Recombinant Ochrobactrum anthropi Nucleoside diphosphate kinase (ndk)

What is Ochrobactrum anthropi and why is it significant in research?

Ochrobactrum anthropi is an aerobic, Gram-negative, motile bacillus that belongs to the α-Proteobacteria class. It has gained significant research attention as an emerging opportunistic and nosocomial pathogen. O. anthropi was previously known as "Achromobacter group Vd" and is characterized as oxidase-positive, urease-positive, and non-lactose-fermenting . It is increasingly recognized in hospital-acquired infections, particularly in immunocompromised patients. The organism presents unique research opportunities due to its ability to interact with eukaryotic cells and its emerging pathogenic status in healthcare settings .

What is Nucleoside diphosphate kinase (ndk) and what is its function in O. anthropi?

Nucleoside diphosphate kinase (ndk) is an enzyme identified in the cytoplasmic fraction of O. anthropi . This enzyme catalyzes the transfer of terminal phosphate groups from nucleoside triphosphates to nucleoside diphosphates, playing a critical role in nucleotide metabolism and cellular energetics. In O. anthropi, ndk has been specifically identified during early growth phases through proteomic studies with a Mowse score of 64, indicating a high confidence in protein identification . The enzyme contributes to the bacterium's metabolic processes and energy management, serving as a component in the complex network of enzymes supporting bacterial growth and adaptation.

How is O. anthropi ndk characterized in proteomic studies?

In gel-based liquid chromatography-mass spectrometry (GeLC-MS) analysis, O. anthropi ndk has been positively identified with the following characteristics:

• Mowse score: 64 (indicating a high confidence level in the identification)

• Cellular localization: Cytoplasmic

• Signal peptide presence: None detected

• SecretomeP prediction: Negative

• emPAI value: 0.156

• Protein molar percentage: 0.615%

This proteomic characterization indicates that ndk is expressed during early growth phases and constitutes approximately 0.615% of the soluble protein content by molar fraction during this phase, suggesting its importance in early cellular processes.

How does the expression pattern of ndk in O. anthropi differ between growth phases, and what does this suggest about its physiological role?

Proteomic analysis using the Exponentially Modified Protein Abundance Index (emPAI) methodology has identified that ndk in O. anthropi is uniquely expressed during early growth phases, with an emPAI value of 0.156 and constituting approximately 0.615% of the molar protein fraction . The enzyme's presence specifically in early growth and absence in late growth suggests a phase-dependent role.

This expression pattern indicates ndk likely supports crucial nucleotide metabolism processes during rapid cellular division and DNA synthesis that characterize early bacterial growth. The absence in late growth phase may indicate either downregulation when growth slows or a shift in metabolic priorities as the bacterium transitions to stationary phase. This growth phase-specific expression could be connected to energy conservation strategies or adaptation to changing environmental conditions as cultures mature.

What challenges exist in distinguishing recombinant O. anthropi ndk from other bacterial nucleoside diphosphate kinases?

Distinguishing recombinant O. anthropi ndk from other bacterial ndks presents several challenges due to structural and sequence similarities. O. anthropi belongs to a group of bacteria that share high genetic homology with other genera, particularly Brucella spp., making protein differentiation complex .

The challenges include:

• Sequence homology: High sequence similarity with ndks from related α-Proteobacteria

• Structural conservation: The nucleoside diphosphate kinase family shows conserved structural features across bacterial species

• Biochemical properties: Similar catalytic mechanisms and substrate preferences

• Identification methodology: Standard biochemical tests often prove insufficient, as seen in the misidentification of O. anthropi itself as Ralstonia paucula

To address these challenges, researchers should employ:

• Molecular approaches such as 16S rRNA and recA analysis for accurate identification

• Mass spectrometry with high-resolution capabilities for proteomic validation

• Epitope tagging of recombinant proteins to facilitate distinction from native proteins

• Species-specific antibody development targeting unique epitopes of O. anthropi ndk

How might O. anthropi ndk be involved in the pathogen's survival against host immune responses?

While the search results don't directly address ndk's role in immune evasion, the proteomic analysis of O. anthropi provides context for potential mechanisms. The study identified that in late phase growth, O. anthropi expresses proteins under the control of the oxyR regulon, which is induced in response to oxidative stress and linked to pathogen survival against host immunity reactions .

Though ndk was not explicitly identified among these stress-response proteins, nucleoside diphosphate kinases in other pathogens have been implicated in:

• Maintaining nucleotide pools during oxidative stress conditions

• Supporting DNA repair mechanisms when faced with host-induced DNA damage

• Contributing to energy homeostasis during immune challenge

• Potentially participating in signaling pathways that regulate virulence factor expression

The cytoplasmic localization of O. anthropi ndk suggests it primarily functions in intracellular metabolic processes, but its activities could indirectly support pathogenicity by ensuring metabolic fitness during host interactions .

What expression systems are most effective for producing recombinant O. anthropi ndk?

For optimal expression of recombinant O. anthropi ndk, researchers should consider several expression systems based on the protein's characteristics and experimental needs:

• E. coli-based expression systems:

  • BL21(DE3) strain with pET vector systems for high-yield cytoplasmic expression

  • Rosetta or CodonPlus strains if O. anthropi codon usage differs significantly from E. coli

  • Fusion tag strategies (His-tag, GST, MBP) to facilitate purification and enhance solubility

• Expression conditions:

  • Induction parameters: IPTG concentration (0.1-1.0 mM)

  • Temperature optimization: Lower temperatures (16-25°C) may improve proper folding

  • Growth media: Rich media (LB, TB) for high biomass or minimal media for isotope labeling

• Protein extraction considerations:

  • Given the cytoplasmic localization of ndk in O. anthropi , standard cell lysis methods (sonication, French press) should be effective

  • Buffer optimization to maintain enzyme activity (typically containing Mg²⁺ as a cofactor)

• Purification strategy:

  • Affinity chromatography based on fusion tags

  • Ion exchange chromatography exploiting the protein's pI characteristics

  • Size exclusion as a polishing step

The choice of expression system should consider the downstream applications and required protein purity, activity, and yield.

What are the optimal conditions for assaying recombinant O. anthropi ndk enzymatic activity?

Based on general nucleoside diphosphate kinase properties and the information about O. anthropi proteomics , the following assay conditions would be recommended:

Standard Enzyme Activity Assay Conditions:

Given that O. anthropi can survive in diverse environments as an opportunistic pathogen , assaying the enzyme under various pH conditions (6.0-9.0) and salt concentrations (0-500 mM NaCl) would provide valuable insights into its adaptability and potential role in pathogen survival.

How can researchers effectively purify recombinant O. anthropi ndk while maintaining its structural integrity and activity?

Effective purification of recombinant O. anthropi ndk requires a strategy that preserves both structural integrity and enzymatic activity:

Recommended Purification Protocol:

• Cell lysis optimization:

  • Gentle lysis methods like enzymatic cell wall degradation or mild sonication

  • Inclusion of protease inhibitors to prevent degradation

  • Buffer composition: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT

• Multi-step purification strategy:

  • Initial capture: Immobilized metal affinity chromatography (IMAC) for His-tagged ndk

  • Intermediate purification: Ion exchange chromatography based on predicted pI

  • Polishing: Size exclusion chromatography to remove aggregates and ensure homogeneity

• Activity preservation measures:

  • Maintain 5-10% glycerol throughout purification to stabilize protein structure

  • Include 1-5 mM MgCl₂ in all buffers to maintain active site integrity

  • Process samples at 4°C to minimize thermal denaturation

  • Consider adding ATP (0.1-0.5 mM) to stabilize the enzyme's conformation

• Quality assessment:

  • Size exclusion chromatography to confirm oligomeric state

  • Circular dichroism to verify secondary structure integrity

  • Activity assays at each purification step to track specific activity

  • Mass spectrometry to confirm protein identity and detect potential modifications

The proteomic analysis indicates that O. anthropi ndk is a cytoplasmic protein without signal peptides , suggesting it should be relatively stable during purification procedures designed for soluble proteins.

What structural analysis techniques are most informative for characterizing O. anthropi ndk?

For comprehensive structural characterization of recombinant O. anthropi ndk, researchers should employ a combination of techniques:

Recommended Structural Analysis Techniques:

Given the emPAI value of 0.156 reported for O. anthropi ndk , obtaining sufficient quantities of pure protein for structural studies may require optimization of recombinant expression systems. The protein appears to be expressed at moderate levels in the bacterium's early growth phase, suggesting it should be amenable to recombinant production in sufficient quantities for structural studies.

How can studies on O. anthropi ndk contribute to understanding this emerging opportunistic pathogen?

Studies on O. anthropi ndk can provide valuable insights into this emerging pathogen through several research avenues:

• Metabolic network mapping:
  Characterizing ndk's role within O. anthropi's metabolic network could help identify essential pathways for bacterial survival and growth. The proteomic study has already identified that ndk is expressed during early growth phases , suggesting its importance in establishing bacterial colonies.

• Adaptation mechanisms:
  O. anthropi is increasingly recognized as a nosocomial pathogen capable of causing infections in immunocompromised patients . Understanding how ndk supports bacterial adaptation to hospital environments and host conditions could reveal survival strategies.

• Comparative proteomics:
  The proteomic analysis that identified ndk provides a foundation for comparing O. anthropi protein expression with other pathogens. Such comparisons could identify unique or conserved features that contribute to its emerging pathogenic status.

• Diagnostic development:
  Given the difficulty in identifying O. anthropi through conventional methods , ndk and other characteristic proteins could serve as biomarkers for improved diagnostics. The study noted that O. anthropi was initially misidentified as Ralstonia paucula, highlighting the need for better identification methods .

• Virulence factor correlation:
  While ndk itself may not be a virulence factor, its role in supplying nucleotides could support the expression of actual virulence factors, particularly those induced during host infection.

What insights can comparative analysis of O. anthropi ndk with other bacterial ndks provide about evolutionary relationships?

Comparative analysis of O. anthropi ndk with ndks from other bacterial species can provide valuable evolutionary insights:

• Phylogenetic relationships:
  O. anthropi belongs to the α-Proteobacteria and shows high genetic similarity to Brucella species . Comparing ndk sequences could help resolve fine-scale phylogenetic relationships, particularly within closely related genera that are otherwise difficult to distinguish.

• Functional conservation vs. specialization:
  While the core catalytic function of ndks is conserved across bacteria, substrate preferences and regulatory mechanisms may vary. The specific expression pattern of O. anthropi ndk in early growth phases suggests possible specialized temporal regulation.

• Horizontal gene transfer assessment:
  Analyzing ndk sequence divergence patterns can help identify potential horizontal gene transfer events, which may contribute to the adaptation of O. anthropi to new ecological niches, including healthcare environments.

• Structural evolution:
  Comparing predicted or determined structures of O. anthropi ndk with other bacterial ndks could reveal conserved structural elements essential for function versus variable regions that might confer species-specific properties.

• Host-pathogen co-evolution:
  As an opportunistic human pathogen , O. anthropi may have evolved specific ndk features in response to host environments. Comparative analysis with ndks from obligate pathogens versus environmental bacteria could highlight adaptations related to pathogenicity.

The high sequence similarity between O. anthropi and Brucella species, as demonstrated by 16S rRNA and recA analysis , suggests that detailed molecular comparisons of functional proteins like ndk could provide additional markers for taxonomic classification and evolutionary studies.

What are common challenges in working with recombinant O. anthropi proteins and their solutions?

Researchers working with recombinant O. anthropi proteins, including ndk, frequently encounter several challenges:

The proteomic analysis of O. anthropi has successfully identified numerous proteins including ndk , demonstrating that these challenges can be overcome with appropriate methodological approaches.

How can researchers address and interpret contradictory data when studying O. anthropi ndk function?

When confronted with contradictory data regarding O. anthropi ndk function, researchers should implement a systematic approach to resolution:

• Validation of protein identity:

  • Confirm protein identity using multiple methods beyond standard biochemical tests

  • Implement 16S rRNA sequencing and recA analysis as demonstrated effective for O. anthropi identification

  • Verify protein sequence by mass spectrometry

• Experimental condition assessment:

  • Evaluate growth phase impacts on protein expression, as O. anthropi shows distinct proteomic profiles between early and late growth phases

  • Control for environmental variables (pH, temperature, media composition)

  • Document precise experimental conditions to identify variables causing contradictory results

• Methodological reconciliation:

  • Compare assay methodologies when contradictory functional data emerge

  • Consider the impact of protein tags, buffer compositions, and detection methods

  • Implement multiple orthogonal assays to triangulate true functional parameters

• Biological context interpretation:

  • Interpret ndk function within the broader context of O. anthropi metabolism

  • Consider potential moonlighting functions beyond canonical nucleoside diphosphate kinase activity

  • Evaluate potential regulatory mechanisms that might create context-dependent functional profiles

• Statistical approach:

  • Conduct sufficient biological and technical replicates to establish statistical confidence

  • Apply appropriate statistical tests to determine if differences are significant

  • Consider Bayesian approaches to weigh contradictory evidence

This systematic approach acknowledges that as an emerging pathogen , our understanding of O. anthropi proteins continues to evolve, and apparent contradictions often represent opportunities for deeper insights.

What are promising research avenues for studying O. anthropi ndk's potential role in pathogenesis?

Several promising research directions could elucidate O. anthropi ndk's role in pathogenesis:

• Temporal expression profiling during infection:
  The proteomic analysis has established that ndk is expressed during early growth phase in culture , but its expression pattern during actual host infection remains unexplored. Time-course studies during infection models could reveal if ndk expression correlates with specific stages of pathogenesis.

• Stress response coordination:
  Investigate potential links between ndk and the oxyR regulon, which was identified in late phase growth and linked to oxidative stress response . While ndk wasn't among the identified oxyR-regulated proteins, its role in nucleotide metabolism might indirectly support stress responses.

• Host-pathogen interaction studies:
  Explore whether O. anthropi ndk, despite being primarily cytoplasmic , might interact with host factors either through secretion mechanisms or following bacterial lysis, potentially influencing host cell signaling.

• Knockout/knockdown phenotype characterization:
  Generate ndk-deficient O. anthropi strains to assess impacts on:

  • Growth kinetics and metabolic fitness

  • Survival during host immune responses

  • Biofilm formation capability

  • Antibiotic susceptibility profiles

• Comparative virulence studies:
  Compare wildtype versus ndk-modified strains in models of opportunistic infection, focusing on the types of infections documented for O. anthropi, such as catheter-associated infections, endocarditis, and infections in immunocompromised patients .

• Small molecule inhibitor screening:
  Identify specific inhibitors of O. anthropi ndk as potential therapeutic leads, leveraging any structural or functional differences from human ndks.

These research directions acknowledge that while O. anthropi is considered to have relatively low virulence, it is increasingly recognized as a problematic opportunistic pathogen, particularly in healthcare settings .

How might multi-omics approaches enhance our understanding of O. anthropi ndk in cellular networks?

Multi-omics approaches offer powerful strategies to contextualize O. anthropi ndk within cellular networks:

• Integrated proteogenomics:
  Building on the existing proteomic characterization , combining genomic, transcriptomic, and proteomic data could:

  • Map the regulatory elements controlling ndk expression

  • Identify co-regulated genes that function alongside ndk

  • Detect post-translational modifications affecting ndk function

• Metabolomics integration:
  As ndk plays a role in nucleotide metabolism, metabolomic profiling could:

  • Quantify changes in nucleotide pools correlated with ndk expression levels

  • Identify metabolic bottlenecks or overflow pathways when ndk activity is altered

  • Map the ripple effects of ndk modulation throughout the metabolic network

• Interactomics approaches:
  Protein-protein interaction studies could reveal:

  • Direct binding partners of O. anthropi ndk

  • Participation in multi-enzyme complexes

  • Potential moonlighting functions beyond canonical enzymatic activity

• Systems biology modeling:
  Mathematical modeling integrating multi-omics data could:

  • Predict the systemic impact of ndk perturbation

  • Identify emergent properties not obvious from single-omics studies

  • Simulate ndk behavior under various environmental conditions

• Comparative multi-omics:
  Extending the approach across growth conditions and related species could:

  • Differentiate core versus condition-specific roles of ndk

  • Identify evolutionary adaptations in ndk network integration

  • Reveal potential therapeutic targets unique to pathogenic species

The proteomic study has already demonstrated distinct protein expression profiles between growth phases , suggesting that temporal multi-omics approaches would be particularly informative for understanding dynamic network roles of ndk.