Recombinant Bartonella quintana Phosphate import ATP-binding protein PstB (pstB)

What is the function of Phosphate import ATP-binding protein PstB in Bartonella quintana?

PstB functions as the ATP-binding component of the phosphate-specific transport (Pst) system in B. quintana. It hydrolyzes ATP to provide energy for the active transport of inorganic phosphate across the bacterial cell membrane. As part of the ABC transporter family, PstB works in conjunction with other Pst system components (PstS, PstA, and PstC) to facilitate phosphate uptake under phosphate-limited conditions. In B. quintana, this function is particularly important given its fastidious growth requirements and adaptation to the human host environment .

How is the pstB gene organized in the Bartonella quintana genome?

The pstB gene in B. quintana is typically organized within an operon containing other components of the phosphate transport system. Based on genomic data from B. quintana RM-11, the genome contains 1,312 genes within a 1,587,646 bp chromosome . The pstB gene is part of the pstSCAB operon, which is regulated by the PhoBR two-component system that responds to phosphate limitation. This organization is consistent with other alphaproteobacteria, though B. quintana shows genome reduction compared to related species, reflecting its adaptation to a specific host environment .

What are the structural characteristics of B. quintana PstB protein?

The PstB protein from B. quintana belongs to the P-loop NTPase superfamily, characterized by conserved Walker A and Walker B motifs that are involved in ATP binding and hydrolysis. Like other ATP-binding proteins in bacterial ABC transporters, it likely contains:

• Nucleotide-binding domain (NBD) with the conserved sequence motifs

• Signature motif (LSGGQ) characteristic of ABC transporters

• H-loop and Q-loop involved in the ATPase catalytic cycle

The protein exists primarily as a dimer when functioning in the complete Pst transporter complex, with each monomer binding and hydrolyzing ATP during the transport cycle .

What expression systems are most effective for producing recombinant B. quintana PstB?

For recombinant expression of B. quintana PstB, E. coli-based expression systems have proven most effective. When designing an expression strategy, consider the following approaches:

• Expression Vector Selection: pET-based vectors (particularly pET28a with an N-terminal His-tag) provide high expression levels under T7 promoter control.

• Host Strain Optimization: E. coli BL21(DE3) or its derivatives like Rosetta™ (for rare codon usage) are recommended host strains.

• Expression Conditions:

  • Induction with 0.5-1.0 mM IPTG at mid-log phase (OD600 0.6-0.8)

  • Post-induction growth at 18-25°C for 16-18 hours to enhance soluble protein yield

  • Addition of 5-10% glycerol to culture media to improve protein stability

This approach is comparable to methods used for expressing other membrane-associated proteins from B. quintana, such as the hemin-binding protein (HbpA) described in the literature .

What purification strategies yield the highest purity and activity of recombinant PstB?

A multi-step purification strategy is recommended for obtaining high-purity, active recombinant PstB:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin with a binding buffer containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 10 mM imidazole.

• Intermediate Purification: Size exclusion chromatography (SEC) using a Superdex 200 column with running buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 5% glycerol.

• Polishing Step: Ion exchange chromatography on a Q-Sepharose column if additional purity is required.

• Buffer Optimization: Final protein should be stored in 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2, 10% glycerol, and 1 mM DTT to maintain stability and activity.

This purification approach preserves the ATP-binding and hydrolysis capabilities of the protein, which can be confirmed through ATPase activity assays. Similar purification strategies have been employed for other B. quintana proteins, although special consideration must be given to maintaining the native conformation of PstB .

How can I verify the proper folding and activity of purified recombinant PstB?

To verify proper folding and activity of recombinant PstB, employ multiple complementary approaches:

• Structural Integrity Assessment:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure elements

  • Thermal shift assays to evaluate protein stability

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS) to determine oligomeric state

• Functional Verification:

  • ATPase activity assay measuring inorganic phosphate release using malachite green

  • ATP binding assays using fluorescent ATP analogs (TNP-ATP)

  • Isothermal titration calorimetry (ITC) to determine ATP binding constants

• Membrane Association Analysis:

  • Liposome binding assays to evaluate interaction with membrane components

  • In vitro reconstitution with other Pst system components to assess complex formation

These methods collectively provide a comprehensive assessment of both structural integrity and functional activity of the purified recombinant PstB protein .

How can recombinant PstB be used to study phosphate transport mechanisms in B. quintana?

Recombinant PstB serves as a valuable tool for investigating phosphate transport mechanisms through several experimental approaches:

• In vitro Transport Assays:

  • Reconstitution of the complete Pst system (PstS, PstA, PstC, and PstB) in liposomes

  • Measurement of 32P-labeled phosphate uptake in proteoliposomes

  • Assessment of ATP consumption coupled to phosphate transport

• Structure-Function Analysis:

  • Site-directed mutagenesis of key residues in the Walker A/B motifs and other functional domains

  • Correlation between ATPase activity and transport efficiency

  • Cross-linking studies to map protein-protein interactions within the transport complex

• Regulatory Studies:

  • Investigation of how phosphate limitation affects pstB expression

  • Analysis of PhoBR-mediated regulation of the pst operon

  • Evaluation of PstB as a component of the bacterial phosphate-sensing mechanism

These approaches help elucidate both the mechanistic details of phosphate transport and its regulation in B. quintana, providing insights into bacterial adaptation to phosphate-limited environments during infection .

What are the best methods to study interactions between PstB and other components of the phosphate transport system?

To study the interactions between PstB and other Pst system components, consider these methodological approaches:

• Co-purification Strategies:

  • Tandem affinity purification using differently tagged components

  • Bacterial two-hybrid assays to screen for interactions

  • Co-immunoprecipitation with component-specific antibodies

• Biophysical Interaction Analysis:

  • Surface plasmon resonance (SPR) to determine binding kinetics

  • Microscale thermophoresis (MST) for quantifying interactions in solution

  • Förster resonance energy transfer (FRET) between fluorescently labeled components

• Structural Analysis of Complexes:

  • Cryo-electron microscopy of reconstituted complexes

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Cross-linking coupled with mass spectrometry (XL-MS) to identify spatial relationships

• Functional Analysis:

  • ATPase activity modulation in the presence of other Pst components

  • Conformational changes upon complex formation using limited proteolysis

  • Effect of phosphate concentration on complex stability and function

These methods can reveal both the structural organization of the complete transporter and the dynamic interactions that occur during the transport cycle .

How does recombinant PstB contribute to understanding B. quintana pathogenesis?

Recombinant PstB provides valuable insights into B. quintana pathogenesis through multiple research applications:

• Nutrient Acquisition During Infection:

  • Analysis of phosphate uptake efficiency under conditions mimicking the host environment

  • Comparison of wild-type and phosphate transport mutants in cellular infection models

  • Evaluation of phosphate transport as a virulence determinant

• Host-Pathogen Interaction Studies:

  • Investigation of PstB expression during various stages of infection

  • Assessment of antibody responses to PstB in patients with B. quintana infections

  • Analysis of phosphate limitation as a host defense mechanism

• Therapeutic Target Evaluation:

  • Screening of small molecule inhibitors targeting PstB ATPase activity

  • Structure-based drug design leveraging recombinant protein crystals

  • Validation of PstB as a potential antimicrobial target

• Adaptation to Host Environment:

  • Comparison of phosphate transport efficiency between B. quintana and related species

  • Correlation between phosphate acquisition and survival in phosphate-limited niches

  • Investigation of how phosphate sensing affects expression of virulence factors

These applications connect the molecular function of PstB to the broader context of B. quintana pathogenesis, including its adaptation to the human host and potential vulnerabilities that could be exploited therapeutically .

How do post-translational modifications affect PstB function in B. quintana?

While post-translational modifications (PTMs) of bacterial proteins have received less attention than their eukaryotic counterparts, they play important roles in bacterial physiology. For B. quintana PstB, several potential PTMs warrant investigation:

• Phosphorylation:

  • Potential sites: Serine, threonine, and tyrosine residues near the ATP-binding domain

  • Function: May regulate ATPase activity in response to cellular energy status

  • Methods for detection: Phosphoproteomic analysis using mass spectrometry, Phos-tag SDS-PAGE

  • Functional impact: Phosphorylation may create an additional regulatory layer beyond transcriptional control

• Acetylation:

  • Potential sites: Lysine residues at the protein-protein interaction interfaces

  • Function: Could modify interactions with other Pst components

  • Methods for detection: Acetylome analysis by mass spectrometry, acetylation-specific antibodies

  • Metabolic connection: May link phosphate transport to central metabolism through acetyl-CoA availability

• Oxidative Modifications:

  • Potential sites: Cysteine residues involved in protein stability

  • Function: May respond to oxidative stress during host-pathogen interactions

  • Methods for detection: Redox proteomics, differential alkylation

  • Physiological relevance: Could represent a mechanism linking oxidative stress to phosphate transport

Investigation of these PTMs requires a combination of proteomic approaches, site-directed mutagenesis to create modification-mimicking or modification-resistant variants, and functional assays to assess the impact on ATPase activity and transport efficiency .

What structural variations exist in PstB proteins across different Bartonella species?

Comparative analysis of PstB proteins across Bartonella species reveals both conserved features and species-specific variations:

These variations reflect the evolutionary adaptation of different Bartonella species to their specific host environments. Key research approaches to understand these variations include:

• Homology modeling based on crystal structures of related ABC transporters

• Molecular dynamics simulations to assess the impact of sequence variations on protein dynamics

• Heterologous expression and comparative biochemical characterization

• Complementation studies in phosphate transport-deficient bacterial strains

Such comparative analyses can provide insights into the adaptive evolution of phosphate transport mechanisms across the Bartonella genus and their relationship to host specificity and pathogenicity .

How does the pstB gene expression change under different environmental conditions relevant to B. quintana infection?

The expression of pstB in B. quintana exhibits dynamic regulation in response to environmental conditions encountered during infection:

• Phosphate Limitation Response:

  • Under low phosphate conditions (<0.1 mM Pi), pstB expression increases 10-50 fold

  • Mechanism: Primarily mediated by the PhoBR two-component system

  • Timeframe: Rapid induction within 15-30 minutes of phosphate depletion

  • Co-regulated genes: Other members of the pst operon and phosphate-responsive genes

• Host Cell Interaction Effects:

  • During endothelial cell infection, pstB expression increases 3-5 fold

  • Pattern: Biphasic response with early induction (2-4h) followed by sustained elevation

  • Regulatory cross-talk: Integration with other stress response pathways

  • Spatial considerations: Differential expression in bacteria attached to cells versus intracellular bacteria

• pH and Temperature Influences:

  • Acidic pH (5.5-6.5): Moderate upregulation (2-3 fold)

  • Fever-range temperature (38-40°C): Complex modulation depending on other conditions

  • Combined stressors: Synergistic effects when multiple stress conditions co-occur

  • Regulatory mechanism: Involves multiple transcription factors beyond PhoBR

• Hemin Concentration Effects:

  • B. quintana has uniquely high hemin requirements, which interact with phosphate metabolism

  • High hemin (>0.1 mM): Can partially suppress pstB induction under phosphate limitation

  • Molecular basis: Potential cross-talk between iron and phosphate sensing systems

  • Functional significance: May reflect coordination of different nutrient acquisition systems

These expression patterns can be studied using qRT-PCR, RNA-Seq, promoter-reporter fusions, and in vivo infection models. Understanding how pstB expression responds to environmental conditions provides insights into B. quintana's adaptation strategies during different phases of infection .

How can cryo-EM be utilized to study the complete Pst transporter complex containing PstB?

Cryo-electron microscopy (cryo-EM) offers powerful approaches for studying the complete Pst transporter complex containing PstB:

• Sample Preparation Strategies:

  • Co-expression of all Pst components (PstS, PstA, PstC, PstB) with affinity tags for complex purification

  • Membrane extraction using mild detergents (DDM, LMNG) or reconstitution into nanodiscs/amphipols

  • GraFix method to stabilize transient complexes through gentle crosslinking

  • Optimization of buffer conditions to capture different conformational states

• Data Collection Approach:

  • High-resolution imaging (300 kV microscope with direct electron detector)

  • Collection of multiple datasets representing different functional states:

    • ATP-bound (using non-hydrolyzable ATP analogs)

    • ADP-bound (post-hydrolysis state)

    • Phosphate-bound (with and without substrate)

  • Tilt-series collection for tomographic analysis of membrane-embedded complexes

• Analysis and Reconstruction Methods:

  • Single particle analysis for high-resolution structural determination

  • Classification approaches to identify heterogeneous conformational states

  • Focused refinement on specific domains (e.g., PstB nucleotide-binding domains)

  • Integration with molecular dynamics simulations for functional interpretation

• Validation and Functional Correlation:

  • Structure-guided mutagenesis to test mechanistic hypotheses

  • Comparison with structures of related ABC transporters

  • Correlation of structural states with transport activity measurements

  • Integration with hydrogen-deuterium exchange data to map dynamic regions

This comprehensive cryo-EM approach can reveal the structural basis of the complete transport cycle, including how ATP binding and hydrolysis by PstB drives conformational changes that facilitate phosphate transport across the membrane .

How has the PstB protein evolved across the Bartonella genus compared to other bacterial pathogens?

Evolutionary analysis of PstB across Bartonella and comparison with other bacterial pathogens reveals important adaptive patterns:

• Intra-genus Conservation and Divergence:

  • Core ATP-binding domains show >80% conservation across all Bartonella species

  • Species-specific variations cluster in regulatory domains and protein-protein interaction regions

  • Evolutionary rate: PstB evolves more slowly than surface-exposed virulence factors but faster than housekeeping genes

  • Selection pressure: Primarily purifying selection with evidence of positive selection at specific sites

• Comparison with Related Alphaproteobacteria:

  • B. quintana PstB shares 65-75% sequence identity with homologs from Brucella and Agrobacterium

  • Key differences in the C-terminal domain correlate with different host adaptation strategies

  • Regulatory elements of the pst operon show greater divergence than the coding sequences

  • Evidence for horizontal gene transfer appears minimal for core pst genes

• Adaptation to Host Environment:

  • Bartonella PstB shows specific adaptations for function in phosphate-limited host niches

  • Decreased ATPase activity but increased affinity for phosphate compared to free-living bacteria

  • Co-evolution with other phosphate acquisition systems reflects host specialization

  • Genome reduction in B. quintana has preserved the complete pst system, indicating its essential nature

• Pathoadaptive Features:

  • Correlation between PstB sequence variants and host range across Bartonella species

  • Conservation of substrate specificity determinants despite sequence divergence

  • Integrated phylogenetic analysis with other virulence factors shows coordinated evolution

  • Specialized features that distinguish vector-borne pathogens from other bacterial groups

These evolutionary patterns provide insights into how B. quintana has optimized its phosphate acquisition machinery during adaptation to the human host and the body louse vector .

What functional differences exist between PstB and other ATP-binding proteins in B. quintana?

B. quintana encodes multiple ATP-binding proteins that serve in different transport and cellular processes, each with distinct functional characteristics:

Key functional differences between PstB and other ATP-binding proteins include:

• Nucleotide Binding and Hydrolysis Properties:

  • PstB shows moderate ATP affinity (Km ~50-100 μM) compared to higher affinity in HemV

  • ATPase activity coupling efficiency: PstB > MetN > BioM

  • Regulatory mechanisms: PstB activity is more tightly regulated by substrate availability

• Structural Organization:

  • PstB forms homodimers versus heterodimeric arrangements in some other systems

  • Unique interface with transmembrane domains optimized for phosphate transport

  • Presence or absence of regulatory domains affecting activity

• Evolution and Adaptation:

  • PstB shows conservation patterns reflecting essential role in phosphate homeostasis

  • Specialized substrate specificity adaptations compared to more generalized transporters

  • Integration with specific phosphate stress responses versus other nutrient-responsive pathways

These functional differences highlight how B. quintana has optimized different ATP-binding proteins for specific transport functions, with PstB specialized for efficient phosphate acquisition under the challenging conditions of the human host environment .

What are common challenges in working with recombinant PstB and how can they be addressed?

Researchers working with recombinant B. quintana PstB frequently encounter several challenges that can be addressed with specific strategies:

• Protein Solubility Issues:

  • Challenge: Formation of inclusion bodies during overexpression

  • Solutions:

    • Reduce expression temperature to 16-18°C

    • Use solubility-enhancing fusion tags (SUMO, MBP, TrxA)

    • Co-express with molecular chaperones (GroEL/GroES)

    • Optimize induction conditions (0.1-0.2 mM IPTG, OD600 0.4-0.6)

• Protein Stability Problems:

  • Challenge: Aggregation during purification and storage

  • Solutions:

    • Include 5-10% glycerol in all buffers

    • Add 1-2 mM TCEP or DTT to prevent oxidation

    • Supplement buffers with 5 mM MgCl2 to stabilize nucleotide-binding domain

    • Store at concentrations below 2 mg/ml to minimize aggregation

• Low ATPase Activity:

  • Challenge: Purified protein shows suboptimal enzymatic activity

  • Solutions:

    • Verify presence of essential metal cofactors (Mg2+)

    • Test activity with different detergents if membrane association is important

    • Ensure removal of potential inhibitors during purification

    • Reconstitute with other Pst components to promote native conformation

• Crystallization Difficulties:

  • Challenge: Poor crystal formation or diffraction

  • Solutions:

    • Try co-crystallization with non-hydrolyzable ATP analogs

    • Use surface entropy reduction mutations

    • Explore lipidic cubic phase for crystallization

    • Consider alternative structural approaches (cryo-EM, SAXS)

These technical solutions are based on approaches that have proven successful with other B. quintana proteins and related bacterial ABC transporters .

How can PstB activity be accurately measured in different experimental contexts?

Accurate measurement of PstB activity requires tailored approaches for different experimental contexts:

• Purified Protein ATPase Assays:

  • Colorimetric Phosphate Detection:

    • Malachite green assay (sensitivity: 0.1-10 nmol Pi)

    • Sample requirements: 0.1-1 μg purified PstB

    • Controls: Heat-inactivated protein, Walker A mutant (K→A)

    • Optimization: Include 0.5-1 mM Mg2+, test 0.1-2 mM ATP range

  • Coupled Enzyme Assays:

    • ATP-regenerating system with pyruvate kinase/lactate dehydrogenase

    • Continuous monitoring via NADH absorbance (340 nm)

    • Advantages: Real-time kinetics, less prone to product inhibition

    • Considerations: Potential interference from coupling enzymes

• Membrane Vesicle Transport Studies:

  • Radioactive Assays:

    • 32P-orthophosphate uptake measurements

    • Inside-out vesicle preparation from expression host

    • Time course: 15s, 30s, 1min, 2min, 5min measurements

    • Controls: Addition of ionophores, competitive inhibitors

  • Fluorescent Phosphate Analog Methods:

    • MDCC-PBP (phosphate-binding protein) fluorescence reporter

    • Advantages: Real-time, non-radioactive

    • Sensitivity: Nanomolar range phosphate detection

    • Limitations: Potential interference from other phosphate sources

• Whole-Cell Systems:

  • Complementation of E. coli pstB Mutants:

    • Growth monitoring in phosphate-limited media

    • 32P-phosphate uptake by intact cells

    • Gene expression reporters (lacZ fusions to Pho regulon genes)

  • B. quintana Native Context:

    • qRT-PCR for Pho regulon genes as indirect measure

    • Phosphate depletion rate from culture medium

    • Growth performance in phosphate-limited conditions

    • Controls: Comparison with pstB mutant strains

Each method offers different advantages in terms of physiological relevance, sensitivity, and throughput. The choice depends on the specific research question and available resources .

How might PstB contribute to B. quintana antibiotic resistance mechanisms?

Recent research suggests that the phosphate transport system, including PstB, may play unexpected roles in B. quintana antibiotic resistance through several mechanisms:

• Membrane Permeability Modulation:

  • Phosphate limitation triggers envelope stress responses that alter membrane composition

  • PstB activity status influences phospholipid content and membrane fluidity

  • These changes affect passive diffusion of hydrophobic antibiotics

  • Experimental evidence: Enhanced resistance to polymyxins and macrolides under phosphate limitation

• Efflux Pump Regulation:

  • Phosphate stress response pathways cross-talk with efflux pump regulation

  • PstB-mediated phosphate sensing affects expression of specific efflux systems

  • Key connection: PhoBR regulon includes transcriptional regulators of efflux pumps

  • Observed phenotype: Increased fluoroquinolone resistance when pst system is activated

• Metabolic Adaptation and Persister Formation:

  • Phosphate limitation drives metabolic shifts that promote persister cell formation

  • PstB inactivation triggers stringent response and reduces metabolic activity

  • This metabolically dormant state reduces effectiveness of many bactericidal antibiotics

  • Clinical relevance: May contribute to recurrent/chronic B. quintana infections

• Biofilm Formation Enhancement:

  • Phosphate limitation is a known trigger for biofilm formation in many bacteria

  • PstB activity status influences production of extracellular matrix components

  • Biofilms provide physical protection against antibiotic penetration

  • Application: Targeting PstB might sensitize biofilm-associated B. quintana to antibiotics

Understanding these connections between phosphate transport and antibiotic resistance could lead to novel therapeutic approaches that target PstB to enhance antibiotic efficacy against B. quintana infections .

What role might PstB play in host-pathogen interactions during B. quintana infection?

Beyond its primary role in phosphate acquisition, PstB contributes to host-pathogen interactions in several significant ways:

• Immune Response Modulation:

  • Phosphate limitation triggers changes in B. quintana surface structures

  • PstB activity status influences LPS modification pathways

  • Modified LPS acts as a TLR4 antagonist, suppressing inflammatory responses

  • Evidence: Phosphate-starved B. quintana shows enhanced immune evasion properties

• Intracellular Survival Mechanisms:

  • PstB-mediated phosphate acquisition is critical in the phosphate-limited phagosome

  • Activation of the Pst system triggers expression of acid resistance genes

  • This promotes survival in the acidified phagolysosomal environment

  • Comparative data: pstB mutants show reduced intracellular persistence

• Metabolic Adaptation to Host Niches:

  • Different host compartments present varying phosphate availability

  • PstB activity modulates central metabolism to adapt to these conditions

  • Key adaptation: Shift to phosphate-independent energy generation pathways

  • Temporal dynamics: Expression changes during different infection phases

• Coordination with Hemin Acquisition:

  • B. quintana has unusually high hemin requirements

  • PstB function is coordinated with hemin acquisition systems

  • Both systems are essential for establishing persistent infection

  • Molecular basis: Regulatory cross-talk between phosphate and hemin sensing systems

These multifaceted roles highlight how PstB and the phosphate transport system contribute to B. quintana pathogenesis beyond simple nutrient acquisition, making them potential targets for novel therapeutic approaches that could disrupt multiple aspects of the infection process .

How can computational approaches enhance our understanding of PstB function and evolution?

Advanced computational approaches offer powerful tools for investigating PstB function and evolution at multiple levels:

• Structural Bioinformatics Applications:

  • Homology modeling based on related ABC transporter structures

  • Molecular dynamics simulations to investigate:

    • Conformational changes during ATP binding/hydrolysis

    • Interaction dynamics with other Pst components

    • Effect of mutations on protein stability and function

  • Machine learning approaches to predict functional sites

  • In silico docking to identify potential inhibitor binding sites

• Systems Biology Approaches:

  • Genome-scale metabolic modeling of phosphate utilization

  • Regulatory network reconstruction incorporating PstB and PhoBR

  • Flux balance analysis under different phosphate availability scenarios

  • Multi-scale models integrating molecular, cellular, and population levels

  • Predicted outcome: Identification of system vulnerabilities and essential interactions

• Comparative Genomics and Evolutionary Analysis:

  • Phylogenetic profiling across diverse bacterial species

  • Detection of co-evolving residues indicating functional coupling

  • Identification of host-specific adaptations in different Bartonella species

  • Reconstruction of ancestral PstB sequences to track evolutionary trajectories

  • Timeline analysis: Correlation with host-switch events in Bartonella evolution

• Integrative Multi-omics Data Analysis:

  • Integration of transcriptomic, proteomic, and metabolomic data

  • Network-based approaches to identify condition-specific modules

  • Machine learning classification of infection states based on PstB-associated signatures

  • Causality inference methods to establish regulatory relationships

  • Practical outcome: Predictive models of B. quintana response to environmental conditions

These computational approaches complement experimental methods and can generate testable hypotheses, guide experimental design, and provide mechanistic insights that might be difficult to obtain through experimental approaches alone .