Recombinant Pseudomonas putida Nickel import ATP-binding protein NikE (nikE)

How does the NikE protein relate to other components of nickel transport systems in P. putida?

The NikE protein is part of a complete ABC transporter system encoded by the gene cluster pedA1A2BC in P. putida. This system consists of four genes encoding a putative permease (pedC), an ATP-binding protein (pedB), a YVTN beta-propeller repeat protein (pedA2), and a periplasmic substrate-binding protein (pedA1). While efflux systems typically comprise transmembrane domains and nucleotide-binding domains, ABC-dependent import systems additionally require a substrate-binding protein for functional transport. As NikE is the ATP-binding component, it provides the energy needed for the translocation of nickel across the membrane, working in concert with the other components .

How is NikE expression regulated in response to environmental conditions?

NikE expression is upregulated in response to zinc toxicity, as demonstrated in transcriptome studies. When P. putida is exposed to elevated zinc levels, nickel uptake systems including NikE are significantly upregulated to counter the nickel defect induced by zinc toxicity. This response occurs because zinc can replace nickel ions in their enzymes, creating a functional nickel deficiency that must be compensated for by increased nickel import. This regulation represents an important metal homeostasis mechanism in P. putida, allowing the bacterium to maintain appropriate intracellular concentrations of different metal ions even under stress conditions .

What are the most effective expression systems for producing recombinant P. putida NikE protein?

The most effective expression systems for NikE production depend on research objectives and downstream applications. For standard biochemical and structural studies, E. coli-based expression systems remain the most common choice due to their high yield and simplicity. The following approaches have proven effective:

For optimal expression, consider using the pET expression system with T7 promoter in E. coli or integrating the gene into one of the seven rRNA-encoding rrn operons in P. putida KT2440, which have been demonstrated to be excellent sites for heterologous gene expression .

What are the critical factors that affect the solubility and activity of recombinant NikE protein?

Several factors can significantly impact NikE solubility and activity:

• Metal supplementation: Cultivation media should be supplemented with trace metals and cofactors. The presence of nickel in the medium is particularly important for proper folding of NikE.

• Expression temperature: Lower temperatures (16-25°C) often improve solubility compared to standard 37°C expression.

• Induction conditions: For IPTG-inducible systems, concentrations between 0.1-0.5 mM are typically optimal, with higher concentrations potentially leading to inclusion body formation.

• Buffer composition: Purification buffers should contain stabilizing agents such as glycerol (10-15%) and should be supplemented with low concentrations of nickel ions (1-5 μM) to maintain the protein's native conformation.

• ATP/ADP presence: Including ATP or ADP (1-2 mM) in purification and storage buffers can stabilize the nucleotide-binding domain.

In transcriptomic studies of P. putida under zinc stress, researchers observed that maintaining appropriate metal homeostasis and intracellular redox status are crucial for protein function and stability, suggesting that these aspects should also be considered when working with isolated NikE protein .

How can researchers validate the functional activity of purified recombinant NikE?

Functional validation of purified NikE can be achieved through multiple complementary approaches:

• ATP hydrolysis assay: Measure the ATPase activity using colorimetric methods (malachite green) or coupled enzyme assays (pyruvate kinase/lactate dehydrogenase system) to monitor phosphate release. Authentic NikE should show increased ATPase activity in the presence of the transport substrate (nickel ions).

• Transport assays: Reconstitute NikE along with other components of the nickel transport system in proteoliposomes and measure nickel uptake using radiolabeled 63Ni or fluorescent nickel chelators.

• Binding assays: Isothermal titration calorimetry (ITC) or microscale thermophoresis (MST) can quantify the binding affinity of NikE for both ATP and nickel ions.

• Complementation studies: Express the recombinant NikE in P. putida strains with deleted endogenous nikE and assess restoration of nickel-dependent phenotypes, similar to studies performed with other metal transport systems .

• Thermal shift assays: Differential scanning fluorimetry to evaluate protein stability in the presence of different ligands (ATP, ADP, nickel) and buffer conditions.

How can genomic integration approaches be optimized for stable expression of recombinant NikE in P. putida?

For stable genomic integration and optimal expression of nikE in P. putida, researchers should consider:

• Integration site selection: The seven rRNA-encoding rrn operons of P. putida KT2440 have been identified as exceptional sites for heterologous gene integration. Research has demonstrated that prodigiosin production (used as a reporter) was mainly dependent on (i) the individual rrn operon where the gene cluster was inserted and (ii) the distance between the rrn promoter and the inserted biosynthetic genes .

• Integration methodology: Use Tn5 transposon-based chromosomal integration or site-specific recombination techniques. For precise targeting, the markerless gene deletion method described by Graf and Altenbuchner can be adapted for insertion .

• Promoter considerations: Expression levels are influenced by the distance between the rrn promoter and the inserted gene. For nikE, optimizing this distance can significantly affect protein yield.

• Strain selection: Consider using genome-reduced strains like P. putida EM42, which have been engineered to remove non-essential genomic regions that might interfere with heterologous expression .

• Validation of integration: Verify correct chromosomal integration by colony PCR using primers specific to the integration site and the nikE gene. Additionally, employ functional assays to confirm expression and activity of the integrated NikE protein .

Recent studies have identified a set of genomic landing pads in P. putida KT2440 with consistent expression patterns under diverse experimental conditions, providing stable sites for heterologous gene integration with predictable expression levels .

What approaches can be used to study the interactions between NikE and other metal transport systems in P. putida?

Investigating the interactions between NikE and other metal transport systems requires multi-level approaches:

• Transcriptomic analysis: RNA-Seq under varying metal conditions can reveal co-regulation patterns between nikE and other metal transporters. Research has shown that under zinc stress, both nickel import systems and arsenate resistance systems are upregulated, suggesting complex metal cross-talk .

• Protein-protein interaction studies:

  • Pull-down assays using tagged NikE to identify interacting partners

  • Bacterial two-hybrid screening to detect direct protein interactions

  • Cross-linking mass spectrometry to map interaction interfaces

• Metal competition assays: Measuring nickel uptake in the presence of varying concentrations of other metals can reveal competitive or cooperative interactions between transport systems.

• Genetic approaches:

  • Construction of double/triple knockouts of different metal transport systems

  • Suppressor mutant screens to identify genetic interactions

  • CRISPR interference to create tunable repression of multiple transporter genes simultaneously

• Metabolomic profiling: Analysis of metal-dependent metabolites can reveal downstream effects of altered metal transport activity and help identify metabolic connections between different metal utilization pathways.

Research on P. putida has demonstrated that zinc can replace nickel ions in enzymes, creating a functional deficiency that triggers upregulation of nickel import systems. Similar relationships likely exist with other metals, making this a rich area for investigation .

How do variations in the genomic context affect the expression and function of integrated nikE genes in different P. putida strains?

Genomic context significantly impacts nikE expression and function in different P. putida strains:

• Locus-dependent effects: Recent research has identified substantial variation in expression levels depending on the genomic integration site. While reproducibility of expression within specific landing pads is high, msfGFP signals varied strongly between different landing pads, confirming a strong influence of the genomic context .

• Strain-specific differences: Comparison between P. putida KT2440 and the genome-reduced strain EM42 revealed that while EM42 shows improved growth characteristics in some contexts, it does not universally outperform KT2440 for heterologous expression. Transcriptomic analysis revealed that genome reduction had global effects on transcript levels .

• Regulatory interference: The presence of nearby global regulators or small RNAs can affect expression. P. putida KT2440 harbors three known members of the CsrA/RsmA family of post-transcriptional regulators (RsmA, RsmE, and RsmI), which can bind to various transcripts and affect their expression .

• Chromosome topology: The three-dimensional organization of the bacterial chromosome creates expression domains with different accessibility to transcription machinery. Integration into highly condensed regions may reduce expression efficiency.

• Insulation requirements: To prevent genomic read-through from flanking promoters under changing cultivation conditions, integration constructs should include strong terminators. Research has employed insulated probe sensors using double terminators from phage M13 and E. coli K12 rrnD1 to study locus-dependent effects on recombinant gene expression .

These findings highlight that genomic "hot" and "cold" spots exist, causing strong promoter-independent variations in gene expression, making the genomic context an essential parameter when designing integrable genomic cassettes for tailored heterologous expression .

What strategies can address low expression yields of recombinant NikE protein?

When facing low expression yields of recombinant NikE, researchers should implement the following strategies:

• Optimize codon usage: Adapt the nikE coding sequence to the preferred codon usage of the expression host. Codon optimization can significantly improve translation efficiency, especially when expressing P. putida genes in E. coli.

• Evaluate promoter strength: Test different promoter systems. For instance, in P. putida, integration into different rrn operons has shown varied expression levels. The distance between the rrn promoter and the inserted gene also significantly affects expression levels .

• Supplement growth media: Cultivation in media supplemented with trace metals and thiamine has been shown to significantly increase cell density and protein production. In one study, supplementation increased OD600 from 2 to 8 and protein titer from 0.2 g/L to 0.8 g/L .

• Modify culture conditions:

  • Reduce growth temperature (16-25°C) after induction

  • Use lower inducer concentrations (0.1-0.2 mM IPTG)

  • Extend expression time (24-48 hours)

  • Test different growth phases for induction

• Improve protein solubility:

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J)

  • Add solubility tags (MBP, SUMO, TRX)

  • Include stabilizing agents in lysis buffer (10-15% glycerol, 1-5 μM nickel)

• Consider genomic integration: For P. putida expression systems, chromosomal integration into specific rrn operons has demonstrated high stability and strong expression. Recombinant strains showed high stability upon subculturing for many generations .

How can researchers differentiate between authentic NikE function and the activities of other ATP-binding proteins in experimental systems?

Distinguishing authentic NikE function from other ATP-binding proteins requires multiple control experiments and specific assays:

• Metal specificity profiling: Compare ATPase stimulation across multiple metals (Ni²⁺, Co²⁺, Fe²⁺, Zn²⁺, Cu²⁺). Authentic NikE should show highest activity with nickel, while other metal-specific transporters will have different profiles.

• Site-directed mutagenesis: Introduce mutations in the conserved Walker A motif (G-X-X-G-X-G-K-S/T) that should abolish ATP binding and hydrolysis. These mutants serve as negative controls to confirm that observed activity is due to NikE.

• Substrate competition assays: Perform transport or binding assays in the presence of competing substrates. For example, nickel transport by NikE should be inhibited by excess unlabeled nickel but less affected by other metal ions.

• Comparative studies with related systems: Include parallel experiments with other characterized metal transport ATP-binding proteins, such as those for zinc or iron transport, to establish specificity profiles.

• Antibody-based approaches: Use specific antibodies against NikE to immunoprecipitate the protein complex before activity assays, eliminating interference from other ATP-binding proteins.

• Heterologous expression in knockout strains: Express recombinant NikE in strains lacking endogenous metal transport systems to eliminate background activity and cross-complementation.

Research on P. putida has shown that metal transport systems can be regulated in response to the availability of other metals, such as the upregulation of nickel import systems under zinc stress, highlighting the interconnected nature of metal homeostasis .

What control experiments are essential when studying NikE function in the context of metal stress responses?

When investigating NikE function during metal stress responses, these critical control experiments must be included:

• Metal specificity controls:

  • Include multiple metal ions at equimolar concentrations

  • Use metal chelators (EDTA, EGTA) to establish baseline activity

  • Include experiments with metal mixtures to detect synergistic or antagonistic effects

• Genetic controls:

  • Compare wild-type strains with nikE deletion mutants

  • Use point mutants with altered metal binding or ATP hydrolysis capacity

  • Include strains with deletions in other metal transport systems to identify compensatory mechanisms

• Environmental variable controls:

  • Test responses across different growth phases

  • Assess pH dependency of nickel transport (pH can alter metal solubility and transporter function)

  • Control for oxygen levels, as oxidative stress interacts with metal homeostasis

• Media composition controls:

  • Use defined minimal media with controlled metal content

  • Consider trace contamination in media components

  • Test multiple carbon sources, as metabolic state can affect metal requirements

• Transcriptional controls:

  • Monitor expression of nikE and other metal transporters using reporter fusions (e.g., luxCDABE fusions as used in P. putida REE-switch studies)

  • Quantify mRNA levels of nikE and related genes under various metal stress conditions

Research has demonstrated that different levels of zinc stress strongly affect the transcription of genes from multiple categories: metal transport genes, membrane homeostasis genes, oxidative-stress-responding genes, and genes associated with basic cellular metabolism, highlighting the complex interconnections that must be controlled for .

How might advanced genetic modification of P. putida strains enhance the study and application of NikE and related metal transport systems?

The future of NikE research will likely benefit from advanced genetic modification strategies:

• Genome minimization approaches: Building on the P. putida EM42 genome-reduced strain, further streamlined strains could eliminate competing metal transport systems or non-essential ATP-consuming processes. The rational removal of 300 genes spanning 4.3% of the entire P. putida KT2440 genome has already enhanced desirable traits in strain EM383, resulting in improved growth properties and metabolic vigour .

• Synthetic biology standardization: Implementing standardized genetic parts for metal transport studies would allow more systematic investigation of NikE function. This could include a library of characterized promoters, ribosome binding sites, and reporter systems specifically optimized for metal transport research.

• Multi-omics integration platforms: Development of strains with integrated sensors for real-time monitoring of metal homeostasis, coupled with inducible expression systems for NikE variants, would allow dynamic studies of metal transport processes.

• CRISPR-based regulation systems: Implementing CRISPRi for tunable repression or CRISPRa for enhanced expression of nikE and related genes would enable precise control over the metal transport network, facilitating the study of complex interactions.

• Biosensor development: Engineering P. putida strains with NikE-based biosensors could enable the detection of nickel in environmental samples or monitoring of intracellular nickel pools during various physiological states.

Comparing this approach to the successful implementation of the 21 kb prodigiosin gene cluster from Serratia marcescens in P. putida, researchers could develop standardized integration methods specifically optimized for metal transport systems .

What novel applications of recombinant NikE could emerge from comprehensive understanding of its structure-function relationship?

Advanced understanding of NikE's structure-function relationship could enable several innovative applications:

• Engineered metal transport systems: Creating chimeric transporters with altered metal specificity could enable the bioaccumulation of valuable metals or remediation of contaminated environments. This builds on P. putida's natural ability to adapt to sites polluted with toxic chemicals .

• Synthetic metalloenzyme development: NikE's metal binding domains could be adapted to create artificial metalloenzymes with novel catalytic activities, potentially enabling new biocatalytic processes.

• Metal-dependent gene regulatory circuits: Engineered NikE variants could serve as components of synthetic gene circuits that respond to specific metal concentrations, enabling programmable cellular responses to environmental conditions.

• Targeted protein delivery systems: The ATP-dependent transport mechanism of NikE could potentially be repurposed for the delivery of non-native substrates, such as therapeutic peptides or signaling molecules.

• Improved bioremediation strains: Enhanced understanding of NikE function could facilitate the development of P. putida strains with improved capacity for metal sequestration and detoxification for environmental applications.

• Bioprocessing applications: NikE knowledge could contribute to the development of strains with improved nickel utilization efficiency for industrial biocatalysis, particularly for reactions requiring nickel-dependent enzymes.

This aligns with broader research showing P. putida's considerable potential for biotechnological applications in agriculture, biocatalysis, bioremediation, and bioplastic production .

How might the interaction between NikE and rare earth elements inform new biotechnological applications?

Recent research on rare earth element (REE) utilization in P. putida opens intriguing possibilities for NikE applications:

• REE-dependent regulation systems: The discovery that copper, zinc, and particularly iron availability influences REE-switch regulation in P. putida suggests complex interactions between different metal transport systems. Understanding how NikE functions within this network could enable the development of sophisticated metal-sensing and accumulation systems .

• Bioleaching and biomining technologies: Insights into how NikE and other transporters respond to REEs could inform the development of bioleaching technologies for rare earth recovery from electronic waste or low-grade ores.

• Lanthanide-dependent enzyme systems: The REE-utilizing pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase system in P. putida demonstrates the existence of REE-dependent enzymes. Similar principles might apply to nickel-dependent systems, opening possibilities for novel biocatalytic applications.

• Biomonitoring applications: P. putida strains with modified NikE systems could be developed as biosensors for environmental monitoring of both traditional heavy metals and REEs.

• Synthetic lanthanide biochemistry: Understanding the interplay between traditional transition metals like nickel and REEs could enable the development of synthetic biological systems that utilize lanthanides for novel functions.

Research has demonstrated that the ABC-type transporter system encoded by the gene cluster pedA1A2BC (which shares characteristics with nickel transport systems) is essential for efficient growth with low lanthanide concentrations, suggesting similar mechanisms might be involved in both traditional and rare earth metal transport .