Recombinant Rhodopirellula baltica Putative Holliday junction resolvase (RB8076)

What is Rhodopirellula baltica and why is it significant as a model organism?

Rhodopirellula baltica (R. baltica) SH 1T is a marine bacterium isolated from the Kiel Fjord (Baltic Sea) and taxonomically grouped within the bacterial phylum Planctomycetes. It is significant as a model organism because members of this group are abundant in aquatic habitats and play important roles in carbon cycling . R. baltica exhibits several unique properties including peptidoglycan-free proteinaceous cell walls, intracellular compartmentalization, and reproduction via budding, which results in a life cycle comprised of motile and sessile morphotypes similar to Caulobacter crescentus . The genome of R. baltica contains numerous interesting features, including a high number of sulfatase genes, carbohydrate-active enzymes, and a distinctive C1-metabolism pathway, making it valuable for studying specialized bacterial metabolism and adaptation .

What is RB8076 and what is its function in Rhodopirellula baltica?

RB8076 is a putative Holliday junction resolvase (EC= 3.1.-.-) encoded by the R. baltica genome . Holliday junction resolvases are enzymes that catalyze the resolution of Holliday junctions, which are four-way DNA structures that form during genetic recombination and DNA repair processes. Based on the annotation, RB8076 is likely involved in DNA recombination and repair mechanisms within R. baltica. The protein consists of 173 amino acids and has been assigned the UniProt accession number Q7UG74 . While annotated as a "putative" resolvase, this suggests that its specific function has been predicted based on sequence homology but may require further experimental validation.

How does RB8076 expression change throughout the R. baltica life cycle?

Research on R. baltica's life cycle has shown that its gene expression profiles change significantly across different growth phases. While specific data on RB8076 expression is limited, transcriptomic studies revealed that during the late stationary phase, R. baltica increases the expression of genes related to DNA recombination and genome rearrangement . For instance, multiple genes coding for transposases, integrases, and recombinases (including RB10096, RB11303, RB11750, RB1190, RB3144, RB4826, RB5887, RB7388, RB12239, RB2186, RB6736, RB9907, RB6167, RB7389, and RB934) show increased expression . This suggests that DNA recombination enzymes like RB8076 might play crucial roles during stress conditions or stationary phase growth, potentially facilitating genome rearrangements to enable efficient transcription during unfavorable conditions .

How does RB8076 compare structurally and functionally to other bacterial Holliday junction resolvases?

While comprehensive comparative data specific to RB8076 is not available in the provided search results, researchers can approach this question through bioinformatic analysis and experimental validation. Holliday junction resolvases generally belong to a superfamily of nucleases that share structural similarities despite sequence divergence. The full sequence of RB8076 provided in the search results can be used for:

• Sequence alignment with characterized resolvases from other bacterial species

• Structural prediction using homology modeling approaches

• Identification of conserved catalytic residues and DNA-binding motifs

These analyses would help determine whether RB8076 belongs to the RuvC family (common in most bacteria), the RusA family, or represents a more divergent type of resolvase. The unique evolutionary position of Planctomycetes makes this comparison particularly interesting, as RB8076 might possess distinctive structural features adapted to R. baltica's unusual cellular compartmentalization and genome organization.

What is the potential role of RB8076 in R. baltica's genome rearrangements during stress conditions?

Transcriptomic studies of R. baltica have shown that under stress conditions or in the late stationary phase, the organism expresses many genes coding for transposases, integrases, and recombinases, suggesting that genome rearrangements take place during these conditions . The upregulation of these genes may be necessary to enable efficient transcription during stressful phases, especially considering the scarcity of operon structures in the R. baltica genome . As a putative Holliday junction resolvase, RB8076 could play a crucial role in these genome reorganization processes by resolving recombination intermediates that form during DNA rearrangement events. Additionally, some of the upregulated recombination-related genes (RB11750, RB12239, RB2186, RB9907, RB7389, and RB934) have also been found active in response to non-physiological conditions of temperature and salinity, indicating their involvement in general stress responses . Research exploring the potential interactions between RB8076 and these other recombination factors would provide valuable insights into R. baltica's stress adaptation mechanisms.

How should researchers design experiments to study RB8076 activity in vitro?

When designing experiments to study RB8076 activity in vitro, researchers should consider the following methodological approach:

• Substrate preparation: Synthesize or prepare Holliday junction DNA structures using complementary oligonucleotides that can form the four-way junction required for resolvase activity testing.

• Activity assay design: The basic assay should include:

  • Purified recombinant RB8076 protein

  • Synthetic Holliday junction DNA substrate

  • Appropriate buffer conditions (typically containing Mg²⁺ or Mn²⁺ as cofactors)

  • Controls including heat-inactivated enzyme and known resolvases like E. coli RuvC

• Analysis methods: Resolution products can be analyzed using:

  • Gel electrophoresis (preferably using radioactively or fluorescently labeled DNA substrates)

  • Quantification of cleavage products to determine reaction kinetics

• Variables to test:

  • Metal ion dependency (Mg²⁺, Mn²⁺, Ca²⁺)

  • pH and salt concentration optimization

  • Sequence specificity at the cleavage site

  • Effects of DNA structure variations

• Data collection: Document reaction rates, substrate preferences, and cofactor requirements to characterize the enzymatic properties of RB8076.

Following the principles of proper experimental design , researchers should ensure appropriate controls, replication, and statistical analysis to obtain reliable and valid results.

What are the recommended approaches for studying RB8076 function in the context of R. baltica's life cycle?

To study RB8076 function throughout R. baltica's life cycle, researchers should consider a comprehensive experimental approach:

• Growth condition establishment: Cultivate R. baltica in defined mineral medium with glucose as the sole carbon source, mimicking the conditions used in previous life cycle studies .

• Temporal sampling: Collect samples at key life cycle points:

  • Early exponential phase (dominated by swarmer and budding cells)

  • Mid-exponential phase

  • Transition phase (mix of single cells, budding cells, and rosettes)

  • Early stationary phase

  • Late stationary phase (dominated by rosette formations)

• Expression analysis: Monitor RB8076 expression using:

  • qRT-PCR for targeted gene expression analysis

  • RNA-seq for genome-wide transcriptomic profiling

  • Western blotting to detect protein levels

• Functional genomics approaches:

  • Create RB8076 knockout or knockdown strains if genetic manipulation systems are available for R. baltica

  • Complement with wild-type or mutant versions of RB8076

  • Analyze phenotypic changes across the life cycle phases

• Morphological correlation: Use microscopy to correlate RB8076 expression with morphological changes and cell cycle progression

This experimental design follows a time-course approach similar to previous studies that revealed differential expression of genes through R. baltica's growth phases , but with specific focus on RB8076 and its potential role in genome maintenance.

What methodological considerations should be addressed when studying the interaction of RB8076 with other DNA recombination proteins?

When investigating potential interactions between RB8076 and other DNA recombination proteins in R. baltica, researchers should consider the following methodological approaches:

• Identification of interaction partners:

  • Conduct co-immunoprecipitation (Co-IP) experiments using antibodies against RB8076 or epitope-tagged versions

  • Perform pull-down assays with purified RB8076 as bait

  • Use yeast two-hybrid or bacterial two-hybrid systems to screen for interactions

• Validation of interactions:

  • Confirm direct protein-protein interactions using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC)

  • Verify co-localization in vivo using fluorescence microscopy with tagged proteins

  • Assess functional interactions through reconstituted in vitro systems

• Functional consequences:

  • Test whether identified interaction partners affect RB8076 enzymatic activity

  • Examine if interaction partners influence substrate specificity or reaction kinetics

  • Investigate how these interactions change under different growth conditions or stress

• Expression correlation analysis:

  • Compare expression patterns of RB8076 with other recombination proteins throughout the growth cycle

  • Focus particularly on the proteins whose genes showed coordinated expression during stress conditions (RB11750, RB12239, RB2186, RB9907, RB7389, and RB934)

These methodological considerations adhere to the principles of experimental design highlighted in search results , ensuring that the experiments are properly controlled, replicable, and address the specific research questions about protein-protein interactions in DNA recombination processes.

How should researchers analyze enzyme kinetics data for RB8076?

When analyzing enzyme kinetics data for RB8076, researchers should follow these methodological steps:

• Determination of initial reaction rates:

  • Measure the formation of products at multiple time points using gel electrophoresis quantification

  • Calculate initial velocities (v₀) for each substrate concentration

  • Ensure linearity of the reaction during the measurement period

• Kinetic parameter determination:

  • Plot reaction velocity versus substrate concentration

  • Fit data to appropriate enzyme kinetic models:

    • Michaelis-Menten equation for standard hyperbolic kinetics

    • Hill equation if cooperative binding is observed

  • Calculate key parameters:

    • Km (Michaelis constant): substrate concentration at half-maximal velocity

    • kcat (turnover number): maximum number of substrate molecules converted per enzyme molecule per unit time

    • kcat/Km: catalytic efficiency

• Comparative analysis:

  • Compare kinetic parameters of RB8076 with those of well-characterized Holliday junction resolvases

  • Assess effects of reaction conditions (pH, temperature, salt concentration) on kinetic parameters

• Statistical validation:

  • Perform experiments in triplicate at minimum

  • Calculate standard errors for all kinetic parameters

  • Use statistical tests to determine significance of differences under varying conditions

Following proper experimental design principles , this systematic approach to kinetic data analysis will provide robust characterization of RB8076 enzymatic activity and allow for meaningful comparisons with other resolvases.

What approaches can be used to interpret RB8076 expression data in the context of R. baltica's stress response?

To interpret RB8076 expression data in the context of R. baltica's stress response, researchers should consider the following analytical framework:

• Temporal expression pattern analysis:

  • Map RB8076 expression changes across growth phases and stress conditions

  • Compare expression patterns with known stress response genes

  • Construct time-course expression profiles to identify co-regulated genes

• Comparative analysis with known stress-responsive elements:

  • Compare RB8076 expression patterns with the 15 genes coding for transposases, integrases, and recombinases that show upregulation during stress

  • Focus particularly on the six genes (RB11750, RB12239, RB2186, RB9907, RB7389, and RB934) that respond to both stationary phase and non-physiological conditions of temperature and salinity

• Regulatory network analysis:

  • Identify potential transcription factor binding sites in the RB8076 promoter region

  • Look for shared regulatory elements among co-expressed genes

  • Construct potential regulatory networks based on expression correlation

• Functional correlation:

  • Analyze whether changes in RB8076 expression coincide with cellular morphological transitions (e.g., from single cells to rosette formations)

  • Correlate expression changes with measured DNA recombination or repair activities

  • Examine whether expression correlates with genome rearrangement events

This analytical approach aligns with the methodologies used in previous studies of R. baltica gene expression and follows sound principles of experimental design and data interpretation .

How can researchers integrate RB8076 function data with broader genomic and proteomic datasets from R. baltica?

To integrate RB8076 functional data with broader genomic and proteomic datasets from R. baltica, researchers should implement the following methodological framework:

• Multi-omics data integration:

  • Align RB8076 expression data with:

    • Transcriptomic datasets from growth phases and stress conditions

    • Proteomic profiles from life cycle analyses

    • Metabolomic data if available

  • Use bioinformatic tools designed for multi-omics data integration

• Network analysis approaches:

  • Construct gene co-expression networks including RB8076

  • Build protein-protein interaction networks based on experimental and predicted interactions

  • Identify functional modules that include RB8076

• Pathway enrichment analysis:

  • Determine which biological pathways are enriched among genes co-regulated with RB8076

  • Focus on DNA repair, recombination, and stress response pathways

  • Compare with the COG (Cluster of Orthologous Group) class distributions observed in previous studies

• Comparative analysis across growth conditions:

  • Create a data integration table similar to the format below, comparing RB8076 with other recombination-related genes:

This integration approach follows sound principles of experimental design and data analysis , allowing researchers to place RB8076 function within the broader context of R. baltica's cellular processes and stress responses.

What are the key considerations for future research on RB8076 and related resolvases in Planctomycetes?

Future research on RB8076 and related resolvases in Planctomycetes should consider:

• Evolutionary significance: Investigate how RB8076 compares with resolvases from other bacterial phyla, particularly given the unique evolutionary position of Planctomycetes.

• Structure-function relationships: Determine the three-dimensional structure of RB8076 and identify critical residues for catalysis and substrate recognition.

• Life cycle regulation: Explore how RB8076 activity is regulated throughout R. baltica's complex life cycle and under various environmental stresses.

• Role in genome plasticity: Investigate whether RB8076 contributes to the genomic rearrangements observed during stress responses and how this relates to R. baltica's adaptation mechanisms.

• Biotechnological applications: Assess the potential of RB8076 as a molecular tool for DNA manipulation, given R. baltica's biotechnologically promising features .