Recombinant Bartonella henselae Protein RecA (recA)

What are the primary functions of RecA in B. henselae?

RecA in B. henselae serves as a central component of the homologous recombination machinery . Its primary functions likely include:

• Catalyzing DNA strand exchange during homologous recombination

• Participating in DNA repair processes, particularly following damage

• Potentially regulating cellular responses to DNA damage, although the complete SOS system present in E. coli may differ in B. henselae

• Contributing to genetic diversity through facilitation of recombination events, which may be particularly important given the observed genetic microdiversity among B. henselae strains

How conserved is the RecA protein among Bartonella species and other bacteria?

RecA proteins are highly conserved across bacterial species, typically showing sequence similarity of 70-80% or higher . While specific comparative data for Bartonella species RecA is limited, the core functional domains are likely preserved across the genus. Researchers should consider that despite this conservation, species-specific variations may exist, particularly in regions involved in protein-protein interactions or regulatory functions. For comparison, H. pylori RecA shows approximately 62% identity with E. coli RecA, while maintaining its essential functions .

Does B. henselae RecA undergo post-translational modifications similar to H. pylori RecA?

Based on studies of H. pylori RecA, which undergoes post-translational modifications affecting its electrophoretic mobility and function, B. henselae RecA may experience similar modifications . In H. pylori:

• RecA migrates at an apparent size of 40 kDa when expressed in its native host but at 38 kDa when expressed in E. coli

• These modifications are linked to genes involved in glycosylation and lipopolysaccharide biosynthesis

• Site-directed mutagenesis of a putative glycosylation site (T189M) results in unmodified RecA protein

• The modifications are necessary for full RecA function in DNA repair

Researchers studying B. henselae RecA should investigate whether similar modifications occur and how they might impact protein function.

How is recA expression regulated in B. henselae during stress conditions?

While specific information on B. henselae recA regulation is limited, researchers should consider that:

• In many bacteria, recA expression is upregulated in response to DNA damage as part of the SOS response

• H. pylori recA is cotranscribed with a downstream enolase gene (eno), and mutations in eno affect bacterial sensitivity to DNA-damaging agents

• The regulation of recA expression in B. henselae may involve unique mechanisms adapted to its lifestyle as an intracellular pathogen

Experimental approaches should include measuring recA expression using qPCR or RNA-seq following exposure to various stressors, including DNA-damaging agents, oxidative stress, and host cell interaction.

What are the optimal conditions for expressing and purifying recombinant B. henselae RecA?

Based on approaches used for RecA proteins from other bacterial species, researchers should consider:

Expression system optimization:

• E. coli BL21(DE3) or similar strains are typically suitable for RecA expression

• Expression vectors with N-terminal or C-terminal His-tags facilitate purification

• Lower induction temperatures (16-25°C) may improve protein solubility

• IPTG concentrations of 0.1-0.5 mM are typically sufficient

Purification protocol:

• Affinity chromatography using Ni-NTA resin for His-tagged RecA

• Ion exchange chromatography as a secondary purification step

• Size exclusion chromatography for highest purity

• Verify protein identity by Western blot and/or mass spectrometry

Researchers should be aware that RecA expressed in E. coli may lack post-translational modifications that occur in the native host, potentially affecting its properties .

How can I assess the DNA-binding and ATPase activities of purified B. henselae RecA?

Functional characterization of RecA typically includes:

DNA-binding assays:

• Electrophoretic mobility shift assays (EMSA) with single-stranded DNA

• Fluorescence anisotropy with fluorescently labeled oligonucleotides

ATPase activity assays:

• Malachite green assay to detect released phosphate

• Coupled enzyme assays linking ATP hydrolysis to NADH oxidation

Strand exchange assays:

• Prepare homologous single-stranded and double-stranded DNA substrates

• Incubate with RecA in the presence of ATP

• Analyze products by gel electrophoresis

What strategies can be employed to create and verify recA knockout mutants in B. henselae?

Creating recA knockout mutants presents challenges due to RecA's essential role in DNA repair. Approaches include:

Knockout strategies:

• Allelic exchange using suicide vectors carrying antibiotic resistance markers

• CRISPR-Cas9 based genome editing for precise modifications

• Conditional knockouts if complete deletion proves lethal

Verification methods:

• PCR confirmation of the genetic modification

• Western blot analysis to confirm absence of the protein

• Phenotypic testing, particularly increased sensitivity to DNA-damaging agents

• Complementation studies to confirm phenotypes are specifically due to recA deletion

How does B. henselae RecA compare functionally to RecA proteins from other pathogens?

Comparative studies between RecA proteins from different bacterial species can provide insights into species-specific adaptations:

Researchers should consider expressing recombinant RecA from multiple species under identical conditions to directly compare their biochemical properties and activities.

What is the potential of B. henselae RecA as a diagnostic target compared to other B. henselae proteins?

Current diagnostic approaches for B. henselae infection focus on other proteins like Pap31 . Comparative considerations include:

Researchers should evaluate RecA's antigenicity using sera from infected patients and compare results with established targets like Pap31.

How might RecA function contribute to the genetic diversity observed in B. henselae populations?

RecA's role in homologous recombination makes it a potential contributor to B. henselae genetic diversity:

• B. henselae shows an unusually high degree of microdiversity among strains generated by homologous recombination

• RecA facilitates the integration of foreign DNA acquired through horizontal gene transfer

• This recombination activity may contribute to strain variability and adaptation to different hosts

• Post-translational modifications of RecA might fine-tune recombination rates or substrate specificity

Research approaches might include analyzing recombination frequencies in strains with wild-type versus modified RecA proteins.

How does DNA damage affect the interaction between RecA and other DNA repair proteins in B. henselae?

Investigation of RecA's interaction network should consider:

• Identifying proteins that co-immunoprecipitate with RecA before and after DNA damage

• Determining whether post-translational modifications affect these protein-protein interactions

• Examining the localization of RecA within the bacterial cell during normal growth and stress conditions

• Comparing the DNA repair protein network in B. henselae with that of better-characterized bacteria

What role might RecA play in B. henselae response to antibiotic treatment?

Given RecA's importance in DNA repair, it likely influences bacterial responses to antibiotics that damage DNA:

• RecA-dependent DNA repair may contribute to survival following exposure to fluoroquinolones or other DNA-targeting antibiotics

• The lack of a canonical SOS response in B. henselae may result in different RecA-mediated responses compared to E. coli

• Inhibition of RecA function could potentially sensitize B. henselae to certain antibiotics

Experimental designs should include exposing wild-type and recA-deficient B. henselae to various antibiotics and measuring survival, mutation rates, and development of resistance.

How might recombinant chimeric proteins incorporating RecA epitopes enhance diagnostic or vaccine development for B. henselae?

Drawing from research on chimeric proteins for B. henselae diagnosis :

• Chimeric proteins combining epitopes from multiple B. henselae antigens have shown promise for diagnostic applications

• Recombinant chimeric proteins demonstrated high specificity but lower sensitivity in serological tests

• Integration of RecA epitopes into chimeric constructs could potentially enhance diagnostic performance

• In silico epitope prediction tools can identify immunogenic regions of RecA for inclusion in chimeric constructs

Researchers developing such constructs should validate their performance against gold standard tests like IFA, which remains the reference method for Bartonella detection .