Recombinant Rhodopirellula baltica DNA repair protein recO (recO)

How does Rhodopirellula baltica RecO compare to RecO proteins from other bacterial species?

The Rhodopirellula baltica RecO protein shares functional similarities with RecO proteins from other bacterial species, but with some distinct characteristics. Unlike Escherichia coli RecO, which interacts with the C-terminal tail of single-stranded DNA binding protein (SSB-Ct), R. baltica RecO likely employs different mechanisms for DNA binding and protein interactions .

Structurally, R. baltica RecO appears more similar to Mycobacterium smegmatis RecO (MsRecO) and Deinococcus radiodurans RecO (DrRecO), both of which possess 4×Cys zinc finger motifs that E. coli RecO lacks. This structural difference suggests that R. baltica RecO may utilize zinc-dependent mechanisms for DNA binding, similar to MsRecO, which has been shown to have zinc-stimulated DNA binding and strand annealing activities .

The functional conservation of RecO across diverse bacterial species underscores its evolutionary importance in DNA repair processes, despite variations in specific interaction mechanisms and structural features.

What are the optimal storage and handling conditions for recombinant R. baltica RecO protein?

For optimal preservation of recombinant R. baltica RecO protein activity, storage conditions should be carefully maintained:

Storage Temperature:

• Liquid form: -20°C/-80°C with a typical shelf life of 6 months

• Lyophilized form: -20°C/-80°C with an extended shelf life of up to 12 months

Reconstitution Protocol:

• Briefly centrifuge the vial before opening to collect contents at the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is typically recommended)

• Aliquot for long-term storage to avoid repeated freeze-thaw cycles

Working Conditions:

• For short-term use, store working aliquots at 4°C for up to one week

• Avoid repeated freezing and thawing as this significantly reduces protein activity

• When using for experiments, maintain the protein in appropriate buffer conditions that preserve its structural integrity

These handling guidelines are crucial for maintaining protein stability and ensuring reproducible experimental results when working with R. baltica RecO.

What experimental assays can be used to study RecO protein activity in vitro?

Several experimental approaches can be employed to study R. baltica RecO activity in vitro, based on methods established for homologous RecO proteins:

DNA Binding Assays:

• Fluorescence anisotropy using fluorescein-labeled oligonucleotides (e.g., 5'-labeled dT15)

• Electrophoretic mobility shift assays (EMSA) to detect protein-DNA complex formation

• DNA binding can be measured under various conditions (e.g., with/without zinc, with/without RecR)

Strand Annealing Activity Assays:

• Using complementary single-stranded DNA oligonucleotides with fluorescent labels

• Monitoring annealing through FRET (Förster Resonance Energy Transfer) between donor and acceptor fluorophores on complementary strands

• Comparing annealing rates in the presence and absence of SSB to evaluate RecO's ability to overcome SSB inhibition

RecA Loading Assays:

• ATP hydrolysis assays using coupled enzymatic reactions to measure RecA's DNA-dependent ATPase activity

• FRET-based strand exchange assays to monitor RecA-mediated homologous recombination

• These assays can compare RecA activity with and without RecO/RecOR to assess recombination mediator function

When designing these experiments, it's important to consider the potential role of zinc in R. baltica RecO activity, as zinc has been shown to stimulate DNA binding in homologous proteins with zinc finger motifs.

How does the zinc-dependency of RecO affect its DNA repair mechanisms in R. baltica?

The zinc-dependency of RecO in R. baltica likely represents a critical regulatory mechanism for its DNA repair functions. Based on studies of homologous RecO proteins:

Dual DNA Binding Mechanisms:
RecO proteins with zinc finger motifs (like those in MsRecO) can interact with DNA through two distinct mechanisms:

• A zinc-dependent pathway primarily involved in strand annealing activities

• A zinc-independent pathway regulated by interaction with RecR during recombination

Regulatory Implications:
This dual regulation system allows the same protein to participate in different DNA repair pathways depending on cellular conditions. In R. baltica, zinc may serve as a molecular switch that directs RecO activity toward strand annealing versus recombination mediator functions .

Structural Basis:
The 4×Cys zinc finger motif present in R. baltica RecO likely undergoes conformational changes upon zinc binding that alter its DNA binding properties. This structural flexibility enables RecO to adapt its function to specific DNA repair contexts .

Ecological Context:
As a marine bacterium, R. baltica lives in environments where zinc concentrations can fluctuate. The zinc-dependency of RecO might represent an adaptation that links DNA repair activity to environmental conditions, potentially allowing the organism to modulate its DNA repair capacity in response to changing metal availability in marine habitats.

Understanding this zinc-dependency is crucial for interpreting RecO function in the cellular context of R. baltica and may provide insights into the evolution of DNA repair mechanisms in marine bacteria.

What is the relationship between RecO and other components of the DNA repair machinery in R. baltica?

RecO functions as part of an interconnected network of DNA repair proteins in R. baltica, with several key relationships:

RecO-RecR Interaction:

• RecR stimulates DNA binding of zinc-depleted RecO, suggesting a regulatory relationship where RecR can compensate for the absence of zinc

• The RecOR complex likely serves as the functional unit that facilitates RecA loading onto SSB-coated ssDNA during homologous recombination

RecO-SSB Interaction:

• Unlike E. coli RecO, R. baltica RecO likely does not interact directly with the C-terminal tail of SSB

• Despite this lack of direct interaction, RecO and RecOR complexes can still function with SSB-ssDNA, suggesting alternative mechanisms for overcoming SSB inhibition

• This represents an evolutionary divergence in how RecO functions across bacterial species

RecO-RecA Relationship:

• RecO and RecOR function as recombination mediator proteins (RMPs) that help load RecA onto ssDNA

• This loading activity is essential for homologous recombination processes that repair DNA double-strand breaks and restart stalled replication forks

• The mechanism likely involves formation of a complex between SSB-ssDNA and RecO/RecOR without direct protein-protein interactions with RecA

Integration in DNA Repair Pathways:

• RecO participates in both homologous recombination (HR) and single-strand annealing (SSA) pathways

• These pathways represent alternative mechanisms for repairing DNA double-strand breaks

• The specific pathway choice may depend on cellular conditions and the nature of the DNA damage

This networked relationship with multiple DNA repair components allows RecO to contribute to genome stability through various mechanisms, highlighting its central role in the DNA repair machinery of R. baltica.

How can RecO be used to study cell cycle regulation in R. baltica?

RecO can serve as a valuable tool for investigating cell cycle regulation in R. baltica, particularly in relation to DNA repair dynamics across different growth phases:

Cell Cycle-Dependent Expression Analysis:

• Monitoring RecO expression levels throughout the R. baltica life cycle using transcriptomic and proteomic approaches

• Correlating RecO expression with different morphological states (swarmer cells, budding cells, rosettes) that characterize R. baltica's complex life cycle

• Gene expression studies have shown that R. baltica undergoes significant transcriptional reprogramming during different growth phases, with distinctive patterns during exponential growth versus stationary phase

DNA Repair Capacity Across Growth Phases:

• Analyzing RecO activity in relation to growth phase-dependent stress responses

• During transition to stationary phase, R. baltica upregulates various stress-related genes including those involved in oxidative stress response, which may interact with RecO-mediated repair pathways

• The differential regulation of as many as 863 genes (12% of the genome) during late stationary phase suggests complex regulatory networks that may include DNA repair systems

Morphotype-Specific Functions:

• Investigating whether RecO activity differs between the motile swarmer cells and sessile rosette formations of R. baltica

• Surface attachment and rosette formation during stationary phase may create unique DNA damage scenarios that require RecO activity

• Specific proteins regulated during growth on solid surfaces could potentially interact with RecO-mediated repair pathways

By linking RecO function to cell cycle progression and morphological differentiation in R. baltica, researchers can gain insights into how DNA repair mechanisms are integrated with the unique developmental program of this planctomycete.

What techniques can be used to study RecO protein localization within the compartmentalized cell structure of R. baltica?

Studying RecO localization in R. baltica presents unique challenges and opportunities due to the organism's distinctive compartmentalized cell architecture. The following techniques can be employed:

Fluorescent Protein Fusion Approaches:

• Construction of RecO-fluorescent protein fusions (using GFP or tdTomato) for in vivo localization studies

• Transformation protocols for R. baltica have been developed, including chemical transformation methods that could be used to introduce RecO-fluorescent protein constructs

• Plasmid vectors such as pRK415 have been tested in R. baltica and could serve as backbone for RecO-fluorescent protein expression constructs

Immunolocalization Techniques:

• Development of specific antibodies against R. baltica RecO

• Immunogold labeling combined with electron microscopy to precisely localize RecO within the pirellulosome (intracellular compartment) or other cellular regions

• This approach would build upon established proteome analysis techniques that have successfully identified proteins localized to different compartments in R. baltica

Subcellular Fractionation Studies:

• Separation of membrane and cytoplasmic fractions to determine RecO distribution

• Particular attention to the pirellulosome, which contains most housekeeping proteins lacking signal peptides

• Comparative analysis with the 146 proteins containing predicted signal peptides identified in previous proteome studies

Protoplast Formation Combined with Localization:

• Utilizing the developed protoplast formation protocol for R. baltica

• This approach could help distinguish between cell wall/membrane-associated RecO and cytoplasmic/pirellulosome-localized RecO

• The protocol using lysozyme treatment and osmotic pressure provides a unique tool for studying protein localization in relation to cell wall structures

These techniques would help determine whether RecO is primarily localized to the pirellulosome or other compartments, providing insights into the spatial organization of DNA repair processes in this compartmentalized bacterium.

How can comparative analysis of RecO from R. baltica and other bacterial species inform evolutionary studies of DNA repair mechanisms?

Comparative analysis of RecO proteins across bacterial species offers a powerful approach to understanding the evolution of DNA repair mechanisms:

Structural Diversity Analysis:

• Compare the zinc finger motifs in R. baltica RecO with those in other bacterial species (e.g., Mycobacterium smegmatis, Deinococcus radiodurans)

• Analyze the evolutionary significance of the absence of direct SSB-Ct interaction in R. baltica RecO compared to E. coli RecO

• Evaluate how these structural differences reflect adaptation to different ecological niches

Functional Conservation Mapping:

• Identify conserved regions critical for RecO function across phylogenetically diverse bacteria

• Map species-specific variations to functional differences in DNA repair strategies

• Determine whether marine bacteria like R. baltica share common adaptations in their RecO proteins

Ecological Adaptation Assessment:

• Correlate RecO structural features with ecological parameters (e.g., marine vs. terrestrial habitats)

• Evaluate whether zinc-dependency in RecO proteins relates to metal availability in different environments

• Analyze how RecO variations contribute to DNA repair efficiency under different environmental stressors

Phylogenetic Reconstruction:

• Use RecO sequence data to complement 16S rRNA-based phylogenies of Planctomycetes

• Assess whether RecO evolution correlates with the evolution of cell compartmentalization in this phylum

• The diversity revealed through phylogenetic analysis of over 60 Rhodopirellula isolates suggests potential diversity in their DNA repair machinery as well

This comparative approach could reveal how DNA repair mechanisms evolved in response to the unique challenges faced by different bacterial lineages and provide insights into the evolutionary history of genome stability maintenance systems.

What are the key methodological considerations for expressing and purifying functionally active recombinant R. baltica RecO?

Expressing and purifying functionally active R. baltica RecO requires careful attention to several methodological aspects:

Expression System Selection:

• Yeast expression systems have been successfully used for R. baltica RecO production

• E. coli-based expression systems might require optimization of codon usage to account for the GC-rich genome of R. baltica

• Consider testing both systems to determine which yields the highest activity and solubility

Tag Design Considerations:

• The tag type should be determined during the manufacturing process based on optimal expression and purification results

• Consider using cleavable tags to obtain native protein for functional studies

• Evaluate the impact of N-terminal versus C-terminal tags on RecO activity

Metal Supplementation:

• Include zinc during expression and purification if maintaining zinc-dependent functions is critical

• Evaluate the use of TCEP (tris(2-carboxyethyl)phosphine) instead of DTT for maintaining reduced cysteines in zinc finger motifs

• Consider parallel purification strategies with and without zinc to obtain protein preparations suitable for different functional studies

Purity Assessment:

• Use SDS-PAGE to confirm purity (target >85%)

• Consider additional characterization by mass spectrometry to verify the intact mass and potential post-translational modifications

• Functional assays (DNA binding, strand annealing) should be used to confirm activity of the purified protein

Storage Optimization:

• For maximum stability, store as aliquots with 50% glycerol at -80°C

• Avoid repeated freeze-thaw cycles that significantly reduce protein activity

• Consider lyophilization for extended shelf-life (up to 12 months compared to 6 months for liquid form)

Adhering to these methodological considerations will help ensure that the recombinant R. baltica RecO maintains its native functional properties and provides reliable results in downstream applications.

What are the challenges in studying RecO function in the context of R. baltica's unique cell biology?

Investigating RecO function in R. baltica presents several unique challenges related to the organism's distinctive cell biology:

Compartmentalized Cell Architecture:

• R. baltica possesses a compartmentalized cell structure with an intracellular compartment called the pirellulosome, which complicates understanding of where DNA repair processes occur

• Determining whether RecO operates within specific compartments requires specialized localization techniques

• The absence of predictable signal peptides for many proteins complicates prediction of RecO localization

Complex Life Cycle:

• R. baltica exhibits distinct morphological forms including swarmer cells, budding cells, and rosette formations

• This complex life cycle may involve stage-specific regulation of DNA repair systems

• RecO function might vary between motile swarmer cells and sessile rosette aggregates, requiring stage-specific analyses

Genetic Manipulation Limitations:

• Until recently, genetic tools for R. baltica were limited, making functional genetic studies challenging

• While transformation protocols have been developed, efficiency remains relatively low

• Creating RecO deletion or modification strains for in vivo functional studies requires further optimization of genetic tools

Growth and Cultivation Requirements:

• R. baltica has specific growth requirements and relatively slow growth rates (optimal temperature 28-30°C)

• Synchronizing cultures for cell cycle studies is difficult, complicating time-resolved analysis of RecO function

• The strict aerobic nature of R. baltica may create specific oxidative damage scenarios that influence RecO activity

Limited Comparative Models:

• As a member of the Planctomycetes, R. baltica is phylogenetically distant from well-studied model bacteria

• Extrapolating findings from E. coli or B. subtilis RecO studies may not be appropriate

• The distinct ecological niche of R. baltica as a marine organism may have driven unique adaptations in its DNA repair systems

Addressing these challenges will require interdisciplinary approaches combining molecular genetics, cell biology, and biochemistry adapted specifically for the unique properties of R. baltica.

How might understanding RecO function contribute to broader insights about DNA repair in marine bacteria?

Understanding RecO function in R. baltica has significant implications for our broader knowledge of DNA repair in marine bacteria:

Environmental Adaptation Mechanisms:

• Marine environments pose unique challenges including UV radiation, pressure variations, and fluctuating salinity

• RecO function in R. baltica may reveal how DNA repair systems have adapted to these marine-specific stressors

• Comparative studies with terrestrial bacteria could highlight marine-specific innovations in DNA repair pathways

Ecological Resilience Factors:

• DNA repair efficiency contributes to bacterial survival and persistence in marine ecosystems

• Understanding how R. baltica maintains genome integrity could explain its successful colonization of diverse marine habitats

• RecO function may contribute to the organism's ability to withstand environmental fluctuations typical of coastal and open ocean environments

Biogeochemical Cycling Implications:

• As contributors to biomass remineralization, Planctomycetes like R. baltica play important roles in marine biogeochemical cycles

• Efficient DNA repair systems may support their ecological functions by ensuring population stability under stress conditions

• RecO-mediated genome stability could be linked to the maintenance of metabolic capabilities essential for carbon and nitrogen cycling

Evolutionary Perspectives:

• Planctomycetes represent an evolutionarily distinct bacterial lineage

• The study of RecO in R. baltica provides insights into the evolution of DNA repair systems across different bacterial phyla

• Unique aspects of RecO function might represent either ancestral traits or specialized adaptations to the marine environment

Biotechnological Applications:

• Understanding RecO's mechanisms for maintaining DNA integrity in marine conditions could inspire biomimetic approaches to DNA preservation

• R. baltica's DNA repair systems might have applications in developing stress-resistant strains for marine biotechnology

• The zinc-dependent properties of RecO could inform the development of metal-regulated molecular tools

This research contributes to our fundamental understanding of how essential cellular processes like DNA repair have evolved in the marine environment, which covers over 70% of Earth's surface and hosts enormous microbial diversity.

What is an optimized protocol for assessing the DNA binding activity of recombinant R. baltica RecO?

Optimized Protocol for RecO DNA Binding Assay

This protocol is designed to quantitatively assess the DNA binding activity of recombinant R. baltica RecO protein under various conditions, including testing zinc-dependent and RecR-dependent DNA binding mechanisms.

Materials Required:

• Purified recombinant R. baltica RecO protein (>85% purity)

• Purified recombinant R. baltica RecR protein (optional, for RecR-dependent studies)

• Fluorescein 5′-labeled dT15 oligonucleotide (or similar ssDNA substrate)

• Buffer A: 50 mM NaCl, 25% (v/v) glycerol, 50 mM HEPES pH 7.5, 1 mM TCEP

• Buffer B: 100 mM NaCl, 25% (v/v) glycerol, 50 mM HEPES pH 7.5, 1 mM TCEP

• Zinc acetate solution (100 mM stock)

• EDTA solution (500 mM stock)

• 96-well black plates with clear bottom

• Fluorescence plate reader with polarization capability

Procedure:

• Sample Preparation:

  • Prepare RecO protein in Buffer A or B at 2 μM concentration

  • For zinc-depleted conditions, treat RecO with 1 mM EDTA and 1 mM DTT, followed by extensive dialysis against buffer containing 1 mM TCEP

  • For RecR-dependent studies, prepare RecR protein in the same buffer

• DNA Substrate Preparation:

  • Dilute fluorescein 5′-labeled dT15 oligonucleotide to 10 nM in appropriate buffer

  • Heat to 95°C for 2 minutes and cool slowly to room temperature

• Binding Reaction Setup:

  • In a 96-well plate, add 50 μL of fluorescein-labeled DNA (5 nM final concentration)

  • Prepare a dilution series of RecO protein (0-500 nM final concentration)

  • For zinc-dependent studies, include parallel reactions with 10 μM Zn(OAc)2

  • For RecR-dependent studies, include 8 μM RecR protein

  • Total reaction volume: 100 μL

• Incubation:

  • Incubate reactions at room temperature for 15 minutes in the dark

  • For time-course studies, measure at 5, 15, 30, and 60 minutes

• Measurement:

  • Measure fluorescence anisotropy with excitation at 485 nm and emission at 528 nm

  • Take multiple readings (at least 3) for each well and average the values

• Data Analysis:

  • Plot anisotropy values against RecO concentration

  • Calculate apparent Kd values by fitting to an appropriate binding model

  • Compare binding affinities under different conditions (±zinc, ±RecR)

• Controls:

  • Negative control: buffer only (no protein)

  • Non-specific binding control: BSA at equivalent concentrations

  • Buffer control to assess contribution of buffer components to anisotropy

This protocol allows for quantitative assessment of the DNA binding properties of RecO under conditions that probe its zinc-dependent and RecR-dependent functions, providing insights into the dual mechanisms of RecO activity in DNA repair processes .

What experimental approaches can detect the interaction between RecO and other components of the DNA repair machinery in R. baltica?

Experimental Approaches for Detecting RecO Interactions

The following methodologies can be employed to investigate interactions between RecO and other components of the DNA repair machinery in R. baltica:

1. In Vitro Protein-Protein Interaction Assays:

Co-immunoprecipitation (Co-IP):

• Express RecO with an affinity tag (His, GST, etc.)

• Incubate with R. baltica cell lysate or purified potential interaction partners

• Isolate RecO using affinity resin and analyze co-precipitated proteins by mass spectrometry

• Specific controls should include tag-only baits and proteins known not to interact with RecO

Surface Plasmon Resonance (SPR):

• Immobilize purified RecO on a sensor chip

• Flow potential interaction partners (RecR, RecA, SSB) over the surface

• Measure binding kinetics (kon and koff) and calculate binding affinity (KD)

• Test interactions under various conditions (±zinc, different salt concentrations)

Fluorescence Resonance Energy Transfer (FRET):

• Label RecO and potential partners with appropriate fluorophore pairs

• Monitor FRET signal upon mixing proteins

• Perform competition assays with unlabeled proteins to confirm specificity

• This approach is particularly useful for detecting conformational changes upon interaction

2. DNA-Protein Complex Analysis:

Electrophoretic Mobility Shift Assay (EMSA):

• Incubate labeled DNA with RecO and potential interaction partners

• Analyze complex formation by native gel electrophoresis

• Super-shift assays using specific antibodies can confirm the presence of specific proteins in complexes

• Compare DNA binding in the presence and absence of SSB to evaluate SSB displacement

DNA Protection Assays:

• Treat RecO-DNA complexes with nucleases

• Analyze protection patterns to identify binding sites

• Compare protection patterns in the presence of different protein combinations

• This approach can reveal cooperative binding or altered DNA accessibility

3. Cellular Localization and Interaction Studies:

Bacterial Two-Hybrid System:

• Adapt bacterial two-hybrid systems for use in R. baltica

• Create fusion constructs of RecO and potential interaction partners

• Monitor reporter gene activation as indicator of protein interaction

• This approach allows testing of interactions in a cellular context

Fluorescence Microscopy with Protein Fusions:

• Express fluorescently tagged RecO in R. baltica cells

• Co-express potential interaction partners with different fluorescent tags

• Analyze co-localization during normal growth and after DNA damage induction

• Quantify co-localization using appropriate image analysis tools

4. Functional Assays for Complex Formation:

ATP Hydrolysis Assays:

• Monitor RecA's DNA-dependent ATPase activity

• Compare activity in the presence of various combinations of RecO, RecR, and SSB

• Altered ATPase activity can indicate successful loading of RecA onto DNA by RecO/RecOR

Strand Exchange Assays:

• Measure RecA-mediated strand exchange using FRET-labeled DNA substrates

• Assess the impact of RecO and RecOR on the efficiency and kinetics of strand exchange

• Compare results using zinc-depleted RecO to evaluate the role of zinc in complex formation

These complementary approaches provide a comprehensive toolkit for investigating the network of interactions centered on RecO in the DNA repair machinery of R. baltica, from direct binary interactions to functional consequences in complex reaction systems .

What are the key properties of recombinant R. baltica RecO protein?

Table 1: Physical and Biochemical Properties of Recombinant R. baltica RecO Protein

This table summarizes the key physical, biochemical, and functional properties of recombinant R. baltica RecO protein based on available literature. The dual DNA binding mechanisms (zinc-dependent and RecR-dependent) represent a particularly notable feature that distinguishes this protein's activity in different DNA repair contexts.

How does R. baltica gene expression change throughout its growth cycle, particularly for DNA repair genes?

Table 2: Growth Phase-Dependent Gene Expression in R. baltica with Focus on DNA Repair