Recombinant Dinoroseobacter shibae Malate dehydrogenase (mdh)

What is malate dehydrogenase (mdh) and what is its fundamental role in D. shibae metabolism?

Malate dehydrogenase catalyzes the reversible conversion of malate to oxaloacetate with concurrent reduction of NAD+ to NADH. In D. shibae, this enzyme plays a crucial role in central carbon metabolism, particularly within the tricarboxylic acid (TCA) cycle.

D. shibae utilizes the Entner-Doudoroff pathway instead of the conventional glycolysis pathway due to the absence of phosphofructokinase activity . This metabolic configuration places increased importance on TCA cycle enzymes like mdh for energy generation and biosynthetic precursor production.

Methodologically, researchers studying mdh function in D. shibae should consider:

• Comparative enzyme assays measuring activity under varying pH, temperature, and salt conditions

• Gene knockout or knockdown studies to assess phenotypic effects

• Metabolic flux analysis using isotope-labeled substrates to track carbon flow

• Transcriptomic and proteomic analyses across growth conditions to determine regulation patterns

What expression systems are most effective for producing recombinant D. shibae mdh?

Selecting the appropriate expression system for recombinant D. shibae mdh requires consideration of protein solubility, yield, and post-purification activity.

For prokaryotic expression:

• E. coli BL21(DE3) or its derivatives with pET-based vectors are recommended initial choices

• Expression optimization should include testing multiple temperatures (16-37°C), inducer concentrations, and induction durations

• Fusion tags (His6, GST, MBP) can improve solubility and facilitate purification

• Codon optimization may be necessary as marine bacteria often have different codon usage than E. coli

For challenging expressions:

• Cold-adapted expression hosts may better accommodate enzymes from marine bacteria

• Cell-free protein synthesis systems can bypass toxicity issues

• Periplasmic expression can facilitate proper disulfide bond formation if present

Experimental validation should include:

• Small-scale expression trials with multiple constructs

• Activity assays of crude lysates to confirm functional expression

• Purification trials to assess yield and purity

• Stability testing under various buffer conditions

How can the purity and activity of recombinant D. shibae mdh be assessed?

A comprehensive assessment of recombinant D. shibae mdh should evaluate both purity and functional activity through multiple complementary techniques.

For purity assessment:

• SDS-PAGE with Coomassie or silver staining to visualize protein bands

• Size-exclusion chromatography to evaluate oligomeric state and homogeneity

• Mass spectrometry for accurate molecular weight determination and contaminant detection

• Dynamic light scattering to assess monodispersity

For activity assessment:

• Spectrophotometric assays monitoring the conversion of malate to oxaloacetate by tracking NADH formation at 340 nm

• Determination of specific activity (μmol/min/mg protein) under standardized conditions

• Measurement of kinetic parameters (Km, Vmax, kcat) using varied substrate concentrations

• Assessment of temperature optimum, pH optimum, and thermal stability

A methodical approach should include:

• Comparing activity before and after each purification step to track activity recovery

• Evaluating effects of potential stabilizers or activators

• Assessing activity under conditions that mimic the bacterium's natural marine environment

• Determining the effects of freeze-thaw cycles on enzyme stability

What buffer systems and storage conditions optimize D. shibae mdh stability?

Optimizing storage conditions for recombinant D. shibae mdh requires systematic testing of buffer components and storage parameters to maintain long-term stability.

Buffer composition considerations:

• pH range (typically 7.0-8.0 for bacterial mdh enzymes)

• Buffer type (phosphate, HEPES, or Tris-based)

• Ionic strength (particularly important for enzymes from marine bacteria adapted to saline environments)

• Cofactor inclusion (low concentrations of NADH may stabilize the enzyme)

• Protective additives (glycerol 10-50%, reducing agents like DTT or β-mercaptoethanol)

Storage condition options:

• Short-term (4°C with appropriate stabilizers)

• Medium-term (-20°C with cryoprotectants)

• Long-term (-80°C or lyophilization)

A systematic approach should include:

• Activity retention testing after storage under different conditions

• Assessment of freeze-thaw stability

• Evaluation of protective excipients for lyophilization

• Testing the effects of salt concentration on storage stability, given D. shibae's marine origin

• Monitoring of potential aggregation or precipitation during storage

What are the basic kinetic parameters of D. shibae mdh and how do they compare to other bacterial mdh enzymes?

Understanding the kinetic behavior of D. shibae mdh provides insights into its catalytic efficiency and potential metabolic roles.

Key kinetic parameters to determine include:

• Km values for malate, oxaloacetate, NAD+, and NADH

• Vmax and kcat for forward and reverse reactions

• kcat/Km ratio as a measure of catalytic efficiency

• Substrate specificity (testing structural analogs of malate)

• Potential allosteric effects of metabolites

Comparative analysis methodology:

• Standardized enzyme assays under identical conditions for multiple bacterial mdh enzymes

• Statistical analysis of kinetic parameters across phylogenetic groups

• Correlation of kinetic properties with ecological niches

• Molecular modeling to identify structural determinants of kinetic differences

For D. shibae specifically, examining adaptation to marine conditions should include:

• Effects of salt concentration on kinetic parameters

• pH-activity profiles compared to terrestrial bacteria

• Temperature-activity relationships reflecting adaptation to marine environments

How does mdh activity in D. shibae change during the transition from aerobic to anaerobic conditions?

D. shibae demonstrates remarkable metabolic flexibility, transitioning from aerobic respiration to denitrification when oxygen becomes limited . This adaptation likely involves significant changes in central metabolism, including mdh regulation and activity.

A comprehensive research approach would include:

• Time-resolved enzyme activity measurements:

  • Sampling D. shibae cultures at defined intervals during oxygen depletion

  • Measuring specific mdh activity in cell extracts

  • Correlating activity with oxygen levels and denitrification markers

• Transcriptomic and proteomic analyses:

  • Quantifying mdh gene expression using RT-qPCR during anaerobic transition

  • Measuring mdh protein levels using targeted proteomics

  • Identifying potential post-translational modifications affecting activity

Research findings indicate that during oxygen depletion, D. shibae experiences a metabolic crisis with reduced ATP concentration and growth rate . The central metabolism, including gluconeogenesis and biosynthetic pathways, is transiently reduced until denitrification machinery is established . This suggests that mdh activity may be modulated to balance cellular redox state during this transition.

Metabolic flux analysis using isotope-labeled substrates would further elucidate how carbon flow through mdh-catalyzed reactions changes during aerobic-anaerobic transitions, providing insights into metabolic reconfiguration strategies.

What role might mdh play in D. shibae's adaptation to anoxic environments?

D. shibae adapts to anoxic conditions by establishing nitrate respiration and inducing arginine fermentation . During this adaptation, central metabolism is transiently reduced, and interestingly, poly-3-hydroxybutanoate accumulates, possibly serving as a NADPH sink .

Mdh may play several critical roles in this adaptation process:

• Redox balance maintenance:

  • Modulating NAD+/NADH ratios to support denitrification

  • Adjusting TCA cycle flux to match altered electron transport demands

• Metabolic reconfiguration:

  • Providing oxaloacetate for biosynthetic pathways during metabolic adjustment

  • Supporting gluconeogenesis when needed

  • Potentially participating in anaplerotic reactions

Research approaches should include:

• Creating mdh knockout or conditional mutants and assessing their ability to transition to anoxic growth

• Conducting metabolomic analysis comparing wild-type and mdh-modified strains during oxygen depletion

• Examining potential interactions between mdh and denitrification enzymes

Experimentally, researchers should measure mdh activity, expression, and flux during the establishment of denitrification machinery, which includes periplasmic nitrate reductase NapA, nitrite reductase NirS, nitric oxide reductase NorB, and nitrous oxide reductase NosZ . The timing of mdh regulation relative to the expression of these enzymes would provide insights into metabolic adaptation coordination.

How might mdh contribute to D. shibae's symbiotic and pathogenic interactions with dinoflagellates?

D. shibae exhibits a fascinating "Jekyll-and-Hyde" relationship with the dinoflagellate Prorocentrum minimum, initially providing essential vitamins B12 and B1 in a symbiotic phase before later killing the algae in a pathogenic phase . This transition is determined by a 191 kb plasmid that can be conjugated into other Roseobacters .

Investigating mdh's potential role in this relationship would require:

• Expression analysis:

  • Comparing mdh expression in free-living versus symbiotic states

  • Monitoring temporal changes in mdh activity during symbiotic-to-pathogenic transition

  • Examining mdh regulation in wild-type versus plasmid-cured strains

• Metabolic integration studies:

  • Tracing carbon flow between D. shibae and dinoflagellates using labeled substrates

  • Analyzing how mdh-catalyzed reactions contribute to vitamin synthesis pathways

  • Investigating metabolic crosstalk between the symbionts

• Mutational analysis:

  • Creating mdh variants with altered activity and assessing their impact on symbiotic relationships

  • Testing if mdh activity correlates with virulence

  • Examining if the 191 kb killer plasmid affects mdh expression or regulation

While the search results don't directly connect mdh to the symbiotic process, central metabolism must play a role in sustaining both the production of essential vitamins for the algae and potentially in the later production of compounds involved in algal killing.

Is D. shibae mdh packaged in outer membrane vesicles (OMVs), and what ecological significance might this have?

D. shibae continuously produces outer membrane vesicles during normal growth, with approximately 0.75 vesicles per cell throughout the growth cycle . These OMVs primarily contain outer membrane and periplasmic proteins, and interestingly, also carry DNA enriched around the replication terminus .

To investigate mdh presence in OMVs:

• Proteomics approach:

  • Isolate OMVs using ultracentrifugation as described in previous research

  • Perform LC-MS/MS analysis of vesicle proteins

  • Compare OMV proteome with cellular proteome to identify enriched or depleted proteins

  • Specifically quantify mdh abundance in both fractions

• Activity-based detection:

  • Develop specific activity assays for mdh in intact OMVs

  • Compare specific activity in OMVs versus cellular fractions

  • Investigate if OMV-associated mdh remains functional

The proteomic analysis of D. shibae OMVs has shown that they are dominated by outer membrane and periplasmic proteins . Some abundant vesicle membrane proteins were predicted to be involved in cell division, including those interacting with peptidoglycan (LysM, Tol-Pal, SpoI, lytic murein transglycosylase) .

If mdh were found in OMVs, this could have several ecological implications:

• Extracellular enzymatic activity affecting local malate/oxaloacetate ratios

• Potential metabolic interactions with other marine microorganisms

• Contribution to community metabolic networks in marine environments

• Transfer of metabolic capabilities between bacterial cells

How can recombinant D. shibae mdh be engineered for enhanced stability or altered substrate specificity?

Engineering recombinant D. shibae mdh for improved properties requires a systematic approach combining structural analysis, rational design, and directed evolution.

Methodological framework:

• Structural analysis and target identification:

  • Determine crystal structure or generate homology model of D. shibae mdh

  • Identify catalytic residues, substrate-binding sites, and stability-determining regions

  • Compare with well-characterized mdh enzymes to identify targets for improvement

• Rational design approaches:

  • Site-directed mutagenesis of active site residues to alter substrate specificity

  • Introduction of salt bridges or disulfide bonds to enhance thermostability

  • Surface charge optimization for improved solubility

  • Cofactor specificity engineering (NAD+ vs. NADP+)

• Directed evolution strategies:

  • Development of high-throughput screening assays

  • Error-prone PCR to generate diversity

  • DNA shuffling with other bacterial mdh genes

  • Selection under challenging conditions (temperature, pH, salt)

• Validation and characterization:

  • Detailed kinetic analysis of engineered variants

  • Structural confirmation of designed changes

  • Stability testing under application-relevant conditions

  • In vivo functionality assessment

Potential targets for engineering could include:

• Increasing catalytic efficiency at lower temperatures typical of marine environments

• Enhancing stability to extremes of pH or salt concentration

• Modifying allosteric regulation to optimize performance in biotechnological applications

• Creating variants with altered substrate specificity for synthetic biology applications

What are the optimal conditions for measuring D. shibae mdh activity in vitro?

Establishing standardized conditions for D. shibae mdh activity measurement is essential for reliable kinetic characterization and comparative studies.

A comprehensive optimization protocol should include:

• Buffer system optimization:

  • Testing multiple buffers (phosphate, HEPES, Tris) at various pH values (6.5-9.0)

  • Evaluating ionic strength effects (50-500 mM)

  • Assessing the impact of salt type and concentration (particularly NaCl, given D. shibae's marine origin)

• Cofactor and substrate parameters:

  • Determining optimal NAD+/NADH concentrations

  • Establishing linear ranges for substrate (malate/oxaloacetate) concentrations

  • Evaluating potential activators or inhibitors

• Assay conditions:

  • Temperature optimization (likely 25-30°C based on D. shibae's marine environment)

  • Reaction time course to ensure initial velocity measurements

  • Protein concentration range for linearity

  • Spectrophotometric parameters (wavelength, path length, scanning vs. fixed wavelength)

• Control experiments:

  • No-enzyme controls

  • Heat-inactivated enzyme controls

  • Alternative substrate controls to assess specificity

When developing an mdh activity assay for D. shibae, researchers should consider the bacterium's natural marine environment and its ability to transition between aerobic and anaerobic metabolism . The enzyme may have evolved specific adaptations to these conditions that affect its in vitro behavior.

A standard assay protocol should be validated by demonstrating reproducibility across different enzyme preparations and linearity with respect to time and enzyme concentration.

How can isotopic labeling be used to investigate the in vivo function of mdh in D. shibae during metabolic adaptations?

Isotopic labeling provides powerful insights into metabolic flux through mdh-catalyzed reactions during D. shibae's adaptation to changing environmental conditions.

Experimental design considerations:

• Selection of labeled substrates:

  • [13C]malate to directly track mdh-catalyzed reactions

  • [13C]glucose or [13C]acetate to monitor carbon flow through central metabolism

  • [15N]nitrate for simultaneous tracking of denitrification pathways

• Experimental conditions:

  • Time-course sampling during aerobic to anaerobic transition

  • Comparison of wild-type and mdh mutant strains

  • Parallel cultures with different electron acceptors (O2, NO3-, NO2-)

• Analytical approaches:

  • GC-MS or LC-MS analysis of intracellular metabolites

  • NMR spectroscopy for detailed positional isotope incorporation

  • Metabolic flux calculation using isotopomer distribution

• Data integration:

  • Correlation with transcriptomic and proteomic data

  • Mathematical modeling of central metabolism

  • Comparison with established metabolic models

This approach is particularly relevant for understanding D. shibae's metabolic reconfiguration during oxygen depletion, where the bacterium experiences a metabolic crisis until denitrification is established . Isotopic labeling would reveal how carbon flow through mdh changes during this adaptation process and how it relates to the accumulation of storage compounds like poly-3-hydroxybutanoate .

The experimental design should include appropriate controls and consider the temporal dynamics of metabolic adaptation to capture the transition phases accurately.

What experimental approaches can determine if mdh activity influences membrane vesicle formation in D. shibae?

D. shibae continuously forms outer membrane vesicles during growth, with approximately 0.75 vesicles per cell . Investigating potential connections between mdh activity and vesicle formation requires multifaceted approaches.

Experimental strategy:

• Genetic manipulation:

  • Construction of mdh knockout, knockdown, and overexpression strains

  • Development of catalytically inactive mdh mutants

  • Creation of reporter strains with fluorescently tagged mdh

• Vesicle characterization:

  • Quantification of vesicle production in modified strains

  • Size distribution analysis using electron microscopy and nanoparticle tracking

  • Proteomics and lipidomics comparison of vesicles from wild-type and modified strains

• Metabolic manipulation:

  • Growth with different carbon sources affecting mdh activity

  • Modulation of NAD+/NADH ratios to affect mdh reaction direction

  • Inhibitor studies targeting mdh activity

• Microscopy approaches:

  • Fluorescence microscopy to track mdh localization during vesicle formation

  • Correlative light and electron microscopy

  • Super-resolution techniques to examine co-localization with vesicle formation sites

Research has shown that some abundant D. shibae vesicle membrane proteins are involved in cell division, including those interacting with peptidoglycan during division (LysM, Tol-Pal, SpoI, lytic murein transglycosylase) . Investigating potential interactions between mdh and these proteins could reveal mechanisms connecting central metabolism to vesicle formation.

The experimental design should consider that vesicle formation peaks at the end of exponential growth phase , suggesting that growth stage-specific sampling is essential for capturing relevant interactions.

How can structural studies of D. shibae mdh inform its role in metabolic adaptation?

Structural characterization of D. shibae mdh can provide critical insights into its adaptation mechanisms and functional versatility.

Comprehensive structural investigation would include:

• Structure determination approaches:

  • X-ray crystallography of recombinant mdh

  • Cryo-electron microscopy for oligomeric state visualization

  • NMR spectroscopy for dynamic regions

  • Hydrogen-deuterium exchange mass spectrometry for conformational changes

• Functional correlation studies:

  • Structures with bound substrates, products, and regulators

  • Enzyme variants with altered catalytic properties

  • Comparison of structures under conditions mimicking aerobic versus anaerobic states

• Analytical techniques:

  • Circular dichroism spectroscopy for secondary structure assessment

  • Differential scanning calorimetry for thermal stability

  • Size-exclusion chromatography with multi-angle light scattering for oligomeric state

  • Small-angle X-ray scattering for solution structure

• Computational approaches:

  • Molecular dynamics simulations to assess flexibility and conformational changes

  • Protein-protein interaction modeling

  • Quantum mechanics/molecular mechanics (QM/MM) for reaction mechanism studies

Given D. shibae's metabolic versatility, including its ability to transition between aerobic and anaerobic metabolism , structural studies should examine potential conformational changes or modified interactions that might occur during these transitions. The protein's adaptation to marine environments might also be reflected in salt-specific structural features.

Results from structural studies can guide rational engineering efforts and provide insights into evolutionary adaptations that enable D. shibae's metabolic flexibility.

How should contradictory kinetic data for recombinant D. shibae mdh be interpreted and resolved?

Contradictory kinetic data for recombinant D. shibae mdh requires systematic investigation to identify sources of variation and determine the most reliable parameters.

Resolution framework:

• Methodological standardization:

  • Comparison of assay conditions (buffer, pH, temperature, ionic strength)

  • Evaluation of protein preparation methods (expression system, purification protocol, storage)

  • Assessment of assay detection methods (direct vs. coupled, spectrophotometric vs. fluorometric)

• Statistical analysis:

  • Meta-analysis of multiple independent measurements

  • Outlier detection and identification of systematic biases

  • Calculation of confidence intervals for kinetic parameters

• Experimental validation:

  • Reproduction of contradictory results using identical conditions

  • Systematic variation of single parameters to identify critical factors

  • Complementary techniques to corroborate findings (e.g., isothermal titration calorimetry)

• Biological context consideration:

  • Comparison with in vivo observations

  • Assessment of physiological relevance of experimental conditions

  • Evaluation of potential post-translational modifications affecting activity

Common sources of contradictory kinetic data include:

• Use of different recombinant constructs (e.g., with/without tags)

• Variations in assay temperature or pH

• Different buffer compositions affecting activity

• Presence of inhibitory contaminants in preparations

• Oligomeric state differences between preparations

For D. shibae specifically, the enzyme's marine origin might make it particularly sensitive to ionic strength and salt composition, potentially explaining contradictory results obtained under different buffer conditions.

How can multi-omics data be integrated to understand mdh's role in D. shibae's metabolic network?

Understanding mdh's position and regulation within D. shibae's metabolic network requires integration of multiple omics datasets to build a comprehensive systems-level view.

Integrative analysis framework:

• Data collection and processing:

  • Transcriptomic data for gene expression patterns

  • Proteomic data for protein abundance and modifications

  • Metabolomic data for substrate/product levels

  • Fluxomic data for pathway activities

• Correlation analyses:

  • Calculation of Pearson or Spearman correlations between mdh expression/activity and other metabolic components

  • Network analysis to identify co-regulated genes and proteins

  • Time-lagged correlations to detect regulatory relationships

• Pathway mapping and enrichment:

  • Overlay of multi-omics data on metabolic pathway maps

  • Flux balance analysis incorporating enzyme kinetics

  • Identification of metabolic bottlenecks and critical control points

• Integration with physiological data:

  • Correlation with growth parameters and nutrient utilization

  • Association with adaptation to environmental stressors

  • Connection to ecological observations

For D. shibae, research has shown significant metabolic adaptation during oxygen depletion, with establishment of denitrification machinery and transient reduction in central metabolism . Multi-omics integration should focus on these transition periods to understand how mdh activity is coordinated with these adaptations.

Particular attention should be paid to the relationship between mdh and the accumulation of storage compounds like poly-3-hydroxybutanoate during anoxic conditions , as this may represent an important metabolic strategy for managing reducing equivalents.

What computational approaches can predict the effects of mutations on D. shibae mdh structure and function?

Computational prediction of mutation effects on D. shibae mdh provides a powerful tool for guiding experimental work and understanding structure-function relationships.

Comprehensive predictive framework:

• Sequence-based approaches:

  • Conservation analysis across homologous proteins

  • Evolutionary coupling analysis to identify co-evolving residues

  • Machine learning methods trained on known mutation effects

  • Sequence-based stability predictors (I-Mutant, MUpro)

• Structure-based approaches:

  • Homology modeling if crystal structure is unavailable

  • Molecular dynamics simulations of wild-type and mutant proteins

  • Free energy calculations to estimate stability changes

  • Protein design algorithms to predict compensatory mutations

• Enzyme-specific predictions:

  • Active site geometry and electrostatic analysis

  • Substrate docking and binding energy calculations

  • Transition state modeling for catalytic efficiency prediction

  • Allosteric site identification and communication pathway analysis

• Validation approaches:

  • Retrospective analysis of known mutation effects

  • Consensus predictions from multiple algorithms

  • Sensitivity analysis to identify robust predictions

For D. shibae mdh, computational predictions should consider the enzyme's adaptation to marine environments and its role in metabolic flexibility. Mutations affecting salt tolerance, temperature adaptation, or regulatory interactions would be particularly relevant given the bacterium's ecological niche and metabolic versatility .

The predictive models should be iteratively refined with experimental validation, creating a feedback loop that improves prediction accuracy over time.

How can researchers distinguish between direct and indirect effects of mdh mutations on D. shibae physiology?

Distinguishing direct from indirect effects of mdh mutations on D. shibae physiology requires careful experimental design and comprehensive phenotypic analysis.

Methodological approach:

• Construction of precise genetic variants:

  • Site-directed mutagenesis targeting specific functional residues

  • Catalytically inactive variants that maintain structure

  • Complementation strains to verify phenotype rescue

  • Expression-matched variants to control for protein level effects

• Multi-level phenotypic characterization:

  • Enzyme activity measurements (direct effect)

  • Metabolite profiling to identify pathway perturbations (primary indirect effects)

  • Transcriptomics to detect compensatory responses (secondary indirect effects)

  • Growth and physiological parameters (tertiary indirect effects)

• Time-resolved analysis:

  • Immediate effects following inducible expression/repression

  • Monitoring adaptation responses over time

  • Distinguishing acute from chronic effects

• Comparative analysis:

  • Contrasting effects of different mutations affecting the same property

  • Comparison with inhibitor studies targeting mdh

  • Parallel analysis of mutations in metabolically connected enzymes

For D. shibae specifically, researchers should focus on how mdh mutations affect:

• Adaptation to oxygen depletion and establishment of denitrification

• Accumulation of storage compounds like poly-3-hydroxybutanoate

• Potential symbiotic interactions with dinoflagellates

• Outer membrane vesicle formation and content

The temporal sequence of observed effects can help distinguish direct consequences (occurring immediately) from indirect regulatory or metabolic adaptations (occurring after a delay).

How can isotopomer distribution analysis be used to quantify flux through mdh-catalyzed reactions in D. shibae?

Isotopomer distribution analysis provides detailed insights into metabolic flux through specific reactions, including those catalyzed by mdh, offering a powerful tool for understanding D. shibae metabolism.

Methodological framework:

• Experimental design:

  • Selection of appropriate labeled substrates (e.g., [1-13C]glucose, [U-13C]malate)

  • Steady-state labeling versus dynamic isotope incorporation

  • Sampling strategy for metabolite extraction

  • Consideration of biological replicates and technical controls

• Analytical approaches:

  • GC-MS or LC-MS for isotopomer distribution measurement

  • NMR spectroscopy for positional isotope incorporation

  • Fragment analysis for detailed positional information

  • Isotope dilution analysis for absolute flux quantification

• Data processing and modeling:

  • Correction for natural isotope abundance

  • Application of metabolic flux analysis algorithms

  • Construction of mathematical models incorporating enzyme kinetics

  • Sensitivity analysis to identify key parameters

• Biological interpretation:

  • Comparison of flux distributions under different conditions

  • Integration with enzyme expression and activity data

  • Identification of metabolic control points

  • Correlation with physiological observations

For D. shibae, isotopomer analysis would be particularly valuable for understanding:

• Carbon flow during transitions between aerobic and anaerobic metabolism

• Relative contributions of different pathways during denitrification

• Metabolic interactions during symbiosis with dinoflagellates

• Sources of carbon for storage compounds like poly-3-hydroxybutanoate under anoxic conditions

The analysis should account for D. shibae's use of the Entner-Doudoroff pathway instead of conventional glycolysis , as this affects the pattern of labeled carbons flowing through central metabolism.