Recombinant Lactobacillus johnsonii Recombination protein RecR (recR)

What is Lactobacillus johnsonii and why is it significant for recombinant protein expression?

Lactobacillus johnsonii (Lj) is a probiotic bacterium considered safe for human consumption, making it an attractive vehicle for oral vaccination and protein delivery. Research demonstrates that L. johnsonii can partially survive simulated gastric conditions in vitro, a critical characteristic for oral delivery systems. This survival capability suggests its potential effectiveness as a delivery vehicle for various proteins, including those involved in recombination processes . Additionally, L. johnsonii has been investigated for its ability to reduce intestinal pathogenic burdens and alleviate pro-inflammatory immune responses upon infection with pathogens such as C. jejuni, further highlighting its potential benefits in therapeutic applications .

What is the RecR protein and its role in DNA recombination?

The RecR protein is a critical component of the RecFOR pathway in DNA recombination and repair mechanisms. It functions in concert with RecF and RecO proteins to assist RecA protein during homologous recombination. Specifically, these proteins help RecA overcome the interference caused by single-stranded DNA binding proteins (Ssb) during recombination processes . The RecR protein plays a crucial role in stabilizing the interaction between RecF and RecO, forming a complex that modulates the binding of RecA to single-stranded DNA, which is an essential step in the recombination process. Through these interactions, RecR contributes significantly to maintaining genomic integrity by facilitating proper DNA repair and recombination.

How do the RecF, RecO, and RecR proteins interact to facilitate recombination?

The RecF, RecO, and RecR proteins form a complex in a 1:1:1 molar ratio, appearing to be at least heterotrimeric with an approximate molecular weight of 276 kDa . Research using immunoprecipitation, size-exclusion chromatography, and Ssb protein affinity chromatography has revealed several key interactions: RecF interacts directly with RecO; RecF interacts with RecR only in the presence of RecO to form the RecF-RecO-RecR complex; and RecF interacts with Ssb protein in the presence of RecO . These findings suggest that RecO serves as a mediator for the interactions of RecF with both RecR and Ssb proteins. When RecF, RecO, RecR, and Ssb proteins are incubated together, RecF-RecO-Ssb complexes form with RecR being excluded, unless RecF, RecO, and RecR are preincubated before Ssb addition. This sequential assembly mechanism is critical for the proper functioning of the RecFOR pathway in recombination.

What is the molecular mechanism of the RecF-RecO-RecR complex in overcoming Ssb interference?

The RecF-RecO-RecR complex functions as an "anti-Ssb factor" by helping RecA protein displace Ssb from single-stranded DNA, allowing RecA to bind and initiate homologous recombination . The molecular mechanism appears to involve several steps: First, RecO mediates interactions between RecF and Ssb proteins. Then, RecF stabilizes the association of RecR with the RecO-Ssb complex, potentially through direct interaction with RecR or by altering binding strengths between RecO, RecR, and Ssb. Research suggests that ATP plays a regulatory role in this process, as interactions between RecF and RecO are lost in the presence of ATP . This ATP-dependent modulation may facilitate the dynamic assembly and disassembly of the complex during recombination processes.

The complex appears to target gapped DNA substrates where single-stranded regions are bound by Ssb tetramers. Current models suggest that each Ssb tetramer binds approximately 72 nucleotides, which must be replaced by about 24 RecA molecules (each binding ~3 nucleotides) during recombination, highlighting the complexity of this molecular displacement process .

How does ATP influence the stability and function of the RecF-RecO-RecR complex?

ATP has been shown to significantly impact the interactions within the RecF-RecO-RecR complex. Specifically, binding of RecF to ATP decreases the affinity of RecO toward RecF without affecting its affinity toward Ssb . Conversely, removal of ATP, presumably through hydrolysis by the complex, increases the affinity of RecO toward both RecF and RecR while simultaneously decreasing its affinity toward Ssb. This ATP-dependent modulation of protein interactions likely plays a critical role in regulating the function of the RecF-RecO-RecR complex at gap junctions during presynaptic complex formation. The weak ATPase activity of RecF protein, which is stimulated by RecR, may facilitate the turnover of Ssb tetramers during recombination processes .

What experimental evidence supports the role of RecR in the RecF pathway?

Genetic and biochemical evidence strongly supports the critical role of RecR in the RecF pathway of recombination. Studies have demonstrated that recF, recO, and recR genes act at the same presynaptic step in recombination and repair processes . A mutant RecA protein (recA803) acts as a partial phenotype suppressor for recF, recO, and recR mutations, suggesting that these genes collectively assist RecA in overcoming Ssb interference . Additionally, overexpression experiments have shown that recR can partially suppress recF mutations in a recO+-dependent manner, indicating a functional relationship between these proteins. In vitro experiments demonstrate that while RecO and RecR proteins can assist RecA without RecF protein, RecF appears to play an important targeting role, directing the anti-Ssb activity of RecO and RecR proteins to gDNA substrates .

What techniques are used to study RecR protein interactions in vitro?

Several sophisticated biochemical techniques have been employed to study RecR protein interactions in vitro. Immunoprecipitation techniques have revealed direct interactions between RecF and RecO proteins, as well as the formation of more complex assemblies in the presence of additional proteins . Size-exclusion chromatography has been instrumental in determining the stoichiometry and approximate molecular weight of the RecF-RecO-RecR complex, indicating a heterotrimeric structure of about 276 kDa . Ssb protein affinity chromatography has been used to examine interactions with Ssb, demonstrating that RecO mediates the interaction between RecF and Ssb proteins . Additional techniques likely employed in such studies include:

• Surface plasmon resonance (SPR) for measuring binding kinetics

• Fluorescence resonance energy transfer (FRET) for examining spatial relationships

• Analytical ultracentrifugation for determining complex composition

• Isothermal titration calorimetry for measuring binding energetics

How can recombinant L. johnsonii expressing RecR be constructed and verified?

Construction of recombinant L. johnsonii expressing RecR would typically follow a multi-step process similar to that used for other heterologous proteins in this bacterium. Based on established methodologies, researchers would first clone the recR gene into a suitable expression vector designed for Lactobacillus species. The vector system would need to include appropriate promoters, signal sequences for potential surface display, and selection markers .

Verification of RecR expression could be accomplished through:

• Western blotting using antibodies specific to RecR or to an incorporated tag

• Flow cytometry for surface-displayed RecR

• Functional assays measuring RecR's DNA binding or protein interaction capabilities

• Mass spectrometry to confirm protein identity and post-translational modifications

In previous studies with L. johnsonii, researchers successfully expressed proteinase PrtB and a tetanus toxin mimotope-PrtB fusion protein on the bacterial surface using specialized vector systems . Similar approaches could be adapted for RecR expression.

What data management approaches are recommended for RecR protein studies?

Effective data management is crucial for complex studies involving recombinant proteins and their interactions. Based on established research data management principles, a comprehensive data table structure is recommended that captures all relevant experimental parameters and results . Below is an example data table framework specifically designed for RecR protein interaction studies:

This structured approach ensures that all critical data elements are properly documented, facilitating both ongoing research and future reproducibility .

How might recombinant L. johnsonii expressing RecR benefit DNA repair research?

Recombinant L. johnsonii expressing the RecR protein could serve as a valuable tool for DNA repair research by providing a novel delivery system for introducing functional RecR into various experimental models. The ability of L. johnsonii to survive gastrointestinal conditions makes it particularly promising for in vivo studies . Such a system could be used to investigate the impact of RecR supplementation on DNA repair processes in models with deficient recombination pathways, potentially offering insights into rescue mechanisms for cells with compromised DNA repair capabilities.

Additionally, the surface expression of RecR on L. johnsonii could be utilized to study protein-protein interactions in complex cellular environments, complementing the in vitro studies that have revealed the intricate relationships between RecF, RecO, RecR, and Ssb proteins . This approach might help bridge the gap between biochemical understanding and physiological relevance of these interactions.

What are the potential immunological considerations when using L. johnsonii as a delivery vehicle for RecR?

When using L. johnsonii as a delivery vehicle for RecR or any other protein, several immunological factors must be considered. Previous research has shown that recombinant L. johnsonii can induce both systemic IgG responses and local mucosal immune responses (fecal IgA) against expressed proteins . These immunological responses could potentially interfere with the intended function of the delivered protein if neutralizing antibodies are generated.