Generation of a random transposon mutant library is advantageous in Leptospira as site-directed mutagenesis remains a challenge, especially in pathogenic species. This procedure is typically completed by transformation of Leptospira with a Himar1 containing plasmid via conjugation with Escherichia coli as a donor cell. Here we describe the methodology to generate random transposon mutants in the saprophyte Leptospira biflexa via conjugation of plasmid pSW29T-TKS2 harbored in E. coli β2163. Determination of transposon insertion site by semi-random nested PCR will also be described. A similar methodology may be employed to generate Tn mutants of pathogenic Leptospira species.

Spirochetes are a widely existing group of bacteria with a distinct morphology. Some spirochetes are important human pathogens that utilize periplasmic flagella to achieve motility and host infection. The motors that drive the rotation of periplasmic flagella have a unique spirochete-specific feature, termed the collar, crucial for the flat-wave morphology and motility of the Lyme disease spirochete Borrelia burgdorferi. Here, we deploy cryo-electron tomography and subtomogram averaging to determine high-resolution in-situ structures of the B. burgdorferi flagellar motor. Comparative analysis and molecular modeling of in-situ flagellar motor structures from B. burgdorferi mutants lacking each of the known collar proteins (FlcA, FlcB, FlcC, FlbB, and Bb0236/FlcD) uncover a complex protein network at the base of the collar. Importantly, our data suggest that FlbB not only forms a novel periplasmic ring around the rotor but also acts as a scaffold supporting collar assembly and subsequent recruitment of stator complexes. The complex protein network based on the FlbB ring effectively bridges the rotor and 16 torque-generating stator complexes in each flagellar motor, thus contributing to the specialized motility and lifestyle of spirochetes in complex environments.

The PilZ domain-containing protein, PlzA, is the only known cyclic di-GMP binding protein encoded by all Lyme disease spirochetes. PlzA has been implicated in the regulation of many borrelial processes, but the effector mechanism of PlzA was not previously known. Here, we report that PlzA can bind DNA and RNA and that nucleic acid binding requires c-di-GMP, with the affinity of PlzA for nucleic acids increasing as concentrations of c-di-GMP were increased. A mutant PlzA that is incapable of binding c-di-GMP did not bind to any tested nucleic acids. We also determined that PlzA interacts predominantly with the major groove of DNA and that sequence length and G-C content play a role in DNA binding affinity. PlzA is a dual-domain protein with a PilZ-like N-terminal domain linked to a canonical C-terminal PilZ domain. Dissection of the domains demonstrated that the separated N-terminal domain bound nucleic acids independently of c-di-GMP. The C-terminal domain, which includes the c-di-GMP binding motifs, did not bind nucleic acids under any tested conditions. Our data are supported by computational docking, which predicts that c-di-GMP binding at the C-terminal domain stabilizes the overall protein structure and facilitates PlzA-DNA interactions via residues in the N-terminal domain. Based on our data, we propose that levels of c-di-GMP during the various stages of the enzootic life cycle direct PlzA binding to regulatory targets.

The Borrelia burgdorferi SpoVG protein has previously been found to be a DNA- and RNA-binding protein. To aid in the elucidation of ligand motifs, affinities for numerous RNAs, ssDNAs, and dsDNAs were measured and compared. The loci used in the study were spoVG, glpFKD, erpAB, bb0242, flaB, and ospAB, with particular focus on the untranslated 5' portion of the mRNAs. Performing binding and competition assays yielded that the 5' end of spoVG mRNA had the highest affinity while the lowest observed affinity was to the 5' end of flaB mRNA. Mutagenesis studies of spoVG RNA and ssDNA sequences suggested that the formation of SpoVG-nucleic acid complexes are not entirely dependent on either sequence or structure. Additionally, exchanging uracil for thymine in ssDNAs did not affect protein-nucleic acid complex formation.

SignificanceHow flagella sense complex environments and control bacterial motility remain fascinating questions. Here, we deploy cryo-electron tomography to determine in situ structures of the flagellar motor in wild-type and mutant cells of Borrelia burgdorferi, revealing that three flagellar proteins (FliL, MotA, and MotB) form a unique supramolecular complex in situ. Importantly, FliL not only enhances motor function by forming a ring around the stator complex MotA/MotB in its extended, active conformation but also facilitates assembly of the stator complex around the motor. Our in situ data provide insights into how cooperative remodeling of the FliL-stator supramolecular complex helps regulate the collective ion flux and establishes the optimal function of the flagellar motor to guide bacterial motility in various environments.

Spirochetes are a remarkable group of bacteria with distinct morphology and periplasmic flagella that enable motility in viscous environments, such as host connective tissues. The collar, a spirochete-specific complex of the periplasmic flagellum, is required for this unique spirochete motility, yet it has not been clear how the collar assembles and enables spirochetes to transit between complex host environments. Here, we characterize the collar complex in the Lyme disease spirochete Borrelia burgdorferi. We discover as well as delineate the distinct functions of two novel collar proteins, FlcB and FlcC, by combining subtractive bioinformatic, genetic, and cryo-electron tomography approaches. Our high-resolution in situ structures reveal that the multiprotein collar has a remarkable structural plasticity essential not only for assembly of flagellar motors in the highly curved membrane of spirochetes but also for generation of the high torque necessary for spirochete motility. IMPORTANCE Many spirochetes cause serious human diseases. They are well recognized by their distinct morphology and motility. Spirochete motility is driven by a periplasmic flagellum, which possesses a unique collar essential for flagellar assembly and spirochete motility. Here, we discover two novel collar proteins in the Lyme disease spirochete Borrelia burgdorferi. We demonstrate, for the first time, that the collar is a multiprotein complex with a remarkable plasticity that enables the motor to accommodate the highly curved membrane of spirochetes and generate the high torque necessary for spirochete motility.

Assembly of the bacterial flagellar rod, hook, and filament requires penetration through the peptidoglycan (PG) sacculus and outer membrane. In most β- and γ-proteobacteria, the protein FlgJ has two functional domains that enable PG hydrolyzing activity to create pores, facilitating proper assembly of the flagellar rod. However, two distinct proteins performing the same functions as the dual-domain FlgJ are proposed in δ- and ε-proteobacteria as well as spirochetes. The Lyme disease spirochete Borrelia burgdorferi genome possesses a FlgJ and a PG lytic SLT enzyme protein homolog (BB0259). FlgJ in B. burgdorferi is crucial for flagellar hook and filament assembly but not for the proper rod assembly reported in other bacteria. However, BB0259 has never been characterized. Here, we use cryo-electron tomography to visualize periplasmic flagella in different bb0259 mutant strains and provide evidence that the E580 residue of BB0259 is essential for PG-hydrolyzing activity. Without the enzyme activity, the flagellar hook fails to penetrate through the pores in the cell wall to complete assembly of an intact periplasmic flagellum. Given that FlgJ and BB0259 interact with each other, they likely coordinate the penetration through the PG sacculus and assembly of a functional flagellum in B. burgdorferi and other spirochetes. Because of its role, we renamed BB0259 as flagellar-specific lytic transglycosylase or LTaseBb.

Borrelia burgdorferi is a highly motile spirochete due to its periplasmic flagella. Unlike flagella of other bacteria, spirochetes' periplasmic flagella possess a complex structure called the collar, about which little is known in terms of function and composition. Using various approaches, we have identified a novel protein, BB0326, as a key component of the collar. We show that a peripheral portion of the collar is diminished in the Δbb0326 mutant and restored in the complemented bb0326+ cells, leading us to rename BB0326 as periplasmic flagellar collar protein A or FlcA. The ΔflcA mutant cells produced fewer, abnormally tilted and shorter flagella, as well as diminished stators, suggesting that FlcA is crucial for flagellar and stator assemblies. We provide further evidence that FlcA interacts with the stator and that this collar–stator interaction is essential for the high torque needed to power the spirochete's periplasmic flagellar motors. These observations suggest that the collar provides various important functions to the spirochete's periplasmic flagellar assembly and rotation.

The bacterial flagellar motor can rotate in counterclockwise (CCW) or clockwise (CW) senses, and transitions are controlled by the phosphorylated form of the response regulator CheY (CheY-P). To dissect the mechanism underlying flagellar rotational switching, we use Borrelia burgdorferi as a model system to determine high-resolution in situ motor structures in cheX and cheY3 mutants, in which motors are locked in either CCW or CW rotation. The structures showed that CheY3-P interacts directly with a switch protein, FliM, inducing a major remodeling of another switch protein, FliG2, and altering its interaction with the torque generator. Our findings lead to a model in which the torque generator rotates in response to an inward flow of H + driven by the proton motive force, and conformational changes in FliG2 driven by CheY3-P allow the switch complex to interact with opposite sides of the rotating torque generator, facilitating rotational switching.

The bacterial flagellar motor is a molecular machine that rotates the flagellar filament at high speed. Torque is generated by the stator-rotor interaction coupled to an ion flux through the torque-generating stator. Here, we employed cryo-electron tomography to visualize the intact flagellar motor in the Lyme disease spirochete Borrelia burgdorferi . By analysis of the motor structures of wild-type and stator mutants, we localize the torque-generating units precisely and determine three-dimensional structure of the stator and its interactions with the rotor. Our study shows that the cytoplasmic domains of the stator are packed regularly around the circumference of the flagellar C-ring. The stator-rotor interaction induces a profound conformational change in the C-ring. Analysis of the motors of a less motile motB- D24E mutant and a non-motile motB- D24N mutant, in which the proton translocation is reduced and blocked, respectively, provides evidence that the conformational change of the C-ring is essential for flagellar rotation.

The bacterial flagellar motor is a molecular machine that can rotate the flagellar filament at high speed. The rotation is generated by the stator–rotor interaction, coupled with an ion flux through the torque-generating stator. Here we employed cryo-electron tomography to visualize the intact flagellar motor in the Lyme disease spirochete, Borrelia burgdorferi. By analyzing the motor structures of wild-type and stator-deletion mutants, we not only localized the stator complex in situ, but also revealed the stator–rotor interaction at an unprecedented detail. Importantly, the stator–rotor interaction induces a conformational change in the flagella C-ring. Given our observation that a non-motile mutant, in which proton flux is blocked, cannot generate the similar conformational change, we propose that the proton-driven torque is responsible for the conformational change required for flagellar rotation.

SummarySpirochetes possess a unique periplasmic flagellar motor component called the collar. However, little is known about the composition or function of the flagellar collar proteins. To identify a collar protein, we have inactivated almost all genes annotated as motility‐related in the Borrelia burgdorferi genome and identified only FlbB, which comprises the base of the collar. Since the major components of the collar complex remained unidentified, we took advantage of a protein–protein interaction map developed in another spirochete, Treponema pallidum to identify proteins of unknown function that could be collar proteins. Subsequently, using various comprehensive approaches, we identified a tetratricopeptide repeat protein BB0236 as a potential candidate for the collar. Biochemical assays indicated that FlbB interacts with BB0236. Furthermore, ∆bb0236 mutant analyses indicated that BB0236 is crucial for collar structure assembly, cellular morphology, motility, orientation of periplasmic flagella and assembly of other flagellar structures. Moreover, using comparative motor analyses, we propose how the collar structure is assembled in B. burgdorferi. Together, our studies provide new insights into the organization and the complex assembly inherent to the unique spirochetal collar structure.

Periplasmic flagella are essential for the distinct morphology and motility of spirochetes. A flagella-specific type III secretion system (fT3SS) composed of a membrane-bound export apparatus and a cytosolic ATPase complex is responsible for the assembly of the periplasmic flagella. Here, we deployed cryo-electron tomography (cryo-ET) to visualize the fT3SS machine in the Lyme disease spirochete Borrelia burgdorferi. We show, for the first time, that the cytosolic ATPase complex is attached to the flagellar C-ring through multiple spokes to form the "spoke and hub" structure in B. burgdorferi. This structure not only strengthens structural rigidity of the round-shaped C-ring but also appears to rotate with the C-ring. Our studies provide structural insights into the unique mechanisms underlying assembly and rotation of the periplasmic flagella and may provide the basis for the development of novel therapeutic strategies against several pathogenic spirochetes.

Borrelia burgdorferi, the causative agent of Lyme disease, is a highly motile spirochete, and motility, which is provided by its periplasmic flagella, is critical for every part of the spirochete's enzootic life cycle. Unlike externally flagellated bacteria, spirochetes possess a unique periplasmic flagellar structure called the collar. This spirochete-specific novel component is linked to the flagellar basal body; however, nothing is known about the proteins encoding the collar or their function in any spirochete. To identify a collar protein and determine its function, we employed a comprehensive strategy that included genetic, biochemical, and microscopic analyses. We found that BB0286 (FlbB) is a novel flagellar motor protein, which is located around the flagellar basal body. Deletion of bb0286 has a profound effect on collar formation, assembly of other flagellar structures, morphology, and motility of the spirochete. Orientation of the flagella toward the cell body is critical for determination of wild-type spirochete's wave-like morphology and motility. Here, we provide the first evidence that FlbB is a key determinant of normal orientation of the flagella and collar assembly.

Borrelia burgdorferi possesses a sophisticated and complex chemotaxis system, but how the organism utilizes this system in its natural enzootic life cycle is poorly understood. Of the three CheY chemotaxis response regulators in B. burgdorferi, we found that only deletion of cheY3 resulted in an altered motility and significantly reduced chemotaxis phenotype. Although ΔcheY3 maintained normal densities in unfed ticks, their numbers were significantly reduced in fed ticks compared with the parental or cheY3-complemented spirochetes. Importantly, mice fed upon by the ΔcheY3-infected ticks did not develop a persistent infection. Intravital confocal microscopy analyses discovered that the ΔcheY3 spirochetes were motile within skin, but appeared unable to reverse direction and perform the characteristic backward-forward motility displayed by the parental strain. Subsequently, the ΔcheY3 became 'trapped' in the skin matrix within days of inoculation, were cleared from the skin needle-inoculation site within 96 h post-injection and did not disseminate to distant tissues. Interestingly, although ΔcheY3 cells were cleared within 96 h post-injection, this attenuated infection elicited significant levels of B. burgdorferi-specific IgM and IgG. Taken together, these data demonstrate that cheY3-mediated chemotaxis is crucial for motility, dissemination and viability of the spirochete both within and between mice and ticks.

Two-thirds of all bacterial genomes sequenced to-date possess an organelle for locomotion, referred to as flagella, periplasmic flagella or type IV pili. These genomes may also contain a chemotaxis-signaling system which governs flagellar rotation, thus leading a coordinated function for motility. Motility and chemotaxis are often crucial for infection or disease process caused by pathogenic bacteria. Although motility-associated genes are well-characterized in some organisms, the highly orchestrated synthesis, regulation, and assembly of periplasmic flagella in spirochetes are just being delineated. Recent advances were fostered by development of unique genetic manipulations in spirochetes coupled with cutting-edge imaging techniques. These contemporary advances in understanding the role of spirochetal motility and chemotaxis in host persistence and disease development are highlighted in this review.

Type III secretion systems (T3SS) are widely used by Gram-negative bacteria to assemble flagella required for locomotion and to transport virulence effectors into eukaryotic cells [1]. The central component of these systems is the Type III secretion apparatus, which consists of a group of integral membrane proteins and three soluble proteins include a T3SS associated ATPase. Powered by proton motive force and ATP, the secretion apparatus plays essential role in substrates recognition and export. However, the structure and function of the intact secretion apparatus remain poorly understood at the molecular level.

In nature, the Lyme disease spirochete Borrelia burgdorferi cycles between the unrelated environments of the Ixodes tick vector and mammalian host. In order to survive transmission between hosts, B. burgdorferi must be able to not only detect changes in its environment, but also rapidly and appropriately respond to these changes. One manner in which this obligate parasite regulates and adapts to its changing environment is through cyclic-di-GMP (c-di-GMP) signaling. c-di-GMP has been shown to be instrumental in orchestrating the adaptation of B. burgdorferi to the tick environment. B. burgdorferi possesses only one set of c-di-GMP-metabolizing genes (one diguanylate cyclase and two distinct phosphodiesterases) and one c-di-GMP-binding PilZ-domain protein designated as PlzA. While studies in the realm of c-di-GMP signaling in B. burgdorferi have exploded in the last few years, there are still many more questions than answers. Elucidation of the importance of c-di-GMP signaling to B. burgdorferi may lead to the identification of mechanisms that are critical for the survival of B. burgdorferi in the tick phase of the enzootic cycle as well as potentially delineate a role (if any) c-di-GMP may play in the transmission and virulence of B. burgdorferi during the enzootic cycle, thereby enabling the development of effective drugs for the prevention and/or treatment of Lyme disease.