Recombinant Salmonella typhimurium Putative 2-aminoethylphosphonate import ATP-binding protein PhnT (phnT)

What is the PhnT protein in Salmonella typhimurium and what is its function?

PhnT in Salmonella typhimurium functions as an ABC family traffic ATPase that is part of the 2-aminoethylphosphonate (AEP) transport system. This protein operates within a system that is activated by the Pho regulon under conditions of phosphate deprivation . PhnT works in conjunction with PhnS (a periplasmic binding protein), PhnU, and PhnV (integral membrane channel proteins) to facilitate the import of 2-aminoethylphosphonate across the bacterial membrane .

As an ATP-binding protein, PhnT likely provides the energy required for the transport process through ATP hydrolysis. The protein is part of a sophisticated mechanism that allows Salmonella to utilize alternative phosphorus sources when inorganic phosphate is limited in the environment, which is crucial for bacterial survival under nutrient-restricted conditions.

How is PhnT regulated within the Pho regulon system?

PhnT expression is regulated primarily through the Pho regulon, which becomes activated under phosphate-limiting conditions . The regulatory system includes PhnR, which functions as a transcriptional regulator based on sequence similarities at the protein level . When inorganic phosphate becomes scarce, this regulatory system activates the expression of genes involved in phosphonate metabolism, including phnT.

This regulation ensures that Salmonella only expresses the phosphonate transport machinery when necessary, providing an efficient energy conservation strategy. The coordinated expression of all components in the transport system (PhnS, PhnT, PhnU, and PhnV) allows for the complete assembly of a functional transport complex.

What is the relationship between PhnT and other proteins in the phosphonate transport system?

PhnT functions as part of an integrated system that includes several other proteins:

All these components work in concert to ensure efficient uptake and utilization of 2-aminoethylphosphonate as an alternative phosphorus source .

What are the optimal methods for constructing recombinant S. typhimurium strains expressing PhnT?

Based on established protocols for recombinant S. typhimurium construction, researchers should consider the following methodology:

• Gene amplification: PCR-amplify the phnT gene using appropriate primers that include restriction sites compatible with your chosen expression vector .

• Cloning: Insert the amplified gene into an expression vector such as pET3a, which has been successfully used for similar proteins .

• Transformation: Transform the recombinant plasmid into an expression host such as E. coli BL21(DE3) for initial protein expression testing .

• Verification: Confirm correct insertion and orientation through DNA sequencing.

• Expression optimization: Test different induction conditions (IPTG concentration, temperature, duration) to maximize protein expression.

• Transfer to Salmonella: Once expression is optimized, transfer the confirmed construct into attenuated S. typhimurium strains using electroporation or other appropriate methods .

For viral or antigenic protein expression within Salmonella (as demonstrated with influenza nucleoprotein), consider directing the protein to different cellular compartments (cytoplasmic, periplasmic, or secreted) to assess effects on immune response and protein functionality .

What experimental designs are most effective for studying PhnT function in vitro?

When designing experiments to study PhnT function, researchers should adhere to these fundamental principles:

• Apply the four pillars of experimental design: replication, randomization, blocking, and appropriate experimental unit size .

• For enzymatic activity assays of PhnT, adapt the methodologies used for related proteins:

  • Measure ATP hydrolysis rates using colorimetric phosphate release assays

  • Track substrate transport using radiolabeled AEP

  • Monitor conformational changes upon ATP/substrate binding using fluorescence spectroscopy

• For interaction studies with other transport components:

  • Implement bacterial two-hybrid systems

  • Perform co-immunoprecipitation experiments

  • Use surface plasmon resonance to measure binding kinetics

A typical ATPase activity assay could be set up as follows:

Measure phosphate release over time using malachite green or similar assays to determine ATPase activity .

How can site-directed mutagenesis be used to identify key functional residues in PhnT?

Site-directed mutagenesis is a powerful approach to identify critical residues in PhnT. Based on established protocols for similar proteins, consider the following methodology:

• Target selection: Identify conserved motifs in PhnT, particularly the Walker A and Walker B motifs common to ATP-binding proteins, as well as the Q-loop and signature motif.

• Mutagenesis strategy: Use PCR-based strategies as described in search result , with phnT gene as the template.

• Primer design: Design primers containing the desired mutations, focusing on:

  • Conservative mutations (K→R, D→E) to test charge importance

  • Non-conservative mutations (K→L, R→A) to eliminate function

• Expression and purification: Express and purify mutant proteins following the same protocol as for wild-type PhnT.

• Functional assays: Compare ATPase activity and substrate transport between wild-type and mutant proteins.

From similar studies on related proteins, the following mutations might be informative:

Yields of purified mutant proteins may vary significantly, as observed in similar studies where yields ranged from 3.6 to 19.1 mg/g of cells depending on the specific mutation .

How does PhnT contribute to Salmonella virulence and immune evasion?

The role of PhnT in Salmonella virulence is complex and likely relates to bacterial survival in phosphate-limited environments encountered during infection. Research shows that:

• Salmonella typhimurium can invade non-phagocytic cells and establish an intracellular niche .

• Once inside these cells, bacteria are resistant to recognition by cytotoxic T lymphocytes (CTLs), even when expressing foreign antigens .

• The ability to utilize alternative phosphorus sources through systems like the Phn pathway may contribute to bacterial persistence within host cells.

• Recombinant S. typhimurium strains have been used as vaccine vectors, with proteins expressed in the periplasm being able to prime CTL responses in infected mice .

This suggests a potential dual role for PhnT: supporting bacterial survival under nutrient limitation while potentially contributing to immune modulation. Research using attenuated S. typhimurium strains has shown they can persist for several weeks in Peyer's patches and are internalized by dendritic cells, which are potent antigen-presenting cells . This may explain the immunogenicity of Salmonella vaccine strains.

For researchers investigating this aspect, comparing wild-type and phnT-deficient strains in infection models would be valuable to determine its specific contribution to virulence and immune interaction.

What approaches can be used to purify functional recombinant PhnT protein?

Purification of functional PhnT protein requires careful attention to protein stability and activity preservation. Based on successful purification of related proteins, the following protocol is recommended:

• Expression system: Clone the phnT gene into an expression vector such as pET3a and transform into E. coli BL21(DE3) .

• Culture conditions: Grow transformed cells in appropriate media (like LB with antibiotics) to OD₆₀₀ of 0.6-0.8 before induction.

• Induction: Add IPTG to induce protein expression, typically at concentrations of 0.5-1.0 mM.

• Cell harvesting: Harvest cells by centrifugation (5,000 × g for 15 min) and resuspend in buffer containing:

  • 10 mM KH₂PO₄

  • 1 mM dithiothreitol

  • 5 μM PLP (if a cofactor is required)

  • pH adjusted to 7.5 with KOH

• Cell lysis: Disrupt cells using a French press at 16,000 lb/in² and clarify by centrifugation at 18,000 × g for 30 min at 4°C .

• Initial fractionation: Add ammonium sulfate to 45% saturation with gentle stirring at 0°C, followed by centrifugation .

• Chromatography: Apply the sample to a DEAE-cellulose column and elute with an appropriate buffer gradient .

Expected yields of purified PhnT protein should be approximately 10-15 mg/g of wet cells, based on yields reported for similar proteins . Verification of protein identity can be confirmed by N-terminal sequencing, as the post-translational removal of Met1 has been observed in related proteins .

How can contradictory results in PhnT research be analyzed and reconciled?

Analyzing contradictory results in PhnT research requires a systematic approach to data integration and examination. Based on integrative data analysis methodologies:

• Implement Integrative Data Analysis (IDA) to simultaneously analyze raw data pooled from multiple studies . This approach offers:

  • Economy through reuse of existing data

  • Increased statistical power from combined sample sizes

  • Potential to address questions not answerable by individual studies

  • Opportunity to examine effect similarities across studies

• Apply a phronetic iterative approach to data analysis , which involves:

  • Asking "What are the data telling me?"

  • Reflecting on "What is it I want to know?"

  • Analyzing the relationship between data findings and research questions

• Use iterative analysis by alternating between:

  • Consulting existing theories and predefined questions

  • Examining emergent qualitative findings

  • Gradually narrowing the focus of research

When facing contradictory findings, consider these specific approaches:

• Examine methodological differences between studies:

  • Cell types or strains used

  • Expression systems and purification methods

  • Assay conditions and detection methods

• Conduct parameter setting and negative case analysis to identify boundary conditions where results differ .

• Develop commensurate measures across studies to ensure valid comparisons .

Creating an analysis outline and writing analytical memos can help synthesize findings across disparate studies .

What are the kinetic properties of PhnT and how can they be measured?

The kinetic properties of PhnT as an ATPase can be measured using approaches similar to those applied to related proteins in the ABC transporter family. Based on established methodologies:

• ATPase activity measurement:

  • Monitor inorganic phosphate release using colorimetric assays (malachite green or similar)

  • Measure ADP formation using coupled enzyme assays with pyruvate kinase and lactate dehydrogenase

• Transport kinetics:

  • Use radiolabeled AEP to measure transport rates

  • Monitor AEP uptake in membrane vesicles containing reconstituted PhnT along with PhnS, PhnU, and PhnV

Expected kinetic parameters for PhnT, based on related ABC transporters and the phosphonate metabolic system described in search result , would include:

For accurate kinetic analysis, it's crucial to:

• Establish conditions where ATPase activity is linearly dependent on enzyme concentration and time

• Account for any basal ATPase activity in the absence of transport substrate

• Measure activity across a range of substrate concentrations (both ATP and AEP)

• Analyze data using appropriate models (Michaelis-Menten, Hill equation, etc.)

How can recombinant S. typhimurium expressing modified PhnT be used in vaccine development?

Recombinant S. typhimurium strains have significant potential as vaccine vectors, and modifications to the PhnT system could enhance their efficacy. Research indicates:

• Attenuated S. typhimurium strains can be immunogenic via oral administration while remaining avirulent .

• S. typhimurium can invade non-phagocytic cells and persist intracellularly, making them suitable for delivering antigens to the immune system .

• Protein localization within the bacteria is crucial - periplasmic expression of antigens has been shown to prime CTL responses more effectively than cytoplasmic expression .

For vaccine development using the PhnT system, researchers should consider:

• Creating fusion constructs that link antigens of interest to PhnT or other components of the phosphonate import system.

• Directing antigen expression to different cellular compartments (cytoplasmic, periplasmic, or secreted) to optimize immune response.

• Using attenuated S. typhimurium strains like PhoPc, which has been shown to persist in Peyer's patches and be internalized by dendritic cells .

• Developing multivalent vaccines that combine PhnT-based systems with other Salmonella antigens.

The WHO has noted that vaccine development against non-typhoidal Salmonella is a public health priority, especially given the emergence of multi-drug resistant strains . Current vaccine candidates include live-attenuated, subunit-based, and recombinant antigen-based approaches, with both humoral and cellular immune responses being important for protection .

What computational approaches can predict structure-function relationships in PhnT?

Computational approaches offer powerful tools for predicting structure-function relationships in proteins like PhnT. Researchers should consider:

• Homology modeling:

  • Identify structural templates from related ABC transporters with solved crystal structures

  • Build three-dimensional models of PhnT based on sequence alignment with templates

  • Validate models using energy minimization and Ramachandran plot analysis

• Molecular dynamics simulations:

  • Simulate PhnT behavior in a lipid bilayer environment

  • Model conformational changes during the ATP hydrolysis cycle

  • Predict effects of mutations on protein stability and function

• Protein-protein docking:

  • Model interactions between PhnT and other components of the transport system

  • Predict binding interfaces and key interacting residues

  • Simulate the assembled transport complex

• Machine learning approaches:

  • Train algorithms on known ABC transporter structures and functions

  • Predict functional sites and mechanistic details specific to PhnT

  • Identify potential allosteric regulation sites

For researchers implementing these approaches, it's important to validate computational predictions through experimental methods such as site-directed mutagenesis, cross-linking studies, and functional assays. The integration of computational and experimental data provides the most robust understanding of PhnT structure-function relationships.

How can phronetic iterative analysis improve data interpretation in PhnT research?

The phronetic iterative approach to data analysis offers significant advantages for PhnT research, particularly when dealing with complex datasets or contradictory findings. Based on search result , researchers should:

• Implement an iterative analysis framework that asks:

  • "What are the data telling me?" (engaging with theoretical and field understandings)

  • "What is it I want to know?" (according to research objectives and questions)

  • "What is the dialectical relationship between what the data are telling me and what I want to know?" (refining focus and linking back to research questions)

• Alternate between emic (emergent) readings of data and etic use of existing models and theories .

• Use abduction to construct hypotheses, test them in the field, and revise when necessary .

The practical implementation of this approach involves:

• Coding data iteratively:

  • First-level descriptive coding to categorize basic information

  • Second-level analytic coding to interpret patterns and relationships

• Crafting a qualitative codebook to ensure consistency in data interpretation .

• Writing analytic memos to synthesize findings and develop theoretical insights .

• Engaging in theoretical sampling, negative case analysis, and parameter setting to refine understanding .

This approach is particularly valuable for PhnT research when:

• Integrating findings across different experimental systems

• Reconciling apparently contradictory results

• Developing comprehensive models of PhnT function within the broader context of bacterial physiology and pathogenesis

The phronetic iterative approach allows researchers to jump into data analysis earlier and learn by doing, which can accelerate research progress while maintaining methodological rigor .

What are common challenges in expressing recombinant PhnT and how can they be overcome?

Expression of recombinant PhnT can present several challenges common to membrane-associated proteins. Based on experiences with similar proteins, researchers may encounter:

• Protein insolubility and inclusion body formation:

  • Solution: Optimize induction conditions (lower temperature, reduced IPTG concentration)

  • Alternative: Express as fusion protein with solubility tags (MBP, SUMO, etc.)

• Low expression levels:

  • Solution: Codon optimization for the expression host

  • Alternative: Test different promoter systems or expression hosts

• Protein instability:

  • Solution: Include appropriate cofactors (ATP, Mg²⁺) in all buffers

  • Alternative: Express and purify with interacting partners (PhnS, PhnU, PhnV)

• Functional impairment:

  • Solution: Verify protein folding using circular dichroism or fluorescence spectroscopy

  • Alternative: Implement quality control steps to assess ATPase activity throughout purification

Based on yields reported for related proteins (3.6-19.1 mg/g of cells depending on specific mutations) , researchers should optimize their protocols until achieving at least 10 mg/g of cells for wild-type PhnT.

When troubleshooting expression issues, a systematic approach is recommended:

How can researchers optimize PhnT activity assays for high-throughput screening?

Optimizing PhnT activity assays for high-throughput screening requires balancing sensitivity, reproducibility, and throughput. Based on established methodologies:

• ATPase activity assay optimization:

  • Adapt colorimetric phosphate detection methods to microplate format

  • Implement continuous assays using coupled enzyme systems (pyruvate kinase/lactate dehydrogenase)

  • Optimize buffer conditions for maximum signal-to-noise ratio

• Substrate transport assay adaptation:

  • Develop fluorescent AEP analogs for real-time monitoring

  • Implement membrane vesicle-based assays in multi-well formats

  • Use pH-sensitive fluorescent dyes to detect proton coupling

• Binding assay development:

  • Establish fluorescence polarization assays for ATP or substrate binding

  • Adapt thermal shift assays to detect ligand-induced stabilization

  • Implement surface plasmon resonance for detailed binding kinetics

For high-throughput applications, consider these practical optimizations:

• Miniaturization to 384-well or 1536-well formats to reduce reagent consumption

• Automation of liquid handling to improve reproducibility

• Development of stable reagents that maintain activity during extended screening campaigns

• Implementation of robust statistical methods for hit identification and validation

A typical workflow for high-throughput assay development might include:

What strategies can improve the yield and stability of purified PhnT protein?

Maximizing yield and stability of purified PhnT requires attention to multiple aspects of the expression and purification process. Based on established protocols for similar proteins:

• Expression optimization:

  • Test multiple expression vectors with different promoter strengths

  • Evaluate various E. coli strains (BL21, C41/C43, Rosetta) for optimal expression

  • Implement auto-induction media to achieve higher cell densities

  • Consider fermentation approaches for large-scale production

• Solubility enhancement:

  • Express at lower temperatures (16-20°C) to slow folding and reduce aggregation

  • Include solubility enhancers like sorbitol or arginine in the growth media

  • Co-express with chaperones (GroEL/ES, DnaK/J) to assist proper folding

• Purification optimization:

  • Implement multi-step purification strategies combining:

    • Affinity chromatography (if tagged)

    • Ion exchange chromatography (DEAE-cellulose as used for similar proteins)

    • Size exclusion chromatography for final polishing

  • Optimize buffer conditions:

    • Include stabilizing agents (glycerol, reducing agents)

    • Maintain ATP and Mg²⁺ throughout purification

    • Test various pH conditions and ionic strengths

• Stability enhancement:

  • Identify and control protease activity with inhibitor cocktails

  • Determine optimal storage conditions (temperature, buffer composition)

  • Consider protein engineering approaches to improve intrinsic stability

For long-term storage, researchers should evaluate multiple conditions:

By systematically optimizing these aspects, researchers can achieve higher yields and improved stability for purified PhnT protein.