Recombinant Coxiella burnetii Recombination protein RecR (recR)

What is the function of RecR protein in Coxiella burnetii?

RecR in C. burnetii is a critical component of DNA repair and recombination machinery, functioning in a RecF-like recombination pathway. Research indicates that RecR proteins typically form a complex with RecO to initiate the loading of RecA onto DNA during repair processes . This function is essential for maintaining genomic integrity, particularly in the harsh phagolysosomal environment where C. burnetii replicates within macrophages .

Unlike many bacteria that employ the RecBCD pathway for DNA repair, C. burnetii lacks this system and instead relies on the RecFOR pathway along with the AddAB system . The RecR protein is particularly important in this context as it helps the pathogen survive in oxidative stress conditions encountered during intracellular growth.

How does the genomic context of recR differ in C. burnetii compared to other bacteria?

In Escherichia coli, the recR gene is part of the dnaX-orf12-recR operon, with the translation of recR coupled to orf12 . In contrast, the genomic organization in C. burnetii differs. According to genomic data, recR is identified as gene CBU_0658 in the C. burnetii Nine Mile phase I RSA493 genome . While C. burnetii's recO is part of the rnc-era-recO operon (similar to arrangements in E. coli, Salmonella typhimurium, and Pseudomonas aeruginosa), the recR gene appears to have a distinct genomic context .

This different genomic organization may reflect adaptations in the DNA repair systems of C. burnetii that contribute to its survival within the macrophage environment.

What regulatory mechanisms control recR expression in C. burnetii?

C. burnetii possesses a unique regulatory system for DNA repair genes. Unlike E. coli and many other bacteria, C. burnetii lacks the LexA repressor that typically regulates SOS response genes . This results in constitutive expression of SOS genes, including DNA repair machinery components.

Additionally, while specific data on recR regulation is limited, research shows that AddAB (functionally equivalent to RecBCD) expression is strongly upregulated under oxidative stress conditions in C. burnetii . This suggests that components of the RecFOR pathway, including RecR, might also be regulated in response to environmental stressors, particularly those encountered within the phagolysosomal compartment where the bacterium replicates.

This constitutive SOS expression coupled with stress-inducible DNA repair systems appears to be a unique adaptation that allows C. burnetii to maintain genomic integrity within hostile host environments.

How does the absence of RecBCD in C. burnetii affect RecR function?

C. burnetii lacks the RecBCD pathway found in many bacteria, instead possessing AddAB orthologous genes as functional equivalents . This alteration in DNA repair machinery likely means that RecR has adapted to work efficiently within this modified system.

The functional significance of this adaptation is demonstrated by the fact that C. burnetii AddAB can restore survival after UV, mitomycin C, or methyl methanesulfonate treatment in E. coli RecBCD deletion strains . This suggests that the AddAB system works cooperatively with the RecFOR pathway, potentially elevating the importance of RecR in C. burnetii.

The constitutive expression of SOS genes due to the lack of LexA repressor, coupled with damage-inducible AddAB, creates a unique environment for RecR function that differs fundamentally from more well-studied bacterial systems. This adaptation likely helps C. burnetii maintain genomic integrity in the face of continuous DNA damage within macrophages.

What experimental evidence supports RecR's role in DNA repair in C. burnetii?

While direct experimental evidence specifically for C. burnetii RecR is limited in the provided sources, several findings support its critical role:

• Genomic presence: RecR (CBU_0658) is maintained in the C. burnetii genome despite the organism's reductive evolution as an obligate intracellular pathogen .

• Pathway conservation: C. burnetii possesses a complete set of genes involved in the RecFOR pathway, including recF, recO, and recR, suggesting their functional importance .

• Functional conservation: The RecFOR pathway is known to be involved in the resumption of replication at DNA breaks , a function that would be critical for C. burnetii survival within the hostile phagolysosomal environment.

• Complementation studies: While not specific to RecR, similar proteins like RecA and AddAB from C. burnetii can functionally complement corresponding mutations in E. coli , suggesting conservation of DNA repair mechanisms.

How can the RecR-RecO-RecA interaction be studied in C. burnetii?

Studying the interactions between RecR, RecO, and RecA in C. burnetii requires specialized approaches due to the organism's obligate intracellular lifestyle. Several methodologies can be employed:

Table 1: Methodologies for studying RecR protein interactions

For C. burnetii specifically, recombinant expression of these proteins using systems like the RTS 100 E. coli HY kit has been successful for other C. burnetii proteins . The expressed proteins can then be used in biochemical assays to study their interactions and functions.

What is the significance of RecR in C. burnetii pathogenesis?

RecR likely plays a crucial role in C. burnetii pathogenesis through its contribution to DNA repair and genomic integrity maintenance:

• Intracellular survival: C. burnetii replicates within a parasitophorous vacuole with lysosomal characteristics , exposing it to DNA-damaging agents including reactive oxygen species. RecR, as part of the RecFOR pathway, helps maintain genomic integrity under these stressful conditions.

• Adaptation to oxidative stress: The unique DNA repair adaptations of C. burnetii, including constitutive SOS expression and stress-inducible repair systems, appear specifically evolved for survival within macrophages . RecR is an integral component of these adaptation mechanisms.

• Chronic infection maintenance: C. burnetii can establish persistent infections that may lead to chronic Q fever . The ability to maintain genomic integrity over extended periods within host cells likely depends on efficient DNA repair systems including RecR.

• Virulence modulation: While not directly addressed in the search results, disruption of DNA repair pathways often attenuates virulence in intracellular pathogens by compromising their ability to respond to host-generated stresses.

What is the optimal protocol for expressing and purifying recombinant C. burnetii RecR protein?

Based on successful approaches for other C. burnetii proteins, the following protocol can be adapted for RecR:

Expression system options:

• Cell-free expression: The RTS 100 E. coli HY kit has been successfully used for small-scale expression of C. burnetii proteins, while the RTS 500 ProteoMaster E. coli system is suitable for large-scale production .

• Vector construction: The recR gene (CBU_0658) can be amplified from C. burnetii genomic DNA using PCR with gene-specific primers. The pIVEX2.4d vector with an N-terminal 6-histidine tag has been successfully used for C. burnetii proteins .

• Purification method: Ni-NTA magnetic agarose beads under native conditions can be used for purification . For larger preparations, column chromatography with immobilized metal affinity, followed by ion exchange and size exclusion chromatography might be necessary.

Recommended protocol steps:

• Amplify the recR gene (CBU_0658) from C. burnetii genomic DNA

• Clone into pIVEX2.4d or similar expression vector

• Verify the construct by sequencing

• Express using the RTS cell-free system

• Purify using Ni-NTA under native conditions

• Store in buffer containing 25% glycerol at -80°C

How can the DNA binding activity of C. burnetii RecR be assessed in vitro?

Several complementary approaches can be used to characterize the DNA binding activity of recombinant C. burnetii RecR:

Electrophoretic Mobility Shift Assay (EMSA):

• Prepare various DNA substrates (single-stranded, double-stranded, gapped, etc.)

• Incubate with increasing concentrations of purified RecR

• Analyze complex formation by native gel electrophoresis

• Include controls with RecO and RecF to assess cooperative binding

Fluorescence Anisotropy:

• Use fluorescently labeled DNA substrates

• Measure changes in rotational diffusion upon RecR binding

• Determine binding constants under various conditions

• Test the effects of RecO and other potential partners on binding kinetics

DNA Protection Assays:

• Pre-incubate DNA with RecR

• Challenge with nucleases (DNase I, ExoIII, etc.)

• Analyze protected fragments to determine binding sites and modes

When interpreting results, it's important to consider that RecR typically functions as part of a multiprotein complex, so its activity alone may differ from its physiological function.

What approaches can be used to study RecR function in C. burnetii's intracellular environment?

Studying RecR function within C. burnetii's natural intracellular environment presents unique challenges but can be approached through several methods:

• Genetic manipulation: Although challenging due to C. burnetii's obligate intracellular lifestyle, transposon mutagenesis or targeted gene disruption of recR could reveal its importance in intracellular survival and replication .

• Inducible expression systems: Developing systems for controlled expression of wild-type or mutant recR could help understand its function.

• Infection models: Comparing the intracellular growth of wild-type and recR-deficient C. burnetii in macrophage cell lines or animal models could reveal its importance in pathogenesis.

• Stress response analysis: Exposing infected cells to DNA-damaging agents and monitoring bacterial survival could highlight RecR's role in damage resistance.

• Transcriptomics/proteomics: Analyzing gene/protein expression changes in response to DNA damage could reveal networks involving RecR.

• Fluorescent tagging: Creating RecR fusion proteins with fluorescent tags could allow visualization of its localization during intracellular replication and in response to stress.

How do recombinant RecR protein assays compare with whole-cell experimental approaches?

Both recombinant protein assays and whole-cell approaches offer complementary insights into RecR function:

Table 2: Comparison of experimental approaches for studying RecR

What techniques can be used to identify substrates and partners of RecR in C. burnetii?

Several complementary techniques can be employed to identify the substrates and interaction partners of C. burnetii RecR:

• Affinity purification coupled with mass spectrometry: Using His-tagged RecR as bait to pull down interacting proteins from C. burnetii lysates.

• DNA-protein crosslinking: To identify DNA sequences bound by RecR in vivo.

• Yeast two-hybrid or bacterial two-hybrid screening: To systematically identify protein interaction partners.

• Co-immunoprecipitation: Using antibodies against RecR to pull down interacting partners from infected cell lysates.

• Chromatin immunoprecipitation sequencing (ChIP-seq): To map genome-wide binding sites of RecR, though this would require antibodies or tagged versions of RecR.

• Protein microarrays: Testing RecR binding against arrays of C. burnetii proteins to identify interaction partners.

• Biochemical assays with candidate partners: Testing interactions with predicted partners based on homology to other bacterial systems (RecO, RecF, SSB, RecA).

For C. burnetii specifically, combining in vitro approaches with the proteins expressed using systems like those described for other C. burnetii proteins with targeted validation in infection models would likely yield the most comprehensive understanding.

What are the emerging research questions regarding RecR in C. burnetii?

Several important research questions remain to be explored:

• Structure-function relationships: Determining the three-dimensional structure of C. burnetii RecR and how it differs from other bacterial RecR proteins.

• Regulatory mechanisms: Understanding how recR expression is regulated in the absence of LexA and whether it responds to specific stressors encountered within host cells.

• Role in virulence: Determining whether RecR is essential for C. burnetii pathogenesis and chronic infection.

• Interaction with AddAB: Exploring how RecR functions in relation to the AddAB system rather than the RecBCD system found in other bacteria.

• Potential as a therapeutic target: Investigating whether inhibition of RecR function could attenuate C. burnetii infections.

• Evolutionary adaptations: Understanding how RecR has evolved specifically for C. burnetii's intracellular lifestyle.

• Role in antigenic variation: Investigating whether RecR contributes to genetic diversity and antigenic variation in C. burnetii populations.

How might RecR contribute to C. burnetii's adaptation to diverse host environments?

RecR likely plays a crucial role in C. burnetii's adaptation to diverse host environments through several mechanisms:

• Maintaining genomic integrity: By facilitating DNA repair under various stress conditions encountered across different hosts (humans, livestock) .

• Supporting persistent infection: Contributing to the bacteria's ability to establish chronic infections by ensuring genomic stability over extended periods .

• Adapting to oxidative stress: Helping manage DNA damage from varying levels of oxidative stress encountered in different host cell types .

• Environmental persistence: Contributing to survival in harsh environmental conditions where C. burnetii can persist between hosts .

• Response to treatment pressures: Potentially contributing to recombination events that could influence adaptation to antibiotic treatments.

The unique DNA repair systems of C. burnetii, including the RecFOR pathway, appear to be specifically adapted for survival within diverse host environments, making RecR an important component of the pathogen's success across multiple species.

Could RecR be a potential target for therapeutic intervention against C. burnetii infections?

RecR represents a potentially interesting therapeutic target for several reasons:

• Essential function: DNA repair is likely essential for C. burnetii survival, particularly within the hostile phagolysosomal environment.

• Unique adaptations: C. burnetii's DNA repair systems show specific adaptations different from many other bacteria, potentially allowing selective targeting.

• Intracellular targeting: Inhibitors could potentially be designed to accumulate within the parasitophorous vacuole where C. burnetii replicates.

• Reduced resistance development: Targeting DNA repair may reduce the bacteria's ability to develop resistance mutations.

Challenges to this approach include:

• Developing compounds that can reach the bacteria within its specialized intracellular niche

• Ensuring specificity to avoid affecting human DNA repair systems

• Demonstrating efficacy in chronic infection models where current treatments often fail