Recombinant Beijerinckia indica subsp. indica Nucleoside diphosphate kinase (ndk)

What is the function of nucleoside diphosphate kinase in Beijerinckia indica metabolism?

Nucleoside diphosphate kinase (NDK) in B. indica subsp. indica plays a crucial role in maintaining the cellular balance of nucleoside triphosphates (NTPs) and nucleoside diphosphates (NDPs). The enzyme catalyzes the transfer of a γ-phosphate from NTPs to NDPs via the reaction: N₁TP + N₂DP ↔ N₁DP + N₂TP. This reversible reaction is vital for numerous cellular processes including DNA replication, RNA synthesis, and polysaccharide formation. In the context of B. indica, an organism with a 4.17 Mbp genome encoding 3,784 predicted proteins, NDK likely supports its metabolic versatility as a chemoorganotroph capable of growth on various organic acids, sugars, and alcohols .

Experimental approaches to study NDK function in B. indica typically involve:

• Enzyme activity assays using purified recombinant protein

• Gene knockout studies to observe phenotypic effects

• Metabolomic analysis comparing wild-type and NDK-deficient strains

How does the genomic context of the ndk gene in Beijerinckia indica inform our understanding of its regulation?

The ndk gene in B. indica is found within its 4.17 Mbp genome, which also contains two plasmids of 181,736 and 66,727 bp . While the specific genomic context of the ndk gene isn't detailed in the available literature, we can analyze potential regulatory elements based on what we know about related organisms.

Researchers should consider:

• Examining the upstream region for potential promoter sequences

• Identifying neighboring genes that might be co-regulated

• Using tools like RegPredict to identify potential regulatory sequences similar to those identified in other related bacteria

• Conducting transcriptomic analysis under different growth conditions to understand expression patterns

The G+C content of the B. indica genome is 57.0% , which may influence codon usage and expression efficiency of the ndk gene. Comparative genomic analysis with closely related organisms like Methylocella silvestris (which shares 57% of its genes with B. indica) could provide insights into conservation and regulatory patterns of ndk .

What expression systems are recommended for recombinant production of Beijerinckia indica NDK?

Based on successful approaches with similar proteins, E. coli remains the preferred expression system for recombinant B. indica NDK production, as evidenced by its use for other B. indica proteins like DnaK . The following methodology is recommended:

Expression Protocol:

• Clone the B. indica ndk gene into a suitable expression vector (pET or pBAD series)

• Transform into an E. coli expression strain (BL21(DE3), Rosetta, or Arctic Express)

• Culture in LB medium supplemented with appropriate antibiotics

• Induce protein expression at OD₆₀₀ of 0.6-0.8 with IPTG (0.1-1.0 mM) or arabinose

• Optimize expression by testing different temperatures (16-37°C) and induction times (3-24 hours)

Purification Strategy:

• Harvest cells by centrifugation and lyse using sonication or French press

• Clarify lysate by centrifugation at ≥20,000 × g

• Purify using affinity chromatography (His-tag or GST-tag)

• Further purify by ion-exchange and size-exclusion chromatography

• Assess purity by SDS-PAGE (aiming for >85% purity)

• Verify protein identity by mass spectrometry

Storage recommendations include adding glycerol (5-50% final concentration) and storing at -20°C/-80°C, with a typical shelf life of 6 months for liquid form and 12 months for lyophilized preparations .

How can structural studies of Beijerinckia indica NDK inform inhibitor design for potential antibacterial applications?

Structural studies of B. indica NDK can provide valuable insights for rational inhibitor design, similar to approaches used with NDK from Borrelia burgdorferi . The following methodological approach is recommended:

• Protein Crystallization Strategy:

  • Use purified recombinant B. indica NDK at 10-15 mg/mL

  • Screen crystallization conditions using commercial kits

  • Optimize promising conditions varying precipitant concentration, pH, and additives

  • Attempt co-crystallization with ADP and vanadate to capture transition state complexes

• Structure Determination Protocol:

  • Collect X-ray diffraction data at synchrotron beamlines

  • Process data using XDS or MOSFLM software packages

  • Solve structure by molecular replacement using related NDK structures as templates

  • Refine structure using PHENIX or REFMAC

  • Validate structure using MolProbity

• Structure-Based Inhibitor Design:

  • Identify catalytic residues and substrate binding pockets

  • Perform in silico docking studies to screen potential inhibitors

  • Design transition state analogs based on the ADP-vanadate complex structure

  • Synthesize and test promising candidates using enzyme inhibition assays

The structural information would be particularly valuable given that NDK function has been demonstrated to be important for establishing infection in mouse models for other bacterial species , suggesting potential therapeutic applications.

What approaches can be used to investigate the role of Beijerinckia indica NDK in exopolysaccharide production?

B. indica is known for its abundant production of exoheteropolysaccharide with potential biotechnological applications . Investigating NDK's potential role in this process requires a multifaceted approach:

• Genetic Manipulation Strategies:

  • Generate conditional ndk knockdown strains using CRISPR interference

  • Create point mutations in catalytic residues to modulate NDK activity

  • Complement knockout strains with wild-type or mutant ndk genes

• Analytical Methods for Exopolysaccharide Quantification:

  • Gravimetric analysis after ethanol precipitation

  • Size-exclusion chromatography for molecular weight determination

  • Composition analysis using HPLC or GC-MS after hydrolysis

  • Rheological characterization to assess viscosity properties

• Metabolic Flux Analysis:

  • Use ¹³C-labeled substrates to trace carbon flux through nucleotide sugar pathways

  • Quantify nucleotide sugar precursors using LC-MS/MS

  • Correlate NDK activity with nucleotide sugar pool sizes

  • Model the impact of altered NTP/NDP ratios on exopolysaccharide biosynthesis

Table 1. Potential Impact of NDK Activity on Exopolysaccharide Precursor Availability

How does the NDK of Beijerinckia indica compare functionally with homologs from phylogenetically related methanotrophs?

B. indica is phylogenetically closely related to facultative and obligate methanotrophs of the genera Methylocella and Methylocapsa . A comparative functional analysis of NDKs from these organisms could provide insights into metabolic adaptations and evolutionary relationships:

• Homology Analysis Protocol:

  • Identify NDK homologs in Methylocella silvestris and other related bacteria

  • Perform multiple sequence alignment to identify conserved and divergent regions

  • Conduct phylogenetic analysis to establish evolutionary relationships

  • Map sequence differences onto structural models to predict functional implications

• Comparative Biochemical Characterization:

  • Express and purify NDKs from B. indica and related methanotrophs

  • Determine kinetic parameters (Km, kcat) for various NTP/NDP combinations

  • Assess thermal stability using differential scanning fluorimetry

  • Compare substrate specificity profiles

  • Analyze oligomeric states using size-exclusion chromatography coupled with multi-angle light scattering

• Functional Complementation Studies:

  • Create NDK knockout strains in both B. indica and M. silvestris

  • Cross-complement with heterologous NDKs

  • Assess rescue of growth defects and metabolic parameters

  • Evaluate the impact on specialized metabolic pathways (N₂ fixation vs. methanotrophy)

Given that 57% of the genes in B. indica have homologues in M. silvestris , comparative analysis of their NDKs could reveal adaptations specific to their distinct metabolic lifestyles, despite their similar genome sizes (4.17 versus 4.30 Mbp) and gene counts (3,788 versus 3,917) .

What are the optimal conditions for assaying Beijerinckia indica NDK activity in vitro?

Establishing reliable assay conditions is crucial for consistent characterization of B. indica NDK. The following methodological approaches are recommended:

• Coupled Spectrophotometric Assay:

  • Reaction mixture: 50 mM Tris-HCl (pH 7.5-8.0), 5 mM MgCl₂, 1 mM NDP (GDP or UDP), 1 mM ATP

  • Coupling system: pyruvate kinase (PK) and lactate dehydrogenase (LDH)

  • Substrates: phosphoenolpyruvate (PEP) and NADH

  • Monitor absorbance decrease at 340 nm as NADH is oxidized

  • Calculate activity using NADH extinction coefficient (6,220 M⁻¹cm⁻¹)

• pH and Temperature Optimization:

  • Test pH range from 5.5-9.0 (relevant for acidophilic B. indica)

  • Evaluate temperature range from 25-45°C

  • Determine pH and temperature stability profiles

• Metal Ion Requirements:

  • Test divalent cations (Mg²⁺, Mn²⁺, Ca²⁺) at 1-10 mM concentrations

  • Assess potential inhibitory effects of other metal ions

  • Determine optimal Mg²⁺ concentration for maximal activity

• Substrate Specificity Analysis:

  • Test various NTP donors (ATP, GTP, CTP, UTP)

  • Evaluate different NDP acceptors (ADP, GDP, CDP, UDP)

  • Determine Km and Vmax for each substrate combination

  • Calculate specificity constants (kcat/Km) to identify preferred substrates

Table 2. Expected Optimal Conditions for B. indica NDK Activity

How might B. indica NDK interact with the nitrogen fixation machinery?

Beijerinckia indica is a nitrogen-fixing bacterium with genes involved in N₂ fixation clustered in two genomic islands (10 kb and 51 kb) . Investigating potential interactions between NDK and the nitrogen fixation machinery requires:

• Protein-Protein Interaction Analysis:

  • Perform bacterial two-hybrid screening using NDK as bait

  • Conduct co-immunoprecipitation assays with tagged NDK

  • Apply crosslinking mass spectrometry to identify interaction partners

  • Use surface plasmon resonance to characterize binding kinetics

• Metabolic Nexus Investigation:

  • Analyze NTP requirements of nitrogenase and associated proteins

  • Determine if NDK co-localizes with nitrogen fixation machinery using fluorescent protein fusions

  • Measure nitrogenase activity in strains with altered NDK expression

  • Quantify ATP/GTP levels in wild-type versus NDK-modified strains during active nitrogen fixation

• Transcriptional Co-regulation Analysis:

  • Perform RNA-seq under nitrogen-fixing versus non-fixing conditions

  • Analyze promoter regions for common regulatory elements

  • Conduct ChIP-seq to identify transcription factors that may regulate both systems

  • Use reporter gene fusions to monitor expression patterns

This research direction is particularly intriguing given that nitrogen fixation is an energy-intensive process requiring significant ATP, and NDK's role in maintaining nucleotide triphosphate pools could be critical for sustaining this metabolically demanding process.

What are the best approaches for designing site-directed mutagenesis experiments to study catalytic residues of B. indica NDK?

Site-directed mutagenesis is a powerful approach to investigate the catalytic mechanism of B. indica NDK. The following methodology is recommended:

• Target Residue Selection Strategy:

  • Identify conserved catalytic residues through multiple sequence alignment with well-characterized NDKs

  • Focus on the active site histidine that forms a phosphohistidine intermediate

  • Target residues involved in metal coordination (typically aspartate or glutamate)

  • Examine residues involved in base specificity (purine/pyrimidine discrimination)

• Mutagenesis Design Principles:

  • For catalytic residues: Create H→N mutations to maintain size but eliminate reactivity

  • For metal-binding residues: Use D→N or E→Q substitutions

  • For substrate-binding residues: Introduce conservative and non-conservative changes

  • Consider alanine-scanning mutagenesis for systematic functional mapping

• Experimental Protocol:

  • Use overlap extension PCR or commercial site-directed mutagenesis kits

  • Verify mutations by DNA sequencing

  • Express and purify mutant proteins using the same conditions as wild-type

  • Compare specific activities, substrate affinities, and catalytic efficiencies

  • Perform thermal stability analysis to ensure mutations don't disrupt protein folding

Table 3. Recommended Site-Directed Mutations for B. indica NDK Functional Analysis

What advanced biophysical techniques are most informative for characterizing B. indica NDK structure-function relationships?

Several sophisticated biophysical techniques can provide detailed insights into the structure-function relationships of B. indica NDK:

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • ¹⁵N-¹H HSQC to monitor ligand binding and conformational changes

  • ³¹P NMR to directly observe phosphohistidine intermediate formation

  • Relaxation dispersion experiments to characterize enzyme dynamics

  • Protocol requires expression in minimal media with ¹⁵N and/or ¹³C labeled precursors

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Map solvent accessibility changes upon substrate binding

  • Identify regions undergoing conformational changes during catalysis

  • Compare wild-type and mutant proteins to pinpoint structural perturbations

  • Requires specialized equipment and careful sample preparation to minimize back-exchange

• Time-Resolved X-ray Crystallography:

  • Capture intermediates in the catalytic cycle using techniques like:

    • Temperature-jump triggered reactions

    • Photocaged substrates

    • Microfluidic crystal mixing devices

  • Similar to approaches used with B. burgdorferi NDK , attempt to capture the enzyme bound to ADP and vanadate to mimic the transition state

• Single-Molecule FRET:

  • Introduce fluorescent labels at strategic positions

  • Monitor conformational dynamics in real-time

  • Detect rare or transient conformational states

  • Correlate conformational changes with catalytic events

These techniques can be particularly powerful when used in combination, providing complementary information about structural dynamics, ligand interactions, and catalytic mechanisms.

How can computational approaches enhance our understanding of B. indica NDK function?

Computational methods offer powerful tools for investigating NDK function when combined with experimental approaches:

• Homology Modeling and Molecular Dynamics:

  • Build structural models based on homologous NDKs

  • Perform extended molecular dynamics simulations (>500 ns)

  • Analyze conformational flexibility and identify potential allosteric sites

  • Use enhanced sampling techniques to study conformational transitions

• Quantum Mechanics/Molecular Mechanics (QM/MM) Simulations:

  • Model the phosphoryl transfer reaction mechanism

  • Calculate energy barriers for catalysis

  • Evaluate effects of mutations on transition state stabilization

  • Compare with experimentally determined kinetic parameters

• Network Analysis of Genome Context:

  • Identify genes consistently co-occurring with ndk across related species

  • Construct metabolic models incorporating NDK function

  • Predict phenotypic effects of ndk perturbation

  • Similar to approaches used to analyze other genes in B. indica

• Machine Learning Applications:

  • Develop sequence-based predictors of NDK substrate specificity

  • Use graph neural networks to model protein-ligand interactions

  • Apply deep learning to predict effects of mutations

  • Integrate multi-omics data to understand NDK's role in cellular networks

These computational approaches can guide experimental design, provide mechanistic insights difficult to obtain experimentally, and help interpret experimental data within a broader biological context.

What are the most promising applications of recombinant B. indica NDK in biotechnology?

Several potential biotechnological applications of recombinant B. indica NDK warrant investigation:

• Enzymatic Synthesis of Nucleotide Analogs:

  • Develop protocols for large-scale production of modified nucleotides

  • Optimize reaction conditions for high yield and purity

  • Explore substrate promiscuity for incorporation of non-natural bases

  • Compare efficiency with NDKs from other sources

• Biosensor Development:

  • Create coupled enzyme systems for ATP or GTP detection

  • Develop fluorescent or bioluminescent reporters linked to NDK activity

  • Design immobilized enzyme systems for continuous monitoring

  • Optimize for sensitivity and specificity

• PCR Enhancement Applications:

  • Evaluate utility as a pyrophosphatase alternative in PCR reactions

  • Test ability to regenerate dNTPs during long-range PCR

  • Compare performance with commercial PCR additives

  • Optimize enzyme stability for thermocycling conditions

• Therapeutic Target Validation:

  • Similar to findings with B. burgdorferi NDK , investigate whether B. indica NDK could serve as a model for antibacterial drug development

  • Develop high-throughput screening assays for inhibitor discovery

  • Assess specificity against human NDKs

  • Evaluate effects in cellular and animal infection models

How might NDK activity influence the unique metabolic capabilities of B. indica?

B. indica possesses several distinctive metabolic features that might be influenced by NDK activity:

• Exopolysaccharide Production:

  • Investigate how nucleotide sugar precursor availability affects exopolysaccharide synthesis

  • Determine if exopolysaccharide composition changes with altered NDK activity

  • Study whether NDK co-localizes with polysaccharide biosynthetic machinery

  • Analyze transcriptional co-regulation of ndk with exopolysaccharide biosynthesis genes

• Nitrogen Fixation Efficiency:

  • Quantify ATP consumption during nitrogen fixation

  • Assess whether NDK overexpression enhances nitrogen fixation capacity

  • Investigate potential protein-protein interactions between NDK and nitrogenase components

  • Examine ndk expression patterns during active nitrogen fixation

• Stress Response and Adaptation:

  • Given B. indica's acidophilic nature, study NDK activity and stability at low pH

  • Investigate NDK expression during various stress conditions

  • Determine if NDK plays a role in acid tolerance mechanisms

  • Compare NDK properties with those from neutrophilic bacteria

• Growth on Diverse Carbon Sources:

  • B. indica is capable of growth on various organic acids, sugars, and alcohols

  • Analyze NDK expression across different carbon sources

  • Determine if NDK activity correlates with growth rate on different substrates

  • Investigate potential roles in metabolic flux regulation

Understanding these connections would provide valuable insights into bacterial metabolism and potentially reveal novel regulatory mechanisms linking energy metabolism, nucleotide homeostasis, and specialized metabolic pathways.