Recombinant Pseudomonas putida Zinc import ATP-binding protein ZnuC (znuC)

What is the function of ZnuC in Pseudomonas putida's zinc uptake system?

ZnuC functions as the ATP-binding component (ATPase) of the ZnuABC transport system in Pseudomonas putida. This high-affinity zinc uptake system consists of three main components: ZnuA (periplasmic binding protein), ZnuB (transmembrane domain), and ZnuC (ATPase). Together, they form an ATP-binding cassette (ABC) transporter that imports zinc from the periplasm into the cytoplasm under zinc-limited conditions.

In P. putida, the ZnuC protein hydrolyzes ATP to provide energy for zinc transport through the inner membrane. This system is particularly important when the bacterium encounters environments with extremely limited zinc availability .

How is the expression of znuC regulated in Pseudomonas putida?

The expression of znuC in P. putida is primarily regulated by the zinc uptake regulator (Zur), a transcriptional repressor that binds to specific DNA sequences (Zur boxes) in the promoter regions of zinc uptake genes. The regulation operates as follows:

• Under zinc-replete conditions: Zinc-bound Zur acts as a repressor, binding to the promoter region of the znuABC operon and preventing transcription.

• Under zinc-limited conditions: Zur cannot bind DNA as a repressor, leading to derepression and expression of znuABC genes .

Experimental data from transcriptional studies in various bacterial strains (including E. coli) have shown that znuC promoter activity significantly increases in zinc-depleted media and in Δzur knockout strains, confirming the repressive role of Zur .

What are the structural characteristics of the ZnuABC system in Pseudomonas putida?

The ZnuABC system in P. putida follows the typical structure of bacterial ABC transporters, with each component having specific structural features:

• ZnuA: A periplasmic solute binding protein that specifically binds zinc with high affinity. In some bacteria, ZnuA can bind multiple zinc ions and may contain His-rich domains that facilitate zinc binding.

• ZnuB: A transmembrane domain protein that forms the channel through which zinc is transported across the inner membrane.

• ZnuC: The nucleotide-binding domain (ATPase) that provides energy for transport through ATP hydrolysis.

The system operates through conformational changes triggered by ATP binding and hydrolysis at ZnuC, which allows the transport of zinc bound to ZnuA through the ZnuB transmembrane channel .

What are the recommended approaches for designing knockout experiments to study ZnuC function in P. putida?

When designing knockout experiments to study ZnuC function in P. putida, researchers should consider the following methodological approach:

• Targeting Strategy: For precise deletions of znuC, use a two-step recombination approach utilizing:

  • Suicide plasmids (such as pEMG) containing recognition sequences for I-SceI homing endonuclease

  • Counter-selection with conditional expression of I-SceI to induce double-stranded breaks

  • Homologous recombination for gene deletion

• Newer Methodologies: Consider advanced techniques recently developed for P. putida:

  • RecET-based markerless recombineering systems

  • Thermoinducible single-stranded recombineering

  • CRISPR/Cas9 technologies for efficient genome editing

• Controls and Complementation:

  • Generate both single (ΔznuC) and combined knockouts (ΔznuCΔzntA) to understand interactions with export systems

  • Include complementation studies with plasmid-expressed znuC to confirm phenotypes

  • Use fluorescent reporters to monitor changes in gene expression

• Growth Media Considerations:

  • Use chemically defined media (CDM) with controlled zinc concentrations

  • Compare growth in zinc-replete and zinc-depleted conditions

  • Consider using semi-synthetic cation-defined medium as described by Pederick et al. (2015)

How should zinc stress conditions be established for studying ZnuC expression and function?

For studying ZnuC expression and function under zinc stress conditions, researchers should establish a clear experimental design with carefully controlled zinc concentrations:

• Determining Zinc Concentration Range:

  • Perform growth inhibition assays to establish sublethal zinc concentrations

  • Use zinc concentrations that produce approximately 5%, 40%, and 80% growth inhibition rates

  Zinc Sulfate Concentration	Growth Inhibition in P. putida
  0.1 mmol L⁻¹	No significant effect
  0.2 mmol L⁻¹	~5% inhibition
  1.5 mmol L⁻¹	~40% inhibition
  2.5 mmol L⁻¹	~80% inhibition
  3.0 mmol L⁻¹	Almost complete inhibition

• Exposure Time:

  • Challenge cultures at early exponential phase (approximately 1.0 × 10⁷ CFU mL⁻¹)

  • Collect samples after 1 hour of exposure (about half a generation time)

• RNA Stabilization:

  • Immediately stabilize RNA using RNAprotect reagent after collection

  • Store samples at -80°C before RNA extraction

• Transcriptional Analysis:

  • Use RNA sequencing or qRT-PCR to measure znuC expression levels

  • Compare transcriptional responses at different zinc concentrations

  • Include time course studies to capture dynamic responses

What are the best methods for recombinant expression and purification of P. putida ZnuC protein?

For optimal recombinant expression and purification of P. putida ZnuC protein, researchers should follow these methodological approaches:

• Expression System Selection:

  • Use E. coli BL21(DE3) or similar expression strains

  • Consider codon optimization for the znuC gene if expression is low

  • Use expression vectors with tightly controlled inducible promoters (like pET systems)

• Protein Tagging Strategy:

  • Add a His-tag (6× histidine) for affinity purification

  • Consider placement at either N- or C-terminus based on structural data

  • Include a cleavable tag if native protein is required for functional studies

• Expression Conditions:

  • Optimize induction parameters (IPTG concentration, temperature, time)

  • Use lower temperatures (16-20°C) for induction to enhance solubility

  • Consider zinc supplementation in growth media to maintain protein stability

• Purification Protocol:

  • Use immobilized metal affinity chromatography (IMAC) for His-tagged proteins

  • Include zinc in purification buffers (typically 10-50 μM) to stabilize the protein

  • Perform size exclusion chromatography as a polishing step

  • Confirm protein purity by SDS-PAGE with Coomassie Blue staining

• Functional Verification:

  • Perform ATPase activity assays to confirm functional protein

  • Use circular dichroism to assess proper protein folding

  • Consider zinc binding assays to verify metal-binding properties

How do the zinc uptake systems in P. putida compare with those in other Pseudomonas species?

Comparative analysis of zinc uptake systems reveals both similarities and distinct differences between P. putida and other Pseudomonas species:

• P. putida vs. P. aeruginosa Comparison:

  Feature	P. putida ZnuABC	P. aeruginosa ZnuABC
  System components	ZnuA, ZnuB, ZnuC in ACB operon	ZnuA, ZnuB, ZnuC plus ZnuD (TonB-dependent transporter)
  Regulation	Zur-dependent	Zur-dependent
  Alternative uptake	Potential ZupT homolog (PA4467)	Multiple systems (ZnuABC, HmtA, pseudopaline)
  Knockout effects	Growth defects in zinc limitation	Slight reduction in zinc-depleted conditions
  Additional transporters	Unknown	HmtA (P-type ATPase) in inner membrane

  P. aeruginosa possesses additional zinc acquisition mechanisms including:

  • ZnuD, a TonB-dependent transporter located in the outer membrane

  • HmtA, a P-type ATPase that transports periplasmic zinc into the cytoplasm

  • Pseudopaline system (ZrmABCD) for chelating and importing zinc

• Promoter Analysis and Gene Expression Patterns:

  • In P. putida, the znuC promoter responds more strongly to zinc depletion compared to P. aeruginosa

  • P. putida contains duplicated genes for some zinc transport components with differential inducibility

  • Studies have shown different inducibilities among duplicated genes in P. putida strains

• Regulation Mechanisms:

  • Both species use Zur as the primary regulator

  • Zur boxes (DNA binding motifs) show high conservation across Pseudomonas species

  • P. aeruginosa has additional regulatory mechanisms involving multiple import systems

What methodologies are most effective for studying the interaction between ZnuA and ZnuC in P. putida?

For investigating the interaction between ZnuA and ZnuC in P. putida, researchers should consider these advanced methodological approaches:

• In vitro Protein-Protein Interaction Studies:

  • Surface Plasmon Resonance (SPR): Immobilize one protein (e.g., ZnuA) and measure binding kinetics with the other (ZnuC)

  • Isothermal Titration Calorimetry (ITC): Determine thermodynamic parameters of binding

  • Pull-down Assays: Use tag-based systems (His-tag, GST-tag) to co-purify interacting partners

  • Microscale Thermophoresis (MST): Measure interactions in solution with minimal protein consumption

• Structural Analysis Approaches:

  • X-ray Crystallography: Determine the 3D structure of the ZnuABC complex

  • Cryo-EM: Visualize the entire transporter complex in different conformational states

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

  • FRET/BRET Assays: Monitor protein interactions in real-time with fluorescent labels

• In vivo Approaches:

  • Bacterial Two-Hybrid System: Adapted for membrane proteins

  • Fluorescence in situ Hybridization (FISH): For co-localization studies

  • Split GFP Complementation: Fuse split GFP fragments to ZnuA and ZnuC to visualize interactions

• Genetic and Mutational Analysis:

  • Site-directed Mutagenesis: Target key residues in interaction interfaces

  • Suppressor Mutation Analysis: Identify compensatory mutations

  • Complementation Studies: Use in knockout strains to validate functionality

• System Reconstitution:

  • Proteoliposome Assays: Reconstitute purified components in artificial membranes

  • Zinc Transport Assays: Monitor zinc uptake with fluorescent indicators

How do environmental factors influence ZnuC expression and function in P. putida strains used for bioremediation?

Environmental factors significantly impact ZnuC expression and function in P. putida strains used for bioremediation applications:

• Zinc Concentration Effects:

  • Low environmental zinc triggers znuC upregulation through Zur derepression

  • High zinc concentrations (above 1.5 mmol L⁻¹) cause significant growth inhibition and oxidative stress

  • Optimal zinc ranges for bioremediation applications should be determined for each strain

• pH Impact on Zinc Bioavailability and Transport:

  • Acidic conditions increase zinc solubility but may stress cells

  • Alkaline conditions decrease zinc bioavailability, potentially inducing znuC expression

  • For bioremediation applications, pH stabilization may be necessary to maintain consistent ZnuC function

• Co-occurring Heavy Metals:

  • Other metals (Cd, Cu, Pb) may compete with the zinc transport system

  • Comparative transcriptome analysis reveals cross-regulation between metal resistance systems

  • Engineered P. putida strains may require optimized zinc supplementation when used in multi-metal contaminated sites

• Oxygen Availability:

  • Low oxygen conditions alter metal uptake systems and metabolism in P. putida

  • Recent studies on the streamlined chassis strain P. putida SEM10 show different responses to oxygen limitation compared to wild-type KT2440

  • For anoxic bioremediation applications, consider oxygen effects on zinc transport systems

• Carbon Source Influence:

  • Different carbon sources alter central metabolism and respiratory chains

  • Zinc stress at intermediate levels impacts amino acid metabolism and respiratory chains

  • Optimal carbon source selection can support zinc homeostasis in bioremediation applications

What are the recommended approaches for detecting recombinant P. putida expressing ZnuC in environmental samples?

For detecting recombinant P. putida expressing ZnuC in environmental samples, researchers should employ the following methodological approach:

• Molecular Detection Techniques:

  • PCR-based Methods: Design primers specific to the recombinant znuC construct

  • Quantitative PCR: Measure copy numbers of znuC genes in environmental samples

  • Fluorescence in situ Hybridization (FISH): Use probes targeting both 23S rRNA (for P. putida) and znuC mRNA for simultaneous detection

  FISH Protocol Optimization Parameters:

  Parameter	Recommended Condition	Notes
  Probe for P. putida	Cy3-labeled targeting 23S rRNA	Species-specific identification
  Probe for znuC	Alexa647-labeled targeting znuC mRNA	Gene-specific detection
  Hybridization temperature	Optimized between 45-55°C	Must be experimentally determined
  Detection method	Confocal microscopy	For visualization in complex matrices

• Functional Detection Methods:

  • Reporter Gene Fusion: Construct znuC-lux or znuC-gfp fusions for direct detection

  • Zinc-responsive Biosensors: Develop whole-cell biosensors using the znuC promoter region

  • Metabolic Fingerprinting: Use BIOLOG plates to identify unique metabolic signatures of the recombinant strain

• Cultivation-dependent Methods:

  • Selective Media: Develop media with specific zinc concentrations and antibiotics

  • Colony PCR: Screen colonies for the presence of recombinant znuC

  • Immunological Detection: Develop antibodies against tagged ZnuC protein

• Sample Processing Considerations:

  • For soil/sediment samples: Include appropriate extraction methods to separate cells from particles

  • For water samples: Concentration steps (filtration, centrifugation) may be necessary

  • Consider using internal standards to quantify recovery efficiency

How can researchers analyze the effects of ZnuC mutations on zinc uptake and bacterial physiology?

To analyze the effects of ZnuC mutations on zinc uptake and bacterial physiology, researchers should implement these methodological approaches:

• Mutant Construction Strategy:

  • Site-directed Mutagenesis: Target conserved ATP-binding motifs (Walker A/B)

  • Domain Swapping: Replace domains with homologs from other species

  • Random Mutagenesis: Screen for phenotypes using error-prone PCR

  • CRISPR/Cas9 Genome Editing: For precise chromosomal mutations

• Phenotypic Characterization:

  • Growth Curves: Compare growth in zinc-limited vs. zinc-replete media

  • Zinc Uptake Assays: Measure 65Zn uptake rates in wild-type vs. mutant strains

  • Minimum Inhibitory Concentration (MIC): Determine zinc tolerance

  • Competitive Fitness Assays: Co-culture wild-type and mutant strains

• Molecular and Biochemical Analysis:

  • ATPase Activity Assays: Measure ATP hydrolysis rates of purified proteins

  • Conformational Studies: Assess structural changes using circular dichroism

  • Protein-Protein Interaction Assays: Examine effects on ZnuA and ZnuB interactions

  • Transcriptome Analysis: Identify compensatory responses using RNA-seq

• Advanced Physiological Assessment:

  • Intracellular Zinc Quantification: Use zinc-specific fluorescent probes or ICP-MS

  • Metabolic Flux Analysis: Examine effects on central metabolism

  • Oxidative Stress Markers: Measure ROS levels and antioxidant enzyme activities

  • Respiratory Chain Analysis: Examine oxygen consumption rates (OCR)

• Complementation Strategy:

  • Express wild-type znuC in trans to confirm phenotype causality

  • Use heterologous complementation in E. coli znuC mutants to assess functional conservation

  • Create a complementation series with different mutation variants

What analytical methods are recommended for quantifying intracellular zinc in P. putida expressing recombinant ZnuC?

For accurate quantification of intracellular zinc in P. putida expressing recombinant ZnuC, researchers should employ these analytical approaches:

• Sample Preparation Protocols:

  • Cell Fractionation: Separate periplasmic, membrane, and cytoplasmic fractions

  • Acid Digestion: Use ultra-pure nitric acid for complete sample digestion

  • Avoid Contamination: Use metal-free plasticware and reagents

  • Standardized Washing: Remove extracellular zinc with EDTA washes

• Instrumental Methods:

  • Inductively Coupled Plasma Mass Spectrometry (ICP-MS):

    • Highest sensitivity (detection limits in ppb range)

    • Can distinguish isotopes for tracer studies

    • Provides multi-element analysis

  • Atomic Absorption Spectroscopy (AAS):

    • Good specificity for zinc

    • Simpler operation than ICP-MS

    • Lower sensitivity than ICP-MS

  • X-ray Fluorescence (XRF) Microscopy:

    • Spatial distribution of zinc within cells

    • Non-destructive analysis

    • Requires specialized facilities

• Fluorescent Probes and Sensors:

  • Low-Molecular-Weight Zinc Fluorescent Probes:

    • FluoZin-3 AM (K_d = 15 nM)

    • Zinpyr-1 (K_d = 0.7 nM)

    • ZIMIR (cell membrane-permeable)

  • Genetically Encoded Zinc Sensors:

    • eCALWY sensors (FRET-based)

    • ZapCY sensors (ratiometric)

    • Express in P. putida for real-time monitoring

• Data Normalization Approaches:

  • Cell Number: Count cells or measure OD600

  • Protein Content: Bradford or BCA assay

  • Dry Weight: For consistent comparison between strains

  • Internal Standards: Add known amounts before sample processing

• Validation and Controls:

  • Reference Standards: Use certified reference materials

  • Spike Recovery Tests: Add known amounts of zinc to samples

  • Method Comparison: Validate results using multiple techniques

  • Biological Replicates: Minimum of three independent experiments

How can recombinant P. putida ZnuC be exploited for enhancing bioremediation of zinc-contaminated environments?

Recombinant P. putida ZnuC can be strategically engineered to enhance bioremediation of zinc-contaminated environments through these methodological approaches:

• Engineered Overexpression Strategies:

  • Promoter Engineering: Replace native zur-regulated promoter with constitutive or inducible promoters

  • Codon Optimization: Enhance translation efficiency in P. putida

  • Copy Number Increase: Integrate additional znuC copies into the genome

  • Ribosome Binding Site (RBS) Engineering: Optimize translation initiation

• Functional Enhancement Approaches:

  • Protein Engineering: Modify zinc binding affinity through targeted mutations

  • System Integration: Co-express with metallothioneins or phytochelatins for zinc sequestration

  • Metabolic Engineering: Couple zinc uptake to central metabolism for enhanced energy generation

• Strain Development Considerations:

  • Chassis Selection: Use streamlined strains like P. putida SEM10 with reduced genome

  • Stress Resistance Engineering: Enhance tolerance to oxidative stress induced by high zinc

  • Multi-metal Resistance: Integrate multiple metal resistance systems

  • Biofilm Formation: Engineer strains with enhanced biofilm capabilities for immobilization

• Field Application Methodologies:

  • Immobilization Techniques: Encapsulate cells in polymeric matrices

  • Nutrient Supplementation Strategies: Optimize carbon sources for maximum activity

  • Monitoring Systems: Develop biosensors for real-time monitoring of bioremediation progress

  • Co-culture Approaches: Combine with synergistic microorganisms

• Performance Metrics and Assessment:

  • Zinc Removal Efficiency: Compare with wild-type strains

  • Long-term Stability: Assess genetic stability and performance over time

  • Environmental Impact Assessment: Monitor ecological effects of introduced strains

  • Cost-effectiveness Analysis: Compare with conventional remediation technologies

What are the current methodologies for using ZnuC as a target for antimicrobial development against Pseudomonas species?

Current methodologies for targeting ZnuC in antimicrobial development against Pseudomonas species include:

• Structure-Based Drug Design Approaches:

  • Virtual Screening: Use platforms like ZINC20 database to screen millions of compounds against ZnuC

  • Fragment-Based Drug Discovery: Identify small molecule binders to ZnuC active sites

  • Molecular Docking: Target ATP-binding site or zinc-coordination regions

  • Pharmacophore Modeling: Develop models based on known ZnuC inhibitors

• High-Throughput Screening Strategies:

  • ATPase Inhibition Assays: Screen for compounds that inhibit ATP hydrolysis

  • Growth Inhibition in Zinc-Limited Media: Identify compounds with enhanced activity

  • Reporter-Based Screens: Use znuC-reporter fusions to identify regulators

  • Whole-Cell Screening: Test compound libraries against Pseudomonas strains

• Novel Inhibitor Classes:

  • ATP-Competitive Inhibitors: Target nucleotide binding domain

  • Allosteric Inhibitors: Disrupt conformational changes required for function

  • Metal-Chelating Inhibitors: Compounds that sequester zinc without entering cells

  • Peptide Inhibitors: Designed to disrupt ZnuA-ZnuC interactions

• Combination Therapy Approaches:

  • Zinc Chelator + Conventional Antibiotics: Enhance efficacy of existing drugs

  • Multi-Target Strategies: Simultaneously inhibit multiple zinc homeostasis components

  • Biofilm Disruptors + ZnuC Inhibitors: Address both planktonic and biofilm states

• Validation Methodologies:

  • Target Engagement Assays: Confirm binding to ZnuC protein

  • Resistance Development Studies: Assess potential for resistance emergence

  • Specificity Testing: Evaluate effects on human zinc transporters

  • In vivo Infection Models: Test efficacy in animal models of Pseudomonas infection

How can systems biology approaches be applied to understand the role of ZnuC in P. putida's zinc homeostasis network?

Systems biology approaches can elucidate the complex role of ZnuC in P. putida's zinc homeostasis network through these integrated methodological strategies:

• Multi-omics Integration Approaches:

  • Transcriptomics: RNA-seq to identify gene expression changes across zinc concentrations

  • Proteomics: Quantify protein levels and post-translational modifications

  • Metabolomics: Analyze metabolic shifts in response to zinc availability

  • Fluxomics: Measure metabolic flux changes using labeled substrates

  Integration of these datasets can reveal coordinated responses to zinc stress beyond direct Zur regulation

• Network Analysis Methodologies:

  • Protein-Protein Interaction Networks: Identify ZnuC interactors

  • Regulatory Network Reconstruction: Map transcription factor binding sites

  • Metabolic Network Analysis: Identify connections between zinc homeostasis and central metabolism

  • Signaling Pathway Mapping: Uncover signaling cascades activated by zinc limitation

• Mathematical Modeling Approaches:

  • Kinetic Modeling: Describe dynamics of zinc uptake and distribution

  • Flux Balance Analysis: Predict metabolic adaptations to zinc limitation

  • Dynamic Bayesian Networks: Model temporal responses to changing zinc levels

  • Agent-Based Modeling: Simulate single-cell behaviors in heterogeneous environments

• Experimental Design for Systems Approaches:

  • Time-course Experiments: Capture dynamic responses to zinc perturbations

  • Dose-response Studies: Systematically vary zinc concentrations

  • Genetic Perturbation Arrays: Combine znuC mutations with other system components

  • Environmental Gradient Analysis: Study responses across multiple variables simultaneously

• Data Analysis and Visualization Tools:

  • Network Visualization Software: Cytoscape, STRING

  • Pathway Enrichment Analysis: Identify over-represented functional categories

  • Machine Learning Approaches: Identify patterns in complex datasets

  • Model Simulation Tools: COPASI, CellDesigner for dynamic modeling

What are the optimal conditions for using recombinant P. putida ZnuC to enhance protein production in biotechnology applications?

For optimizing recombinant P. putida ZnuC to enhance protein production in biotechnology applications, researchers should consider these methodological parameters:

• Zinc Supplementation Strategy:

  • Optimal Concentration: 25 mg/L zinc supplementation has shown significant increases in recombinant protein titers

  • Timing: Add zinc at early exponential phase for maximum effect

  • Form: ZnSO₄ is commonly used and highly soluble

  Comparable effects observed in CHO cells showed:

  Zinc Concentration	EPO Titer Increase	IgG Titer Increase
  Control (no added zinc)	Baseline	Baseline
  25 mg/L (CDM media)	1.7-fold	2.6-fold
  25 mg/L (CDM+A media)	1.8-fold	1.2-fold

  Similar enhancement effects may be achievable in P. putida systems

• Medium Composition Optimization:

  • Base Medium: Chemically defined media (CDM) without chelating agents

  • Carbon Source: Glucose or glycerol at 1-2% for balanced growth

  • Nitrogen Source: Ammonium salts preferred over amino acids

  • Buffer System: MOPS or phosphate buffer at pH 7.0-7.2

  • Other Trace Elements: Balance copper, manganese and iron to prevent antagonism

• Cultivation Parameters:

  • Temperature: 28-30°C optimal for P. putida

  • Dissolved Oxygen: Maintain above 30% saturation

  • pH Control: Maintain at 7.0-7.2 for optimal zinc solubility

  • Feeding Strategy: Fed-batch with glucose feeding to prevent overflow metabolism

  • Induction Parameters: Optimize timing and inducer concentration

• Genetic Engineering Considerations:

  • Promoter Selection: Strong constitutive promoters (e.g., Ptac) for znuC expression

  • Codon Optimization: Adapt to P. putida codon usage preference

  • Vector Stability: Use chromosomal integration for long-term stability

  • Gene Dosage: Optimize copy number for balanced expression

• Process Monitoring and Control:

  • Online Monitoring: Track dissolved oxygen, pH, and substrate concentration

  • Metabolic State Assessment: Monitor respiration quotient

  • Zinc Level Monitoring: Periodic sampling for zinc concentration

  • Protein Expression Monitoring: Online fluorescence for reporter-tagged proteins