Recombinant Photobacterium profundum Fatty acid oxidation complex subunit alpha (fadJ), partial

What is Photobacterium profundum and why is it a valuable model organism?

Photobacterium profundum is a Gram-negative bacterium originally isolated from the Sulu Sea at a depth of 2.5 km. It belongs to the Photobacterium subgroup of the Vibrionaceae family. What makes P. profundum particularly valuable as a model organism is its ability to grow across a wide range of pressures (0.1 MPa to 90 MPa), with optimal growth at 28 MPa and 15°C, classifying it as both a piezophile (thrives under high pressure) and psychrophile (thrives in cold conditions). Unlike many other piezophiles, P. profundum can grow at atmospheric pressure, allowing for easier genetic manipulation and laboratory culture . Its genome consists of two chromosomes and an 80 kb plasmid, making it genetically tractable for research purposes .

What is the role of the fadJ gene in Photobacterium profundum?

The fadJ gene in P. profundum encodes the alpha subunit of the fatty acid oxidation complex, which plays a crucial role in fatty acid metabolism. In deep-sea bacteria, fatty acid metabolism is particularly important for membrane fluidity modulation in response to both high pressure and low temperature. The FadJ protein is involved in catalyzing several steps in the beta-oxidation pathway of fatty acids, converting them to acetyl-CoA for entry into central metabolism. Given P. profundum's ability to shift between respiratory and fermentative metabolism depending on environmental conditions , the fadJ gene product likely plays an important role in energy metabolism adaptation to different pressure environments.

How does the expression of fadJ in P. profundum compare to other bacteria?

While the search results don't specifically address fadJ expression patterns in P. profundum compared to other bacteria, we can infer that as part of metabolic pathways, it may follow similar patterns observed in other differentially expressed proteins. P. profundum shows significant changes in protein expression patterns between atmospheric (0.1 MPa) and high pressure (28 MPa) conditions . For instance, proteins involved in glycolysis/gluconeogenesis are up-regulated at high pressure, while those involved in oxidative phosphorylation are up-regulated at atmospheric pressure . This suggests that fadJ might also show pressure-dependent expression, potentially contributing to the bacterium's ability to adapt its metabolism to different pressure environments.

What are the optimal conditions for expressing recombinant P. profundum fadJ protein?

When working with recombinant P. profundum fadJ, researchers should consider the native conditions of the organism. P. profundum grows optimally at 28 MPa and 15°C , but these conditions are challenging to replicate in standard laboratory settings. For protein expression, it's advisable to use a two-phase approach:

• Initial cloning and construct validation in standard E. coli expression systems at atmospheric pressure

• Expression optimization in pressure-adapted systems for functional studies

For cultivation, marine broth supplemented with 20 mM glucose and 100 mM HEPES buffer (pH 7.5) has been used successfully for P. profundum growth . Anaerobic conditions at 17°C have yielded good results in previous studies . When expressing fadJ specifically, researchers should consider that its native function occurs under high pressure, so protein folding and activity may be affected by expression at atmospheric pressure.

What approaches can be used to study the effect of pressure on recombinant fadJ function?

To study pressure effects on recombinant fadJ function, researchers can employ several approaches:

• Comparative activity assays: Measure enzymatic activity at different pressures using high-pressure bioreactors or pressure vessels similar to those used in previous P. profundum studies .

• Structural analysis: Compare protein structure at different pressures using techniques such as high-pressure NMR or X-ray crystallography.

• Complementation studies: Create fadJ knockout mutants in P. profundum and assess growth under different pressure conditions with wild-type or mutant fadJ variants.

• Label-free proteomic analysis: Similar to the approach used in previous P. profundum studies , compare expression levels and post-translational modifications of native versus recombinant fadJ at different pressures.

• In vitro reconstitution: Reconstitute the fatty acid oxidation complex with recombinant components and test functionality under various pressure conditions.

How should researchers address the challenge of protein stability when working with P. profundum fadJ at different pressures?

When working with P. profundum fadJ at different pressures, protein stability is a critical consideration. Researchers should:

• Utilize pressure-adapted buffer systems: Design buffer compositions that maintain pH stability under pressure changes, similar to the HEPES buffer systems used in P. profundum cultivation .

• Monitor chaperone co-expression: Previous studies have shown that chaperones like GroEL and DnaK are differentially expressed under different pressure conditions in P. profundum . Co-expression of these chaperones may improve recombinant fadJ stability.

• Apply molecular dynamics simulations: Predict pressure effects on protein stability before experimental work.

• Implement gradual pressure adaptation protocols: When shifting between pressure conditions, use step-wise adaptation rather than sudden changes to minimize protein denaturation.

• Include pressure-stabilizing additives: Certain osmolytes and compatible solutes can enhance protein stability under pressure variation.

What purification strategies are most effective for recombinant P. profundum fadJ?

For effective purification of recombinant P. profundum fadJ, researchers should consider:

• Affinity chromatography: Utilize His-tag or other affinity tags that can function under the salt conditions necessary for P. profundum protein stability.

• Cold-adapted purification protocols: Since P. profundum is a psychrophile growing optimally at 15°C , purification should be conducted at lower temperatures (4-15°C) to maintain native conformation.

• Pressure considerations: If possible, perform key purification steps under moderate pressure to maintain native conformation.

• Salt concentration: Maintain appropriate salt concentrations throughout purification, as P. profundum has a requirement for salt .

• Two-step chromatography: Combine affinity chromatography with size exclusion or ion exchange chromatography to achieve higher purity while maintaining native complex formation.

When assessing purity, mass spectrometry approaches similar to those used in previous P. profundum proteomic studies can be effective, with precursor mass tolerance set to approximately 7 ppm and MS/MS tolerance to 0.4 amu .

What analytical techniques are most informative for studying the function of recombinant fadJ in relation to pressure adaptation?

To study recombinant fadJ function in pressure adaptation, the following analytical techniques are particularly informative:

• Label-free quantitative proteomics: This approach has been successfully employed for P. profundum pressure adaptation studies and can reveal how fadJ expression changes in response to pressure.

• Enzyme kinetics under pressure: Using high-pressure stopped-flow devices to measure reaction rates and substrate affinities at various pressures.

• Comparative structural analysis: Techniques such as circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR) can reveal structural changes in the protein at different pressures.

• Membrane association studies: Since fatty acid metabolism is often associated with membrane processes, techniques like fluorescence recovery after photobleaching (FRAP) can reveal how fadJ interacts with membranes under different pressure conditions.

• Metabolomic analysis: Analyzing changes in fatty acid metabolites under different pressure conditions can provide insights into fadJ function in vivo.

How can researchers effectively compare recombinant fadJ function with native expression in P. profundum?

To effectively compare recombinant fadJ function with native expression in P. profundum:

• Parallel proteomics: Apply the same label-free quantitative proteomic approach used in previous P. profundum studies to compare expression levels and post-translational modifications.

• Activity normalization: Develop standardized activity assays that can be performed under identical conditions for both recombinant and native proteins.

• In situ localization: Use fluorescent tagging to compare cellular localization of native versus recombinant fadJ under different pressure conditions.

• Complementation studies: Test whether recombinant fadJ can rescue phenotypes in fadJ-knockout P. profundum strains under various pressure conditions.

• Transcriptomic-proteomic correlation: Compare whether the relationship between mRNA and protein levels follows similar patterns for recombinant and native fadJ, as pressure-specific anticorrelation between transcriptome and proteome has been observed in P. profundum .

How does fadJ contribute to the pressure-dependent metabolic switching observed in P. profundum?

P. profundum shows evidence of metabolic switching between fermentative metabolism at high pressure and respiratory metabolism at atmospheric pressure . The fadJ gene, as part of the fatty acid oxidation complex, may play a critical role in this pressure-dependent metabolic switching through:

• Energy yield modulation: Beta-oxidation provides reduced electron carriers (FADH₂ and NADH) that can feed into either fermentative or respiratory pathways.

• Membrane adaptation: Products of the fatty acid oxidation pathway can be redirected to membrane lipid synthesis, potentially altering membrane fluidity in response to pressure.

• Metabolic flux control: By controlling the availability of acetyl-CoA from fatty acid breakdown, fadJ may influence the balance between different metabolic pathways.

Research approaches to study this include metabolic flux analysis using stable isotope labeling, comparative enzymatic assays under different pressure conditions, and systems biology modeling of pressure-dependent metabolism.

What role does protein-protein interaction play in the function of recombinant fadJ under different pressure conditions?

The function of recombinant fadJ likely depends on protein-protein interactions that may be affected by pressure changes. Research considerations include:

• Complex formation: FadJ functions as part of a multienzyme complex in fatty acid oxidation. Pressure may affect the stability and assembly of this complex.

• Interaction with chaperones: P. profundum shows differential expression of chaperones like GroEL and DnaK under different pressure conditions . These chaperones may interact differently with fadJ based on pressure.

• Membrane protein associations: As a fatty acid metabolism enzyme, fadJ likely associates with membrane proteins, and these associations may be pressure-dependent.

Research methods to investigate these interactions include co-immunoprecipitation under pressure, fluorescence resonance energy transfer (FRET) analysis, and cross-linking mass spectrometry to capture transient interactions under different pressure conditions.

How do post-translational modifications of fadJ differ between high pressure and atmospheric pressure conditions?

Post-translational modifications (PTMs) likely play a significant role in regulating fadJ function under different pressure conditions. Research considerations include:

• Pressure-dependent phosphorylation: Kinase activity may be altered by pressure, leading to different phosphorylation patterns on fadJ.

• Redox-based modifications: The oxidative state differs between high pressure and atmospheric environments, potentially affecting cysteine modifications in fadJ.

• Proteolytic processing: Pressure may affect protein maturation through altered proteolytic processing.

Research approaches should include enrichment strategies for specific PTMs before mass spectrometry analysis, similar to the proteomic methods employed in previous P. profundum studies , but with specific focus on identifying and quantifying modifications on fadJ.

How should researchers address the observed discrepancies between transcriptomic and proteomic data in P. profundum pressure adaptation studies?

Previous studies on P. profundum have highlighted discrepancies between transcriptomic and proteomic data, particularly for stress response proteins . When studying fadJ, researchers should:

• Implement multi-omics approaches: Simultaneously collect transcriptomic, proteomic, and metabolomic data to provide a comprehensive view of fadJ regulation.

• Consider time-course experiments: The anti-correlation between transcriptome and proteome may be due to temporal differences in response. Time-course experiments can capture these dynamics.

• Investigate post-transcriptional regulation: Possible mechanisms like antisense RNA regulation have been suggested to explain transcriptome-proteome discrepancies in P. profundum .

• Account for protein stability differences: Different pressure conditions may affect protein turnover rates, contributing to observed discrepancies.

• Consider technical artifacts: Different pressure conditions may affect sample preparation efficiency for both RNA and protein extraction.

What statistical approaches are most appropriate for analyzing fadJ expression and function data across pressure conditions?

When analyzing fadJ expression and function across pressure conditions, researchers should consider:

• Appropriate transformations: Previous P. profundum studies have used ArcSinH transformation for protein abundances since the detection method can generate near-zero measurements for which log transformation is not ideal .

• ANOVA-based approaches: One-way ANOVA has been successfully applied to transformed protein abundance data in P. profundum studies .

• Multiple testing correction: When analyzing large datasets, use appropriate corrections (e.g., Benjamini-Hochberg) to control false discovery rates.

• Significance thresholds: Consider both statistical significance (p<0.05) and fold change thresholds (absolute ratio ≥1.5) as used in previous studies .

• Multivariate analysis: Given the complex relationship between pressure, temperature, and metabolism, multivariate statistical approaches may reveal patterns not apparent in univariate analyses.

How can researchers effectively distinguish direct pressure effects on fadJ from indirect effects through metabolic or regulatory networks?

Distinguishing direct from indirect pressure effects on fadJ requires sophisticated experimental design:

• In vitro reconstitution: Study purified recombinant fadJ under different pressure conditions to identify direct pressure effects on structure and function.

• Targeted mutagenesis: Introduce specific mutations in pressure-sensitive regions of fadJ to determine which structural elements respond directly to pressure.

• Network analysis: Use systems biology approaches to model how pressure perturbations propagate through metabolic and regulatory networks affecting fadJ.

• Rapid sampling techniques: Develop methods for rapid sampling and protein fixation upon pressure changes to capture immediate effects before regulatory responses occur.

• Comparative genomics: Compare fadJ structure and regulation across related bacteria adapted to different pressure environments to identify conserved pressure-responsive elements.