent punctate signals: immobile dots, mobile dots that exhibited simple diffusion, and mobile dots that exhibited restricted diffusion. When GFP-FliG was expressed in a DeltafliG background, most of the cells were not mobile. When the cells were tethered to a glass side, however, rotating cells were commonly observed and a single fluorescent dot was always observed at the rotational center of the tethered cell. These fluorescent dots were likely positions at which functional GFP-FliG had been incorporated into a flagellar motor. Our results suggest that flagellar basal bodies diffuse in the cytoplasmic membrane until the axial structure and/or other structures assemble.     

Microcinematographic analysis of tethered Leptospira illini.

A model of Leptospira motility was recently proposed. One element of the model states that in translating cells the anterior spiral-shaped end gyrates counterclockwise and the posterior hook-shaped end gyrates clockwise. We tested these predictions by analyzing cells tethered to a glass surface. Leptospira illini was incubated with antibody-coated latex beads (Ab-beads). These beads adhered to the cells, and subsequently some cells became attached to either the slide or the cover glass via the Ab-beads. As previously reported, these cells rapidly moved back and forth across the surface of the beads. In addition, a general trend was observed: cells tethered to the cover glass rotated clockwise around the Ab-bead; cells tethered to the slide rotated counterclockwise around the Ab-bead. A computer-aided microcinematographic analysis of tethered cells indicated that the direction of rotation of cells around the Ab-bead was a function of both the surface of attachment and the shape of the cell ends. The results can best be explained by assuming that the gyrating ends interact with the glass surface to cause rotation around the Ab-beads. The analysis obtained indicates that the hook- and spiral-shaped ends rotate in the directions predicted by the model. In addition, the tethered cell assay permitted detection of rapid, coordinated reversals of the cell ends, e.g., cells rapidly switched from a hook-spiral configuration to a spiral-hook configuration. These results suggest the existance of a mechanism which coordinates the shape of the cell ends of L. illini.     

N-terminal signal region of FliK is dispensable for length control of the flagellar hook

The length of the flagellar hook is regulated; it is 55 +/- 6 nm long in Salmonella. Five genes involved in hook-length regulation are fliK, flhB, fliG, fliM and fliN. The last four genes encode structural components of the protein export apparatus in the flagellar base, whereas FliK is soluble and secreted during flagellar assembly. The role of FliK, however, remains ambiguous. We constructed two kinds of FliK variants: N-terminally truncated FliK protein and FliK N-terminally fused with cyan fluorescent protein (CFP-FliK). Both N-terminally truncated FliK missing the first 99 amino acids (aa) and CFP-FliK fusion variants partially complemented a fliK null (polyhook) mutant to produce cells with filaments, allowing cells to swim; the hooks, however, were not normal but were polyhooks. When the N-terminally defective FliK variants were expressed at high levels, the average polyhook length was shortened coming close to the length of the wild-type hook, independently of the sizes of the FliK variants. These FliK variants were not secreted. CFP-FliK fusion proteins were observed to homogeneously distribute in the cytoplasm. We conclude that FliK does not need to be exported to control hook length and is unlikely to be a ruler; instead, we conclude that FliK controls hook length by the timely switching of secretion modes of the flagellar type III secretion system by the FliK C-terminal domain, and that the N-terminal region is dispensable for hook length control.     

Rhizobium meliloti swims by unidirectional, intermittent rotation of right-handed flagellar helices.

The 5 to 10 peritrichously inserted complex flagella of Rhizobium meliloti MVII-1 were found to form right-handed flagellar bundles. Bacteria swam at speeds up to 60 microns/s, their random three-dimensional walk consisting of straight runs and quick directional changes (turns) without the vigorous angular motion (tumbling) seen in swimming Escherichia coli cells. Observations of R. meliloti cells tethered by a single flagellar filament revealed that flagellar rotation was exclusively clockwise, interrupted by very brief stops (shorter than 0.1 s), typically every 1 to 2 s. Swimming bacteria responded to chemotactic stimuli by extending their runs, and tethered bacteria responded by prolonged intervals of clockwise rotation. Moreover, the motility tracks of a generally nonchemotactic ("smooth") mutant consisted of long runs without sharp turns, and tethered mutant cells showed continuous clockwise rotation without detectable stops. These observations suggested that the runs of swimming cells correspond to clockwise flagellar rotation, and the turns correspond to the brief rotation stops. We propose that single rotating flagella (depending on their insertion point on the rod-shaped bacterial surface) can reorient a swimming cell whenever the majority of flagellar motors stop.     

Length Control of the Flagellar Hook in a Temperature-Sensitive flgE Mutant of Salmonella enterica Serovar Typhimurium

The flagellar hook is a short, curved, extracellular structure located between the basal body and the filament. The hook is composed of the FlgE protein. In this study, we analyzed flagellum assembly in a temperature-sensitive flgE mutant of Salmonella enterica serovar Typhimurium. When the mutant cells were grown at 30°C, they produced flagella of a normal length (71% of the population) and short hooks without filaments (26%). At 37°C, 70% of the basal bodies lacked hooks, and intact flagella made up only 6% of the population. Mutant cells secreted monomeric FlgE in abundance at 37°C, suggesting that the mutant FlgE protein might be defective in polymerization at higher temperatures. The average length of the hooks in intact filaments was 55 nm, whereas after acid treatment, it was 45 nm. SDS-PAGE analysis of the hook-basal body showed that HAP1 was missing in the mutant but not in the wild type. We concluded that hook length in the mutant is controlled in the same way as in the wild type, but the hook appeared short after acid treatment due to the lack of HAP1. We also learned that the true length of the hook is possibly 45 nm, not 55 nm, as has been believed.     

Ethylene-mediated enhancement of apical hook formation in etiolated Arabidopsis thaliana seedlings is gibberellin dependent

Dark-grown Arabidopsis seedlings develop an apical hook by differential elongation and division of hypocotyl cells. This allows the curved hypocotyl to gently drag the apex, which is protected by the cotyledons, upwards through the soil. Several plant hormones are known to be involved in hook development, including ethylene, which causes exaggeration of the hook. We show that gibberellins (GAs) are also involved in this process. Inhibition of GA biosynthesis with paclobutrazol (PAC) prevented hook formation in wild-type (WT) seedlings and in constitutive ethylene response (ctr)1-1, a mutant that exhibits a constitutive ethylene response. In addition, a GA-deficient mutant (ga1-3) did not form an apical hook in the presence of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC). Analysis of transgenic Arabidopsis seedlings expressing a green fluorescent protein (GFP)-repressor of ga1-3 (RGA) fusion protein suggested that ACC inhibits cell elongation in the apical hook by inhibition of GA signaling. A decreased feedback of GA possibly causes an induction of GA biosynthesis based upon the expression of genes encoding copalyl diphosphate synthase (CPS; GA1) and GA 2-oxidase (AtGA2ox1). Furthermore, expression of GASA1, a GA-response gene, suggests that differential cell elongation in the apical hook might be a result of differential GA-sensitivity.     

Analysis of a flagellar filament cap mutant reveals that HtrA serine protease degrades unfolded flagellin protein in the periplasm of Borrelia burgdorferi

Unlike external flagellated bacteria, spirochetes have periplasmic flagella (PF). Very little is known about how PF are assembled within the periplasm of spirochaetal cells. Herein, we report that FliD (BB0149), a flagellar cap protein (also named hook‐associated protein 2), controls flagellin stability and flagellar filament assembly in the Lyme disease spirochete Borrelia burgdorferi. Deletion of fliD leads to non‐motile mutant cells that are unable to assemble flagellar filaments and pentagon‐shaped caps (10 nm in diameter, 12 nm in length). Interestingly, FlaB, a major flagellin protein of B. burgdorferi, is degraded in the fliD mutant but not in other flagella‐deficient mutants (i.e., in the hook, rod, or MS‐ring). Biochemical and genetic studies reveal that HtrA, a serine protease of B. burgdorferi, controls FlaB turnover. Specifically, HtrA degrades unfolded but not polymerized FlaB, and deletion of htrA increases the level of FlaB in the fliD mutant. Collectively, we propose that the flagellar cap protein FliD promotes flagellin polymerization and filament growth in the periplasm. Deletion of fliD abolishes this process, which leads to leakage of unfolded FlaB proteins into the periplasm where they are degraded by HtrA, a protease that prevents accumulation of toxic products in the periplasm.     

Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems

Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems.     

The Flagellar Soluble Protein FliK Determines the Minimal Length of the Hook in Salmonella enterica Serovar Typhimurium

The length of the flagellar hook is controlled by the soluble protein FliK. FliK is structurally divided into two halves with distinct functions; the N-terminal half determines hook length, while the C-terminal half switches the secretion substrate specificity, consequently terminating hook elongation. FliK properly achieves both functions only when it is secreted. In a previous paper, we showed that a temperature-sensitive flgE mutant of Salmonella enterica serovar Typhimurium, SJW2219, produced basal bodies with short hooks (average length, 25 nm) at 37°C. In this study, we show that the mutant cells grown at 37°C secrete FliK but not flagellin (FliC), indicating that FliK is abortively secreted into the medium when the hook is shorter than 30 nm. In contrast, FliK unfailingly switches the gate modes when the hook is longer than 30 nm. Taking the FliC, FliK, and FlgM secretion patterns into account, we conclude that FliK determines the minimal length of the hook. We will discuss how FliK detects the critical switching point of the secretion gate.     

Rod-to-Hook Transition for Extracellular Flagellum Assembly Is Catalyzed by the L-Ring-Dependent Rod Scaffold Removal

In Salmonella, the rod substructure of the flagellum is a periplasmic driveshaft that couples the torque generated by the basal body motor to the extracellular hook and filament. The rod subunits self-assemble, spanning the periplasmic space and stopping at the outer membrane when a mature length of ∼22 nm is reached. Assembly of the extracellular hook and filament follow rod completion. Hook initiation requires that a pore forms in the outer membrane and that the rod-capping protein, FlgJ, dislodges from the tip of the distal rod and is replaced with the hook-capping protein, FlgD. Approximately 26 FlgH subunits form the L-ring around the distal rod that creates the pore through which the growing flagellum will elongate from the cell body. The function of the L-ring in the mature flagellum is also thought to act as a bushing for the rotating rod. Work presented here demonstrates that, in addition to outer membrane pore formation, L-ring formation catalyzes the removal of the FlgJ rod cap. Rod cap removal allows the hook cap to assemble at the rod tip and results in the transition from rod completion in the periplasm to extracellular hook polymerization. By coupling the rod-to-hook switch to outer membrane penetration, FlgH ensures that hook and filament polymerization is initiated at the appropriate spatial and temporal point in flagellar biosynthesis.     

Compliance of bacterial polyhooks measured with optical tweezers.

In earlier work, a single-beam gradient force optical trap ("optical tweezers") was used to measure the torsional compliance of flagella in wild-type cells of Escherichia coli that had been tethered to glass by a single flagellum. This compliance was nonlinear, exhibiting a torsionally soft phase up to 180 degrees, followed by a torsionally rigid phase for larger angles. Values for the torsional spring constant in the soft phase were substantially less than estimates based on the rigidity determined for isolated flagellar filaments. It was suggested that the soft phase might correspond to wind-up of the flagellar hook, and the rigid phase to wind-up of the stiffer filament. Here, we have measured the torsional compliance of flagella on cells of an E. coli strain that produces abnormally long hooks but no filaments. The small-angle compliance of these cells, as determined from the elastic rebound of the cell body after wind-up and release, was found to be the same as for wild-type cells. This confirms that the small-angle compliance of wild-type cells is dominated by the response of the hook. Hook flexibility is likely to play a useful role in stabilizing the flagellar bundle.     

Unidirectional, intermittent rotation of the flagellum of Rhodobacter sphaeroides.

The single flagellum of the photosynthetic bacterium Rhodobacter sphaeroides was found to be medially located on the cell body. Observation of free-swimming bacteria, and bacteria tethered by their flagellar filaments, revealed that the flagellum could only rotate in the clockwise direction; switching of the direction of rotation was never observed. Flagellar rotation stopped periodically, typically several times a minute for up to several seconds each. Reorientation of swimming cells appeared to be the result of Brownian rotation during the stop periods. The flagellar filament displayed polymorphism; detached and nonrotating filaments were usually seen as large-amplitude helices of such short wavelength that they appeared as flat coils or circles, whereas the filaments on swimming cells showed a normal (small-amplitude, long-wavelength) helical form. With attached filaments, the transition from the normal to the coiled form occurred when the flagellar motor stopped rotating, proceeding from the distal end towards the cell body. It is possible that both the relaxation process and the smaller frictional resistance after relaxation may act to enhance the rate of reorientation of the cell. The transition from the coiled to the normal form occurred when the motor restarted, proceeding from the proximal end outwards, which might further contribute to the reorientation of the cell before it reaches a stable swimming geometry.     

Homogeneous noncompetitive assay of protein via Förster-resonance-energy-transfer with tryptophan residue(s) as intrinsic donor(s) and fluorescent ligand as acceptor

Homogeneous noncompetitive assay of a protein in biological samples based on Förster-resonance-energy-transfer (FRET) was proposed by using its tryptophan residues as intrinsic donors and its specific fluorescent ligand as the FRET acceptor that was defined as an analytical FRET probe. Conjugate of a suitable fluorophore, which should have an excitation peak around 340 nm but an excitation valley around 280 nm, with a moiety binding to a protein of interest gave an analytical FRET probe to the protein. To test this method, N-biotinyl-N′-(1-naphthyl)-ethylenediamine (BNEDA) was used as an analytical FRET probe for homogeneous noncompetitive assay of streptavidin (SAV). The occurrence of FRET between the bound BNEDA and tryptophan residues was supported by the modeled geometry of the complex. By excitation at 280 nm, free BNEDA produced negligible fluorescence at 430 nm, but the bound BNEDA produced much higher stable fluorescence at 430 nm after 2 min of binding reaction. The competitive binding between BNEDA and biotin gave the dissociation constant of (16 ± 3) fM for BNEDA (n = 3). By excitation at 280 nm, fluorescence at 430 nm of reaction mixtures containing 32.0 nM BNEDA responded linearly to SAV subunit concentrations ranging from 0.40 to 30.0 nM with the desirable resistance to common interferences in biological samples. Therefore, by using tryptophan residue(s) in a protein of interest as intrinsic donor(s) and its fluorescent ligand as the corresponding FRET acceptor, this homogeneous noncompetitive assay of the protein in biological samples was effective and advantageous.     

Direct observation of speed fluctuations of flagellar motor rotation at extremely low load close to zero

The bacterial flagellar motor accommodates ten stator units around the rotor to produce large torque at high load. But when external load is low, some previous studies showed that a single stator unit can spin the rotor at the maximum speed, suggesting that the maximum speed does not depend on the number of active stator units, whereas others reported that the speed is also dependent on the stator number. To clarify these two controversial observations, much more precise measurements of motor rotation would be required at external load as close to zero as possible. Here, we constructed a Salmonella filament-less mutant that produces a rigid, straight, twice longer hook to efficiently label a 60 nm gold particle and analyzed flagellar motor dynamics at low load close to zero. The maximum motor speed was about 400 Hz. Large speed fluctuations and long pausing events were frequently observed, and they were suppressed by either over-expression of the MotAB stator complex or increase in the external load, suggesting that the number of active stator units in the motor largely fluctuates near zero load. We conclude that the lifetime of the active stator unit becomes much shorter when the motor operates near zero load.     

Fusion expression of cecropin B-like antibacterial peptide in <Emphasis Type="Italic">Escherichia coli</Emphasis> and preparation of its antiserum

Antibacterial peptides have a broad range of antibacterial properties that makes them highly toxic for expression in Escherichia coli. For prepare an antiserum to detect these peptides, we developed a cecropin B mutant with a green fluorescent protein fusion partner resulting in high expression of a 37 kDa fusion peptide in E. coli with a yield of 7.9 mg/l culture medium after purification on Ni-IDA resin. Guinea pigs when immunized with the fusion peptide produced a specific antiserum which titers in excess of 1:25,600.     

Pausing of flagellar rotation is a component of bacterial motility and chemotaxis.

When bacterial cells are tethered to glass by their flagella, many of them spin. On the basis of experiments with tethered cells it has generally been thought that the motor which drives the flagellum is a two-state device, existing in either a counterclockwise or a clockwise state. Here we show that a third state of the motor is that of pausing, the duration and frequency of which are affected by chemotactic stimuli. We have recorded on video tape the rotation of tethered Escherichia coli and Salmonella typhimurium cells and analyzed the recordings frame by frame and in slow motion. Most wild-type cells paused intermittently. The addition of repellents caused an increase in the frequency and duration of the pauses. The addition of attractants sharply reduced the number of pauses. A chemotaxis mutant which lacks a large part of the chemotaxis machinery owing to a deletion of the genes from cheA to cheZ did not pause at all and did not respond to repellents by pausing. A tumbly mutant of S. typhimurium responded to repellents by smooth swimming and to attractants by tumbling. When tethered, these cells exhibited a normal rotational response but an inverse pausing response to chemotactic stimuli: the frequency of pauses decreased in response to repellents and increased in response to attractants. It is suggested that (i) pausing is an integral part of bacterial motility and chemotaxis, (ii) pausing is independent of the direction of flagellar rotation, and (iii) pausing may be one of the causes of tumbling.     

Fluorescence imaging using a fluorescent protein with a large Stokes shift

Keima is a far-red fluorescent protein endowed with a large Stokes shift. It absorbs light maximally at around 440 nm and emits maximally at around 620 nm. While the original Keima is obligately tetrameric (tKeima), the dimeric and monomeric versions (mKeima and dKeima, respectively) have been generated. More recently, a tandem dimer of Keima (tdKeima) has been developed as the brightest version. Here we describe examples, which show the usefulness of Keima for dual-color fluorescence imaging technologies, such as fluorescence cross-correlation spectroscopy (FCCS) and two-photon laser scanning microscopy (TPLSM). Keima can be used in conjunction with existing fluorescent proteins in which the Stokes shift is much smaller, with the idea that while two fluorescent proteins are excited by a single laser each will fluoresce a different color.     

Method of localized removal of cells using a bolt‐clamped Langevin transducer with an ultrasonic horn

Cell isolation by eliminating undesirable cell aggregations or colonies with low activity is essential to improve cell culture efficiency. Moreover, when creating tissues from induced pluripotent stem cells, residual undifferentiated cells must be removed to prevent tumor formation in vivo. Here, we evaluated the use of ultrasonic irradiation, which can apply energy locally without contact, and proposed a method to eliminate cells in a small area of culture by ultrasonic irradiation from a Langevin transducer. We constructed a device that incorporated a bolt‐clamped 19.84 kHz Langevin transducer with an ultrasonic horn and determined the optimal conditions for stable elimination of cells in small areas of a 35‐mm culture dish. The optimal conditions were as follows: number of cycles = 400, clearance distance = 1 mm, volume of medium = 4 mL, and distance from the center of culture surface = 0 mm. The mean cell elimination area under these conditions was 0.097 mm². We also evaluated the viability of neighboring cells after ultrasonic irradiation by fluorescent staining and found that most cells around the elimination area survived. These findings suggest that the proposed method has potential for localized elimination of cells without the need for contact with the cell surface.     

Structural analysis of the Leptospiraceae and Borrelia burgdorferi by high-voltage electron microscopy.

Spirochetes are an evolutionary and structurally unique group of bacteria. Outermost is a membrane sheath (OS), and within this sheath are the protoplasmic cell cylinder (PC) and periplasmic flagella (PFs). The PFs are attached at each end of the PC and, depending on the species, may or may not overlap in the center of the cell. The precise location of the PFs within the spirochetal cells is unknown. The PFs could lie along the cell axis. Alternatively, the PFs could wrap around the PC in either a right- or a left-handed sense. To understand the factors that cause the PFs to influence cell shape and allow the cells to swim, we determined the precise location of the PFs in the Leptospiraceae (Leptonema illini) and Borrelia burgdorferi. Our approach was to use high-voltage electron microscopy and analyze the three-dimensional images obtained from thick sections of embedded cells. We found that a single PF in L. illini is located in a central channel 29 nm in diameter running along the helix axis of the right-handed PC. The presence of the PFs is associated with the end being hook shaped. The results obtained agree with the current model of Leptospiraceae motility. In B. burgdorferi, which forms a flattened wave, the relationship between the PFs and the PC is more complicated. A multistrand ridge 67 nm in diameter, which was shown to be composed of PFs by cross-sectional and mutant analysis, was found to extend along the entire length of the cell. We found that the PFs wrapped around the PC in a right-handed sense. However, the PFs formed a left-handed helix in space. The wavelength of the cell body and the helix pitch of the PFs were found to be identical (2.83 microm). The results obtained were used to propose a model of B. burgdorferi motility whereby backward-propagating waves, which gyrate counterclockwise as viewed from the back of the cell, are generated by the counterclockwise rotation of the internal PFs. Concomitant with this motion, the cell is believed to rotate clockwise about the body axis as shown for the Leptospiraceae.