Recombinant Chlamydophila caviae Recombination protein RecR (recR)

What is the role of RecR in Chlamydophila caviae's homologous recombination pathways?

RecR is a crucial component of the RecFOR pathway for homologous recombination in C. caviae. It functions as part of a multimeric complex containing RecA, single-stranded DNA binding protein (SSB), RecF, and RecO that mediates the pairing of single-stranded DNA with homologous DNA . This pathway is essential for DNA repair and genetic exchange.

In Chlamydia species including C. caviae, homologous recombination consists of two major pathways - the RecBCD pathway and the RecFOR pathway - both utilizing the RecA protein to facilitate DNA exchange between complementary sequences and single-strand DNA . RecR specifically works in conjunction with RecF and RecO to load RecA onto single-stranded DNA gaps, promoting strand invasion and subsequent recombination events. This machinery is developmentally regulated in Chlamydia species and plays a vital role in their ability to repair DNA damage and potentially acquire genetic diversity.

How can researchers express and purify recombinant C. caviae RecR protein for in vitro studies?

Methodology for expression and purification of recombinant C. caviae RecR typically follows these steps:

• Gene amplification: PCR amplification of the recR gene from C. caviae genomic DNA using specific primers that include appropriate restriction sites.

• Cloning: Insertion of the amplified gene into an expression vector (commonly pET systems) containing an inducible promoter and affinity tag (6xHis is frequently used).

• Expression system: Transformation into E. coli expression strains such as BL21(DE3) that lack certain proteases and contain the T7 RNA polymerase gene.

• Protein expression optimization:

• Purification strategy: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin, followed by size-exclusion chromatography to ensure high purity.

• Protein quality assessment: SDS-PAGE, Western blotting, and mass spectrometry to confirm identity and purity.

• Functional testing: DNA-binding assays and interaction studies with other recombination proteins .

What structural characteristics define the RecR protein in C. caviae?

The RecR protein in C. caviae shares structural similarities with RecR proteins from other bacterial species. Key structural features include:

• Oligomerization domains: RecR typically forms tetramers or dimers in solution, which is critical for its function in DNA repair .

• DNA binding region: Contains motifs that facilitate interaction with DNA, particularly single-stranded DNA gaps.

• Protein interaction interfaces: Specific domains that mediate interactions with RecF and RecO to form the functional RecFOR complex.

• Conserved regions: Sequence analysis shows that chlamydial RecR contains highly conserved amino acid sequences compared to RecR from other bacteria, suggesting evolutionary preservation of critical functional domains .

• Walker motifs: Some RecR proteins contain ATP-binding motifs, though the role of ATP hydrolysis in RecR function may vary between species.

No complete crystal structure of C. caviae RecR has been published to date, but structural predictions based on homology modeling with other bacterial RecR proteins suggest similar domain organization and functional regions.

What experimental approaches can be used to study the interaction between RecR and other recombination proteins in C. caviae?

Several sophisticated approaches can be employed to characterize RecR interactions with RecF, RecO, and other proteins:

• Bacterial two-hybrid systems: Allows for in vivo detection of protein-protein interactions by fusing proteins of interest to separate domains of a transcription factor.

• Co-immunoprecipitation (Co-IP): Using antibodies against RecR or epitope-tagged versions to pull down protein complexes from C. caviae lysates, followed by mass spectrometry to identify interacting partners.

• Surface plasmon resonance (SPR): Quantitative measurement of binding kinetics between purified RecR and other recombination proteins.

• Fluorescence resonance energy transfer (FRET): Tagging RecR and potential partners with fluorescent proteins to visualize interactions in real-time within living cells.

• Crosslinking mass spectrometry: Identifies specific amino acid residues involved in protein-protein interactions.

• Genetic complementation studies: In recombination-deficient strains, testing the ability of mutant RecR constructs to restore recombination function provides insights into critical domains for interaction .

• Chromatin immunoprecipitation sequencing (ChIP-seq): Identifies DNA binding sites of RecR complexes genome-wide.

The challenge with C. caviae is its obligate intracellular nature, requiring careful experimental design that often involves genetic transformation systems as described in recent research .

What methodological approaches are optimal for studying RecR function in the context of C. caviae's obligate intracellular lifestyle?

Studying RecR in obligate intracellular pathogens like C. caviae presents unique challenges requiring specialized approaches:

• Shuttle vector transformation systems: Recent research has successfully established transformation systems for C. caviae using shuttle vectors comprising the cryptic plasmid of C. caviae, the pUC19 origin of replication, beta-lactamase, and genes for heterologous expression of fluorescent proteins . This system provides a foundation for genetic manipulation of C. caviae to study RecR.

• Conditional expression systems: Employing inducible promoters to control RecR expression levels at different stages of the developmental cycle.

• Cell culture infection models: Using guinea pig epithelial cells as the preferred host cell system for C. caviae studies.

• Fluorescent protein tagging: Creating RecR-GFP fusions to track protein localization during the developmental cycle.

• Single-cell analysis techniques: Combining microscopy with cell sorting to study RecR dynamics in individual infected cells.

• CRISPR interference (CRISPRi): While full CRISPR-Cas9 editing remains challenging in Chlamydia, CRISPRi approaches can downregulate RecR expression without complete gene deletion.

• Heterologous expression systems: Expressing C. caviae RecR in more genetically tractable organisms like E. coli or yeast to study specific biochemical properties.

The transformation protocol that successfully works for C. caviae involves a 30-minute incubation in 50 mM CaCl₂ at room temperature, followed by co-incubation with trypsinized cells for 20 minutes (Protocol B) . This provides a methodological foundation for genetic manipulation studies of RecR.

How does RecR contribute to C. caviae's DNA repair mechanisms during stress responses?

RecR's role in DNA repair during stress conditions in C. caviae likely follows these key mechanisms:

• Oxidative stress response: During oxidative damage caused by host immune responses, RecR works with RecF and RecO to load RecA onto damaged DNA sites, facilitating repair of oxidative lesions.

• Replication fork restart: When DNA replication is halted by damage, the RecFOR complex helps to process stalled replication forks and promote their restart.

• SOS response regulation: Although Chlamydia lack a canonical SOS system, RecR likely participates in alternative damage response pathways.

• Development-specific activity: RecR activity may be regulated during the transition between elementary bodies (EBs) and reticulate bodies (RBs) to address different DNA repair needs .

The histone-like protein Hc1 in Chlamydia is involved in the condensation of the chlamydial nucleoid and inhibits RecA activity. Interestingly, Hc1 only inhibits RecA's repair activity but not its recombinational activity . This suggests a complex regulatory mechanism that may influence how RecR functions within the RecFOR pathway during stress responses.

Experimental approaches to study this include:

• Stress induction experiments with oxidative agents, UV radiation, or antibiotics

• Quantitative RT-PCR to measure RecR expression changes during stress

• Immunofluorescence microscopy to track RecR localization during the stress response

• ChIP-seq to identify RecR binding sites across the genome under different stress conditions

What experimental design considerations are critical when investigating recombination hotspots in C. caviae and the role of RecR in these regions?

Investigating recombination hotspots in C. caviae requires careful experimental design:

• Whole-genome sequencing approach: Using next-generation sequencing to identify regions of high sequence diversity that may indicate recombination hotspots .

• Comparative genomics framework: Analysis of multiple C. caviae isolates from diverse geographical and pathological origins to identify polymorphic regions .

• Recombination detection algorithms: Employing specialized software (e.g., RDP4, ClonalFrameML) that can detect recombination events and distinguish them from point mutations.

• Controlled co-infection experiments: Following the methodology established in , researchers can:

  • Create differently antibiotic-resistant strains of C. caviae

  • Co-infect cell cultures with these strains

  • Apply selective pressure to identify recombinants

  • Use whole-genome sequencing to map recombination breakpoints

• Machine learning approaches: As demonstrated in , unsupervised machine learning algorithms can be applied to observe the sequence landscape of recombination proteins across phylogenetic distances.

• RecR binding site analysis: ChIP-seq experiments targeting RecR to identify its binding locations relative to potential recombination hotspots.

• Experimental controls: Include both positive controls (known recombining Chlamydia species) and negative controls to validate experimental systems.

• Statistical analysis plan: Prepare for potential biases in recombination detection by using appropriate statistical methods to distinguish true recombination events from sequencing or assembly artifacts .

For optimal experimental design, researchers should implement randomization, replication, and blocking when applicable to control for extraneous variables that might influence results .

What are the challenges and solutions for expressing functional recombinant C. caviae RecR in heterologous systems for structural studies?

Expression of functional recombinant C. caviae RecR in heterologous systems presents several challenges:

Challenges:

• Protein solubility: RecR may form inclusion bodies when overexpressed in E. coli.

• Proper folding: The intracellular environment of Chlamydia differs from expression hosts, potentially affecting protein folding.

• Post-translational modifications: Potential modifications in native C. caviae may be absent in heterologous systems.

• Oligomerization: RecR typically forms functional tetramers or dimers, which may not assemble correctly in heterologous systems.

• Protein stability: Purified RecR may have limited stability for crystallization or NMR studies.

Solutions and Methodology:

• Expression optimization strategies:

• Refolding protocols: If inclusion bodies form, develop optimized denaturation and refolding protocols using gradual dialysis or on-column refolding.

• Structural biology approaches:

  • X-ray crystallography with various crystallization conditions

  • Cryo-electron microscopy for complex structures

  • NMR for dynamics studies of smaller domains

  • Small-angle X-ray scattering (SAXS) for solution structure

• Functional validation: Develop DNA binding assays and ATPase activity measurements to confirm that recombinant protein maintains native functionality.

• Protein engineering: Create truncated versions or specific point mutations to enhance stability while maintaining functional domains.

These approaches have been successfully applied to other recombination proteins and can be adapted specifically for C. caviae RecR .

How can researchers design experiments to elucidate the potential role of RecR in C. caviae's virulence and host adaptation?

To investigate RecR's potential contributions to C. caviae virulence and host adaptation, researchers can employ these experimental approaches:

• Controlled gene expression studies:

  • Create RecR knockdown strains using antisense RNA or CRISPRi

  • Develop inducible overexpression systems for RecR

  • Measure the impact on growth rates, infectious yield, and developmental cycle progression

• Animal infection models:

  • Utilize the guinea pig ocular infection model, which represents a natural host for C. caviae

  • Compare wild-type and RecR-modified strains for differences in colonization, pathology, and immune response

  • Evaluate the importance of RecR in persistence and reinfection scenarios

• Stress adaptation experiments:

  • Subject C. caviae to various stressors relevant to the host environment (oxidative stress, nutrient limitation, pH changes)

  • Compare survival and adaptation of wild-type and RecR-modified strains

  • Analyze transcriptomic and proteomic changes to identify RecR-dependent responses

• Co-infection dynamics:

  • Utilize the recently developed GFP-expressing C. caviae strain to track bacterial dissemination in vivo

  • Study potential genetic exchange in mixed infections

  • Investigate if RecR influences co-infection dynamics and genetic exchange rates

• Host cell interaction studies:

  • Examine differences in inclusion formation, development, and host cell response between wild-type and RecR-modified strains

  • Investigate potential impacts on immune evasion mechanisms

  • Analyze host transcriptomic responses to identify RecR-dependent pathways

The successful transformation of C. caviae with fluorescent protein markers provides a valuable tool for these investigations, enabling researchers to track bacteria during infection and potentially observe recombination events in real-time . Additionally, analyzing clinical isolates for RecR sequence variations and correlating with virulence phenotypes could provide insights into its role in natural infections .