Recombinant Bdellovibrio bacteriovorus Recombination protein RecR (recR)

What is RecR protein and what is its significance in B. bacteriovorus?

RecR is a DNA recombination protein that functions as part of the RecFOR pathway, which is critical for DNA repair and homologous recombination in bacteria. In B. bacteriovorus, RecR likely plays an essential role in maintaining genomic integrity during its predatory lifecycle, particularly during the intensive DNA replication that occurs inside prey bacteria. The predatory lifecycle of B. bacteriovorus involves attack-phase cells entering other Gram-negative bacteria, growing filamentously in the prey periplasm, and septating after about 4 hours . This unique lifestyle likely places special demands on DNA repair and recombination systems.

How does RecR function in relation to other recombination proteins?

RecR typically functions in conjunction with RecF and RecO to facilitate loading of RecA onto single-stranded DNA. In B. bacteriovorus, RecA (Bd0512) has been shown to be highly upregulated throughout periplasmic predation, with fold changes >2 for T2h-T5h, peaking in abundance at T3h and T4h . This suggests intensive DNA recombination activity during the predatory cycle. RecR likely works in coordination with RecA, potentially modulating its activity and facilitating strand exchange during the recombination process that occurs during the predatory lifecycle.

What experimental approaches are recommended for studying RecR in B. bacteriovorus?

For studying RecR in B. bacteriovorus, researchers should consider:

• Fluorescent protein tagging, similar to methods used for studying MreB in B. bacteriovorus (e.g., using monomeric teal-fluorescent protein)

• Protein expression analysis throughout the predatory cycle using quantitative proteomics techniques

• Comparative genomic analysis with RecR proteins from non-predatory bacteria

• In vitro assays with recombinant protein to study biochemical properties

• Gene manipulation techniques, considering that essential genes in B. bacteriovorus must be studied through partial inactivation methods rather than complete knockouts

How might expression and activity of RecR vary during the B. bacteriovorus predatory cycle?

Based on quantitative proteomics data from B. bacteriovorus predation, we can infer potential RecR activity patterns:

During the T3h-T4h period when intensive DNA replication and cell division occur, RecR would likely work with RecA to facilitate DNA recombination events necessary for chromosome replication and repair.

What are the structural and functional differences between RecR in B. bacteriovorus and RecR in non-predatory bacteria?

While specific structural data for B. bacteriovorus RecR is not available in the provided sources, comparative analysis suggests potential adaptations to its predatory lifestyle:

• RecR in B. bacteriovorus may have adapted to function during unique growth phases, including filamentous growth within prey periplasm and rapid septation

• The protein may have specialized regulation to accommodate the condensed predatory lifecycle with its intensive DNA replication phase

• Potential structural modifications could exist to coordinate with B. bacteriovorus-specific versions of RecF and RecO

• Unique protein-protein interactions might have evolved to function within the predatory context

A crystal structure determination, similar to the approach used for the bacterial histone HBb (Bd0055) from B. bacteriovorus , would be valuable for identifying any structural adaptations specific to this predatory bacterium.

How does RecR contribute to genome stability during the unique life cycle of B. bacteriovorus?

The predatory lifestyle of B. bacteriovorus presents several challenges to genome stability:

• Rapid entry into prey cells requiring morphological changes

• Filamentous growth within prey periplasm

• Intensive DNA replication to support multiple progeny formation

• Septation and progeny cell formation

RecR likely plays critical roles in:

• Repairing DNA damage that may occur during attack phase or entry

• Supporting homologous recombination during intensive DNA replication

• Ensuring accurate chromosome segregation in coordination with the ParABS system (Bd3906, Bd3905)

• Maintaining genomic integrity during the transition from filamentous growth to individual progeny cells

What expression systems and purification protocols are most effective for producing active recombinant B. bacteriovorus RecR?

For optimal expression and purification of recombinant B. bacteriovorus RecR:

Expression Systems:

• E. coli BL21(DE3) with pET or similar expression vectors

• Consider codon optimization for improved expression

• Use fusion tags (His6, GST, MBP) to facilitate purification

• Test expression at reduced temperatures (16-25°C) to enhance solubility

Purification Protocol:

• Affinity chromatography using tag-specific resins

• Ion exchange chromatography to remove contaminants

• Size exclusion chromatography to obtain homogeneous protein

• Optional tag removal if the tag affects activity

• Activity assessment using DNA binding and RecA interaction assays

Buffer Optimization:

• Test buffers containing 20-50 mM Tris-HCl or HEPES (pH 7.5-8.0)

• Include 100-300 mM NaCl to maintain solubility

• Add 1-5 mM DTT or β-mercaptoethanol to prevent oxidation

• Consider adding 5-10% glycerol for protein stability

• Test divalent cation (Mg2+, Mn2+) requirements for activity

How can recombinant B. bacteriovorus RecR be used to study DNA repair mechanisms specific to predatory bacteria?

Recombinant B. bacteriovorus RecR enables several research approaches:

• Reconstitution of the complete RecFOR pathway in vitro to understand predator-specific aspects of DNA repair

• Comparative biochemical studies with RecR from non-predatory bacteria to identify functional adaptations

• Protein-protein interaction studies to map the DNA repair interactome in B. bacteriovorus

• Development of inhibitors that could specifically target the predatory bacterium's DNA repair system

• Structural studies to determine if RecR has unique features related to the predatory lifestyle

These approaches could reveal adaptations in DNA repair systems that support the predatory lifecycle and potentially identify novel mechanisms for maintaining genome stability during prey invasion and consumption.

What techniques are most effective for analyzing RecR activity during different stages of the B. bacteriovorus predatory cycle?

To analyze stage-specific RecR activity during predation:

Synchronization Methods:

• Use synchronized predatory cultures similar to those described for proteomics studies

• Separate attack-phase cells from bdelloplasts at different time points

Analysis Techniques:

• Stage-specific sampling for quantitative proteomics

• Fluorescent tagging of RecR for live-cell microscopy (similar to MreB-mTFP approaches)

• ChIP-seq to identify RecR binding sites at different predation stages

• Co-immunoprecipitation to detect stage-specific protein interactions

• RT-qPCR to monitor transcriptional changes of recR and related genes

Data Integration:

• Compare RecR activity with known predation phase markers

• Correlate with expression patterns of RecA and other DNA metabolism proteins

• Integrate with cell morphology changes observed during predation

How does the interaction between RecR and RecA in B. bacteriovorus compare to that in model organisms?

The RecA-RecR interaction in B. bacteriovorus may have unique features:

• RecA (Bd0512) shows significant upregulation during predation phases T2h-T5h , suggesting a crucial role during prey consumption

• The timing of RecA peak expression (T3h-T4h) corresponds to intensive DNA replication and septation

• RecR likely modulates RecA activity during these critical phases

A comparative analysis might reveal:

• Differences in binding affinities between RecA and RecR in B. bacteriovorus versus model organisms

• Predator-specific regulatory mechanisms that coordinate RecA-RecR interaction with the predatory lifecycle

• Potential novel interaction partners unique to predatory bacteria

• Adaptations that support rapid DNA recombination during intensive replication inside prey

What role might RecR play in the adaptation of B. bacteriovorus to different prey bacteria?

RecR may contribute to B. bacteriovorus adaptation to diverse prey through:

• Supporting genome plasticity that allows recognition of different prey types

• Facilitating DNA repair after exposure to prey defense mechanisms

• Enabling recombination events that incorporate beneficial genetic material

• Maintaining genomic stability during transitions between different prey environments

The predatory lifestyle requires B. bacteriovorus to interact with diverse bacterial species, potentially exposing it to various DNA-damaging agents. RecR's role in DNA repair would be essential for maintaining genomic integrity across these challenging transitions.

What are the major technical challenges in studying RecR function in B. bacteriovorus?

Researchers face several challenges when investigating RecR in B. bacteriovorus:

• The predatory lifestyle complicates genetic manipulation approaches

• If RecR is essential, complete knockout may not be viable (similar to both MreB proteins being essential)

• The unique growth phases require specialized techniques for stage-specific analysis

• Potential functional redundancy with other DNA repair pathways

• Limited genetic tools compared to model organisms

Potential solutions include:

• Partial inactivation approaches (similar to MreB-mTFP tagging)

• Conditional expression systems

• Careful synchronization of predatory cultures for stage-specific analysis

• Heterologous expression in model organisms for biochemical studies

How can high-throughput approaches be applied to understand the RecR interactome in B. bacteriovorus?

Modern high-throughput techniques can reveal the complete RecR interactome:

Recommended Approaches:

• Proximity-dependent biotin labeling (BioID or TurboID) with RecR as bait

• Affinity purification-mass spectrometry (AP-MS) at different predation stages

• Yeast two-hybrid or bacterial two-hybrid screening against B. bacteriovorus prey library

• Protein microarrays using recombinant RecR to identify binding partners

• Next-generation sequencing approaches to identify RecR binding sites

Data Analysis Framework:

• Network analysis to identify key interaction hubs

• Temporal mapping of interactions across the predatory cycle

• Comparison with interactomes from non-predatory bacteria

• Functional classification of interaction partners

This comprehensive understanding of RecR's interaction network would provide insights into its integrated role in DNA metabolism during predation.

What evolutionary insights might be gained from studying RecR across different predatory bacterial species?

Comparative analysis of RecR across predatory bacteria could reveal:

• Convergent adaptations in DNA repair systems that support predatory lifestyles

• Lineage-specific innovations in RecR structure and function

• Correlation between RecR properties and predatory efficiency or prey range

• Evolutionary history of DNA repair systems in predatory bacteria

This evolutionary perspective would contribute to understanding how DNA repair and recombination systems have adapted to support the unique challenges of bacterial predation.