Recombinant Methanococcus maripaludis Phosphate import ATP-binding protein PstB (pstB)

What is the functional role of PstB in M. maripaludis and how is it structured within the phosphate transport system?

PstB functions as the ATP-binding component of the Pst (phosphate-specific transport) system in M. maripaludis. It is responsible for ATP hydrolysis that energizes the transmembrane channel formed by PstA and PstC, enabling phosphate transport across the cell membrane. The Pst system consists of four primary components:

• PstS: Periplasmic phosphate-binding protein

• PstA and PstC: Transmembrane channel proteins

• PstB: Membrane-bound ATP hydrolysis site

The system is regulated by PhoU, a regulatory protein that interacts with PstB and PhoB to control phosphate import . PstB is particularly crucial for phosphate uptake under phosphate-limiting conditions, when the entire pst operon is upregulated .

Unlike some other organisms that have two Pst systems, M. maripaludis contains multiple Pst phosphate uptake systems distributed across three distinct operons, demonstrating functional redundancy that likely enhances the organism's ability to acquire phosphate under varying environmental conditions .

How can researchers express recombinant PstB in M. maripaludis for functional studies?

To express recombinant PstB in M. maripaludis, researchers can utilize several methodological approaches:

• Phosphate-Regulated Expression System:

  • Clone the pstB gene under the control of the native pst promoter (Ppst)

  • This promoter responds to phosphate limitation with 4-6 fold increased expression when medium phosphate drops to growth-limiting concentrations

  • Utilize this system to decouple growth from heterologous gene expression without adding inducers

• Shuttle Vector Construction:

  • Use pMEV5mT-based vectors (with terminator) to separate transcription of gene inserts from antibiotic resistance markers

  • Replace the constitutive PhmvA promoter with Ppst by PCR amplification using appropriate primers containing restriction sites (NdeI, HindIII)

  • Maintain the recombinant plasmids in M. maripaludis with 2.5 μg/mL puromycin

• Transformation Protocol:

  • Pre-methylate plasmid DNA with PstI methylase to overcome M. maripaludis' native PstI restriction system, which increases transformation efficiency by at least 4-fold

  • Use optimized polyethylene glycol (PEG) transformation method, which can achieve transformation frequencies of approximately 1.8×10^5 transformants/μg DNA

The phosphate-regulated expression system is particularly advantageous for potentially toxic proteins, as expression is activated between mid-log and early stationary phase, minimizing growth inhibition .

What are the key components of the pst promoter in M. maripaludis and how do they regulate gene expression?

The pst promoter (Ppst) in M. maripaludis contains several key regulatory elements that control phosphate-dependent gene expression:

Bioinformatic analysis revealed that the 243 bp intergenic sequence between MMP1094 and MMP1095 contains the Ppst promoter. This region includes conserved predicted cis-2 BRE and TATA box elements, along with an AT-rich region upstream of the BRE. The sequence analysis also identified both direct and inverted repeats within and immediately downstream of the AT-rich region, as well as a potential second BRE and TATA box that may contribute to expression regulation .

When designing expression constructs using this promoter, researchers should note that changes to the factor B recognition element and start codon had no significant impact on expression levels, while modifications to the transcription start site and 5' UTR resulted in differential protein production while maintaining phosphate-dependent regulation .

How does phosphate concentration affect pstB expression in M. maripaludis, and what methods can quantify this relationship?

Phosphate concentration has a significant inverse relationship with pstB expression in M. maripaludis. Under phosphate-limiting conditions (typically 40-80 μM Pi), expression from the pst promoter increases 4-6 fold compared to high phosphate conditions (800 μM Pi) . This regulatory response allows M. maripaludis to enhance phosphate uptake capacity when this essential nutrient becomes scarce.

Methods to quantify this relationship include:

• Western Blot Analysis:

  • Grow recombinant M. maripaludis strains under varying phosphate concentrations (e.g., 40, 80, and 800 μM)

  • Prepare cell-free extracts and perform SDS-PAGE

  • Transfer proteins to membranes and probe with antibodies against tags (e.g., FLAG) attached to recombinant PstB

  • Quantify band intensity to determine relative expression levels

  Research findings using this method have shown that FLAG-tagged proteins under pst promoter control are expressed 2.6-3.3 fold higher in low phosphate (40-80 μM) compared to high phosphate (800 μM) conditions .

• Quantitative Real-Time PCR (qPCR):

  • Isolate RNA from cultures grown under different phosphate concentrations

  • Synthesize cDNA and perform qPCR with pstB-specific primers

  • Normalize to reference genes for accurate comparison

  Similar experiments with PstB1 in other organisms showed mRNA levels increased significantly over 64 hours in cells cultured without added phosphate and decreased in cells exposed to high phosphate concentrations (12.8 mM) compared to normal conditions (0.8 mM) .

• Reporter Gene Assays:

  • Fuse the pst promoter to reporter genes such as β-glucuronidase

  • Measure activity using substrates like 4-Nitrophenyl β-D-glucuronide (4-NPG)

  • Compare activity levels across phosphate concentrations

These methodologies provide complementary approaches to characterize the phosphate-dependent regulation of pstB expression in M. maripaludis.

What approaches can be used to create pstB knockouts in M. maripaludis, and how do cells compensate for the loss?

Several advanced genetic approaches can be used to create pstB knockouts in M. maripaludis:

• CRISPR/Cas12a-Based Genome Editing:

  • Design CRISPR RNA (crRNA) targeting the pstB gene

  • Include homology arms (500-1000 bp) flanking the target site

  • Transform M. maripaludis with the CRISPR/Cas12a plasmid

  • Verify knockout by PCR and restriction digestion

  This system has demonstrated deletion efficiencies of up to 95% despite M. maripaludis' hyperpolyploidy . Transformation efficiency is approximately 5 times lower with 500 bp homology arms compared to 25 bp, but no significant difference exists between 500 and 1000 bp arms .

• Homologous Recombination:

  • Design constructs containing selectable markers flanked by regions homologous to the pstB gene

  • Transform using optimized PEG-based methods

  • Select transformants using appropriate antibiotics

Regarding cellular compensation for pstB loss, studies of similar systems in other organisms provide insight. When PstB1 was knocked out in Nostoc punctiforme, significant compensatory upregulation of pstB2, pstB3, and pstB4 mRNA levels was observed, particularly under phosphate starvation conditions . This redundancy explains why the knockout did not significantly affect cellular phosphate accumulation or growth in most conditions.

In M. maripaludis, similar redundancy likely exists across the multiple Pst phosphate uptake systems distributed among three distinct operons . Researchers should therefore investigate the expression changes in all pstB homologs following knockout of a single gene to fully understand the compensatory mechanisms.

How can researchers optimize cultivation conditions for M. maripaludis to study phosphate-dependent gene expression?

Optimizing cultivation conditions for M. maripaludis requires careful control of multiple parameters, particularly when studying phosphate-dependent gene expression:

• Medium Preparation:

  • Use minimal formate or H₂/CO₂ medium

  • For phosphate-limiting conditions, reduce K₂HPO₄ from 800 μM (standard) to 40-80 μM

  • Acid-wash culture vessels (1% HCl overnight) to remove residual phosphate

  • Seal vessels with rubber stoppers and aluminum crimp seals

• Growth Conditions:

  • Maintain anaerobic environment (80% N₂/20% CO₂ atmosphere)

  • Incubate at 37°C

  • For phosphate starvation studies, pregrow cultures in low-Pi medium before experimental inoculation

  • Use 4% inoculum for experimental cultures

• Scale-up Options:

  • Small-scale: 28 mL culture tubes or 160 mL culture bottles with 28 mL tube side-arms

  • Medium-scale: 1.5 L reactors with optimized agitation and harvesting methods

  • Large-scale: 22 L stainless steel bioreactors (15 L working volume) have achieved a 300-fold scale-up from serum bottles

• Growth Monitoring:

  • Track optical density at 600 nm (OD₆₀₀) using spectrophotometry

  • Under optimized conditions, specific growth rates of 0.16 h⁻¹ and maximum OD₅₇₈ values of 3.4 have been achieved

• Antibiotic Selection:

  • For maintaining plasmids, add 2.5 μg/mL puromycin to the medium

  • For plasmid curing, grow without antibiotics then select on solid medium containing 0.25 mg/mL 6-azauracil

When comparing growth and gene expression under different phosphate concentrations, ensure all other conditions remain consistent to isolate phosphate effects. Using optimized protocols has reduced the time to reach peak optical density by half compared to traditional methods .

How does the PstB-mediated phosphate transport system interact with methanogenesis pathways in M. maripaludis?

The interaction between PstB-mediated phosphate transport and methanogenesis in M. maripaludis represents a critical regulatory nexus:

• Energetic Requirements:

  • PstB functions as the ATP hydrolysis site in the Pst system, requiring energy that must be balanced with methanogenesis energy demands

  • Methanogenesis produces ATP through chemiosmotic coupling, providing the energy needed for phosphate transport

• Expression Coordination:

  • The phosphate-regulated pst promoter has been successfully used to express key methanogenic enzymes including:

    • Methyl-coenzyme M reductase (MCR) - achieved ~6% of total protein in cell-free extract, 140% increase over constitutive promoter expression

    • MmpX (methanogen marker protein 10) - an S-adenosyl methionine-dependent arginine methylase responsible for MCR post-translational modifications

• Regulatory Overlap:

  • Phosphate limitation triggers not only upregulation of the Pst system but also metabolic adaptations in central carbon metabolism pathways connected to methanogenesis

  • Unlike other regulated systems such as nitrogen-dependent (nif) and temperature-dependent (fla) promoters, phosphate regulation via the pst promoter doesn't cause large changes in global gene expression

• Physiological Connections:

  • Phosphate is essential for energy-carrying molecules (ATP) and key coenzymes used in methanogenesis

  • By decoupling growth from protein expression, the phosphate-regulated system provides a useful tool for expressing potentially toxic components of the methanogenesis pathway

The controlled expression of methanogenic enzymes via the phosphate-regulated pst promoter enables researchers to study these interactions without the confounding effects seen with other regulatory systems, making it particularly valuable for understanding the metabolic integration of phosphate transport and methanogenesis.

What advantages does the pst promoter offer over other expression systems for recombinant protein production in M. maripaludis?

The pst promoter offers several significant advantages over alternative expression systems for recombinant protein production in M. maripaludis:

The pst promoter is particularly valuable because it automatically decouples growth from heterologous gene expression without requiring external inducers. As cultures grow and deplete available phosphate, the promoter activates, initiating protein expression as growth naturally slows due to phosphate limitation .

This system has demonstrated superior performance for expressing potentially toxic proteins like the arginine methyltransferase MmpX, which showed only low expression levels under the constitutive hmvA promoter but high expression using the phosphate-regulated pst promoter .

Moreover, the pst promoter is the only regulatory system in M. maripaludis where the operon was the only one with significantly changed transcription during limitation, minimizing unwanted pleiotropic effects on cell physiology that could confound experimental results .

What methodological approaches can be used to study the kinetics of phosphate uptake mediated by recombinant PstB in M. maripaludis?

Studying phosphate uptake kinetics mediated by recombinant PstB requires specialized techniques adapted for anaerobic archaeal systems:

• Radioactive Phosphate Uptake Assays:

  • Grow cultures to mid-log phase under desired phosphate conditions

  • Harvest and wash cells to remove residual phosphate

  • Resuspend in buffer containing ³²P-labeled orthophosphate

  • Sample at defined intervals and filter cells to separate from medium

  • Quantify cell-associated radioactivity to determine uptake rates

  • Compare wild-type, overexpression, and knockout strains

• Continuous Culture Methods:

  • Establish phosphate-limited chemostat cultures

  • Maintain steady-state growth at defined dilution rates

  • Measure residual phosphate concentrations

  • Calculate uptake rates based on mass balance principles

  • This approach allows precise control of phosphate limitation intensity

• Phosphate Depletion Measurements:

  • Monitor phosphate disappearance from the medium over time

  • Use colorimetric assays (e.g., molybdate blue method) to quantify phosphate

  • Calculate uptake rates from the depletion curves

  • This method has been used to demonstrate that overexpression of pstB1 increases phosphate uptake rates

• Functional Complementation Studies:

  • Express M. maripaludis pstB in heterologous hosts (e.g., E. coli PstB knockout mutants)

  • Measure restoration of phosphate uptake capacity

  • This approach has confirmed PstB1 functionality in phosphate transport

• Intracellular Phosphate Quantification:

  • Digest cells to release total phosphate

  • Quantify using standard phosphate determination methods

  • Compare accumulation between wild-type and genetically modified strains

  • This can reveal whether altered uptake affects steady-state phosphate levels

These methodological approaches can be combined to develop a comprehensive understanding of how PstB variants affect phosphate transport kinetics in M. maripaludis under various environmental conditions.

How do researchers identify and characterize different PstB homologs in M. maripaludis?

Identifying and characterizing PstB homologs in M. maripaludis requires a combination of bioinformatic and experimental approaches:

• Genome Sequence Analysis:

  • Search the M. maripaludis genome for PstB homologs using BLAST or similar tools

  • Identify conserved motifs characteristic of ABC transporter ATP-binding domains

  • Map genomic context to identify association with other Pst system components

  • This approach has revealed multiple Pst phosphate uptake systems distributed across three distinct operons in M. maripaludis

• Phylogenetic Analysis:

  • Align amino acid sequences of putative PstB homologs

  • Construct phylogenetic trees to establish evolutionary relationships

  • Compare with characterized PstB proteins from related organisms

  • Identify conserved and divergent regions that may relate to functional differences

• Structural Prediction and Analysis:

  • Use homology modeling to predict three-dimensional structures

  • Identify ATP-binding sites and interfaces with other Pst components

  • Compare structural features among homologs to infer functional distinctions

• Expression Pattern Analysis:

  • Perform quantitative PCR to measure expression levels of different pstB homologs

  • Compare expression patterns under varying phosphate concentrations

  • Studies in related systems have shown differential regulation of pstB homologs, with some responding more strongly to phosphate limitation

• Genetic Complementation Tests:

  • Create knockout strains for individual pstB homologs

  • Test whether expression of other homologs can restore function

  • Evaluate growth and phosphate uptake phenotypes

  • This approach has demonstrated functional redundancy among PstB homologs

• Heterologous Expression and Biochemical Characterization:

  • Express individual PstB homologs with affinity tags

  • Purify proteins and characterize ATP hydrolysis activity

  • Compare kinetic parameters (Km, Vmax) among homologs

  • Assess interaction with other Pst system components

These comprehensive approaches allow researchers to distinguish between homologs with potentially overlapping but distinct functions in phosphate transport under different environmental conditions.

What is the relationship between PstB function and the phosphate regulon in M. maripaludis?

The relationship between PstB function and the phosphate regulon in M. maripaludis involves complex regulatory interactions:

• Regulatory Framework:

  • The Pst system in bacteria and archaea typically functions both as a phosphate transporter and as a sensory complex that regulates the phosphate (Pho) regulon

  • In this dual role, PstB not only provides energy for phosphate transport but also participates in signaling phosphate availability to regulatory systems

• Signal Transduction:

  • PstB interacts with PhoU, a regulatory protein that serves as an intermediary between the Pst system and two-component regulatory systems

  • Under phosphate-sufficient conditions, the Pst-PhoU complex inhibits PhoR activity, preventing activation of the Pho regulon

  • When phosphate is limiting, this inhibition is relieved, allowing PhoR to phosphorylate PhoB (or archaeal equivalent), activating phosphate-responsive genes

• Transcriptional Response:

  • In M. maripaludis, the pst operon itself is the primary transcriptional target responding to phosphate limitation

  • Unlike in other organisms where numerous genes are regulated by phosphate, M. maripaludis shows a more focused response

  • During phosphate-limited growth, the pst operon was the only one whose transcription significantly changed

• Regulatory Efficiency:

  • This focused regulation makes the pst promoter especially valuable for recombinant protein expression

  • The limited pleiotropic effects mean that changes in gene expression are specifically related to phosphate transport rather than broad metabolic adjustments

• Experimental Implications:

  • When manipulating PstB expression for recombinant protein production, researchers should consider potential feedback effects on the entire phosphate regulatory network

  • PstB overexpression might potentially alter normal phosphate sensing, affecting the regulation of the native pst operon

This understanding of PstB's dual transport and regulatory roles is critical for designing effective experimental systems that utilize the pst promoter for controlled gene expression without disrupting cellular phosphate homeostasis.

What recent advances in genetic tools have improved the study of PstB and phosphate transport in M. maripaludis?

Recent advances in genetic tools have significantly enhanced the ability to study PstB and phosphate transport in M. maripaludis:

• CRISPR/Cas12a-Based Genome Editing:

  • A groundbreaking toolbox developed for M. maripaludis with deletion efficiency up to 95%

  • Overcomes challenges posed by the organism's hyperpolyploidy

  • Enables precise targeting of pstB genes with minimal off-target effects

  • Allows introduction of marker-free mutations for clean genetic analysis

• Improved Shuttle Vectors:

  • Development of smaller, more efficient shuttle vectors like pAW42

  • Provides up to 7000-fold increase in transformation efficiency compared to earlier pURB500-based vectors

  • Facilitates easier genetic manipulation of M. maripaludis S2 strain

  • Incorporation of terminators to separate transcription of gene inserts from antibiotic resistance markers, minimizing pleiotropic effects

• Expression Vectors with Tunable Promoters:

  • Characterization of the phosphate-responsive pst promoter enabling regulated gene expression

  • Development of rational modifications to the 5' UTR resulting in differential protein production while maintaining regulation

  • Creation of expression vectors combining the pst promoter with affinity tags (3XFLAG, Twin Strep) for protein purification and detection

• Optimized Transformation Protocols:

  • Enhanced PEG-based transformation methods achieving frequencies of 1.8×10^5 transformants/μg DNA

  • Pre-methylation strategies to overcome the native PstI restriction system

  • These improvements have made genetic manipulation of M. maripaludis more accessible and efficient

• Scale-up Cultivation Technologies:

  • Development of protocols for growing M. maripaludis in 22L bioreactors (300-fold scale-up)

  • Methods for maintaining controlled phosphate limitation in larger culture volumes

  • These advances enable production of sufficient biomass for biochemical and structural studies of phosphate transport components

These technological advances collectively provide researchers with unprecedented capabilities to manipulate pstB genes, study their expression patterns, and characterize the resulting phenotypes in M. maripaludis, accelerating our understanding of archaeal phosphate transport mechanisms.

How do researchers measure the impact of PstB variants on phosphate-dependent gene expression and cellular physiology?

Measuring the impact of PstB variants on phosphate-dependent gene expression and cellular physiology requires integrated experimental approaches:

• Transcriptomic Analysis:

  • RNA-Seq to compare global transcriptional responses between wild-type and PstB variant strains

  • qRT-PCR targeting specific phosphate-responsive genes

  • Analysis of both immediate-early responses and adaptive changes after prolonged phosphate limitation

  • These approaches can reveal whether PstB variants alter normal phosphate sensing and signaling

• Reporter Gene Systems:

  • Fusion of phosphate-responsive promoters (including the pst promoter itself) to reporter genes

  • Measurement of reporter activity across varying phosphate concentrations

  • Comparison between wild-type and PstB variant backgrounds

  • This approach directly quantifies changes in transcriptional regulation

• Proteomic Analysis:

  • Quantitative proteomics to identify proteins differentially expressed in PstB variants

  • Western blotting with antibodies against known phosphate-responsive proteins

  • Particular focus on other components of the Pst system and related phosphate transport mechanisms

  • These methods reveal post-transcriptional effects not captured by RNA analysis

• Phosphate Uptake Kinetics:

  • Measurement of initial rates of phosphate uptake across a range of phosphate concentrations

  • Determination of Km and Vmax parameters for different PstB variants

  • Assessment of uptake inhibition by phosphate analogs

  • These studies directly quantify functional differences in transport activity

• Growth Phenotyping:

  • Comparative growth curves under various phosphate concentrations

  • Determination of growth rates, lag phases, and final cell densities

  • Competition experiments between wild-type and variant strains

  • These approaches reveal the physiological consequences of altered phosphate transport

• Cellular Phosphate Content Analysis:

  • Determination of total cellular phosphate levels

  • Fractionation to measure distribution between soluble and macromolecular pools

  • Time-course analysis during phosphate limitation and recovery

  • These measurements connect transport activity to intracellular phosphate homeostasis

• Metabolomic Profiling:

  • Quantification of phosphate-containing metabolites (ATP, GTP, phosphosugars, etc.)

  • Analysis of changes in central carbon metabolism

  • Measurement of methanogenesis intermediates and products

  • These studies reveal how altered phosphate transport affects broader metabolic networks

What challenges exist in purifying and characterizing recombinant PstB from M. maripaludis for structural studies?

Purifying and characterizing recombinant PstB from M. maripaludis for structural studies presents several significant challenges:

• Expression Optimization:

  • Balancing expression levels to avoid toxicity while obtaining sufficient yields

  • The phosphate-regulated pst promoter offers advantages here, with induction levels of 4-6 fold under phosphate limitation

  • Addition of affinity tags can potentially interfere with folding or activity

  • N-terminal vs. C-terminal tag placement must be empirically tested

• Membrane Association:

  • PstB is membrane-associated and interacts with transmembrane components PstA and PstC

  • Extraction requires careful optimization of detergent types and concentrations

  • Maintaining native conformation during solubilization is challenging

  • Consideration of whether to co-purify with interacting partners

• Anaerobic Purification Requirements:

  • As a protein from a strict anaerobe, PstB may be oxygen-sensitive

  • Purification must be performed under strictly anaerobic conditions

  • This necessitates specialized equipment and techniques

  • All buffers must be degassed and include reducing agents

• Functional State Preservation:

  • ATP binding and hydrolysis activity must be maintained during purification

  • Conformational dynamics are essential for function and structural studies

  • Appropriate nucleotide binding states must be stabilized for analysis

  • Activity assays must be optimized for archaeal proteins

• Scale-up Challenges:

  • Obtaining sufficient biomass from an anaerobic archaeon is difficult

  • Scale-up to bioreactors (up to 22L) has been achieved but requires specialized equipment

  • Maintaining consistent expression conditions at larger scales

  • Ensuring homogeneity of phosphate limitation for promoter induction

• Crystallization and Structure Determination:

  • Membrane-associated proteins are notoriously difficult to crystallize

  • Lipid composition differs in archaea, potentially affecting protein-detergent interactions

  • Sample homogeneity is critical but challenging to achieve

  • Alternative approaches like cryo-electron microscopy may be preferable

• Post-translational Modifications:

  • Potential archaeal-specific modifications must be preserved and characterized

  • Expression in heterologous hosts may not recapitulate native modifications

  • Mass spectrometry methods must be optimized for archaeal proteins

To address these challenges, researchers have developed specialized expression systems using the native pst promoter combined with affinity tags (3XFLAG, Twin Strep) for protein purification and detection . These systems have proven successful for other challenging archaeal proteins like methyl-coenzyme M reductase and arginine methyltransferase MmpX, suggesting their potential utility for PstB structural studies .

How can systems biology approaches integrate PstB function into genome-scale metabolic models of M. maripaludis?

Systems biology approaches offer powerful frameworks for integrating PstB function into genome-scale metabolic models of M. maripaludis:

• Expanding Existing Models:

  • The current genome-scale metabolic model (iMM518) can be enhanced to better represent phosphate transport and metabolism

  • This requires integration of:

    • Detailed PstB-mediated transport kinetics

    • Phosphate-dependent regulatory interactions

    • Energy coupling between phosphate transport and methanogenesis

• Flux Balance Analysis (FBA):

  • Implement phosphate uptake constraints based on experimental measurements

  • Predict growth phenotypes under varying phosphate availability

  • Simulate the metabolic consequences of PstB variants or knockouts

  • Identify potential compensatory pathways activated in response to phosphate limitation

• Integration of Multi-omics Data:

  • Incorporate transcriptomic data showing phosphate-dependent expression changes

  • Use proteomic data to constrain enzyme abundance parameters

  • Include metabolomic measurements of phosphate-containing compounds

  • This multi-layered approach creates more realistic models of cellular responses

• Regulatory Network Modeling:

  • Develop mathematical descriptions of the phosphate regulon

  • Include feedback between phosphate transport, sensing, and gene expression

  • Model the dynamics of adaptation to changing phosphate availability

  • Integrate with the broader regulatory network controlling methanogenesis

• Community-Level Modeling:

  • Extend models to include M. maripaludis interactions with other organisms

  • Simulate phosphate competition in mixed microbial communities

  • Model the ecological implications of phosphate transport efficiency

  • Predict community-level responses to phosphate limitation

• Bioprocess Optimization Applications:

  • Use models to optimize cultivation conditions for heterologous protein production

  • Predict optimal harvesting times based on phosphate depletion dynamics

  • Design media formulations that balance growth and protein expression

  • Develop scale-up strategies for bioreactor cultivation

• Experimental Validation Cycle:

  • Generate model-driven hypotheses about phosphate transport and metabolism

  • Design targeted experiments to test these predictions

  • Refine models based on experimental outcomes

  • Iterate this cycle to progressively improve model accuracy