Recombinant Chlamydophila caviae Holliday junction ATP-dependent DNA helicase RuvB (ruvB)

What is the function of RuvB in Chlamydophila caviae?

RuvB in C. caviae functions as an ATP-dependent DNA helicase that catalyzes branch migration of Holliday junctions during homologous recombination. This protein plays a crucial role in DNA repair and genetic exchange mechanisms. Similar to other bacterial RuvB proteins, C. caviae RuvB works in conjunction with RuvA to recognize and process Holliday junctions, which are essential intermediates in homologous recombination. The RuvB protein provides the motor force for branch migration while RuvA provides specificity by binding to the Holliday junction structure . This molecular machinery is critical for maintaining genomic integrity in C. caviae and facilitating genetic diversity through recombination events.

How does the quaternary structure of RuvB relate to its function?

RuvB assembles into a spiral staircase-like hexameric ring that encircles double-stranded DNA. This architectural arrangement is fundamental to its function as a molecular motor that drives Holliday junction migration. Recent cryo-EM structural studies reveal that four of the six protomers in the RuvB hexamer directly contact the DNA backbone, with a translocation step size of 2 nucleotides . The asymmetric assembly of the RuvB hexamer explains the 6:4 stoichiometry observed in the RuvB/RuvA complex that coordinates Holliday junction migration in bacteria . This structural configuration enables the sequential hydrolysis of ATP at distinct positions within the hexamer, generating the mechanical force required to propel the DNA strands through the complex during branch migration.

What is known about the genomic context of the ruvB gene in C. caviae?

The ruvB gene is part of the core genome of Chlamydophila caviae, which consists of 1,173,390 nucleotides with an additional plasmid of 7,966 nucleotides . The gene is preserved among chlamydial species as part of the essential DNA repair and recombination machinery. In C. caviae, as in other Chlamydia species, the presence of the ruv genes indicates functional RecBCD and RecFOR pathways involved in the formation and resolution of Holliday junctions . The genomic stability of the ruvB gene across Chlamydiaceae highlights its evolutionary importance, despite the significant genomic plasticity observed in other regions of these bacterial genomes, particularly in the replication termination region (RTR).

What expression systems are most effective for producing recombinant C. caviae RuvB protein?

For recombinant expression of C. caviae RuvB, E. coli-based systems utilizing pET vectors with T7 promoters have proven most effective. When expressing RuvB, consider these methodological approaches:

• Expression optimization:

  • Use BL21(DE3) or Rosetta(DE3) strains to address codon bias

  • Induce with 0.5-1.0 mM IPTG at OD600 0.6-0.8

  • Lower induction temperature to 18-25°C for 16-18 hours to enhance solubility

• Purification strategy:

  • Employ a dual-tag approach (His6 and MBP tags) for enhanced purity

  • Include 5-10% glycerol and 1-5 mM ATP in all buffers to stabilize the hexameric structure

  • Use size exclusion chromatography as a final purification step to isolate homogeneous hexamers

The primary challenge in RuvB expression is maintaining the protein in its native oligomeric state, which is essential for functional studies. ATP presence in purification buffers is critical as it promotes stable hexamer formation and prevents aggregation that commonly occurs with this motor protein.

How can researchers effectively assay the ATP-dependent branch migration activity of RuvB?

To assay the ATP-dependent branch migration activity of RuvB, researchers should implement a multi-faceted approach:

Holliday Junction Branch Migration Assay:

• Substrate preparation:

  • Construct synthetic Holliday junctions using four oligonucleotides (60-80 nucleotides each)

  • Label one strand with a fluorophore and its complementary strand with a quencher

  • Anneal to form mobile Holliday junctions

• Reaction conditions:

  • Buffer: 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 50 mM NaCl

  • ATP concentration: 1-5 mM

  • RuvB concentration: 50-200 nM (hexamer)

  • RuvA concentration (if included): 50-200 nM (tetramer)

• Detection methods:

  • Real-time fluorescence measurement as branch migration brings fluorophore and quencher into proximity

  • Gel-based assays using 10% native PAGE to visualize branch migration products

  • Stopped-flow kinetic analysis for measuring initial rates

ATP-dependent activity can be confirmed by comparing results with non-hydrolyzable ATP analogs (ATPγS) and ADP controls. This methodology allows quantitative assessment of RuvB function under various conditions, enabling detailed mechanistic studies .

What approaches can be used to study RuvB-RuvA interactions in C. caviae?

To investigate RuvB-RuvA interactions in C. caviae, researchers can employ these complementary methodologies:

Biochemical interaction studies:

• Co-immunoprecipitation using antibodies against either RuvA or RuvB

• Pull-down assays using recombinant tagged proteins

• Surface plasmon resonance to determine binding kinetics and affinities

• Analytical ultracentrifugation to characterize complex formation

Structural approaches:

• Cryo-electron microscopy of RuvAB-Holliday junction complexes

• X-ray crystallography of co-crystallized components

• Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

Functional assays:

• Branch migration assays comparing RuvB alone versus RuvAB complex

• ATP hydrolysis assays to measure how RuvA modulates RuvB ATPase activity

• DNA binding assays using electrophoretic mobility shift assays

Research has demonstrated that RuvA significantly enhances the specificity of RuvB for Holliday junctions, reducing the required concentration of RuvB by approximately 50-fold . This interaction is critical for proper targeting of the branch migration machinery to recombination intermediates, preventing non-specific interaction of RuvB with other DNA structures.

How does the sequential ATP hydrolysis mechanism in RuvB contribute to branch migration directionality?

The sequential ATP hydrolysis mechanism in RuvB hexamers creates a coordinated power stroke that drives unidirectional branch migration. Based on recent structural studies, the process involves:

• Nucleotide state progression:

  • The hexameric RuvB exhibits different nucleotide-binding states among its subunits

  • ATP binding, hydrolysis, and release occur at distinct, singular positions within the hexamer

  • This creates a "sequential model" where each subunit progresses through the ATP cycle in a defined order

• Conformational coupling:

  • ATP binding induces conformational changes that strengthen DNA contacts

  • Hydrolysis triggers a power stroke that moves DNA by 2 nucleotides

  • ADP release resets the subunit for the next cycle

• Coordinated action:

  • The asymmetric assembly ensures that only one subunit hydrolyzes ATP at a time

  • This prevents futile ATP consumption and maintains directional movement

This highly coordinated mechanism ensures processive branch migration with minimal energy expenditure. The directionality appears to be reinforced by the architecture of the RuvAB complex, where RuvA binding to the Holliday junction orients the RuvB hexamers to promote branch migration in a specific direction .

What is known about the differences in RuvB function between C. caviae and other Chlamydiaceae?

While the core function of RuvB is conserved across Chlamydiaceae, several species-specific differences have been observed:

The differences in recombination rates may reflect ecological adaptations and suggest that while the basic RuvB mechanism is conserved, regulatory elements or interaction partners might vary between species. Interestingly, cross-species recombination studies indicate that fusogenic inclusions form between many Chlamydia species, but not between Chlamydia and Chlamydophila caviae, suggesting potential differences in recombination machinery functionality or compatibility .

How does the RuvB mechanism bypass DNA lesions during recombinational repair?

RuvB's ability to bypass DNA lesions during recombinational repair represents a critical function for maintaining genomic integrity. This process involves:

• Lesion recognition and processing:

  • When replication encounters DNA damage (e.g., UV-induced thymine dimers), replication fork collapse can occur

  • RecA-mediated strand exchange forms Holliday junctions at these sites

  • RuvAB complexes are recruited to these junctions

• Bypass mechanism:

  • RuvB provides sufficient ATP-driven mechanical force to push the Holliday junction through damaged DNA regions

  • The hexameric motor can generate enough torque to overcome structural distortions caused by lesions

  • This enables branch migration to continue despite the presence of DNA damage

• Coordination with repair processes:

  • As RuvAB migrates the junction past the lesion, it creates an opportunity for other repair proteins to access the damage

  • The resolution of the Holliday junction by RuvC completes the repair process

This lesion bypass capability is particularly important in obligate intracellular bacteria like C. caviae, which have limited DNA repair pathways compared to free-living bacteria. The RuvAB complex thus serves as a critical component of the DNA damage tolerance mechanisms in these organisms, allowing for genome maintenance despite environmental stresses and replication errors .

How does horizontal gene transfer influence RuvB function across Chlamydia species?

Horizontal gene transfer (HGT) has significant implications for RuvB function across Chlamydia species, although the ruvB gene itself appears to be relatively conserved as part of the core genome. The influence occurs primarily through:

• Conservation patterns:

  • Core recombination machinery genes like ruvB show high conservation across Chlamydiaceae

  • This suggests strong selective pressure to maintain RuvB function despite HGT events in other genomic regions

• Species-specific recombination rates:

  • Studies have demonstrated variable recombination rates across Chlamydia species

  • C. psittaci, C. pneumoniae, and C. suis exhibit higher recombination rates than C. trachomatis lineages

  • These differences may reflect variation in RuvB regulation or interaction partners rather than in the core enzyme itself

• Impact on genomic context:

  • The replication termination region (RTR or plasticity zone) is a hotspot for genome variation and HGT events

  • While ruvB is not located in this region, the genomic context in which RuvB operates varies significantly between species

  • For example, C. caviae contains unique genes not found in other chlamydial species, including toxin genes and bacteriophage insertions

Interestingly, laboratory experiments have demonstrated that horizontal gene transfer can occur between some Chlamydia species but not between Chlamydophila caviae and other chlamydial species. This suggests potential incompatibilities in the recombination machinery or DNA recognition systems that may influence how RuvB functions in interspecies contexts .

What role does RuvB play in the acquisition of antibiotic resistance in Chlamydia species?

RuvB plays a critical though indirect role in the acquisition of antibiotic resistance in Chlamydia species by facilitating the homologous recombination events necessary for horizontal gene transfer of resistance determinants. This process involves:

• Resistance acquisition mechanisms:

  • Laboratory experiments have demonstrated transfer of tetracycline resistance (tet(C)) between C. suis and other chlamydial species including C. trachomatis and C. muridarum

  • These transfers occurred through two distinct mechanisms:
    a) Integration of a 40-kb fragment into a single ribosomal operon creating a merodiploid structure (in C. trachomatis)
    b) Classical double-crossover event transferring 99 kb of DNA (in C. muridarum)

• RuvB's mechanistic contribution:

  • As the motor protein driving Holliday junction migration, RuvB is essential for completing recombination events that incorporate resistance genes

  • The efficiency of RuvB-mediated branch migration likely influences the frequency of successful resistance gene acquisition

  • RuvB's ability to process Holliday junctions formed during co-infection with multiple strains enables genetic exchange

• Species-specific variation:

  • While no stable tetracycline resistance has been reported in clinical C. trachomatis strains, it was successfully transferred in laboratory settings

  • The inability to establish recombination between Chlamydophila caviae and other chlamydial species suggests molecular barriers that may limit the spread of resistance to C. caviae

This research has significant public health implications, suggesting that antibiotic resistance genes could potentially spread among human chlamydial pathogens through RuvB-mediated recombination, though such events have not yet been documented in clinical settings .

How do the RuvAB and RecA pathways interact in Chlamydia species?

The interaction between RuvAB and RecA pathways in Chlamydia species represents a sophisticated coordination system for DNA repair and recombination:

• Functional relationship:

  • RecA mediates the initial strand invasion and pairing steps of homologous recombination

  • RuvAB acts downstream of RecA, processing the Holliday junctions formed during RecA-mediated recombination

  • Experimental evidence shows that purified RuvA and RuvB stimulate RecA-mediated strand exchange and heteroduplex DNA formation

• Mechanistic handoff:

  • RecA forms a nucleoprotein filament on single-stranded DNA and catalyzes strand invasion

  • This creates a Holliday junction intermediate

  • RuvA recognizes and binds to this structure

  • RuvB is recruited by RuvA to provide motor function for branch migration

  • The process does not involve direct protein-protein interaction between RecA and RuvAB; rather, RuvAB acts directly on the DNA structures created by RecA

• Chlamydia-specific characteristics:

  • Chlamydial RecA shows moderate recombinational activity compared to other bacteria

  • It possesses low efficiency in response to DNA damage by UV radiation

  • The histone-like protein Hc1 in Chlamydia inhibits RecA's repair activity but interestingly does not inhibit its recombinational function

  • This selective inhibition may represent a specialization that balances genomic stability with beneficial recombination events

The coordinated action of these pathways is essential for maintaining genomic integrity in Chlamydia species, which as obligate intracellular bacteria have limited DNA repair mechanisms compared to free-living bacteria .

What are common problems in purifying active recombinant C. caviae RuvB, and how can they be addressed?

Researchers frequently encounter several challenges when purifying active recombinant C. caviae RuvB. Here are the most common issues and their solutions:

• Protein aggregation:

  • Problem: RuvB tends to form non-functional aggregates during expression and purification

  • Solutions:
    a) Add 5-10% glycerol to all purification buffers
    b) Maintain 1-5 mM ATP in all buffers to stabilize the hexameric form
    c) Keep temperature at 4°C throughout purification
    d) Consider fusion partners like MBP that enhance solubility

• Low ATPase activity:

  • Problem: Purified RuvB shows minimal ATP hydrolysis

  • Solutions:
    a) Verify protein folding using circular dichroism
    b) Test activity in the presence of RuvA and Holliday junction DNA
    c) Ensure Mg²⁺ is present at 5-10 mM concentration
    d) Check for inhibitory contaminants using activity assays with varying protein concentrations

• Incomplete hexamer formation:

  • Problem: RuvB fails to assemble into functional hexamers

  • Solutions:
    a) Use analytical size exclusion chromatography to assess oligomeric state
    b) Pre-incubate with ATP before activity assays
    c) Try varying salt concentrations (50-200 mM NaCl) to optimize assembly
    d) Consider crosslinking approaches to stabilize hexamers

• Expression toxicity in E. coli:

  • Problem: RuvB expression is toxic to host cells

  • Solutions:
    a) Use tight expression control with T7-lac or arabinose-inducible systems
    b) Lower induction temperature to 16-18°C
    c) Reduce inducer concentration
    d) Try specialized E. coli strains like C41(DE3) designed for toxic protein expression

By addressing these common issues methodically, researchers can significantly improve the yield and activity of recombinant C. caviae RuvB preparations, enabling more reliable biochemical and structural studies.

How can researchers distinguish between RuvB-specific effects and other recombination factors in experimental systems?

Distinguishing RuvB-specific effects from those of other recombination factors requires careful experimental design and appropriate controls:

• Genetic approaches:

  • Generate conditional ruvB mutants/knockdowns when possible

  • Compare with mutations in other recombination genes (recA, ruvA, ruvC)

  • Use complementation studies with wild-type and mutant alleles

  • Create point mutations in key functional domains (Walker A/B motifs) to specifically disrupt ATPase activity

• Biochemical separation:

  • Purify individual components for in vitro reconstitution experiments

  • Conduct order-of-addition experiments to determine sequence of action

  • Use specific inhibitors when available:
    a) ATPase inhibitors for RuvB
    b) DNA binding competitors for RuvA
    c) Nuclease inhibitors for RuvC

• Specific activity assays:

  • Branch migration assays for RuvB/RuvAB

  • Junction resolution assays for RuvC

  • DNA pairing assays for RecA

  • Comparative analysis of rates and efficiencies

• Control experiments:

  • Use ATPase-deficient RuvB mutants (K68A equivalent) as negative controls

  • Compare activity with and without RuvA

  • Test on various DNA substrates (Holliday junctions vs. linear DNA)

  • Include non-chlamydial RuvB proteins as reference standards

By implementing these strategies, researchers can isolate RuvB-specific activities and accurately attribute observed effects to the appropriate components of the recombination machinery, avoiding misinterpretation of experimental results caused by overlapping functions or contaminating activities.

What are the best approaches for studying RuvB in the context of intact Chlamydia given their obligate intracellular lifestyle?

Studying RuvB in intact Chlamydia presents unique challenges due to their obligate intracellular lifestyle. Here are optimal approaches:

• Genetic manipulation strategies:

  • Transformation with recombinant plasmids based on the pBBR1 vector system, which has been shown to work in Chlamydia

  • Use of chemical mutagenesis followed by selection for specific phenotypes

  • CRISPR/Cas9 adaptation for chlamydial systems

  • Conditional expression systems using tetracycline-inducible promoters

  • Fluorescent protein tagging of RuvB for localization studies

• Co-infection approaches:

  • Utilize co-infection of mammalian cells with different chlamydial strains to study recombination

  • Create selectable markers (such as antibiotic resistance) to isolate recombinants

  • Use fluorescently labeled strains to visualize fusion of inclusions during co-infection

  • Employ PCR-based detection methods to identify recombination junctions

• Microscopy techniques:

  • Immunofluorescence using anti-RuvB antibodies to track protein localization during infection cycle

  • Super-resolution microscopy to visualize RuvB-DNA interactions

  • Live-cell imaging with fluorescently tagged RuvB to monitor dynamics

  • Correlative light and electron microscopy to link function to ultrastructure

• Specialized culture models:

  • Polarized epithelial cell models that better mimic natural infection sites

  • Long-term infection models to study recombination under persistent infection conditions

  • Cell lines expressing fluorescent DNA damage markers to correlate with RuvB activity

  • Co-culture systems allowing for genetic exchange monitoring

These approaches enable the study of RuvB function within its natural biological context while overcoming the limitations imposed by Chlamydia's obligate intracellular lifestyle and historically limited genetic tractability. Recent advances in chlamydial genetics, particularly the development of transformation systems using vectors like pBVR1 (based on pBBR1 from Bordetella pertussis), have significantly expanded the available toolkit for such studies .

What are promising directions for structural studies of C. caviae RuvB?

Several promising directions for structural studies of C. caviae RuvB could significantly advance our understanding of this molecular motor:

• High-resolution cryo-EM analysis:

  • Capture RuvB in different nucleotide-bound states to visualize the conformational changes during ATP hydrolysis cycle

  • Determine structures of complete RuvAB-Holliday junction complexes at sub-3Å resolution

  • Visualize the structural transitions during branch migration using time-resolved cryo-EM

• Single-molecule structural techniques:

  • Implement FRET-based approaches to monitor conformational changes in real-time

  • Apply atomic force microscopy to visualize RuvB hexamer assembly on DNA

  • Use DNA origami platforms to control the spatial arrangement of RuvB substrates

• Comparative structural biology:

  • Determine structures of RuvB from multiple Chlamydia species to identify species-specific features

  • Map structural elements that prevent recombination between C. caviae and other Chlamydia species

  • Identify potential interaction interfaces with species-specific partners

• Functional structural biology:

  • Develop methods to visualize RuvB dynamics within chlamydial inclusions

  • Create fluorescent biosensors to monitor RuvB activity in vivo

  • Apply correlative light and electron microscopy to link structural observations to functional outcomes in infected cells

These approaches would provide crucial insights into how RuvB's structure determines its function in DNA recombination and repair, potentially revealing novel aspects of chlamydial biology and pathogenesis. The resulting structural information could also guide the development of specific inhibitors for basic research and potentially therapeutic applications.

How might studying RuvB advance our understanding of chlamydial evolution and pathogenesis?

Studying RuvB provides a unique window into chlamydial evolution and pathogenesis through several interconnected mechanisms:

• Evolutionary insights:

  • RuvB's role in recombination directly influences genetic diversity and adaptation

  • Comparative analysis of RuvB across Chlamydiaceae can reveal evolutionary relationships and selective pressures

  • The varying recombination rates observed in different species (high in C. pneumoniae and C. suis, variable in C. trachomatis) suggest adaptation to different ecological niches

  • Understanding how recombination machinery contributed to the acquisition of virulence factors could explain pathogenic divergence

• Genomic plasticity mechanisms:

  • RuvB-mediated recombination contributes to the variability seen in the replication termination region (RTR)

  • This plasticity zone contains numerous virulence-associated genes, including toxins in C. caviae

  • The presence of bacteriophage insertions and toxin genes specific to C. caviae suggests RuvB may have facilitated ancient horizontal gene transfer events

• Host adaptation:

  • Recombination rates appear correlated with host range and tissue tropism

  • For example, C. trachomatis strains show differing recombination patterns: ocular strains show less recombination than urogenital strains, while the LGV lineage shows minimal recombination

  • These differences may reflect how recombination machinery, including RuvB, has been optimized for different host environments

• Potential therapeutic targets:

  • As an essential component of DNA repair, RuvB could represent a novel target for anti-chlamydial therapeutics

  • Inhibitors that disrupt RuvB function might sensitize Chlamydia to DNA-damaging agents

  • Understanding species-specific differences in RuvB could enable targeted approaches against particular chlamydial pathogens

By exploring these aspects of RuvB biology, researchers can gain insight into the fundamental processes that have shaped chlamydial genomes and continue to influence their evolution and interaction with hosts. This knowledge could ultimately lead to new strategies for controlling chlamydial infections and mitigating their impact on human and animal health.

What techniques could enable real-time visualization of RuvB-mediated branch migration in living cells?

• Fluorescent protein fusions and biosensors:

  • CRISPR-based tagging of endogenous RuvB with split fluorescent proteins

  • Development of conformational biosensors that change FRET efficiency upon RuvB activation

  • ATPase activity sensors that generate fluorescent signals upon ATP hydrolysis

  • Engineered Holliday junction structures with fluorophore-quencher pairs that separate during branch migration

• Advanced microscopy techniques:

  • Lattice light-sheet microscopy to reduce phototoxicity while achieving high spatiotemporal resolution

  • Super-resolution approaches like PALM/STORM to overcome diffraction limits

  • 4D imaging (3D + time) with deconvolution to track dynamic processes

  • Fluorescence correlation spectroscopy to measure RuvB diffusion and binding kinetics

• Substrate visualization strategies:

  • Fluorescent DNA intercalators that preferentially bind Holliday junctions

  • Sequence-specific fluorescent probes that track branch migration progress

  • DNA damage-inducible fluorescent reporters to monitor recombination events

  • Quantum dot-labeled DNA substrates introduced via cell-penetrating peptides

• Hybrid approaches for chlamydial systems:

  • Cell-free extracts from infected cells to reconstitute activity in controlled settings

  • Permeabilized cell systems that maintain inclusion integrity while allowing substrate access

  • Microinjection of fluorescent substrates into inclusions

  • Optogenetic control of recombination initiation to synchronize events for imaging

Implementation of these techniques would require significant adaptation for the unique challenges of the chlamydial inclusion environment. Success would provide unprecedented insights into the spatial and temporal dynamics of RuvB activity during the chlamydial developmental cycle, potentially revealing how recombination contributes to chlamydial adaptation and pathogenesis. Such approaches could also elucidate whether RuvB-mediated processes occur throughout the developmental cycle or are confined to specific stages, such as during RB division or stress response.

What are the key takeaways about C. caviae RuvB for researchers entering this field?

For researchers entering the field of C. caviae RuvB study, several key takeaways should guide initial experimental design and interpretation:

• Fundamental properties:

  • RuvB functions as an ATP-dependent DNA helicase that drives Holliday junction branch migration

  • It assembles into a hexameric ring structure that encircles DNA

  • The motor activity requires ATP hydrolysis and works in concert with RuvA for specificity

  • Four of the six protomers contact DNA with a translocation step size of 2 nucleotides

• Evolutionary context:

  • RuvB is part of the core genome conserved across Chlamydiaceae

  • Despite this conservation, recombination rates vary significantly between chlamydial species

  • C. caviae contains unique genomic elements including toxin genes and bacteriophage insertions not present in other species

  • Recombination appears to be incompatible between C. caviae and other Chlamydia species, suggesting molecular barriers

• Experimental considerations:

  • Expressing active recombinant RuvB requires careful optimization to maintain hexameric structure

  • Activity assays should include both RuvA and appropriate DNA substrates

  • ATP and magnesium are essential cofactors for activity

  • The obligate intracellular lifestyle of Chlamydia presents unique challenges for in vivo studies

• Relevance to chlamydial biology:

  • RuvB-mediated recombination contributes to genomic plasticity, particularly in the replication termination region

  • The recombination machinery facilitates horizontal gene transfer, including antibiotic resistance genes

  • Understanding RuvB function provides insights into chlamydial evolution and adaptation

These foundational concepts provide a starting point for researchers to develop sophisticated hypotheses and experimental approaches to further our understanding of this important molecular motor in chlamydial biology.

How does understanding C. caviae RuvB inform broader questions in molecular microbiology?

Understanding C. caviae RuvB contributes to broader questions in molecular microbiology through several conceptual frameworks:

• Evolution of molecular motors:

  • The RuvB hexameric ATPase represents a model system for understanding how ATP hydrolysis is converted to mechanical force

  • The sequential ATP hydrolysis mechanism observed in RuvB illustrates principles likely shared by other hexameric ATPases across diverse bacterial species

  • Comparing RuvB across species reveals how molecular motors adapt to different genomic contexts while maintaining core functionality

• DNA repair and recombination across bacterial phyla:

  • The chlamydial RecBCD and RecFOR pathways demonstrate how bacteria with reduced genomes maintain essential DNA repair functions

  • Understanding how obligate intracellular bacteria manage genomic integrity provides insights into minimal requirements for bacterial survival

  • The RuvABC system illustrates conservation of core DNA repair machinery despite significant genomic streamlining in obligate intracellular bacteria

• Horizontal gene transfer mechanisms:

  • RuvB's role in facilitating recombination between chlamydial species demonstrates principles of horizontal gene transfer relevant to bacterial evolution broadly

  • The barriers preventing recombination between C. caviae and other chlamydial species highlight molecular mechanisms that maintain species boundaries

  • These insights inform our understanding of bacterial speciation and the spread of antibiotic resistance

• Structure-function relationships in enzymes:

  • The asymmetric assembly of RuvB and its sequential ATP hydrolysis provide a model for studying allostery in multi-subunit enzymes

  • The ability to bypass DNA lesions demonstrates how molecular motors overcome structural impediments

  • These principles extend to diverse molecular machines across all domains of life

By examining C. caviae RuvB through these conceptual lenses, researchers can connect specific findings to fundamental principles in molecular microbiology, enhancing our understanding of both chlamydial biology and broader biological processes.

What technical advances would most benefit research on C. caviae RuvB function?

Several technical advances would substantially accelerate research on C. caviae RuvB function:

• Improved genetic manipulation systems:

  • Development of more efficient transformation protocols specifically for C. caviae

  • Refinement of CRISPR/Cas9 systems adapted for chlamydial biology

  • Creation of conditional knockdown/knockout systems for essential genes like ruvB

  • Site-specific recombination systems for precise genomic integration

• Advanced structural biology techniques:

  • Time-resolved cryo-EM to capture transient states during ATP hydrolysis and branch migration

  • Single-particle approaches to study heterogeneity in RuvAB-Holliday junction complexes

  • Integrative structural biology combining multiple techniques (X-ray crystallography, NMR, SAXS, cryo-EM)

  • Computational methods to model conformational changes during the reaction cycle

• Single-molecule biophysics:

  • Optical and magnetic tweezers optimized for studying RuvB motor function

  • Single-molecule FRET to monitor conformational changes during catalysis

  • High-throughput single-molecule approaches to gather statistically robust datasets

  • Microfluidic platforms for precise control of reaction conditions

• Cell biology innovations:

  • Improved host cell systems that better mimic natural infection sites

  • Non-invasive methods to deliver substrates and probes into chlamydial inclusions

  • Long-term live-cell imaging systems compatible with chlamydial developmental cycle

  • Methods to isolate and analyze inclusion contents without disrupting ongoing processes