The unique paradigm of spirochete motility and chemotaxis.
ABSTRACT: Spirochete motility is enigmatic: It differs from the motility of most other bacteria in that the entire bacterium is involved in translocation in the absence of external appendages. Using the Lyme disease spirochete Borrelia burgdorferi (Bb) as a model system, we explore the current research on spirochete motility and chemotaxis. Bb has periplasmic flagella (PFs) subterminally attached to each end of the protoplasmic cell cylinder, and surrounding the cell is an outer membrane. These internal helix-shaped PFs allow the spirochete to swim by generating backward-moving waves by rotation. Exciting advances using cryoelectron tomography are presented with respect to in situ analysis of cell, PF, and motor structure. In addition, advances in the dynamics of motility, chemotaxis, gene regulation, and the role of motility and chemotaxis in the life cycle of Bb are summarized. The results indicate that the motility paradigms of flagellated bacteria do not apply to these unique bacteria.
Project description:Leptospira are spirochete bacteria distinguished by a short-pitch coiled body and intracellular flagella. Leptospira cells swim in liquid with an asymmetric morphology of the cell body; the anterior end has a long-pitch spiral shape (S-end) and the posterior end is hook-shaped (H-end). Although the S-end and the coiled cell body called the protoplasmic cylinder are thought to be responsible for propulsion together, most observations on the motion mechanism have remained qualitative. In this study, we analyzed the swimming speed and rotation rate of the S-end, protoplasmic cylinder, and H-end of individual Leptospira cells by one-sided dark-field microscopy. At various viscosities of media containing different concentrations of Ficoll, the rotation rate of the S-end and protoplasmic cylinder showed a clear correlation with the swimming speed, suggesting that these two helical parts play a central role in the motion of Leptospira. In contrast, the H-end rotation rate was unstable and showed much less correlation with the swimming speed. Forces produced by the rotation of the S-end and protoplasmic cylinder showed that these two helical parts contribute to propulsion at nearly equal magnitude. Torque generated by each part, also obtained from experimental motion parameters, indicated that the flagellar motor can generate torque >4000 pN nm, twice as large as that of Escherichia coli. Furthermore, the S-end torque was found to show a markedly larger fluctuation than the protoplasmic cylinder torque, suggesting that the unstable H-end rotation might be mechanically related to changes in the S-end rotation rate for torque balance of the entire cell. Variations in torque at the anterior and posterior ends of the Leptospira cell body could be transmitted from one end to the other through the cell body to coordinate the morphological transformations of the two ends for a rapid change in the swimming direction.
Project description:Many species of bacteria are motile, but their migration mechanisms are considerably diverse. Whatever mechanism is used, being motile allows bacteria to search for more optimal environments for growth, and motility is a crucial virulence factor for pathogenic species. The spirochete Leptospira, having two flagella in the periplasmic space, swims in liquid but has also been previously shown to crawl over solid surfaces. The present motility assays show that the spirochete movements both in liquid and on surfaces involve a rotation of the helical cell body. Direct observations of cell-surface movement with amino-specific fluorescent dye and antibody-coated microbeads suggest that the spirochete attaches to the surface via mobile, adhesive outer membrane components, and the cell body rotation propels the cell relative to the anchoring points. Our results provide models of how the spirochete switches its motility mode from swimming to crawling.
Project description:FlgJ plays a very important role in flagellar assembly. In the enteric bacteria, flgJ null mutants fail to produce the flagellar rods, hooks, and filaments but still assemble the integral membrane-supramembrane (MS) rings. These mutants are nonmotile. The FlgJ proteins consist of two functional domains. The N-terminal rod-capping domain acts as a scaffold for rod assembly, and the C-terminal domain acts as a peptidoglycan (PG) hydrolase (PGase), which allows the elongating flagellar rod to penetrate through the PG layer. However, the FlgJ homologs in several bacterial phyla (including spirochetes) often lack the PGase domain. The function of these single-domain FlgJ proteins remains elusive. Herein, a single-domain FlgJ homolog (FlgJ(Bb)) was studied in the Lyme disease spirochete Borrelia burgdorferi. Cryo-electron tomography analysis revealed that the flgJ(Bb) mutant still assembled intact flagellar basal bodies but had fewer and disoriented flagellar hooks and filaments. Consistently, Western blots showed that the levels of flagellar hook (FlgE) and filament (FlaB) proteins were substantially decreased in the flgJ(Bb) mutant. Further studies disclosed that the decreases of FlgE and FlaB in the mutant occurred at the posttranscriptional level. Microscopic observation and swarm plate assay showed that the motility of the flgJ(Bb) mutant was partially deficient. The altered phenotypes were completely restored when the mutant was complemented. Collectively, these results indicate that FlgJ(Bb) is involved in the assembly of the flagellar hook and filament but not the flagellar rod in B. burgdorferi. The observed phenotype is different from that of flgJ mutants in the enteric bacteria.
Project description:Spirochete bacteria, including important pathogens, exhibit a distinctive means of swimming via undulations of the entire cell. Motility is powered by the rotation of supercoiled 'endoflagella' that wrap around the cell body, confined within the periplasmic space. To investigate the structural basis of flagellar supercoiling, which is critical for motility, we determined the structure of native flagellar filaments from the spirochete <i>Leptospira</i> by integrating high-resolution cryo-electron tomography and X-ray crystallography. We show that these filaments are coated by a highly asymmetric, multi-component sheath layer, contrasting with flagellin-only homopolymers previously observed in exoflagellated bacteria. Distinct sheath proteins localize to the filament inner and outer curvatures to define the supercoiling geometry, explaining a key functional attribute of this spirochete flagellum.
Project description:The spirochete which causes Lyme disease, Borrelia burgdorferi, has many features common to other spirochete species. Outermost is a membrane sheath, and within this sheath are the cell cylinder and periplasmic flagella (PFs). The PFs are subterminally attached to the cell cylinder and overlap in the center of the cell. Most descriptions of the B. burgdorferi flagellar filaments indicate that these organelles consist of only one flagellin protein (FlaB). In contrast, the PFs from other spirochete species are comprised of an outer layer of FlaA and a core of FlaB. We recently found that a flaA homolog was expressed in B. burgdorferi and that it mapped in a fla/che operon. These results led us to analyze the PFs and FlaA of B. burgdorferi in detail. Using Triton X-100 to remove the outer membrane and isolate the PFs, we found that the 38.0-kDa FlaA protein purified with the PFs in association with the 41.0-kDa FlaB protein. On the other hand, purifying the PFs by using Sarkosyl resulted in no FlaA in the isolated PFs. Sarkosyl has been used by others to purify B. burgdorferi PFs, and our results explain in part their failure to find FlaA. Unlike other spirochetes, B. burgdorferi FlaA was expressed at a lower level than FlaB. In characterizing FlaA, we found that it was posttranslationally modified by glycosylation, and thus it resembles its counterpart from Serpulina hyodysenteriae. We also tested if FlaA was synthesized in a spontaneously occurring PF mutant of B. burgdorferi (HB19Fla-). Although this mutant still synthesized flaA message in amounts similar to the wild-type amounts, it failed to synthesize FlaA protein. These results suggest that, in agreement with data found for FlaB and other spirochete flagellar proteins, FlaA is likely to be regulated on the translational level. Western blot analysis using Treponema pallidum anti-FlaA serum indicated that FlaA was antigenically well conserved in several spirochete species. Taken together, the results indicate that both FlaA and FlaB comprise the PFs of B. burgdorferi and that they are regulated differently from flagellin proteins of other bacteria.
Project description:In order to clear the body of infecting spirochetes, phagocytic cells must be able to get hold of them. In real-time phase-contrast videomicroscopy we were able to measure the speed of Borrelia burgdorferi (Bb), the Lyme spirochete, moving back and forth across a platelet to which it was tethered. Its mean crossing speed was 1,636 microm/min (N = 28), maximum, 2800 microm/min (N = 3). This is the fastest speed recorded for a spirochete, and upward of two orders of magnitude above the speed of a human neutrophil, the fastest cell in the body. This alacrity and its interpretation, in an organism with bidirectional motor capacity, may well contribute to difficulties in spirochete clearance by the host.
Project description:Leptospira spp. are spirochete bacteria that possess periplasmic flagella (PFs) underneath the outer membrane; each flagellum is attached to each end of the protoplasmic cylinder. PFs of Leptospira have a coiled shape that bends the end of the cell body. However, the molecular mechanism by which multiple flagellar proteins organize to form the distinctively curled PF of Leptospira remains unclear. Here we obtained a slow-motility mutant of L. biflexa MD4-3 by random insertion mutagenesis using a Himar1 transposon. In MD4-3, the gene encoding the flagellar sheath protein, flagellar-coiling protein A (FcpA), which was recently identified in L. interrogans, was inactivated. As with L. interrogans ?fcpA strains, the L. biflexa ?fcpA strain lacked a distinct curvature at both ends of the cell body, and its motility was significantly reduced as compared with that of the wild-type strain. PFs isolated from the ?fcpA strain were straight and were thinner than those isolated from the wild-type strain. Western blot analysis revealed that flagellar proteins FlaA1, FlaA2, FlaB1, and FlaB2 were expressed in the ?fcpA strain but the flagellar proteins, except for FlaB2 were not incorporated in its PFs. Immunoprecipitation assay using anti-FcpA antiserum demonstrated that FcpA associates with FlaA2 and FlaB1. The association between FcpA and FlaA2 was also verified using pull-down assay. The regions of FlaA2 and FlaB1 interacting with FcpA were determined using a bacterial two-hybrid assay. These results suggest that FcpA together with FlaA2, produces coiling of PF of the Leptospira, and the interaction between the sheath and core filament may be mediated by FcpA and FlaB1.
Project description:Spirochete periplasmic flagella (PFs), including those from Brachyspira (Serpulina), Spirochaeta, Treponema, and Leptospira spp., have a unique structure. In most spirochete species, the periplasmic flagellar filaments consist of a core of at least three proteins (FlaB1, FlaB2, and FlaB3) and a sheath protein (FlaA). Each of these proteins is encoded by a separate gene. Using Brachyspira hyodysenteriae as a model system for analyzing PF function by allelic exchange mutagenesis, we analyzed purified PFs from previously constructed flaA::cat, flaA::kan, and flaB1::kan mutants and newly constructed flaB2::cat and flaB3::cat mutants. We investigated whether any of these mutants had a loss of motility and altered PF structure. As formerly found with flaA::cat, flaA::kan, and flaB1::kan mutants, flaB2::cat and flaB3::cat mutants were still motile, but all were less motile than the wild-type strain, using a swarm-plate assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis indicated that each mutation resulted in the specific loss of the cognate gene product in the assembled purified PFs. Consistent with these results, Northern blot analysis indicated that each flagellar filament gene was monocistronic. In contrast to previous results that analyzed PFs attached to disrupted cells, purified PFs from a flaA::cat mutant were significantly thinner (19.6 nm) than those of the wild-type strain and flaB1::kan, flaB2::cat, and flaB3::cat mutants (24 to 25 nm). These results provide supportive genetic evidence that FlaA forms a sheath around the FlaB core. Using high-magnification dark-field microscopy, we also found that flaA::cat and flaA::kan mutants produced PFs with a smaller helix pitch and helix diameter compared to the wild-type strain and flaB mutants. These results indicate that the interaction of FlaA with the FlaB core impacts periplasmic flagellar helical morphology.
Project description:Most investigators have assumed that the periplasmic flagella (PFs) of Borrelia burgdorferi are composed of only one flagellin protein. The PFs of most other spirochete species are complex: these PFs contain an outer sheath of FlaA proteins and a core filament of FlaB proteins. During an analysis of a chemotaxis gene cluster of B. burgdorferi 212, we were surprised to find a flaA gene homolog with a deduced polypeptide having 54 to 58% similarity to FlaA from other spirochetes. Like other FlaA proteins, B. burgdorferi FlaA has a conserved signal sequence at its N terminus. Based on reverse transcription-PCR and primer extension analysis, this flaA homolog and five chemotaxis genes constitute a motility-chemotaxis operon. Immunoblots using anti-FlaA serum from Treponema pallidum and a lysate of B. burgdorferi showed strong reactivity to a protein of 38.0 kDa, which is consistent with the expression of flaA in growing cells.
Project description:The spirochete endoflagellum is a unique motility apparatus among bacteria. Despite its critical importance for pathogenesis, the full composition of the flagellum remains to be determined. We have recently reported that FcpA is a novel flagellar protein and a major component of the sheath of the filament of the spirochete Leptospira. By screening a library of random transposon mutants in the spirochete Leptospira biflexa, we found a motility-deficient mutant harboring a disruption in a hypothetical gene of unknown function. Here, we show that this gene encodes a surface component of the endoflagellar filament and is required for typical hook- and spiral-shaped ends of the cell body, coiled structure of the endoflagella, and high velocity phenotype. We therefore named the gene fcpB for flagellar-coiling protein B. fcpB is conserved in all members of the Leptospira genus, but not present in other organisms including other spirochetes. Complementation of the fcpB- mutant restored the wild-type morphology and motility phenotypes. Immunoblotting with anti-FcpA and anti-FcpB antisera and cryo-electron microscopy of the filament indicated that FcpB assembled onto the surface of the sheath of the filament and mostly located on the outer (convex) side of the coiled filament. We provide evidence that FcpB, together with FcpA, are Leptospira-specific novel components of the sheath of the filament, key determinants of the coiled and asymmetric structure of the endoflagella and are essential for high velocity. Defining the components of the endoflagella and their functions in these atypical bacteria should greatly enhance our understanding of the mechanisms by which these bacteria produce motility.