Recombinant Pseudomonas stutzeri Putative phosphite transport system permease protein htxE (htxE)

What is htxE and how does it function within the P. stutzeri phosphorus metabolism system?

htxE is a putative permease protein that forms part of the htxABCDEFGHIJKLMN operon in Pseudomonas stutzeri. This protein functions as a component of a binding protein-dependent transport system specifically involved in phosphite uptake. The htx operon encodes a complete pathway for hypophosphite oxidation to phosphate, with htxE serving as one of the membrane components facilitating transport across the cell membrane .

The broader htx system works in conjunction with other phosphorus acquisition pathways in P. stutzeri. Current research indicates that htxA encodes a hypophosphite-2-oxoglutarate dioxygenase that oxidizes hypophosphite to phosphite, while the remaining htx components (including htxE) facilitate further metabolism of phosphite to phosphate .

How does the htx operon differ from the phn operon in P. stutzeri?

P. stutzeri possesses two distinct C—P lyase operons: the htx and phn operons, which have partially overlapping but distinct functions:

Deletion studies have shown that both operons individually support growth on methylphosphonate, but only the phn operon efficiently supports growth on aminoethylphosphonate and phosphite. Notably, neither operon supports growth on other phosphonate compounds like glyphosate or phenylphosphonate .

What is the genetic organization of the htx operon and what is known about its regulation?

The htx operon consists of genes htxABCDEFGHIJKLMN that are cotranscribed based on gene organization. Reverse transcription-PCR with total RNA has verified the presence of intergenic sequences, confirming the operon structure .

While the complete regulatory mechanism has not been fully characterized in the available research, phosphorus limitation likely serves as a key trigger for htx operon expression, as is common for genes involved in alternative phosphorus acquisition pathways. The precise regulatory elements, including promoter sequences and potential repressor or activator binding sites, represent important targets for future research.

What expression systems are most effective for producing recombinant htxE protein for functional and structural studies?

For membrane proteins like htxE, optimal expression requires screening multiple systems. Based on current high-throughput protein production methodologies, researchers should consider:

Researchers should employ sticky-end PCR methods to generate DNA products with appropriate restriction sites that can be cloned into multiple fusion protein expression vectors without requiring restriction digestion of PCR products . This approach allows for high-efficiency (>95%) directional cloning into different fusion protein expression vectors using universal restriction sites .

What high-throughput methods can be applied to optimize htxE expression and purification?

Based on current high-throughput experimentation (HTE) techniques for membrane proteins:

• Parallel expression screening:

  • Use 96-well format for bacterial cultures (~1.5 mL)

  • Test multiple induction temperatures (16°C, 25°C, 30°C)

  • Vary inducer concentrations systematically

  • Screen multiple detergents for extraction efficiency

• Solubility assessment:

  • Perform high-speed centrifugation in 96-tube format

  • Analyze supernatant and pellet fractions using multiwell SDS-PAGE

  • Quantify relative soluble expression across conditions

• Purification optimization:

  • Employ parallel affinity purification using magnetic beads

  • Test buffer conditions systematically (pH, salt, additives)

  • Evaluate stabilizing agents for membrane protein stability

This high-throughput approach enables testing of multidimensional hypotheses and collection of large datasets, leading to more rapid optimization than traditional methods .

How can structure-function relationships in htxE be effectively investigated?

Investigating structure-function relationships in htxE requires integrated approaches:

• Bioinformatic analysis:

  • Transmembrane topology prediction

  • Identification of conserved residues across related transporters

  • Homology modeling based on structurally characterized bacterial permeases

• Systematic mutagenesis:

  • Alanine-scanning mutagenesis of predicted functional regions

  • Creation of chimeric proteins with related transporters

  • Site-directed mutagenesis of conserved residues

• Functional characterization:

  • Transport assays using radiolabeled phosphite

  • Growth complementation studies in knockout strains

  • In vitro reconstitution in proteoliposomes

• Structural studies:

  • Detergent screening for stability and homogeneity

  • Crystallization trials or cryo-EM analysis

  • Hydrogen-deuterium exchange mass spectrometry for dynamics

Current research indicates that complementation studies in deletion mutants provide valuable insights into structure-function relationships, as demonstrated with other components of the phosphorus metabolism system in P. stutzeri .

What methods are most effective for measuring phosphite transport activity mediated by htxE?

Robust characterization of htxE-mediated phosphite transport can be achieved through:

• Whole-cell transport assays:

  • Expression of htxE in transport-deficient strains

  • Measurement of ³²P-labeled phosphite uptake

  • Kinetic analysis to determine Km and Vmax values

  • Competition assays with structural analogs

• Genetic complementation:

  • Construction of Δhtx and/or Δphn strains

  • Expression of wild-type or mutant htxE variants

  • Growth assessment on phosphite as sole phosphorus source

  • Quantification of growth rates and yields

• Biochemical characterization:

  • Purification of complete transport complex components

  • Reconstitution in proteoliposomes

  • Measurement of ATP hydrolysis coupled to transport

  • Assessment of substrate specificity

The search results indicate that deletion of both htx and phn operons abolishes growth on methylphosphonate and aminoethylphosphonate, providing a clear phenotype for complementation studies .

How can one distinguish between the contributions of htxE and other components of the phosphite transport system?

Distinguishing the specific role of htxE within the transport system requires:

• Component-specific knockouts:

  • Generation of precise htxE deletion mutations

  • Construction of strains with mutations in other htx components

  • Creation of conditional expression systems for individual components

• Protein-protein interaction studies:

  • Co-immunoprecipitation with tagged htxE

  • Bacterial two-hybrid assays to map interaction partners

  • In vitro binding assays with purified components

• Domain swapping experiments:

  • Creation of chimeric proteins with corresponding components from related systems

  • Expression in appropriate knockout backgrounds

  • Functional assessment of chimeric transport systems

Research has demonstrated that the phn operon can support growth on phosphite, while the htx operon provides only limited phosphite utilization capacity . This differential functionality provides a basis for distinguishing the specific contributions of htxE through targeted genetic manipulations.

What approaches can resolve contradictory findings in htxE functional studies?

To address contradictory findings, researchers should systematically:

• Standardize experimental conditions:

  • Use defined minimal media with controlled phosphorus content

  • Standardize growth phases for functional assays

  • Control expression levels using calibrated induction systems

• Employ multiple complementary methods:

  • Combine genetic, biochemical, and physiological approaches

  • Use both in vivo and in vitro systems

  • Apply both qualitative and quantitative measurements

• Consider strain-specific effects:

  • Compare results across multiple P. stutzeri strains

  • Examine chromosomal context effects on expression

  • Evaluate potential compensatory mechanisms

• Statistical analysis:

  • Perform sufficient biological and technical replicates

  • Apply appropriate statistical tests

  • Consider meta-analysis approaches for conflicting literature

When studying phosphorus metabolism pathways, it is crucial to account for potential cross-talk between pathways and the influence of growth conditions on regulatory networks .

What is the most efficient strategy for generating htxE knockout mutants in P. stutzeri?

Based on successful genetic manipulation approaches for phosphorus metabolism genes:

• Deletion strategy:

  • Amplify ~500 bp regions upstream and downstream of htxE

  • Join fragments using overlap extension PCR or Gibson Assembly

  • Clone into a suicide vector with appropriate selection markers

  • Select for double crossover events using counter-selection

Example primer design strategy based on published successful deletions:

This approach has been successfully applied for creating deletions in other phosphorus metabolism operons in P. stutzeri .

How can recombinational cloning methods be optimized for htxE manipulation?

While traditional recombinational cloning (RC) using cre-lox or Int/Xis/IHF systems can be applied to htxE, certain limitations must be addressed:

• Limitations of standard RC approaches:

  • Potential aberrant recombination products

  • Required translation fusions of recombination sites

  • Potentially detrimental effects of longer translation fusions

• Optimized approach for htxE:

  • Employ conventional cloning with shorter translation fusions

  • Use sticky end PCR method to generate products with appropriate restriction sites

  • Implement directional cloning without restriction digestion of PCR products

• Vector design considerations:

  • Incorporate removable tags for purification

  • Include protease cleavage sites to remove fusion partners

  • Optimize codon usage for expression host

The sticky end PCR method generates DNA products with sticky ends (such as 5′ EcoRI and 3′ XhoI) directly, enabling high-efficiency cloning without restriction digestion of PCR products .

What are the best methods for verifying htxE expression and localization in recombinant systems?

To confirm proper expression and localization:

• Expression verification:

  • Western blotting with antibodies against tags or htxE

  • RT-qPCR for transcript quantification

  • Mass spectrometry of membrane fractions

• Localization studies:

  • Membrane fractionation and Western blotting

  • Fluorescence microscopy with fluorescent protein fusions

  • Immunogold electron microscopy

• Functional verification:

  • Complementation of growth defects in knockout strains

  • Transport assays with radiolabeled substrates

  • In vitro reconstitution studies

• High-throughput screening:

  • Analysis of total cell lysates by centrifugation in 96-tube format

  • Assessment of membrane vs. soluble fractions by SDS-PAGE

  • Parallel testing of multiple constructs and conditions

Current research shows that approximately 80% of genes screened in high-throughput systems show high levels of expression in at least one fusion protein construct, suggesting that screening multiple constructs is essential for success .

How conserved is htxE across different species of Pseudomonas and related bacteria?

Investigating htxE conservation requires systematic comparative genomic approaches:

• Sequence similarity analysis:

  • BLAST searches against bacterial genome databases

  • Multiple sequence alignment of htxE homologs

  • Calculation of sequence identity and similarity metrics

• Phylogenetic analysis:

  • Construction of phylogenetic trees of htxE homologs

  • Comparison with species phylogeny to identify horizontal gene transfer

  • Identification of selection signatures (dN/dS ratios)

• Operon structure comparison:

  • Assessment of gene synteny around htxE

  • Identification of operon structure conservation or rearrangements

  • Evaluation of regulatory element conservation

The search results indicate that P. stutzeri contains two distinct C—P lyase operons (htx and phn), suggesting gene duplication and functional divergence have occurred during evolution . This provides an excellent system for studying the evolution of phosphorus acquisition pathways.

What can be learned from comparing htxE with homologous phosphite transporters in other organisms?

Comparative analysis of htxE with homologs provides insights into:

• Functional conservation and divergence:

  • Identification of universally conserved residues essential for function

  • Recognition of clade-specific residues indicating functional specialization

  • Detection of convergent evolution in unrelated phosphite transporters

• Substrate specificity determinants:

  • Correlation of sequence variations with known substrate preferences

  • Identification of putative substrate-binding residues

  • Design of chimeric transporters to test specificity hypotheses

• Structural insights:

  • Prediction of conserved structural features

  • Identification of variable regions that may confer specific functions

  • Recognition of potential interaction interfaces with partner proteins

The presence of both htx and phn operons in P. stutzeri, with their distinct but overlapping functionalities, provides a natural system for studying the evolution of substrate specificity in phosphorus transport systems .

How has the htx system evolved in relation to environmental phosphorus availability?

Understanding the evolutionary drivers of htx system diversity requires:

• Ecological distribution analysis:

  • Survey of htxE presence across bacteria from diverse environments

  • Correlation with environmental phosphorus availability

  • Assessment of co-occurrence with other phosphorus acquisition systems

• Experimental evolution approaches:

  • Laboratory evolution under phosphite-selective conditions

  • Characterization of adaptive mutations in the htx system

  • Fitness assessment of htx variants in different phosphorus regimes

• Population genomics:

  • Analysis of htxE polymorphisms in natural populations

  • Identification of signatures of selection

  • Assessment of horizontal gene transfer events

Research has demonstrated that P. stutzeri possesses remarkably versatile phosphorus acquisition capabilities, including the ability to metabolize hypophosphite via the htx system, suggesting adaptation to environments where alternative phosphorus sources are available .

How does htxE integrate into the broader phosphorus regulatory network in P. stutzeri?

Understanding the systems-level integration of htxE requires:

• Transcriptional regulation analysis:

  • RNA-seq under varying phosphorus conditions

  • ChIP-seq to identify transcription factor binding sites

  • Promoter dissection through reporter assays

• Metabolic network mapping:

  • Flux analysis of phosphorus compounds through different pathways

  • Identification of metabolic bottlenecks and regulatory nodes

  • Computational modeling of phosphorus acquisition network

• Protein interaction network:

  • Identification of htxE-interacting proteins

  • Characterization of multiprotein complexes involved in phosphite transport

  • Analysis of post-translational modifications affecting htxE function

The search results indicate that P. stutzeri possesses multiple phosphorus acquisition systems that may be coordinately regulated, including the ptx operon encoding phosphite:NAD oxidoreductase and the htx operon with its C—P lyase activity .

How can computational modeling enhance our understanding of htxE function and evolution?

Computational approaches provide powerful tools for htxE research:

• Structural modeling:

  • Homology modeling based on related transporters

  • Molecular dynamics simulations to study conformational changes

  • Docking studies to predict substrate interactions

• Systems biology modeling:

  • Kinetic modeling of phosphite transport and metabolism

  • Genome-scale metabolic modeling of phosphorus utilization

  • Evolutionary simulations to reconstruct htxE history

• Machine learning applications:

  • Prediction of functional residues from sequence analysis

  • Classification of transporter subfamilies based on sequence features

  • Integration of diverse data types to predict htxE interactions

When studying complex phosphorus acquisition systems like those in P. stutzeri, computational models can integrate experimental data and generate testable hypotheses about system behavior under different conditions .

How can htxE research contribute to understanding phosphorus cycling in natural environments?

htxE research has significant implications for environmental microbiology:

• Environmental monitoring:

  • Development of molecular markers for htxE detection in environmental samples

  • Quantification of htxE-containing bacteria in diverse ecosystems

  • Correlation with phosphite availability and turnover

• Phosphorus cycling studies:

  • Assessment of phosphite as an overlooked phosphorus reservoir

  • Quantification of bacterial phosphite oxidation rates

  • Modeling of phosphite contributions to phosphorus biogeochemical cycles

• Microbial ecology:

  • Characterization of niche specialization based on phosphorus acquisition

  • Competition studies between organisms with different phosphorus acquisition strategies

  • Investigation of phosphite-based microbial interactions

The discovery that P. stutzeri possesses dedicated systems for phosphite utilization suggests that phosphite may be a more significant component of environmental phosphorus cycling than previously recognized .

What potential biotechnological applications exist for engineered htxE variants?

Engineered htxE proteins could enable several biotechnological applications:

• Biosensing technologies:

  • Development of whole-cell biosensors for phosphite detection

  • Creation of protein-based biosensors using htxE components

  • Environmental monitoring of phosphorus compounds

• Bioremediation tools:

  • Engineering enhanced phosphite uptake for phosphorus recovery

  • Development of organisms capable of degrading phosphonate pollutants

  • Creation of phosphite-accumulating organisms for phosphorus capture

• Synthetic biology applications:

  • Design of artificial phosphorus utilization pathways

  • Creation of organisms with altered phosphorus preferences

  • Development of phosphite-dependent containment systems

The detailed understanding of htxE structure-function relationships could enable rational design of variants with enhanced or altered properties for these applications.

What are the most promising future research directions for understanding htxE and related phosphite transport systems?

Key directions for future research include:

• Structural biology:

  • Determination of htxE structure through crystallography or cryo-EM

  • Characterization of conformational changes during transport

  • Elucidation of the complete transport complex architecture

• Systems-level understanding:

  • Comprehensive mapping of phosphorus regulatory networks

  • Characterization of cross-talk between different phosphorus acquisition systems

  • Understanding of ecological significance in natural environments

• Synthetic and chemical biology:

  • Development of specific inhibitors of htxE-mediated transport

  • Creation of synthetic phosphite transport systems with novel properties

  • Engineering of organisms with expanded phosphorus substrate range

• Evolutionary biology:

  • Investigation of the origins and diversification of phosphite transport systems

  • Comparative genomics across diverse bacterial lineages

  • Experimental evolution studies under phosphite selection

The presence of multiple, distinct phosphorus acquisition systems in P. stutzeri makes it an excellent model organism for studying the evolution and diversification of nutrient acquisition pathways .