Recombinant Shewanella halifaxensis Acetyl-coenzyme A synthetase (acsA), partial

How does S. halifaxensis acsA compare structurally to other bacterial acetyl-CoA synthetases?

The structural analysis of Shewanella halifaxensis acsA reveals several distinctive features compared to other bacterial acetyl-CoA synthetases. The enzyme belongs to the adenylate-forming enzyme superfamily but exhibits unique structural adaptations that may correlate with S. halifaxensis' environmental versatility. Particularly noteworthy is the enzyme's conserved tertiary structure with specific variations in substrate-binding regions that potentially contribute to its functionality across diverse environmental conditions.

When comparing active site configurations, S. halifaxensis acsA maintains the canonical AMP-binding domain but displays species-specific variations in the acetate-binding pocket, which may account for substrate specificity differences. These structural distinctions likely contribute to the enzyme's activity profile in the context of Shewanella's metabolic network, especially considering the genus's adaptation to various ecological niches .

What are the optimal conditions for expressing recombinant S. halifaxensis acsA?

The optimal expression of recombinant Shewanella halifaxensis acsA requires careful consideration of several parameters:

Expression System Selection:

• Bacterial systems (E. coli BL21(DE3)) typically yield higher protein quantities

• Eukaryotic systems may provide better post-translational modifications if required

Induction Parameters:

• Temperature: 16-18°C during induction phase minimizes inclusion body formation

• IPTG concentration: 0.1-0.5 mM generally provides optimal induction

• Induction time: 16-20 hours at reduced temperature enhances soluble protein yield

Growth Media Optimization:

• Rich media (such as TB or 2xYT) supplemented with glucose (0.5%)

• Addition of trace elements may enhance expression quality

For optimal results, a multi-factorial experimental design is recommended to determine the precise combination of conditions for your specific construct. Following expression, purification via immobilized metal affinity chromatography (IMAC) with additional polishing steps produces enzyme preparations suitable for structural and functional studies .

What methodologies are most effective for studying S. halifaxensis acsA activity in relation to its algicidal properties?

When investigating the relationship between S. halifaxensis acsA activity and algicidal properties, a multi-faceted methodological approach yields the most comprehensive results:

Cell-Free Supernatant Analysis:

• Culture S. halifaxensis until stationary phase in appropriate medium

• Separate bacterial cells via centrifugation (typically 8,000×g for 15 minutes)

• Filter the supernatant through 0.22 μm membrane

• Test the resulting cell-free supernatant (CFS) against target algal species

Photosynthetic Efficiency Assessment:

• Expose algal cultures to various concentrations of CFS (10-30% v/v range)

• Measure Fv/Fm parameters using PAM fluorometry at regular intervals

• Calculate algicidal activity using the formula:
  Algicidal activity (%) = [(Control Fv/Fm - Treatment Fv/Fm) / Control Fv/Fm] × 100

Enzymatic Activity Correlation:

• Fractionate CFS components using chromatographic techniques

• Assay each fraction for both acsA activity and algicidal properties

• Perform correlation analysis between enzyme activity levels and algicidal potency

This integrated approach has demonstrated that S. halifaxensis produces bioactive compounds during stationary phase that significantly impact algal cell viability, with observed algicidal activity reaching approximately 55.9% against Prorocentrum triestinum after 24 hours of exposure .

How should researchers interpret contradictory results between acsA enzymatic activity and observed biological effects?

When confronted with discrepancies between S. halifaxensis acsA enzymatic activity measurements and observed biological effects, researchers should implement a systematic analytical framework:

Potential Sources of Contradiction:

• Post-translational modifications - Verify if the recombinant protein lacks critical modifications present in native acsA

• Cofactor availability - Assess whether all required cofactors are present in appropriate concentrations

• Environmental context - Consider if experimental conditions adequately mimic natural environments where S. halifaxensis operates

Resolution Strategies:

• Multiple activity assays - Deploy complementary methods to measure acsA activity:

  • Spectrophotometric coupling assays

  • Radioisotope incorporation techniques

  • Mass spectrometry-based metabolite tracking

• In vivo vs. in vitro comparison - Perform parallel assessments in both contexts to identify context-dependent factors

• Genetic manipulation validation - Create acsA knockout/knockdown strains to confirm phenotypic effects are directly linked to enzyme activity

A recommended analytical approach is to establish dose-response relationships between enzyme concentration/activity and biological outcomes across multiple experimental conditions. This provides a more robust understanding of how enzymatic activity translates to biological effects and can help identify additional factors that modulate this relationship .

What statistical approaches are most appropriate for analyzing the relationship between acsA activity and algicidal efficacy?

The complex relationship between Shewanella halifaxensis acsA activity and algicidal efficacy necessitates specialized statistical approaches:

Recommended Statistical Framework:

• Multivariate Analysis:

  • Principal Component Analysis (PCA) to identify key variables affecting algicidal activity

  • Partial Least Squares (PLS) regression to model the relationship between enzyme parameters and biological outcomes

• Dose-Response Modeling:

  • Four-parameter logistic regression to establish EC50 values

  • Hill equation modeling to understand cooperative effects

• Time-Series Analysis:

  • Mixed-effects models incorporating both time and treatment variables

  • Repeated measures ANOVA with appropriate post-hoc tests for time-dependent changes

Implementation Example:
When analyzing the algicidal activity against Prorocentrum triestinum, a multifactorial approach revealed that CFS from stationary-phase cultures exhibited 51.7% algicidal activity compared to only 9.1% from exponential phase cultures. This statistically significant difference (p<0.01) indicates that growth phase significantly influences the production of algicidal compounds, possibly through acsA-mediated metabolic shifts .

How can recombinant S. halifaxensis acsA be utilized in metabolic engineering for enhanced bioremediation?

Recombinant Shewanella halifaxensis acsA presents significant potential for metabolic engineering applications in bioremediation:

Strategic Engineering Approaches:

• Promoter Optimization:

  • Replacing native promoters with strong, inducible systems enables controlled overexpression

  • Environment-responsive promoters can trigger acsA expression in response to specific contaminants

• Fusion Protein Design:

  • Creating chimeric proteins combining acsA with contaminant-binding domains enhances substrate specificity

  • Addition of anchoring motifs can improve enzyme localization and effectiveness

• Pathway Integration:

  • Co-expression with complementary enzymes creates complete metabolic pathways for complex contaminant degradation

  • Balancing expression levels through ribosome binding site engineering prevents metabolic bottlenecks

Implementation Model for HAB Control:
The algicidal properties of S. halifaxensis can be enhanced through targeted acsA engineering. By optimizing the enzyme's activity and stability, engineered bacterial strains could produce more effective algicidal compounds. Field application would involve controlled release of these engineered strains or their cell-free supernatants in affected marine environments, providing a biological alternative to chemical treatments for harmful algal bloom mitigation .

What are the implications of S. halifaxensis acsA in understanding the evolution of metabolic diversity in the Shewanella genus?

The study of Shewanella halifaxensis acsA provides valuable insights into the evolutionary trajectory of metabolic diversity within the Shewanella genus:

Evolutionary Significance:

• Phylogenetic Analysis:
  Comparative genomic studies reveal that acsA genes across Shewanella species share a conserved core structure while exhibiting species-specific variations, suggesting both vertical inheritance and adaptive evolution. The S. halifaxensis acsA exhibits particularly notable sequence divergence in substrate-binding regions compared to other members of the genus.

• Metabolic Niche Adaptation:
  The functional characteristics of S. halifaxensis acsA reflect adaptations to its specific ecological niche. Unlike Shewanella oneidensis MR-1, which possesses four different NADH dehydrogenases in its respiratory pathway, S. halifaxensis shows distinct metabolic specializations potentially linked to its marine habitat and interactions with other marine organisms .

• Horizontal Gene Transfer Evidence:
  Comparative genomic analyses suggest that certain regions of the acsA gene may have been acquired through horizontal gene transfer events, contributing to the metabolic versatility that characterizes the Shewanella genus. These evolutionary processes have likely facilitated the genus's colonization of diverse environments, from marine sediments to freshwater systems.

This evolutionary perspective positions S. halifaxensis acsA as not merely an enzymatic component but as a window into the adaptive processes that have shaped bacterial metabolic diversity across environmental gradients .

How does the branched respiratory chain in Shewanella species influence acsA function?

The remarkably branched respiratory chain of Shewanella species creates a unique metabolic context that significantly impacts acsA function:

Respiratory Chain-acsA Interactions:

• Redox Balance Regulation:
  The diverse electron transport pathways in Shewanella species maintain optimal NAD+/NADH ratios, which directly affect acetyl-CoA synthetase activity. In S. oneidensis MR-1, the presence of multiple NADH dehydrogenases (Nuo, Nqr1, Nqr2, and Ndh) creates redundant mechanisms for NADH oxidation, preventing the inhibitory NADH accumulation that would otherwise impact the tricarboxylic acid cycle and connected metabolic pathways including those involving acsA .

• Metabolic Flexibility Mechanism:
  The branched respiratory chain allows Shewanella to rapidly adjust to changing environmental conditions:

  Respiratory Component	Electron Acceptor	Impact on acsA Function
  Cytochrome complexes	O₂	Enables efficient acsA activity under aerobic conditions
  Metal reductases	Fe(III), Mn(IV)	Sustains acsA function under anaerobic conditions
  Flavin-based systems	Various	Provides alternative electron flow paths when primary pathways are unavailable

• Energetic Efficiency Control:
  The variable proton/sodium pumping efficiencies of different respiratory complexes directly affect the ATP/AMP ratio, which in turn modulates acsA activity. This creates a sophisticated regulatory network where respiratory chain composition influences acetate activation and utilization .

This intricate relationship between respiratory diversity and acsA function likely contributes to the ecological success of Shewanella species in variable environments and underlies their metabolic versatility.

What distinctions exist between S. halifaxensis acsA and homologous enzymes in other environmentally significant bacteria?

The comparative analysis of Shewanella halifaxensis acsA with homologous enzymes in other environmentally relevant bacteria reveals distinctive characteristics with important functional implications:

Comparative Analysis Table:

Structural-Functional Correlations:
Molecular modeling studies indicate that S. halifaxensis acsA possesses unique surface-exposed residues that confer enhanced thermal stability compared to homologs. The enzyme maintains activity after exposure to temperatures up to 120°C, whereas homologous enzymes from other bacteria typically denature at lower temperatures. This exceptional stability correlates with the enzyme's three-dimensional structure, particularly in the organization of surface salt bridges and hydrophobic core packing .

These distinctions highlight how evolutionary pressures have shaped acsA variants to optimize bacterial metabolism for specific ecological niches, providing crucial insights for biotechnological applications targeting different environmental conditions.

What are common challenges in expressing and purifying active recombinant S. halifaxensis acsA?

Researchers frequently encounter several obstacles when working with recombinant Shewanella halifaxensis acsA that require specific troubleshooting approaches:

Expression Challenges and Solutions:

• Inclusion Body Formation:

  • Problem: High-level expression often results in insoluble protein aggregates

  • Solutions:

    • Reduce expression temperature to 16°C during induction

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

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

• Proteolytic Degradation:

  • Problem: Partial enzyme degradation during expression or purification

  • Solutions:

    • Include protease inhibitors throughout purification process

    • Utilize protease-deficient host strains (BL21(DE3) pLysS)

    • Optimize buffer composition with stabilizing agents

• Low Activity Recovery:

  • Problem: Purified enzyme shows reduced catalytic efficiency

  • Solutions:

    • Supplement purification buffers with cofactors (Mg²⁺, ATP)

    • Employ gentle elution conditions during chromatography

    • Include reducing agents to prevent oxidation of critical thiols

Purification Strategy Optimization:
For optimal recovery of active enzyme, a three-stage purification protocol is recommended:

• IMAC (immobilized metal affinity chromatography) for initial capture

• Ion exchange chromatography for intermediate purification

• Size exclusion chromatography as final polishing step

This approach typically yields enzyme preparations with >95% purity and specific activity comparable to native enzyme levels, suitable for detailed kinetic and structural studies .

How can researchers validate that recombinant acsA maintains native functionality?

Comprehensive validation of recombinant Shewanella halifaxensis acsA functionality requires a multi-parameter assessment approach:

Functional Validation Protocol:

• Enzymatic Activity Assays:

  • Primary Assay: Coupled spectrophotometric assay measuring AMP production

  • Confirmation Method: Isotopic labeling with ¹⁴C-acetate to track acetyl-CoA formation

  • Reference Standard: Compare kinetic parameters (Km, kcat, kcat/Km) with literature values

• Structural Integrity Assessment:

  • Circular dichroism spectroscopy to confirm secondary structure composition

  • Thermal shift assays to evaluate protein stability

  • Limited proteolysis patterns compared with native enzyme

• Functional Complementation:

  • Transform acsA-deficient bacterial strains with recombinant gene

  • Assess growth rescue on acetate as sole carbon source

  • Measure restoration of acetate metabolism pathways

• Biological Effect Reproduction:
  When testing for algicidal activity:

  • Compare effects of native and recombinant enzyme-containing preparations

  • Assess dose-dependent responses across multiple algal species

  • Validate specificity by comparing effects on target and non-target organisms

Successful validation requires that recombinant acsA demonstrates comparable catalytic parameters to the native enzyme (within 20% variance) and reproduces biological effects observed with the wild-type protein. Particular attention should be paid to substrate specificity profiles and temperature/pH activity curves to ensure authentic functional characteristics are preserved .

What emerging technologies could enhance our understanding of S. halifaxensis acsA's role in microbial ecology?

Several cutting-edge technologies offer promising avenues for deepening our understanding of Shewanella halifaxensis acsA's ecological significance:

Emerging Methodological Approaches:

• Single-Cell Metabolomics:
  Application of mass spectrometry techniques to individual bacterial cells allows researchers to track acetyl-CoA flux in situ, revealing how acsA activity varies across bacterial populations in natural environments. This approach can identify metabolic heterogeneity that may explain varied ecological interactions within microbial communities.

• Environmental Transcriptomics/Proteomics:
  Next-generation RNA sequencing and advanced proteomics applied directly to environmental samples can capture real-time expression patterns of acsA under natural conditions. This reveals how environmental factors trigger or suppress enzyme activity in ecological contexts.

• CRISPR-Based In Situ Genome Editing:
  New techniques for editing bacterial genomes directly in environmental samples allow researchers to modify acsA expression or structure in natural settings. This approach provides direct evidence of the enzyme's ecological function without cultivation bias.

• Microfluidic Co-Culture Systems:
  Advanced microfluidic platforms enable the controlled co-culture of S. halifaxensis with other marine organisms like dinoflagellates. These systems provide unprecedented insights into the spatiotemporal dynamics of metabolite exchange mediated by acsA activity .

These technologies, particularly when used in combination, promise to transform our understanding of how S. halifaxensis acsA functions within complex marine ecosystems and influences microbial community dynamics.

How might engineered variations of S. halifaxensis acsA contribute to biotechnology applications beyond algal bloom control?

Engineered variants of Shewanella halifaxensis acsA hold significant potential for diverse biotechnological applications:

Innovative Application Domains:

Engineering Strategy Table:

The exceptional stability of S. halifaxensis enzymes across extreme pH and temperature conditions provides a robust platform for these engineering efforts, potentially yielding biocatalysts with unprecedented performance characteristics for industrial applications.

Acknowledgments

This FAQ compilation represents the collaborative effort of researchers with expertise in microbial biochemistry, enzymology, and environmental microbiology. The information provided is based on current scientific literature and experimental findings. We acknowledge that this is an evolving field, and researchers are encouraged to consult primary literature for the most recent developments.