Recombinant Photobacterium profundum 3-phosphoshikimate 1-carboxyvinyltransferase (aroA)

What is Photobacterium profundum and why is it significant for studying pressure adaptation?

Photobacterium profundum is a deep-sea bacterium isolated from the Sulu Sea at a depth of 2.5 km in 1984. This γ-proteobacterium is a close relative of Vibrio cholerae and serves as an established model for studying high-pressure adaptation . P. profundum SS9 is piezophilic, meaning it grows better at elevated hydrostatic pressure than at atmospheric pressure, with the ability to grow at pressures ranging from 0.1 MPa to 90 MPa (optimal pressure: 28 MPa) . It is also psychrotolerant, growing at temperatures from <2°C to >20°C (optimal temperature: 15°C) . These characteristics make it an excellent model organism for investigating molecular mechanisms of adaptation to extreme deep-sea environments.

What is 3-phosphoshikimate 1-carboxyvinyltransferase (aroA) and what function does it serve?

The 3-phosphoshikimate 1-carboxyvinyltransferase enzyme, also known as aroA or EPSPS (5-enolpyruvylshikimate-3-phosphate synthase), catalyzes a critical step in the shikimate pathway. Specifically, it transfers the enolpyruvyl moiety of phosphoenolpyruvate (PEP) to the 5-hydroxyl of shikimate-3-phosphate (S3P) to produce enolpyruvyl shikimate-3-phosphate and inorganic phosphate . This enzymatic reaction is essential for the biosynthesis of aromatic amino acids in bacteria. In P. profundum, aroA appears to be differentially regulated under varying pressure conditions, suggesting its involvement in pressure adaptation mechanisms .

How does hydrostatic pressure affect aroA gene expression in deep-sea bacteria?

Transcriptomic analyses have revealed that aroA expression is significantly affected by hydrostatic pressure in deep-sea bacteria. In Desulfovibrio hydrothermalis, a related piezophilic bacterium, the aroA gene shows decreased expression (log2 fold change of -2.888) when grown at in situ pressure (26 MPa) compared to atmospheric pressure (0.1 MPa) . This downregulation appears to be part of a broader response affecting aromatic amino acid metabolism genes under high-pressure conditions. Similar pressure-responsive regulation has been observed in P. profundum, where transcriptome-level studies have demonstrated that regulation at the gene expression level plays a crucial role in hydrostatic pressure adaptation .

What expression systems are most suitable for producing recombinant P. profundum aroA?

While specific expression systems for P. profundum aroA are not detailed in the search results, general principles for recombinant protein expression can be applied. For heterologous expression, Escherichia coli is often the system of choice due to its well-established protocols and high yield potential . When expressing recombinant proteins from a piezophilic organism like P. profundum, researchers should consider:

• Codon optimization for the host organism

• Addition of purification tags (e.g., His-tag)

• Expression temperature optimization (potentially lower temperatures)

• Induction conditions that minimize the formation of inclusion bodies

• Use of specialized strains designed for expression of proteins from AT-rich genomes

For functional studies that require pressure conditions, expression in a native or related piezophilic host might be necessary to ensure proper folding and activity.

What are the optimal growth conditions for cultivating P. profundum for experimental studies?

P. profundum requires specific growth conditions that reflect its deep-sea origin. Based on research protocols described in the literature, the following conditions are recommended:

For pressure experiments, specialized equipment such as stainless steel pressure vessels is required, with careful attention to removing air bubbles before pressurization . When monitoring pressure sensitivity, researchers typically calculate a pressure sensitivity ratio by comparing growth at different pressures (e.g., 45 MPa vs. 0.1 MPa) .

How can I design effective protocols for purifying recombinant P. profundum aroA?

Although specific purification protocols for P. profundum aroA are not provided in the search results, a general purification strategy can be outlined based on common approaches for similar enzymes:

• Cell lysis: Use mechanical disruption (e.g., sonication, French press) in a buffer optimized for protein stability (typically containing reducing agents and protease inhibitors)

• Initial clarification: Remove cell debris by centrifugation (20,000 × g, 30 min)

• Affinity chromatography: If using a His-tagged construct, purify using Ni-NTA resin with imidazole gradient elution

• Ion exchange chromatography: Further purify using anion or cation exchange depending on the protein's isoelectric point

• Size exclusion chromatography: Remove aggregates and achieve final purification

• Quality control: Assess purity by SDS-PAGE (aim for >90% purity) and verify identity by mass spectrometry or western blotting

When working with proteins from piezophilic organisms, consider performing purification steps at lower temperatures (4-10°C) and testing the effect of different buffer compositions on protein stability.

What assays can be used to determine the activity of recombinant P. profundum aroA?

Several approaches can be used to assess the enzymatic activity of recombinant aroA:

• Spectrophotometric assays: Monitor the consumption of PEP at 340 nm using a coupled enzyme system with pyruvate kinase and lactate dehydrogenase

• HPLC-based methods: Directly measure the formation of enolpyruvyl shikimate-3-phosphate or the disappearance of substrates

• Colorimetric phosphate release assays: Quantify the inorganic phosphate released during the reaction

• Radiometric assays: Use radiolabeled substrates to track product formation with high sensitivity

To evaluate pressure effects on enzyme activity, these assays must be adapted for high-pressure conditions using specialized equipment. Researchers should establish baseline kinetic parameters (Km, Vmax, kcat) at atmospheric pressure before investigating pressure effects. When comparing enzymatic activities, it's essential to consider both thermodynamic and kinetic aspects of the reaction under different pressure conditions.

How does pressure affect transcriptional regulation of aroA in P. profundum?

Transcriptomic analyses of P. profundum have revealed complex patterns of gene expression in response to pressure changes. While specific details about aroA regulation are not fully described in the search results, studies on related systems indicate that pressure-responsive genes often show coordinated expression patterns . In Desulfovibrio hydrothermalis, aroA and other genes involved in aromatic amino acid metabolism show similar patterns of downregulation under high pressure, suggesting they may be co-regulated .

Potential mechanisms for pressure-responsive transcriptional regulation include:

• Dedicated pressure-sensing transcription factors

• Two-component signal transduction systems responding to pressure-induced cellular changes

• Global regulators that coordinate multiple stress responses

• Small RNAs that modulate gene expression post-transcriptionally

RNA-seq analyses of P. profundum have identified numerous previously unknown regulatory features, including 460 putative small RNA genes and an unexpectedly high number of genes (992) with large 5'-UTRs that could harbor cis-regulatory RNA structures . These findings suggest that pressure adaptation involves sophisticated regulatory mechanisms beyond simple transcriptional control.

How can structural biology approaches contribute to understanding pressure adaptation in P. profundum aroA?

Structural biology techniques can provide crucial insights into the molecular basis of pressure adaptation in P. profundum aroA. Potential approaches include:

• X-ray crystallography or cryo-EM: Determine the three-dimensional structure of aroA to identify unique structural features that might contribute to pressure adaptation

• High-pressure X-ray crystallography: Directly observe structural changes under pressure conditions

• NMR spectroscopy: Investigate protein dynamics and conformational changes at different pressures

• Molecular dynamics simulations: Model the behavior of the protein under various pressure conditions to identify key residues involved in pressure adaptation

• Comparative structural analysis: Compare the structure of P. profundum aroA with homologs from non-piezophilic organisms to identify adaptive differences

These structural investigations could reveal specific adaptations such as changes in protein flexibility, altered patterns of internal cavities, modified surface charge distributions, or pressure-responsive conformational states that maintain enzymatic function under deep-sea conditions.

What are the methodological challenges in studying enzyme function under high-pressure conditions?

Investigating enzymatic activity under high pressure presents several significant challenges:

• Technical equipment limitations: Standard laboratory equipment is not designed to operate under high pressure, requiring specialized apparatus that can withstand pressures up to 90 MPa while allowing spectroscopic measurements

• Sample containment: Ensuring proper sealing and containment of samples without introducing artifacts (e.g., air bubbles) that could affect measurements

• Real-time monitoring: Difficulties in real-time monitoring of reactions under pressure, often requiring innovative approaches or indirect measurements

• Pressure effects on assay components: Accounting for pressure effects on substrates, cofactors, and detection systems independent of enzyme activity

• Temperature control: Maintaining consistent temperature during pressure experiments, as compression typically generates heat

• Data interpretation: Distinguishing between pressure effects on protein structure/function versus effects on the reaction equilibrium or kinetics

Researchers studying P. profundum aroA under pressure must develop specialized methodologies that address these challenges while maintaining experimental rigor and reproducibility.

How can site-directed mutagenesis be used to investigate pressure-adaptation mechanisms in P. profundum aroA?

Site-directed mutagenesis represents a powerful approach for investigating the molecular basis of pressure adaptation in P. profundum aroA. A systematic mutagenesis strategy might include:

• Comparative sequence analysis: Identify amino acid residues that differ between piezophilic and non-piezophilic aroA homologs

• Structure-based predictions: Target residues in regions likely to affect pressure sensitivity, such as active site flexibility, subunit interfaces, or volume-change sensitive areas

• Reciprocal mutations: Introduce residues from non-piezophilic homologs into P. profundum aroA and vice versa to test their contribution to pressure adaptation

• Conservative versus non-conservative mutations: Assess the effect of maintaining versus changing physicochemical properties of key residues

• Domain swapping: Exchange entire domains between piezophilic and non-piezophilic enzymes to identify regions critical for pressure adaptation

For each mutant, researchers should compare enzymatic activity, stability, and structural properties across a range of pressure conditions. This approach can identify specific residues or regions that contribute to the maintenance of catalytic function under high pressure, providing insights into the molecular mechanisms of piezophilic adaptation.

How can systems biology approaches provide insights into the role of aroA in pressure adaptation?

Systems biology approaches can provide a comprehensive understanding of aroA's role in pressure adaptation by integrating multiple levels of biological information:

• Multi-omics integration: Combine transcriptomics, proteomics, and metabolomics data to map changes in aroA expression, protein abundance, and related metabolite levels under different pressure conditions

• Regulatory network reconstruction: Identify transcription factors, small RNAs, and other regulatory elements that control aroA expression in response to pressure

• Metabolic flux analysis: Quantify changes in metabolic flux through the shikimate pathway under different pressure conditions

• Protein-protein interaction mapping: Identify physical interactions between AroA and other proteins that might be pressure-dependent

This integrative approach could reveal unexpected connections between aroA and other cellular processes involved in pressure adaptation. For example, RNA-seq studies of P. profundum have already identified complex expression patterns and potential regulatory structures that could coordinate pressure responses .

What are promising future research directions for understanding aroA function in deep-sea adaptation?

Future research on P. profundum aroA could explore several promising directions:

• Comparative studies across piezophilic species: Investigate aroA function in multiple deep-sea organisms to identify common adaptation mechanisms

• Evolution of pressure adaptation: Trace the evolutionary history of aroA in deep-sea bacteria to understand how pressure adaptation emerged

• Synthetic biology applications: Engineer pressure-adapted aroA variants for biotechnological applications requiring high-pressure conditions

• In situ studies: Develop methodologies to study aroA function in simulated deep-sea environments that combine multiple parameters (pressure, temperature, salinity)

• Climate change impacts: Investigate how changing ocean conditions might affect aroA function and pressure adaptation in deep-sea microorganisms

These research directions could not only advance our understanding of deep-sea adaptation but also provide insights into fundamental principles of protein evolution and function under extreme conditions.