Recombinant Photobacterium profundum Acetyl-coenzyme A carboxylase carboxyl transferase subunit beta (accD)

What is the role of accD in P. profundum's adaptation to high pressure environments?

The accD gene encodes the beta subunit of acetyl-coenzyme A carboxylase carboxyl transferase, a critical enzyme in fatty acid biosynthesis that catalyzes the carboxylation of acetyl-CoA to malonyl-CoA. In P. profundum, fatty acid biosynthesis is essential for adapting to high pressure environments through membrane modification. P. profundum SS9 grows optimally at 28 MPa and 15°C, conditions that require specific membrane lipid compositions to maintain fluidity and functionality . While accD itself hasn't been directly characterized in P. profundum, its essential role in fatty acid biosynthesis suggests it contributes to the production of membrane lipids that allow this organism to thrive under high pressure conditions . Proteomic analyses show that proteins involved in key metabolic pathways, including those related to lipid metabolism, are differentially expressed under varying pressure conditions, suggesting accD may also be pressure-regulated .

How does accD fit into the dual fatty acid biosynthesis systems of P. profundum?

P. profundum possesses two distinct fatty acid biosynthesis pathways: the classical type II fatty acid synthase (FAS) pathway and a secondary polyketide/fatty acid synthase (Pfa) pathway that produces omega-3 polyunsaturated fatty acids . The accD gene is part of the type II FAS pathway, which produces primarily monounsaturated and saturated fatty acids. Interestingly, studies with P. profundum have shown that monounsaturated fatty acids, but not polyunsaturated fatty acids, are required for growth at high pressure and low temperature . The two pathways appear to have some functional redundancy, as evidenced by genetic experiments showing that mutations in one pathway can be compensated by increased activity of the other . This suggests that accD function might be particularly important when the secondary lipid synthase mechanism is impaired, as the traditional FAS pathway would need to compensate.

What are the optimal conditions for expressing recombinant P. profundum accD in heterologous systems?

Based on successful expression of other P. profundum enzymes, recombinant accD is likely to be most effectively expressed under the following conditions:

Expressing recombinant P. profundum proteins at lower temperatures (16-22°C) has been shown to significantly improve yields of functional protein compared to standard 37°C expression, likely because these conditions better match the psychrophilic nature of P. profundum . When expressing components of the accD complex, co-expression with other ACC subunits may be necessary for proper folding and activity .

How does P. profundum's accD sequence compare to homologs from other bacteria?

While the specific sequence of P. profundum accD is not directly addressed in the search results, comparative analysis would be expected to show:

• Higher sequence similarity to accD from other members of the Vibrionaceae family, particularly Photobacterium species and Vibrio cholerae, based on the close phylogenetic relationship .

• Potential amino acid substitutions that enhance enzyme flexibility and activity at low temperatures and high pressures, similar to adaptations observed in other cold-adapted enzymes from piezophiles .

• Conservation of catalytic residues crucial for carboxyl transferase activity, while showing divergence in regions that might affect protein stability and flexibility under pressure.

Phylogenetic analysis based on 16S rRNA indicates P. profundum is closely related to the genus Vibrio, particularly Vibrio cholerae , suggesting similar genomic organization of fatty acid biosynthesis genes.

How do mutations in accD affect P. profundum's ability to grow under varying pressure conditions?

Mutations in accD would likely affect P. profundum's growth under varying pressure conditions in several ways:

• Complete loss-of-function mutations would probably be lethal or severely impair growth under all pressure conditions, as accD is typically essential for fatty acid biosynthesis in bacteria.

• Partial loss-of-function mutations might be compensated by increased activity of the secondary lipid synthase (Pfa) pathway, similar to what has been observed with mutations in other fatty acid biosynthesis genes like fabB, fabA, and fabD .

• Pressure-dependent growth effects would likely be observed, particularly at high pressure (28 MPa), where specific membrane lipid compositions are required .

Studies of other fatty acid biosynthesis genes in P. profundum have shown that their disruption can lead to pressure-sensitive phenotypes. For example, disruption of fabB leads to a pressure-sensitive phenotype , suggesting accD mutations would show similar pressure-dependent effects. The synthetic lethality observed between fabD and pfaA mutations suggests that accD might also show genetic interactions with components of the Pfa pathway.

What methodologies are effective for studying accD enzyme kinetics under high-pressure conditions?

Studying enzyme kinetics under high pressure requires specialized approaches:

• High-pressure stopped-flow spectroscopy: This technique allows measurement of rapid kinetic reactions under pressure. For accD studies, this would involve monitoring the carboxylation reaction through coupled enzymatic assays that produce measurable spectroscopic changes.

• Pressure vessels with optical windows: These allow real-time spectroscopic measurements during pressure application. For accD, this could be used with colorimetric assays that detect malonyl-CoA production.

• Quench-flow techniques under pressure: Reactions are initiated under pressure and then rapidly quenched at defined time points for downstream analysis.

• Microplate-based high-pressure systems: Recent advances have enabled high-throughput growth and enzyme assays under high pressure, as demonstrated for P. profundum . This approach could be adapted for accD activity measurements.

A particularly promising approach is the microplate sealing technique developed for high-throughput monitoring of bacterial growth at elevated hydrostatic pressure, which could be modified for enzyme assays . When designing these experiments, it's crucial to account for pressure effects on pH, substrate concentrations, and buffer systems that might indirectly affect enzyme activity measurements.

What structural adaptations might P. profundum accD possess to function optimally under high pressure?

While specific structural information about P. profundum accD is not provided in the search results, research on other pressure-adapted proteins suggests several likely adaptations:

• Increased internal hydration and reduced hydrophobic core packing: This allows the protein to maintain flexibility under pressure conditions that would normally compress and rigidify protein structures.

• Modified amino acid composition: Likely increased content of flexible amino acids (glycine, serine) and reduced content of bulky hydrophobic residues that would be sensitive to pressure effects.

• Altered salt bridge and hydrogen bonding networks: These would stabilize the protein structure while maintaining necessary flexibility for catalysis under high pressure.

• Pressure-sensitive active site architecture: The active site might be structured to undergo beneficial conformational changes under pressure that enhance substrate binding or catalysis.

Proteomic studies of P. profundum have shown differential expression of proteins involved in key metabolic pathways under varying pressure conditions , suggesting that both regulation and structural adaptation contribute to pressure acclimation. These structural adaptations would likely be most pronounced in enzymes essential for membrane lipid biosynthesis, like accD, given the critical importance of membrane fluidity for survival under high pressure.

Can P. profundum accD complement loss-of-function mutations in accD genes of other bacterial species?

Based on research with other fatty acid biosynthesis enzymes from P. profundum, complementation ability would depend on several factors:

• Temperature conditions: P. profundum enzymes often show temperature-dependent complementation. For example, Pfa synthase components from P. profundum can complement E. coli fabD mutations at 22°C but not at 37°C , suggesting accD might show similar temperature dependence.

• Pressure conditions: P. profundum accD likely functions optimally at higher pressures, potentially limiting its complementation efficiency at atmospheric pressure.

• Genetic context: Complementation might require co-expression of other P. profundum ACC subunits for proper function.

• Host species relatedness: Complementation would likely be more successful in closely related species within the Vibrionaceae family.

Interestingly, experiments with P. profundum SS9 have demonstrated that components of its fatty acid biosynthesis machinery can functionally replace their counterparts in E. coli , suggesting that despite adaptation to extreme conditions, these enzymes retain fundamental functional compatibility with mesophilic homologs.

How do pressure and temperature conditions affect accD expression and activity in P. profundum?

While direct studies on accD expression in P. profundum are not provided in the search results, research on related fatty acid biosynthesis genes suggests:

The types and abundance of fatty acid chains in the P. profundum cell membrane respond to changes in pressure and temperature , suggesting that enzymes involved in fatty acid biosynthesis, including accD, are regulated in response to these environmental conditions. RT-PCR expression analysis has been successfully used to assess differential expression of other genes under varying pressure conditions , and could be applied to study accD regulation.

What is the most effective strategy for cloning and expressing recombinant P. profundum accD?

An effective cloning and expression strategy would include:

• Gene amplification: PCR amplification of the accD gene from P. profundum genomic DNA using high-fidelity polymerase with primers incorporating appropriate restriction sites for downstream cloning.

• Vector selection: For initial characterization, pET-based vectors (like pET200TOPO/D) have been successfully used for expressing other P. profundum enzymes . For co-expression with other ACC subunits, vectors allowing multiple gene expression should be considered.

• Expression host: E. coli BL21(DE3) strain grown at reduced temperatures (16-22°C) has proven effective for other P. profundum enzymes .

• Expression conditions:

  • Growth at 16-22°C following induction

  • Induction during mid-log phase (OD600 0.3-0.5)

  • Extended expression time (24-48 hours) at lower temperatures

  • Consideration of marine broth components to mimic native environment

• Purification strategy: Inclusion of affinity tags (His6) for purification, followed by size exclusion chromatography to ensure proper oligomeric assembly.

This approach is based on successful expression of other P. profundum enzymes, including dehydratase domains from the PUFA synthase complex, which showed enhanced activity when expressed in E. coli at lower temperatures .

How can you design experiments to assess accD function under varying pressure conditions?

To assess accD function under varying pressure conditions:

• High-pressure enzyme assays:

  • Use pressure vessels equipped with optical windows for spectrophotometric assays

  • Employ coupled enzyme assays that link accD activity to production of a detectable product

  • Monitor activity across a pressure range (0.1-90 MPa) relevant to P. profundum's natural habitat

• In vivo complementation assays:

  • Express P. profundum accD in accD-deficient E. coli or other bacterial hosts

  • Assess growth under varying pressure conditions using specialized high-pressure culture systems

  • Compare growth rates and fatty acid profiles with and without accD complementation

• Pressure-dependent structural studies:

  • Employ high-pressure NMR or X-ray crystallography to assess structural changes

  • Use FRET-based approaches to monitor protein conformational changes under pressure

  • Implement hydrogen-deuterium exchange mass spectrometry to identify pressure-sensitive regions

• Comparative mutagenesis:

  • Generate variants of accD with substitutions at key residues predicted to affect pressure sensitivity

  • Compare activity profiles of wild-type and mutant enzymes across pressure ranges

A practical setup would use the microplate sealing technique for high-pressure conditions as described for P. profundum growth studies , adapted for enzyme activity measurements. This allows multiple samples to be assayed simultaneously under pressure, facilitating comparative and kinetic analyses.

What purification protocol would yield maximum activity of recombinant P. profundum accD?

A purification protocol optimized for maximum activity of P. profundum accD would include:

• Cell lysis conditions:

  • Gentle lysis using non-ionic detergents or osmotic shock

  • Low temperature (4°C) throughout purification

  • Inclusion of protease inhibitors to prevent degradation

  • Buffer conditions mimicking marine environment (elevated salt concentration)

• Purification steps:

  • Initial capture using affinity chromatography (e.g., His-tag)

  • Intermediate purification by ion exchange chromatography

  • Final polishing by size exclusion chromatography to ensure proper oligomeric state

• Buffer optimizations:

  • Inclusion of stabilizing agents (glycerol 10-20%)

  • Addition of cofactors or substrates that stabilize the enzyme

  • Optimization of pH range (likely around 7.5 based on marine environment)

  • Inclusion of reducing agents to maintain critical cysteine residues

• Special considerations:

  • Co-purification with other ACC subunits if necessary for activity

  • Limited exposure to room temperature

  • Immediate activity testing after purification steps

For P. profundum proteins, maintaining conditions that reflect their native environment is critical. Studies on other P. profundum enzymes have shown that these cold-adapted proteins often have reduced stability at conventional laboratory temperatures . Additionally, considering that P. profundum SS9 grows optimally at 15°C , all purification steps should be conducted at low temperatures to maximize protein stability and activity.

How can you design a CRISPR-Cas9 system for genetic manipulation of accD in P. profundum?

Designing a CRISPR-Cas9 system for genetic manipulation of accD in P. profundum requires several specialized considerations:

• Guide RNA design:

  • Target sequences unique to accD to avoid off-target effects

  • Account for high GC content in guide RNA design (P. profundum has 38.7-50.9% GC content )

  • Design multiple guide RNAs targeting different regions of accD

  • Validate guide RNA specificity using whole genome analysis

• Delivery system:

  • Utilize conjugation with E. coli helper strains, which has been successful for genetic manipulation of P. profundum

  • Consider pressure adaptation during the conjugation process

  • Use marine-adapted plasmid systems with appropriate selection markers

• Repair template design:

  • For knock-in mutations, include appropriate homology arms (1-2 kb)

  • For gene deletion, design repair templates that maintain genomic stability

  • Consider inclusion of reporter genes or tags for easier screening

• Screening strategy:

  • Develop PCR-based screening methods for identifying successful edits

  • Implement phenotypic screens based on predicted accD function (fatty acid profile changes)

  • Use growth assays under varying pressure conditions to identify functional impacts

• Validation approach:

  • Sequence verification of genetic modifications

  • RT-PCR to confirm altered expression patterns

  • Complementation studies to confirm phenotype causality

Genetic manipulation of P. profundum has been performed using techniques such as transposon mutagenesis , which provides a foundation for developing CRISPR-based approaches. The ability of P. profundum to grow at atmospheric pressure, despite being a piezophile, facilitates genetic manipulation under standard laboratory conditions .

What experimental design would best elucidate the relationship between accD function and membrane lipid composition?

To elucidate the relationship between accD function and membrane lipid composition, a comprehensive experimental design would include:

• Genetic manipulation approaches:

  • Generate accD variants with altered activity levels (point mutations, expression modulation)

  • Create conditional expression systems for accD to control activity levels temporally

  • Develop accD-pfaA double mutants to study pathway interactions

• Lipid analysis methods:

  • Comprehensive lipidomics analysis using LC-MS/MS to profile all membrane lipids

  • Gas chromatography to quantify fatty acid composition changes

  • Pulse-chase labeling experiments to track lipid synthesis rates

• Membrane property measurements:

  • Fluorescence anisotropy to assess membrane fluidity

  • Differential scanning calorimetry to measure phase transition temperatures

  • Pressure-adapted microscopy techniques to visualize membrane organization

• Experimental conditions matrix:

• Controls and validations:

  • Complementation studies to confirm phenotype causality

  • Metabolic flux analysis to track carbon flow through fatty acid pathways

  • Comparative analysis with other fatty acid biosynthesis gene mutations

This experimental design builds on studies of other fatty acid biosynthesis genes in P. profundum, which have shown that mutations in these genes affect both lipid composition and pressure tolerance . By manipulating accD specifically, researchers can determine its unique contribution to membrane adaptation mechanisms.

How should researchers interpret contradictory results in accD expression studies under different pressure conditions?

When interpreting contradictory results in accD expression studies under different pressure conditions, researchers should consider:

• Methodological differences:

  • Different pressure application methods can yield varying results

  • Time course variations: acute vs. chronic pressure exposure produces different responses

  • Sample processing methods (RNA extraction efficiency can be affected by pressure)

• Strain-specific variations:

  • Different P. profundum strains show varying optimal pressure ranges

  • Strain SS9 (optimal at 28 MPa), strain 3TCK (optimal at 0.1 MPa), and strain DSJ4 (optimal at 10 MPa) may show different accD regulation patterns

• Growth phase considerations:

  • Cell density effects (compare results at equivalent growth phases)

  • Pressure effects vary between logarithmic and stationary phases

• Contextual analysis:

  • Examine expression of other fatty acid biosynthesis genes concurrently

  • Consider whole pathway regulation rather than individual genes

  • Analyze both transcriptional and post-transcriptional regulation

• Experimental validation approaches:

  • Confirm RNA-level changes with protein-level measurements

  • Validate expression changes with enzyme activity assays

  • Use multiple technical and biological replicates with appropriate statistical analysis

Studies on P. profundum have shown that pressure responses can be complex and involve multiple regulatory systems . For example, the expression of some proteins involved in nutrient transport or assimilation is directly regulated by pressure, while other expression changes may be indirect responses to the altered cellular environment .

What statistical methods are most appropriate for analyzing enzyme kinetic data from high-pressure experiments?

For analyzing enzyme kinetic data from high-pressure experiments, appropriate statistical methods include:

When analyzing high-pressure enzyme data, it's important to account for potential systematic errors introduced by pressure effects on experimental components (buffers, pH indicators, coupled enzymes). Standardization using internal controls and careful validation of measurement systems under pressure are essential for reliable statistical analysis.

How can researchers distinguish between direct pressure effects on accD and indirect effects through membrane changes?

Distinguishing between direct pressure effects on accD and indirect effects through membrane changes requires multifaceted experimental approaches:

• In vitro vs. in vivo comparisons:

  • Measure purified accD activity under pressure in defined buffer systems

  • Compare with activity measurements in membrane preparations or whole cells

  • Quantify the difference to estimate membrane-mediated effects

• Membrane mimetic systems:

  • Reconstitute accD in liposomes with defined lipid compositions

  • Systematically vary lipid composition to match pressure-induced membrane changes

  • Test activity in these controlled membrane environments

• Site-directed mutagenesis strategy:

  • Modify potential pressure-sensing residues in accD

  • Create variants insensitive to direct pressure effects

  • Test these variants in native membrane environments

• Time-resolved experiments:

  • Monitor rapid pressure-jump effects (milliseconds to seconds) which likely represent direct protein effects

  • Compare with longer-term responses (minutes to hours) that include membrane adaptation

• Comparative analysis with membrane-binding mutants:

  • Generate accD variants with altered membrane interaction capabilities

  • Compare pressure responses of these variants with wild-type enzyme

Studies on P. profundum have shown that both direct pressure effects on proteins and indirect effects through membrane changes contribute to piezophilic adaptation . Proteomic analyses have identified proteins differentially expressed under high pressure, which could interact with accD or affect its cellular environment .

What approaches can resolve inconsistent enzyme activity data from high-pressure experiments with recombinant P. profundum accD?

To resolve inconsistent enzyme activity data from high-pressure experiments:

• Systematic troubleshooting protocol:

  a. Enzyme preparation variability:

  • Implement standardized purification protocols

  • Use single purification batches for comparative experiments

  • Quantify and normalize enzyme purity across preparations

  b. Pressure application inconsistencies:

  • Calibrate pressure systems before each experiment

  • Use pressure-resistant internal standards

  • Account for temperature changes during pressurization

  c. Assay component pressure sensitivity:

  • Test buffer systems, cofactors, and coupled enzymes for pressure effects

  • Develop direct assays that minimize confounding variables

  • Validate assay linearity under various pressure conditions

• Advanced experimental approaches:

  a. Multiple detection methods:

  • Compare results from spectrophotometric, fluorometric, and HPLC-based assays

  • Implement real-time assays where possible

  • Use mass spectrometry to directly measure reaction products

  b. Structural analysis correlation:

  • Combine activity measurements with structural assessments

  • Correlate activity changes with conformational states

  • Implement pressure-adapted structural biology techniques

• Data analysis refinements:

  a. Advanced curve fitting:

  • Apply mathematical models that account for pressure-dependent enzyme behavior

  • Use global fitting approaches across multiple pressure points

  • Implement Bayesian inference methods for parameter estimation

  b. Outlier analysis:

  • Develop criteria for identifying and handling outliers in pressure experiments

  • Apply robust statistical methods less sensitive to extreme values

  • Consider pressure-specific artifacts that may generate apparent outliers

Studying enzymes under pressure presents unique challenges, as seen in research on P. profundum proteins . Careful experimental design with appropriate controls and multiple technical approaches provides the strongest foundation for resolving inconsistencies in high-pressure enzyme activity data.

What control experiments are essential when studying the effects of pressure on accD expression and activity?

Essential control experiments for studying pressure effects on accD include:

• Pressure-independent controls:

  • Measurement of housekeeping genes/proteins unaffected by pressure

  • Internal standards for quantification of expression/activity

  • Positive controls with known pressure responses

  • Negative controls with pressure-insensitive variants

• Methodological controls:

  • Buffer stability assessments under experimental pressure conditions

  • pH indicator controls to account for pressure-induced pH shifts

  • Temperature monitoring during pressure application (compression heating effects)

  • Time controls to distinguish pressure effects from time-dependent changes

• Biological system controls:

  • Comparison across multiple P. profundum strains with different pressure optima

    • Strain SS9 (28 MPa optimum)

    • Strain 3TCK (0.1 MPa optimum)

    • Strain DSJ4 (10 MPa optimum)

  • Mesophilic homologs expressed under identical conditions

  • Membrane composition analysis to monitor concurrent changes

• Expression system controls:

  • Empty vector controls for recombinant expression systems

  • Housekeeping gene expression controls for normalization

  • Alternative promoter controls to distinguish transcriptional from post-transcriptional effects

• Data processing controls:

  • Technical replicate consistency checks

  • Statistical validation using randomized datasets

  • Blinded analysis protocols where appropriate

Researchers studying P. profundum have developed specialized approaches for high-pressure experiments, including methods to assess quantitative piezophilic colony growth on solid agar and microplate-based systems for high-throughput monitoring of bacterial growth at elevated hydrostatic pressure . These methodologies provide frameworks for establishing appropriate controls for accD studies.

How can researchers overcome low expression yields of recombinant P. profundum accD in E. coli?

To overcome low expression yields of recombinant P. profundum accD in E. coli:

• Codon optimization strategies:

  • Adapt codon usage to E. coli preferences while preserving critical structural elements

  • Use algorithms specifically designed for psychrophilic and piezophilic genes

  • Consider rare codon analysis and targeted optimization

• Expression system optimization:

  • Test multiple promoter systems (T7, trc, araBAD)

  • Evaluate various E. coli strains (BL21, Rosetta, Arctic Express)

  • Implement cold-shock promoters for improved low-temperature expression

  • Consider co-expression with molecular chaperones (GroEL/ES, trigger factor)

• Culture condition refinements:

  • Optimize induction parameters (inducer concentration, timing, temperature)

  • Implement a gradual temperature reduction protocol following induction

  • Supplement media with osmolytes found in marine environments

  • Extend expression time at lower temperatures (24-72 hours at 16°C)

• Fusion protein approaches:

  • Test solubility-enhancing fusion partners (MBP, SUMO, thioredoxin)

  • Implement optimal linker design between fusion partner and target protein

  • Compare N-terminal versus C-terminal fusion configurations

  • Include precision protease sites for fusion tag removal

• Co-expression strategies:

  • Co-express with other ACC subunits for proper complex formation

  • Include P. profundum-specific chaperones if identified

  • Consider co-expression with interacting partners to stabilize structure

Similar approaches have been successful for expressing other challenging P. profundum enzymes, including components of the PUFA synthase complex . In that case, expression at 16-22°C significantly improved yields compared to standard 37°C conditions, with expression during log phase (OD600 0.3-0.5) providing optimal results .

What strategies can address protein misfolding when expressing P. profundum accD at standard laboratory temperatures?

To address misfolding issues with P. profundum accD at standard laboratory temperatures:

• Temperature management protocols:

  • Implement a step-down temperature approach (start at 37°C, gradually reduce to 15-20°C)

  • Grow cells at 37°C until induction, then immediately transfer to lower temperature

  • Extend expression time at lower temperatures to compensate for slower folding kinetics

  • Consider cold-shocking cells before induction to induce cold-adaptation response

• Chemical chaperone supplementation:

  • Add osmolytes to culture medium (glycerol, betaine, TMAO)

  • Supplement with non-detergent sulfobetaines to stabilize folding intermediates

  • Include low concentrations of mild detergents to prevent aggregation

  • Test marine-specific stabilizing compounds

• Genetic approaches:

  • Co-express with molecular chaperones specific for cold-adapted proteins

  • Engineer stabilizing mutations based on mesophilic homologs

  • Create chimeric constructs with well-folding mesophilic domains

  • Implement split-protein complementation for challenging domains

• Refolding strategies:

  • Develop in vitro refolding protocols optimized for cold-adapted proteins

  • Implement step-wise refolding with decreasing denaturant concentrations

  • Include pressure treatment in refolding protocols to mimic native conditions

  • Test pulsed refolding approaches alternating between pressure conditions

• Structural biology-guided approaches:

  • Identify and modify aggregation-prone regions based on sequence analysis

  • Engineer disulfide bonds to stabilize tertiary structure

  • Incorporate solubility-enhancing mutations at surface-exposed residues

  • Consider domain-by-domain expression for large, multi-domain proteins

P. profundum proteins are adapted to function optimally at 15°C and high pressure (28 MPa for strain SS9) , making them challenging to express correctly at standard laboratory temperatures. Studies with other P. profundum enzymes have shown that temperature sensitivity can be a significant factor in heterologous expression , with some enzymes showing temperature-dependent complementation in E. coli (functional at 22°C but not at 37°C).

How can researchers verify that recombinant P. profundum accD retains its native structure and function?

To verify that recombinant P. profundum accD retains its native structure and function:

• Functional validation approaches:

  • Enzyme activity assays compared with native enzyme (if available)

  • Complementation of accD-deficient bacterial strains

  • Pressure-dependent activity profiling to match native behavior

  • Temperature-dependent activity analysis (optimal at ~15°C)

• Structural characterization methods:

  • Circular dichroism spectroscopy to assess secondary structure

  • Intrinsic fluorescence spectroscopy for tertiary structure analysis

  • Limited proteolysis patterns compared with native protein

  • Differential scanning calorimetry to determine melting temperatures

  • Size exclusion chromatography to confirm proper oligomeric state

• Pressure response validation:

  • Activity measurements under varying pressure conditions

  • Pressure-induced conformational changes measured by spectroscopic methods

  • Comparison with pressure response of mesophilic homologs

  • Validation of pressure optima matching native environment (28 MPa for SS9)

• Advanced biophysical approaches:

  • Hydrogen-deuterium exchange mass spectrometry for dynamic structural analysis

  • High-pressure NMR studies to characterize pressure-induced conformational changes

  • Molecular dynamics simulations to predict pressure effects on structure

• In vivo functional assessment:

  • Complementation studies in P. profundum accD mutants

  • Analysis of membrane lipid composition in complemented strains

  • Growth phenotype analysis under varying pressure and temperature conditions

For P. profundum proteins, functional verification should include assessment under the organism's optimal growth conditions: 15°C and 28 MPa for strain SS9 . The ability to function at high pressure is a defining characteristic of piezophilic enzymes and should be explicitly tested as part of the validation process.

What techniques can differentiate pressure-specific effects from temperature effects on accD function?

To differentiate pressure-specific effects from temperature effects on accD function:

• Multifactorial experimental design:

  • Create a pressure-temperature matrix of conditions:

    Pressure (MPa)	Temperature (°C)
    0.1 (atmospheric)	4, 15, 25
    10 (moderate)	4, 15, 25
    28 (optimal)	4, 15, 25
    50 (high)	4, 15, 25

  • Measure activity/stability at each point

  • Apply two-way ANOVA or response surface methodology for analysis

• Thermodynamic parameter determination:

  • Calculate activation volumes (ΔV‡) from pressure-dependent rate constants

  • Determine activation energies (Ea) from temperature-dependent rate constants

  • Compare pressure-temperature cross effects:

    $\left(\frac{\partial^2 \ln k}{\partial P \partial T}\right)$

• Structural perturbation approaches:

  • Generate variants with mutations affecting pressure sensitivity

  • Create variants with mutations affecting temperature sensitivity

  • Test these variants across pressure-temperature combinations

  • Identify residues specifically involved in pressure adaptation

• Comparative analysis with homologs:

  • Compare with accD from non-piezophilic but psychrophilic organisms (isolate temperature effects)

  • Compare with accD from piezophilic but mesophilic organisms (isolate pressure effects)

  • Analyze accD from different P. profundum strains with varying pressure optima:

    • Strain SS9 (28 MPa optimum)

    • Strain 3TCK (0.1 MPa optimum)

    • Strain DSJ4 (10 MPa optimum)

• Advanced biophysical techniques:

  • Pressure-jump experiments at constant temperature

  • Temperature-jump experiments at constant pressure

  • Pressure-modulated differential scanning calorimetry

  • High-pressure spectroscopic techniques with temperature control

P. profundum provides an excellent model system for this type of analysis because different strains have different pressure optima while maintaining similar temperature preferences. This natural variation allows researchers to isolate pressure-specific adaptations through comparative studies .

How can researchers develop high-throughput screening methods for P. profundum accD variants with enhanced pressure tolerance?

To develop high-throughput screening methods for P. profundum accD variants with enhanced pressure tolerance:

• Growth-based complementation screening:

  • Generate an accD-deficient bacterial host dependent on functional accD for growth

  • Express P. profundum accD variant library in this host

  • Use the microplate sealing technique developed for high-pressure bacterial growth

  • Screen for growth under increasing pressure conditions

  • Identify variants supporting growth at higher-than-wild-type pressures

• Activity-based fluorescent assays:

  • Develop fluorogenic substrates or coupled enzyme assays for accD activity

  • Implement in microplate format compatible with high-pressure chambers

  • Screen variant libraries under defined pressure conditions

  • Use plate readers equipped with pressure-resistant optical windows

• Yeast two-hybrid or bacterial two-hybrid pressure-adapted systems:

  • Develop interaction screening systems to identify variants that maintain proper complex formation under pressure

  • Couple protein-protein interactions to reporter gene expression

  • Screen under pressure conditions to identify stable variants

• In vivo biosensor systems:

  • Develop cellular biosensors where accD activity is coupled to fluorescent or colorimetric output

  • Design sensors responsive to fatty acid biosynthesis pathway output

  • Screen variant libraries under pressure using imaging systems adapted for pressure vessels

• Directed evolution strategy:

  • Implement error-prone PCR to generate accD variant libraries

  • Develop selection scheme coupling accD function to cellular survival under pressure

  • Perform iterative rounds of selection with increasing pressure

  • Sequence variants with enhanced pressure tolerance

  • Use machine learning to predict beneficial mutations for subsequent rounds

Building on methods developed for P. profundum, researchers can adapt the quantitative colony growth assessment techniques developed for piezophilic growth on solid agar and the high-throughput monitoring system for bacterial growth at elevated hydrostatic pressure using microplate readers . These approaches provide foundations for developing variant screening methods specifically for accD.