Biology subjects

Botting, J. M.

Publications and source records attributed to Botting, J. M..

7 recordsLinked to original sources

bioRxiv2026

Asymmetric architecture and adaptation of Treponema flagella

Spirochetes exhibit a distinctive corkscrew-like motility driven by periplasmic flagella that wrap around the cell body in a supercoiled configuration, yet the structural basis of this unique propulsion remains poorly understood. Here we combine cryo-electron microscopy, cryo-electron tomography, and genetic and biochemical analyses to determine the assembly and adaptation principles of the supercoiled flagellar filament in Treponema denticola, a major periodontal pathogen. Near-atomic structures reveal a glycosylated FlaB flagellin core encased by a previously unrecognized asymmetric sheath. The major sheath protein FlaA forms the bulk of the sheath and mechanically couples to the core through defined interfaces required for efficient motility, whereas four minor sheath proteins (FlaA1, FlaA2, FlaAP1, and FlaAP2) assemble along the concave side of the filament to accommodate intrinsic curvature. Disruption of this asymmetric core-sheath organization compromises force transmission and impairs motility, establishing coordinated asymmetric assembly as a fundamental mechanism underlying spirochetal motility.

molecular biology↗

bioRxiv2025

Assembly and glycosylation of Helicobacter pylori sheathed flagella

The bacterial flagellum is a complex nanomachine essential for motility, colonization, and invasion in diverse species. Helicobacter pylori has evolved elaborate sheathed flagella that enable migration through the highly viscous gastric mucus layer to reach its colonization niche on the gastric epithelium, yet the molecular basis for these unique adaptations has remained elusive. Here, we use in-situ single particle cryo-electron microscopy to determine near-atomic structures of the flagellar filament within the membranous sheath of H. pylori. The major flagellin FlaA constitutes the bulk of the filament, whereas the minor flagellin FlaB contributes critically to the hook-proximal region. Both FlaA and FlaB form a conserved core surrounded by variable surface-exposed domains. Our structures further reveal that pseudaminic-acid glycans decorate these domains, where they mediate inter- and intra-subunit contacts that stabilize the filament and confer a negatively charged surface. Together, these findings support a model in which the filament rotates independently of the membranous sheath to drive H. pylori motility and provide a molecular framework for understanding how the sheathed flagellum enables colonization and persistence within the gastric niche. Significance StatementWe present the first in-situ near-atomic structure of the sheathed flagellar filament in Helicobacter pylori, revealing distinctive adaptations that underpin the pathogens unique motility and persistent infection. Our in-situ structures show that the two flagellins, FlaA and FlaB, assemble into an extended and exceptionally stable filament through an extensive hydrogen-bonding network. Pseudaminic acid glycans decorate the surface-exposed domains, where they stabilize inter-subunit packing and render the surface negatively charged and hydrophilic. These findings, which provide insight into the assembly of the flagellar filament and its relationship to the surrounding sheath, provide a structural framework for developing strategies to disrupt H. pylori motility and infection.

microbiology↗

bioRxiv2025

Near-atomic in-situ architecture and membrane-coupled dynamics of the Vibrio cholerae sheathed flagellum

The sheathed flagellum of Vibrio cholerae is a self-assembling membranous organelle that must coordinate axial assembly, sheath biogenesis, and rapid motor rotation. Here, we determine in-situ near-atomic structures of the sheathed flagellar motor inside intact cells. The motor anchors to the outer membrane through lipidated HL-rings without forming a membrane pore, thereby allowing axial assembly to drive sheath formation. Conserved LP-rings act as slide-rotary bushings that permit high-speed rotation within a dynamic envelope yet can constrict to seal the pore upon stress-induced ejection. We further show that stator activation requires a specific PomB-MotX interaction rather than peptidoglycan engagement. Together, these findings reveal how the distinctive architecture and dynamics of the sheathed flagellum promote V. cholerae motility, environmental survival, and persistent colonization of the human gut.

microbiology↗

bioRxiv2025

In-situ structures of the Legionella Dot/Icm T4SS identify the DotA-IcmX complex as the gatekeeper for effector translocation

The Dot/Icm machine in Legionella pneumophila is one of the most versatile type IV secretion systems (T4SSs), with a remarkable capacity to translocate over 330 different effector proteins across the bacterial envelope into host cells. At least 27 Dot and Icm proteins are required for assembly and function of the system, yet the architecture and activation mechanism remain poorly understood at the molecular level. Here, we deploy cryo-electron microscopy to reveal in-situ structures of the Dot/Icm machine at near-atomic resolution. Importantly, two proteins essential for effector translocation, DotA and IcmX, form a pentameric protochannel at an inactive state. Upon activation, the DotA-IcmX protochannel undergoes extensive rearrangements to form an extended transenvelope passage capable of transporting effector proteins from the bacterial cytoplasm into host cells as revealed by cryo-electron tomography. Collectively, our findings suggest that the DotA-IcmX complex functions as the gatekeeper for effector translocation of the Dot/Icm T4SS.

molecular biology↗

bioRxiv2025

The architecture, assembly, and evolution of a complex flagellar motor

Bacterial flagella drive motility in many species, likely including the last bacterial common ancestor 1,2. Knowledge of flagellar assembly and function has mainly come from studies of Escherichia coli and Salmonella enterica, which have simple flagellar motors 3-7. However, most flagellated bacteria possess complex motors with unique, species-specific adaptations whose mechanisms and evolution remain largely unexplored 8-10. Here, we deploy a multidisciplinary approach to build a near-complete model of the flagellar motor in Campylobacter jejuni, revealing its remarkable complexity in architecture and composition. We identify an E-ring around the MS-ring, a periplasmic cage with two distinctive conformations, and an intricate interaction network between the E-ring and cage. These scaffolds play critical roles in stabilizing and regulating 17 torque-generating stator complexes for optimal motility. In-depth evolutionary analyses uncover the ancient origin and prevalence of the E-ring in flagellated species of the domain Bacteria as well as a unique exaptation of type IV pili components PilMNOPQF in the ancestral motor of the phylum Campylobacterota. Collectively, our studies reveal novel mechanisms of assembly and function in complex flagellar motors and shed light on the evolution of flagella and modern bacterial species.

microbiology↗

bioRxiv2024

Saccharibacteria deploy two distinct Type IV pili, driving episymbiosis, host competition, and twitching motility

All cultivated Patescibacteria, or CPR, exist as obligate episymbionts on other microbes. Despite being ubiquitous in mammals and environmentally, molecular mechanisms of host identification and binding amongst ultrasmall bacterial episymbionts are largely unknown. Type 4 pili (T4P) are well conserved in this group and predicted to facilitate symbiotic interactions. To test this, we targeted T4P pilin genes in Saccharibacteria Nanosynbacter lyticus strain TM7x to assess their essentiality and roles in symbiosis. Our results revealed that N. lyticus assembles two distinct T4P, a non-essential thin pili that has the smallest diameter of any T4P and contributes to host-binding, episymbiont growth, and competitive fitness relative to other Saccharibacteria, and an essential thick pili whose functions include twitching motility. Identification of lectin-like minor pilins and modification of host cell walls suggest glycan binding mechanisms. Collectively our findings demonstrate that Saccharibacteria encode unique extracellular pili that are vital mediators of their underexplored episymbiotic lifestyle.

microbiology↗

bioRxiv2022

Helicobacter pylori FlgV forms a flagellar motor ring structure required for optimal motility

The bacterium Helicobacter pylori has a large flagellar motor that generates significantly higher torque than the archetypical Escherichia coli motor. To understand how H. pylori navigates the viscous environment of the stomach, it is essential to establish how specific motor components contribute to efficient motility. We show here that the protein FlgV, required for motility in Campylobacter jejuni, forms a novel ring associated with the MS and C rings in H. pylori. Deletion of flgV from H. pylori B128 or a highly motile variant of H. pylori G27 (G27M) resulted in reduced motility in soft agar medium. Based on comparative analyses of in-situ flagellar motor structures of H. pylori wild-type and {Delta}flgV mutants, the reduced motility of the {Delta}flgV mutants and the location of the FlgV ring suggest it stabilizes interactions between the MS and C rings and/or plays a role in switching the direction of flagellar rotation. Overall, these results identify a novel motor accessory likely adapted to promote flagellar function for bacterial colonization of high-load environments such as the gastric mucosa.

microbiology↗