Recombinant Rhodopirellula baltica GTPase obg (obg)

What is the basic structure of the Obg GTPase in Rhodopirellula baltica?

Obg proteins belong to the P-loop guanine triphosphatase (GTPase) superfamily that is conserved from bacteria to humans. The protein has a multi-domain structure consisting of: (1) a highly conserved glycine-rich N-terminal domain known as Obg fold, which functions primarily as a protein-protein interaction domain; (2) a central conserved GTPase domain (G domain) with Ras-like folds containing five conserved motifs (G1-G5) responsible for nucleotide binding and hydrolysis; and (3) a less conserved C-terminal domain that varies between species . In R. baltica, the intrinsically disordered C-terminal domain plays an important role in protein interactions and functional regulation .

What are the primary cellular functions of Obg GTPases in bacteria?

Obg GTPases function as molecular switches by cycling between GTP-bound "on" and GDP-bound "off" states to regulate various cellular processes. In bacteria, including R. baltica, Obg plays essential roles in:

• Ribosome maturation and assembly, particularly interacting with the 50S ribosomal subunit

• Control of the cell cycle and DNA replication

• Cell division processes

• Stress response mechanisms, including stringent response

• General stress adaptation pathways

• Energy status monitoring and maintenance

The protein is essential for bacterial viability, making it an attractive target for antimicrobial development .

How does Obg GTPase interact with nucleotides and what effects do these interactions have?

Obg GTPase binds GTP, GDP, and possibly (p)ppGpp with moderate affinity, exhibiting high nucleotide exchange rates but relatively low GTP hydrolysis rates . Nucleotide binding induces conformational changes in the protein, particularly in the switch I and switch II regions located in the G2/G3 motifs. These conformational changes affect Obg's interactions with binding partners, especially the ribosome:

The presence of different nucleotides significantly affects Obg's binding to the 50S ribosomal subunit, suggesting that Obg adjusts its behavior according to changes in the cellular nucleotide pool during different growth phases .

What are the recommended methods for producing recombinant R. baltica Obg protein?

For the expression and purification of recombinant R. baltica Obg (ObgE), the following methodological approach is recommended:

• Gene synthesis and vector construction: The coding sequence can be synthesized based on the R. baltica genome (approximately 335 amino acids for standard Obg proteins) . The gene should be cloned into an expression vector with an appropriate tag (His-tag is commonly used) for purification.

• Expression system: E. coli BL21(DE3) or similar strains are suitable for expression. Culture conditions typically include induction with IPTG (0.1-0.5 mM) at OD600 of 0.6-0.8, followed by growth at 18-25°C for 16-20 hours to minimize inclusion body formation.

• Purification protocol:

  • Affinity chromatography using Ni-NTA for His-tagged proteins

  • Ion exchange chromatography as a secondary purification step

  • Size exclusion chromatography for final polishing and buffer exchange

• Storage buffer: Purified ObgE should be stored in a buffer containing 20-50 mM Tris-HCl (pH 7.5-8.0), 100-200 mM NaCl, 5-10% glycerol, and 1-5 mM DTT or 0.5-1 mM TCEP to maintain protein stability .

The recombinant protein can then be used for various assays including nucleotide binding, GTPase activity assays, and interaction studies.

How can researchers measure the GTPase activity of recombinant Obg?

The GTPase activity of R. baltica Obg can be assessed using several complementary approaches:

• Colorimetric phosphate release assay:

  • Mix purified Obg protein (0.5-5 μM) with GTP (50-200 μM) in reaction buffer containing 50 mM Tris-HCl (pH 7.5), 50-100 mM KCl (essential for optimal activity), 5 mM MgCl2

  • Incubate at 25-37°C for defined time periods

  • Stop reactions with malachite green or similar phosphate detection reagent

  • Measure absorbance at 620-650 nm to quantify released phosphate

• Fluorescence-based assays:

  • Use fluorescent guanine nucleotide analogs like mant-GTP and mant-GDP

  • Detect changes in fluorescence upon nucleotide binding or hydrolysis

  • Measure fluorescence resonance energy transfer (FRET) between the protein and the fluorescent nucleotide

• Radiometric assays:

  • Use [γ-32P]GTP as substrate

  • Separate free phosphate from nucleotides by thin-layer chromatography

  • Quantify released 32P by phosphorimaging or scintillation counting

It's important to note that potassium ions significantly enhance the GTPase activity of many Obg proteins, and this effect should be considered when designing assays .

What techniques are effective for studying Obg-protein interactions in R. baltica?

Several complementary techniques are recommended for investigating Obg-protein interactions in R. baltica:

• Isothermal Titration Calorimetry (ITC):

  • Provides quantitative binding parameters (Kd, ΔH, ΔS)

  • Can reveal biphasic binding patterns characteristic of Obg interactions

  • Requires relatively large amounts of purified proteins (typically 20-200 μM)

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS):

  • Determines absolute molecular weights of protein complexes

  • Confirms complex formation and stoichiometry

  • Differentiates between monomeric and oligomeric states

• Small Angle X-ray Scattering (SAXS):

  • Provides low-resolution structural information about complexes in solution

  • Reveals conformational changes upon complex formation

  • Complements crystallographic studies

• Pull-down assays and co-immunoprecipitation:

  • Identifies interaction partners from cell lysates

  • Can be performed with tagged recombinant Obg as bait

  • Should include appropriate nucleotide conditions (GTP, GDP, or ppGpp)

• X-ray crystallography:

  • Provides atomic-level details of interaction interfaces

  • Requires successful crystallization of the complex

  • Most informative when comparing different nucleotide-bound states

These approaches have been successfully applied to characterize the interaction between E. coli Obg (ObgE) and its binding partners, revealing high-affinity biphasic interactions that are influenced by nucleotide binding and the intrinsically disordered C-terminal domain .

How can Obg GTPase serve as a target for antimicrobial development?

Obg GTPase represents a promising target for novel broad-spectrum antibiotics due to several key properties:

• Essential nature: Obg is essential for the survival of all bacteria, including pathogens like Neisseria gonorrhoeae, Klebsiella pneumoniae, and methicillin-resistant Staphylococcus aureus .

• Conservation: The high conservation of Obg across bacterial species enables broad-spectrum targeting, while sufficient differences from eukaryotic homologs may allow for selectivity .

• Established screening methodology: A robust 384-well GTPase assay has been developed and validated for high-throughput screening of Obg inhibitors, achieving an average Z' value of 0.58 ± 0.02, indicating suitability for large-scale compound screening .

• Secondary assessment tools: Several secondary assays have been developed to evaluate lead compounds:

  • Fluorescence-based binding assays using mant-GTP and mant-GDP

  • GTPase activity assessments with variant Obg proteins containing alterations in G-domains

  • Cross-species activity testing against Obg from multiple pathogens

• Mechanism of action: Obg inhibitors could disrupt multiple essential cellular processes, including ribosome biogenesis, DNA replication, and cell division, potentially reducing the likelihood of resistance development .

When targeting R. baltica Obg for inhibitor development, researchers should focus on compounds that interfere with either nucleotide binding or specific protein-protein interactions that are essential for Obg function.

What is known about the role of Obg in bacterial stress response and persistence?

Obg plays multifaceted roles in bacterial stress response and persistence mechanisms, making it relevant for understanding antimicrobial resistance and bacterial survival under adverse conditions:

• Stringent response regulation:

  • Obg interacts with the alarmone (p)ppGpp, a central mediator of bacterial stress response

  • This interaction affects nucleotide binding and dissociation kinetics

  • Under stress conditions, Obg-ppGpp interaction may redirect cellular resources away from growth toward survival mechanisms

• SOS response modulation:

  • Obg efficiently inhibits the DNA binding ability of interaction partners involved in the SOS stress response

  • This suggests a regulatory role in DNA damage repair and mutagenesis

• Ribosome modulation during stress:

  • The occupancy of Obg on the 50S ribosomal subunit increases over 5-fold in the presence of ppGpp compared to the unbound state

  • Obg is incompatible with fully assembled 70S ribosomes when present in excess, resulting in disassembled subunits

  • This suggests a role in translational reprogramming during stress

• Persistence phenotype:

  • Obg is implicated in bacterial persistence, a phenotypic state characterized by transient antibiotic tolerance

  • Alterations in Obg levels or activity can affect the frequency of persister cell formation

Understanding these mechanisms in R. baltica and other bacterial species could lead to strategies for combating persistent infections and antibiotic tolerance.

How does the intrinsically disordered C-terminal domain of Obg contribute to its function?

The intrinsically disordered C-terminal domain (CTD) of Obg plays several crucial roles in the protein's function, highlighting the importance of structural flexibility in molecular switch mechanisms:

• Protein-protein interactions: The CTD serves as a platform for interactions with various protein partners, expanding Obg's functional repertoire beyond direct GTPase activity. In E. coli Obg (ObgE), the CTD has been demonstrated to be important for high-affinity binding to interaction partners .

• Nucleotide binding modulation: The CTD influences Obg's nucleotide binding and dissociation kinetics. Studies have shown that the interaction between ObgE and its partners affects ObgE's nucleotide handling properties, suggesting regulatory feedback between binding partners and the GTPase cycle .

• Conformational flexibility: The disordered nature of the CTD allows Obg to adopt different conformations depending on its nucleotide-bound state and interaction partners. This flexibility is likely important for Obg's ability to function as a cellular sensor that integrates multiple inputs .

• Species-specific adaptations: While the GTPase domain is highly conserved across bacteria, the CTD shows considerable variation between species, suggesting adaptation to specific cellular contexts and interaction networks. In R. baltica, the CTD may have evolved to optimize interactions with species-specific partners .

Researchers investigating R. baltica Obg should consider generating truncated variants lacking the CTD to assess its contribution to specific functions and interactions in this organism.

How does R. baltica Obg compare with Obg proteins from other bacterial species?

Comparative analysis of Obg proteins across bacterial species reveals both conserved features and species-specific adaptations:

R. baltica, with its large genome of over 7,000 genes, contains a substantial core genome shared with other Rhodopirellula species (around 3,000 genes) but also many strain-specific genes . This genomic context may influence the specific roles and interactions of the Obg protein in R. baltica compared to other bacteria.

While the fundamental mechanisms of Obg function are conserved, the specifics of its regulatory networks likely differ between species, reflecting adaptations to different ecological niches. For instance, R. baltica is a marine bacterium that must adapt to various salinity levels, which may have influenced the evolution of its stress response systems, including those involving Obg .

What insights can genomic and phylogenetic analyses provide about R. baltica Obg evolution?

Genomic and phylogenetic analyses of R. baltica and its Obg protein reveal important evolutionary insights:

• Genomic context: R. baltica SH1(T) possesses a large genome with 7,325 genes, of which around 3,000 form the core genome of the Rhodopirellula genus . This genomic context suggests that Obg may have acquired additional functions or interactions in this organism compared to bacteria with smaller genomes.

• Genus-level diversity: Comparative genomics of Rhodopirellula strains reveals substantial genetic diversity. Individual strains possess a large portion of strain-specific genes, suggesting that even within the genus, Obg may have evolved different interaction networks .

• Operational taxonomic units (OTUs): Multilocus sequence analysis (MLSA) studies have identified at least 13 genetically defined OTUs within the Rhodopirellula genus . This genetic diversity correlates with biogeographic distribution, suggesting that Obg may have adapted to specific environmental conditions in different marine regions.

• Conservation across domains of life: The presence of Obg homologs across bacteria, archaea, and eukaryotes indicates that this GTPase emerged early in cellular evolution. In eukaryotes, related proteins like the developmentally regulated GTP-binding proteins (DRGs) share structural features with bacterial Obg, including the presence of a TGS domain in some family members .

• Horizontal gene transfer: The biogeographic distribution of closely related Rhodopirellula species suggests limited habitat sizes for attached-living bacteria, potentially affecting the evolution of their Obg proteins through restricted horizontal gene transfer opportunities .

These evolutionary considerations are important when designing experiments to study R. baltica Obg, as they provide context for understanding species-specific adaptations and potential functions.

How can Obg studies contribute to understanding the biology of Rhodopirellula baltica and other Planctomycetes?

Research on Obg GTPase can provide significant insights into the unique biology of R. baltica and other Planctomycetes in several ways:

• Cell cycle regulation: Planctomycetes like R. baltica have complex cell cycles and unusual cell division mechanisms. Since Obg is involved in cell cycle regulation in bacteria, studying its function in R. baltica could reveal adaptation of this regulatory system to the distinctive Planctomycete cell biology .

• Environmental adaptation: R. baltica and other Rhodopirellula species show specific biogeographic distribution patterns and adaptations to environmental conditions:

  • Tolerance to varying salinity levels (from 1.15% to 4.6% NaCl)

  • Adaptation to different marine regions in European seas

  • Potential roles in polluted environments, including hydrocarbon-contaminated mangroves

  Studying how Obg contributes to these adaptations could provide insights into stress response mechanisms in these specialized bacteria.

• Metabolic regulation: Analysis of R. baltica's life cycle through transcriptome studies has revealed complex metabolic regulation during different growth phases. During the stationary phase, the bacterium expresses many genes coding for transposases, integrases, and recombinases, suggesting genome rearrangements under stress conditions . Investigating Obg's role in these transitions could illuminate its contribution to metabolic adaptation.

• Evolutionary insights: Planctomycetes have distinctive evolutionary features, including large genomes with extensive strain-specific genes . Comparative studies of Obg across different Planctomycetes could provide insights into the evolutionary history and functional diversification of this essential protein in this unique bacterial phylum.

• Biotechnological applications: Understanding Obg's role in stress response could potentially be leveraged for biotechnological applications, particularly given the ability of some Rhodopirellula strains to degrade hydrocarbons in polluted environments .

What are common challenges in expressing and purifying recombinant R. baltica Obg?

Researchers working with recombinant R. baltica Obg may encounter several technical challenges:

• Solubility issues: Obg proteins can exhibit limited solubility when overexpressed in E. coli.

  • Solution: Optimize expression conditions by reducing induction temperature (16-18°C), decreasing IPTG concentration (0.1-0.2 mM), or using solubility-enhancing fusion tags (SUMO, MBP, or TRX).

  • Alternative: Consider expression in cold-adapted systems or cell-free protein synthesis.

• Nucleotide retention: Purified Obg often co-purifies with bound nucleotides that can affect downstream analyses.

  • Solution: Include additional washing steps with high salt buffer (500 mM NaCl) and/or mild denaturants during affinity purification.

  • Detection: Use HPLC analysis of protein samples to confirm nucleotide content before functional assays.

• Protein instability: The multi-domain structure of Obg, including the intrinsically disordered C-terminal domain, can lead to degradation during purification.

  • Solution: Include protease inhibitors throughout purification and minimize handling time.

  • Storage: Add glycerol (10-20%) and reducing agents, and store protein in small aliquots at -80°C to avoid freeze-thaw cycles.

• Low GTPase activity: R. baltica Obg may exhibit low intrinsic GTPase activity that can be difficult to measure.

  • Solution: Ensure buffer contains optimal potassium concentration (50-100 mM KCl), as potassium ions are essential for Obg GTPase activity in many species.

  • Sensitive assays: Use high-sensitivity methods like radiometric assays or enzyme-coupled continuous assays for detection of low activity.

• Structural heterogeneity: The flexible nature of Obg proteins, particularly the C-terminal domain, can lead to conformational heterogeneity.

  • Solution: For structural studies, consider generating truncated constructs lacking the flexible C-terminal region.

  • Analysis: Use techniques like SAXS that can accommodate conformational flexibility in solution .

How can researchers optimize GTPase activity assays for R. baltica Obg?

Optimizing GTPase activity assays for R. baltica Obg requires attention to several critical parameters:

• Buffer composition:

  • Include 50-100 mM KCl, as potassium ions are essential for optimal activity of many Obg proteins

  • Maintain 5-10 mM MgCl2 for proper coordination of the nucleotide

  • Use pH 7.5-8.0 (Tris-HCl or HEPES buffer systems)

  • Include 1-2 mM DTT or TCEP to maintain reduced state

• Temperature considerations:

  • As R. baltica is a marine bacterium, its Obg may show optimal activity at lower temperatures

  • Test activity at various temperatures (25°C, 30°C, 37°C)

  • For kinetic studies, select a temperature that balances activity with protein stability

• Nucleotide concentration:

  • Use GTP concentrations ranging from 50-500 μM for standard assays

  • For Km determination, test a range from 1-1000 μM GTP

  • Consider the effect of other nucleotides (ppGpp, GDP) as potential regulators

• Assay sensitivity:

  • For colorimetric assays, extend incubation times (up to 60 minutes) to detect low activity

  • Consider using more sensitive methods like coupled-enzyme assays that continuously monitor phosphate release

  • For the highest sensitivity, radiometric assays with [γ-32P]GTP can detect very low activities

• Controls and validations:

  • Include a GTPase-deficient variant (mutations in G1 or G3 motifs) as a negative control

  • Use a known active GTPase (such as Ras or another well-characterized Obg) as a positive control

  • Verify that observed activity is protein-concentration dependent

  • Confirm that activity is inhibited by nucleotide analogs like GTPγS or GDP

By carefully optimizing these parameters, researchers can develop reliable assays for the potentially low intrinsic GTPase activity of R. baltica Obg.

What strategies are effective for detecting and characterizing Obg-protein interactions in complex bacterial systems?

To effectively study Obg-protein interactions in complex bacterial systems like R. baltica, researchers can employ several complementary strategies:

• In vivo crosslinking combined with mass spectrometry:

  • Treat bacterial cultures with membrane-permeable crosslinkers (formaldehyde or DSP)

  • Purify Obg complexes using antibodies or via tagged Obg variants

  • Identify interaction partners by mass spectrometry

  • Compare interaction profiles under different conditions (various growth phases, stress conditions) to identify context-specific interactions

• Bacterial two-hybrid and three-hybrid systems:

  • Adapt bacterial two-hybrid systems for marine bacteria or use heterologous systems

  • Consider three-hybrid approaches to include the effect of nucleotides (GTP, GDP, ppGpp) on interactions

  • Use comprehensive genomic libraries to screen for novel interaction partners

• Proximity-dependent labeling:

  • Express Obg fused to promiscuous labeling enzymes (BioID, APEX2, TurboID)

  • Allow in vivo labeling of proximal proteins

  • Purify and identify labeled proteins by mass spectrometry

  • This approach captures transient and weak interactions difficult to detect by other methods

• Co-immunoprecipitation with nucleotide considerations:

  • Perform immunoprecipitation in the presence of specific nucleotides (GTP, GDP, ppGpp)

  • Use non-hydrolyzable GTP analogs (GTPγS, GMPPNP) to trap specific conformational states

  • Compare interaction profiles across nucleotide conditions to identify state-specific partners

• Protein microarrays and surface plasmon resonance:

  • Create arrays of R. baltica proteins for systematic interaction screening

  • Use surface plasmon resonance or biolayer interferometry to determine binding kinetics

  • Test interactions in the presence of different nucleotides

  • Validate candidate interactions with orthogonal methods

These approaches can help unravel the Obg interactome in R. baltica, providing insights into its roles in ribosome biogenesis, stress response, and other cellular processes.

What are promising future research directions for R. baltica Obg studies?

Several promising research directions could advance our understanding of R. baltica Obg and its functions:

• Comprehensive interactome mapping:

  • Apply modern proteomics approaches to identify the complete set of Obg interaction partners in R. baltica

  • Compare interactomes across different growth conditions, particularly during environmental stress

  • Develop a network model of Obg's role in cellular regulation specific to Planctomycetes

• Structural biology approaches:

  • Determine high-resolution structures of R. baltica Obg in different nucleotide-bound states

  • Investigate the structural basis of Obg interaction with the ribosome in R. baltica

  • Use cryo-electron microscopy to visualize Obg-ribosome complexes in native-like conditions

• Environmental adaptation mechanisms:

  • Investigate how Obg contributes to R. baltica's adaptation to varying salinity levels

  • Explore the role of Obg in the response to pollutants, particularly in hydrocarbon-contaminated environments

  • Compare Obg function across Rhodopirellula strains from different marine environments

• Gene editing and functional genomics:

  • Develop or adapt CRISPR-Cas systems for Planctomycetes to enable precise genetic manipulation

  • Create conditional mutants to study the essential functions of Obg in R. baltica

  • Use transcriptomics to identify genes regulated by Obg under different conditions

• Antimicrobial applications:

  • Screen for specific inhibitors of R. baltica Obg as tools for studying its function

  • Compare binding sites across bacterial Obg proteins to identify conserved targets for broad-spectrum antimicrobials

  • Evaluate natural compounds from marine environments as potential Obg inhibitors

These research directions could significantly advance our understanding of this essential bacterial GTPase while potentially yielding new insights into the unique biology of Planctomycetes and developing novel antimicrobial strategies.

How might systems biology approaches enhance our understanding of Obg networks in R. baltica?

Systems biology approaches offer powerful tools for understanding the complex networks involving Obg in R. baltica:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data to build comprehensive models of Obg's influence on cellular physiology

  • Track system-wide changes in response to perturbations of Obg function

  • Identify emergent properties of Obg regulatory networks not apparent from reductionist approaches

• Network analysis:

  • Construct protein-protein interaction networks centered on Obg

  • Apply graph theory to identify key nodes and regulatory hubs connected to Obg

  • Use network motif analysis to identify recurring regulatory patterns involving Obg

• Mathematical modeling:

  • Develop quantitative models of Obg's GTPase cycle and its regulation by nucleotides and protein partners

  • Create kinetic models of ribosome assembly with Obg as a regulatory component

  • Simulate the effects of environmental stressors on Obg-dependent processes

• Comparative systems biology:

  • Compare Obg networks across multiple Rhodopirellula strains to identify core and variable components

  • Extend comparisons to other bacterial phyla to understand how Obg networks evolved in different lineages

  • Identify system-level adaptations in marine bacteria versus terrestrial or pathogenic species

• Genome-scale metabolic modeling:

  • Incorporate Obg regulation into genome-scale metabolic models of R. baltica

  • Predict how Obg activity affects metabolic flux distributions under different conditions

  • Identify metabolic vulnerabilities that could be targeted by Obg inhibitors

These approaches would help place Obg within the broader context of R. baltica's cellular systems, revealing how this essential GTPase coordinates multiple aspects of bacterial physiology in response to environmental cues.

What potential biotechnological applications might emerge from R. baltica Obg research?

Research on R. baltica Obg could lead to several innovative biotechnological applications:

• Novel antimicrobial development:

  • Design of broad-spectrum antibiotics targeting conserved features of bacterial Obg proteins

  • Development of anti-persister compounds that interfere with Obg's role in bacterial persistence

  • Creation of combination therapies targeting both Obg and interacting cellular components

• Bioremediation tools:

  • Engineering of Rhodopirellula strains with enhanced Obg-mediated stress tolerance for use in bioremediation of contaminated marine environments

  • Development of biosensors based on Obg stress responses to detect environmental pollutants

  • Optimization of R. baltica's natural capacity for hydrocarbon degradation through modification of Obg regulatory networks

• Synthetic biology applications:

  • Utilization of Obg as a molecular switch in synthetic circuits designed for marine bacteria

  • Creation of tunable gene expression systems responding to specific environmental stressors via Obg-dependent pathways

  • Development of growth-controlled bioproduction systems in marine bacteria

• Bioprocess optimization:

  • Engineering of stress-resistant bacterial strains for industrial processes through modification of Obg pathways

  • Design of ribosome engineering strategies based on Obg interactions for enhanced protein production

  • Development of controlled persistence states for long-term maintenance of bacterial cultures

• Diagnostic tools:

  • Creation of diagnostic assays based on Obg function to detect and characterize bacterial pathogens

  • Development of high-throughput screening platforms for Obg inhibitors

  • Design of biosensors that leverage Obg's nucleotide-sensing capabilities

These applications would leverage the fundamental understanding of Obg function in R. baltica while addressing important challenges in antimicrobial resistance, environmental remediation, and sustainable biotechnology.