Recombinant Geobacter sulfurreducens Molybdenum cofactor biosynthesis protein C (moaC)

What is the role of moaC in molybdenum cofactor biosynthesis in G. sulfurreducens?

Molybdenum cofactor biosynthesis protein C (moaC) in G. sulfurreducens is involved in the first step of molybdenum cofactor (Moco) biosynthesis, which converts GTP to cyclic pyranopterin monophosphate (cPMP). This initial step is critical as Moco biosynthesis involves the complex interaction of six proteins across four distinct steps, requiring iron, ATP, and copper as cofactors . In G. sulfurreducens, moaC functions within the conserved biosynthetic pathway that enables the bacterium to synthesize the molybdenum cofactor necessary for enzymes involved in various metabolic processes, including anaerobic respiration.

How does G. sulfurreducens' oxygen tolerance influence molybdenum cofactor biosynthesis?

G. sulfurreducens was originally classified as a strict anaerobe but has since been confirmed to tolerate and even utilize oxygen as a terminal electron acceptor under specific conditions . When oxygen is present at moderate levels (below the maximum specific oxygen uptake rate of 95 mg O₂ g CDW⁻¹ h⁻¹), G. sulfurreducens can grow to the same extent as with fumarate . This oxygen tolerance may significantly impact molybdenum cofactor biosynthesis, as transcriptome analysis reveals different survival strategies depending on oxygen concentration. These strategies include attempting to escape microaerobic areas at low oxygen levels, focusing on rapid oxygen reduction at higher concentrations, or forming protective layers when complete reduction becomes impossible . The oxygen response mechanisms likely influence the expression and activity of molybdenum cofactor biosynthesis proteins, including moaC, which may be differentially regulated under varying oxygen conditions to maintain proper enzyme function.

What genomic features characterize the molybdenum cofactor biosynthesis pathway in G. sulfurreducens?

While G. sulfurreducens possesses a homolog of the moaE gene (GSU2699) encoding the large subunit of molybdopterin synthase, it notably lacks homologs of the small subunit gene . This genomic arrangement suggests a potentially unique configuration of the molybdenum cofactor biosynthesis pathway in G. sulfurreducens. Vestiges of the molybdate (ModE) regulon are detectable in related species such as G. metallireducens, which has lost the global regulatory protein ModE but retained some putative ModE-binding sites while multiplying certain genes involved in molybdenum cofactor biosynthesis . This evolutionary divergence indicates that G. sulfurreducens may employ alternative regulatory mechanisms for controlling molybdenum cofactor biosynthesis compared to other bacteria.

What are the optimal conditions for heterologous expression of recombinant G. sulfurreducens moaC?

For optimal heterologous expression of recombinant G. sulfurreducens moaC, researchers should consider several factors:

Expression System Selection:

• E. coli BL21(DE3) strains are recommended due to their reduced protease activity and efficient T7 RNA polymerase-based expression

• Consider codon optimization for G. sulfurreducens sequences, which have different codon usage patterns than common expression hosts

Culture Conditions:

The microaerobic conditions are particularly important given that G. sulfurreducens has been shown to grow optimally with limited oxygen availability up to its maximum specific oxygen uptake rate of 95 mg O₂ g CDW⁻¹ h⁻¹ . The presence of molybdenum in the growth medium is crucial as it serves as a substrate for the expressed protein's ultimate product.

What experimental approaches are most effective for investigating moaC structure-function relationships?

To investigate structure-function relationships of G. sulfurreducens moaC, researchers should employ a multi-faceted approach:

• X-ray Crystallography and Cryo-EM Analysis:

  • Express recombinant moaC with an N-terminal His-tag for affinity purification

  • Consider protein fusion partners (MBP, SUMO) to enhance solubility

  • Screen multiple crystallization conditions with and without substrate analogs

• Site-Directed Mutagenesis:

  • Target conserved active site residues based on alignment with characterized moaC proteins

  • Employ alanine-scanning mutagenesis to identify essential residues

  • Create conservative mutations to probe specific interactions

• Enzymatic Assays:

  • Develop a coupled assay system to monitor cPMP formation

  • Use LC-MS/MS to detect and quantify reaction intermediates

  • Perform kinetic analyses under varying substrate concentrations and pH/temperature conditions

• In silico Analysis:

  • Generate homology models based on related moaC structures

  • Perform molecular dynamics simulations to examine substrate binding

  • Identify potential protein-protein interaction surfaces

This comprehensive approach allows researchers to correlate structural features with enzymatic function while providing insights into how G. sulfurreducens moaC may differ from homologous proteins in other organisms in terms of substrate specificity, catalytic efficiency, and regulation.

How does the interplay between molybdenum cofactor biosynthesis and oxygen reduction pathways affect G. sulfurreducens metabolism?

The interplay between molybdenum cofactor biosynthesis and oxygen reduction in G. sulfurreducens represents a complex metabolic coordination system:

Molybdenum cofactor biosynthesis is tightly connected to Fe-S cluster synthesis, with several molybdenum enzymes and the biosynthesis process itself depending on Fe-S enzymes . Simultaneously, G. sulfurreducens employs a menaquinol oxidase for oxygen reduction during microaerobic growth . This creates a metabolic intersection where:

• Electron Transfer Competition: Both pathways require electron donors, potentially creating competition under certain conditions.

• Transcriptional Regulation: Transcriptome analysis reveals that G. sulfurreducens employs distinct survival strategies depending on oxygen concentration , which likely includes differential regulation of molybdenum cofactor biosynthesis genes.

• Metabolic Flux Redistribution: Under microaerobic conditions (when oxygen acts as terminal electron acceptor), the TCA cycle functions as a closed loop , potentially altering the availability of metabolites needed for cofactor synthesis.

To experimentally investigate this interplay, researchers should employ:

• Transcriptomics and proteomics under varying oxygen concentrations

• Metabolic flux analysis using isotope labeling

• Construction of reporter strains with fluorescent proteins linked to the promoters of key genes in both pathways

• Gene knockout studies targeting specific components of either pathway to observe metabolic compensation mechanisms

Understanding this interplay is crucial for optimizing G. sulfurreducens-based biotechnological applications, particularly in environmental settings where oxygen gradients exist.

What purification strategy yields the highest activity of recombinant G. sulfurreducens moaC?

A systematic purification strategy for obtaining high-activity recombinant G. sulfurreducens moaC involves multiple steps:

Step 1: Initial Extraction

• Harvest cells under microaerobic conditions (to maintain protein in native state)

• Use gentle lysis methods (e.g., lysozyme treatment followed by sonication) in buffer containing:

  • 50 mM Tris-HCl (pH 8.0)

  • 150 mM NaCl

  • 10% glycerol

  • 1 mM DTT

  • Protease inhibitor cocktail

Step 2: Initial Purification

Step 3: Secondary Purification

• Size exclusion chromatography using Superdex 75/200 in buffer containing:

  • 25 mM HEPES (pH 7.5)

  • 100 mM NaCl

  • 5% glycerol

  • 0.5 mM DTT

Step 4: Activity Preservation

• Add stabilizing agents to the final preparation:

  • 1 mM DTT (to maintain reduced state)

  • 10% glycerol (cryoprotectant)

  • Consider addition of 10 μM molybdate

Critical Considerations:

• Maintain microaerobic or anaerobic conditions throughout purification, as G. sulfurreducens proteins may be sensitive to high oxygen levels

• Monitor activity at each purification step using a specific enzymatic assay

• Consider tag removal if the affinity tag affects activity

• Storage in small aliquots at -80°C with flash freezing in liquid nitrogen

This approach addresses the unique characteristics of G. sulfurreducens proteins, including potential oxygen sensitivity and the requirement for specific cofactors.

How should researchers design transcriptomic experiments to investigate moaC regulation under different electron acceptor conditions?

For robust transcriptomic investigation of moaC regulation under different electron acceptor conditions:

Experimental Design:

• Growth Conditions:

  • Culture G. sulfurreducens with acetate as carbon source and varying terminal electron acceptors:

    • Fumarate (anaerobic control)

    • Fe(III) citrate (anaerobic, metal reduction)

    • Oxygen at various concentrations (1%, 2%, 5% headspace)

    • No electron acceptor (stress condition)

• Sampling Strategy:

  • Collect samples at multiple growth phases:

    • Early exponential (OD600 ~0.2)

    • Mid-exponential (OD600 ~0.5)

    • Late exponential (OD600 ~0.8)

    • Early stationary phase

• RNA Extraction Protocol:

  • Immediate sample preservation (RNAlater or flash freezing)

  • Phenol-chloroform extraction with DNase treatment

  • Quality control via Bioanalyzer (RIN > 8.0)

• Sequencing Approach:

  • RNA-Seq with rRNA depletion (not poly-A selection)

  • Minimum 20 million reads per sample

  • Include 3-5 biological replicates per condition

Data Analysis Workflow:

• Quality trimming and adapter removal

• Alignment to G. sulfurreducens genome

• Differential expression analysis using DESeq2 or EdgeR

• Co-expression network analysis to identify genes regulated similarly to moaC

• Promoter analysis of co-regulated genes to identify common regulatory elements

Validation Approaches:

• RT-qPCR of moaC and related genes

• Promoter-reporter fusions (if genetic tools available)

• Chromatin immunoprecipitation (ChIP) to identify regulatory proteins

This design specifically addresses the unique physiology of G. sulfurreducens, considering its ability to grow with oxygen as a terminal electron acceptor up to specific concentrations , while providing a comprehensive view of how moaC regulation fits within the broader transcriptional response to different electron acceptors.

What controls should be included when testing the enzymatic activity of purified recombinant moaC?

A comprehensive set of controls is essential when assessing the enzymatic activity of purified recombinant G. sulfurreducens moaC:

Negative Controls:

• Heat-inactivated enzyme (95°C for 10 minutes)

• Reaction mixture without substrate (GTP)

• Reaction mixture without enzyme

• Purified irrelevant protein (e.g., BSA) at equivalent concentration

Positive Controls:

• Commercial moaC from related organism (if available)

• Co-expressed and purified complete molybdenum cofactor biosynthesis pathway proteins (moaA, moaC, moaD, moaE) to confirm activity in the full pathway context

Specificity Controls:

• Substrate analogs (e.g., ATP, GMP) to test substrate specificity

• Inhibitor controls (e.g., metal chelators)

• pH series (pH 6.0-9.0) to determine optimal conditions

Technical Validation Controls:

• Multiple enzyme concentrations to establish linearity of the assay

• Time-course measurements to ensure reaction occurs in linear range

• Internal standards for quantitative analyses

Analytical Method Controls:

When interpreting results, researchers should consider the unique oxygen tolerance of G. sulfurreducens and how it might affect enzyme activity. Additionally, the presence or absence of the molybdate (ModE) regulon components, as observed in comparative genomics between Geobacter species , may influence the regulation and activity of recombinant moaC.

How can researchers resolve contradictory data regarding moaC function in G. sulfurreducens?

When confronted with contradictory data regarding moaC function in G. sulfurreducens, researchers should adopt a systematic approach to data integration and reconciliation:

Step 1: Identify Sources of Contradictions

• Experimental conditions (anaerobic vs. microaerobic growth)

• Strain variations (wild-type vs. engineered strains)

• Methodology differences (in vitro vs. in vivo assays)

• Data analysis approaches

Step 2: Implement Strategic Reconciliation Methods

• Parallel Analysis Strategy:

  • Apply multiple analytical methods to the same biological samples

  • Example: Combine transcriptomics, proteomics, and enzymatic assays on cells grown under identical conditions

• Systematic Variation Approach:

  • Systematically vary one parameter at a time while controlling others

  • Create a matrix of experiments that spans the conditions where contradictions were observed

• Triangulation Method:

  • Use at least three independent methodologies to measure the same phenomenon

  • Example: Assess moaC activity using: (a) direct enzymatic assay, (b) reporter gene fusion, and (c) metabolomic analysis of pathway intermediates

Step 3: Statistical Analysis for Reconciliation

• Apply meta-analysis techniques to integrate multiple datasets

• Use Bayesian approaches to incorporate prior knowledge

• Employ multidimensional analysis to identify patterns across contradictory datasets

As noted in the research literature on contradictory data in mixed methods research, contradictions can arise from "different categories and levels of analysis, as well as contrasting explanatory logics" . These principles apply directly to biochemical and molecular biological data about moaC function, particularly when studying an organism like G. sulfurreducens that exhibits complex responses to environmental conditions .

What bioinformatic approaches are most useful for predicting protein-protein interactions involving moaC?

To predict and analyze protein-protein interactions (PPIs) involving G. sulfurreducens moaC, researchers should employ multiple complementary bioinformatic approaches:

Sequence-Based Methods:

• Co-evolution Analysis:

  • Multiple sequence alignment of moaC across related species

  • Statistical coupling analysis to identify co-evolving residues

  • Direct coupling analysis to predict contact residues

• Domain-Domain Interaction Prediction:

  • Identify conserved interaction domains

  • Query domain interaction databases (DOMINE, 3did)

  • Use hidden Markov models to predict novel interaction domains

Structure-Based Methods:

• Homology Modeling and Docking:

  • Generate structural models based on homologous proteins

  • Perform protein-protein docking simulations

  • Energy minimization of predicted complexes

• Interface Prediction:

  • Surface patch analysis for hydrophobicity, electrostatics

  • Conservation mapping to identify potential interface residues

  • Machine learning-based interface prediction (SPPIDER, IntPred)

Genomic Context Methods:

• Gene Neighborhood Analysis:

  • Examine conserved gene clusters across related species

  • Identify operonic structures containing moaC

• Gene Fusion Detection:

  • Search for fusion events involving moaC and potential partners

  • These often indicate functional interactions

Network-Based Approaches:

• Guilt by Association:

  • Leverage existing PPI networks from model organisms

  • Transfer annotations based on orthology relationships

  • Use network topology to predict functional relationships

• Integration of Multiple Evidence Types:

  • Weighted scoring systems combining different prediction methods

  • Bayesian integration of diverse data sources

For G. sulfurreducens specifically, researchers should focus on interactions with proteins involved in the molybdenum cofactor biosynthesis pathway and potential relationships with proteins involved in oxygen response, as G. sulfurreducens has been shown to tolerate and utilize oxygen under certain conditions . The connection between molybdenum metabolism and Fe-S cluster synthesis also suggests examining potential interactions with proteins involved in iron metabolism.

How should researchers interpret changes in moaC expression during biofilm formation in G. sulfurreducens?

Interpreting changes in moaC expression during G. sulfurreducens biofilm formation requires a nuanced analytical framework that accounts for the unique physiology of this organism:

Fundamental Interpretation Framework:

• Developmental Context:

  • Early biofilm stage: Changes may relate to initial attachment mechanisms

  • Mature biofilm: Alterations likely reflect metabolic adaptation to oxygen gradients

  • Biofilm dispersal: Expression shifts may indicate preparation for planktonic lifestyle

• Metabolic Significance:

  • Increased expression: May indicate enhanced requirement for molybdenum-containing enzymes

  • Decreased expression: Could suggest switch to alternative metabolic pathways

  • Temporal oscillations: May reflect cyclic changes in microenvironmental conditions

• Spatial Considerations:

  • Expression gradients across biofilm depth may correlate with:

    • Oxygen penetration (G. sulfurreducens can utilize oxygen up to specific concentrations )

    • Electron acceptor availability

    • Nutrient gradients

Analytical Approach:

Contextual Integration:

Researchers should interpret moaC expression changes in relation to:

• The three survival strategies G. sulfurreducens employs under different oxygen conditions

• The formation of protective layers when oxygen reduction becomes impossible

• The connection between molybdenum metabolism and Fe-S cluster synthesis

• The potential role of the vestigial molybdate (ModE) regulon components

Changes in moaC expression may indicate shifts in the electron transfer capabilities of the biofilm, particularly given that G. sulfurreducens can utilize oxygen as a terminal electron acceptor up to specific concentrations . This ability suggests that moaC regulation may be part of a broader metabolic adaptation strategy during biofilm development.

What are the most promising approaches for creating moaC knockout or modified strains in G. sulfurreducens?

Creating moaC knockout or modified strains in G. sulfurreducens requires specialized approaches due to the unique characteristics of this organism:

Genetic Modification Strategies:

• CRISPR-Cas9 System Adaptation:

  • Design sgRNAs targeting moaC with minimal off-target effects

  • Optimize Cas9 expression under anaerobic/microaerobic conditions

  • Deliver system via conjugation from E. coli donor strains

  • Include PAM site analysis specific to G. sulfurreducens genome

• Homologous Recombination Approach:

  • Design targeting vectors with:

    • ≥1 kb homology arms flanking moaC

    • Selectable markers functional in G. sulfurreducens

    • Counter-selection markers for markerless deletions

  • Introduce via electroporation under optimized conditions

• Conditional Expression Systems:

  • Develop inducible promoters responsive to:

    • Tetracycline or similar inducers

    • Temperature shifts

    • Specific electron acceptor presence/absence

  • Create moaC under conditional control for essential gene studies

Verification Methods:

Phenotypic Characterization:

• Test growth with different electron acceptors (fumarate, Fe(III), oxygen at various concentrations)

• Measure molybdenum cofactor levels and dependent enzyme activities

• Assess biofilm formation capabilities

• Evaluate survival under oxidative stress conditions

Given that G. sulfurreducens can grow with oxygen as a terminal electron acceptor within specific concentration limits , researchers should carefully consider the oxygen conditions when growing and characterizing moaC mutants. Additionally, the connection between molybdenum metabolism and Fe-S cluster synthesis suggests that moaC modification may have broader metabolic impacts that should be assessed comprehensively.

How might structural studies of moaC contribute to understanding G. sulfurreducens' unique metabolic capabilities?

Structural studies of moaC from G. sulfurreducens would provide significant insights into this organism's distinctive metabolic capabilities:

Fundamental Structural Investigations:

• Comparative Structural Analysis:

  • Determine crystal structure of G. sulfurreducens moaC

  • Compare with structures from strict anaerobes and aerobes

  • Identify unique structural features that may relate to oxygen tolerance

• Substrate Binding Studies:

  • Co-crystallize with substrate analogs or transition state mimics

  • Identify binding pocket adaptations specific to G. sulfurreducens

  • Characterize potential allosteric sites

• Protein-Protein Interaction Interfaces:

  • Map interaction surfaces with other molybdenum cofactor biosynthesis proteins

  • Investigate potential interactions with oxygen-responsive proteins

  • Examine oligomerization states under different oxygen conditions

Functional Implications:

The structural studies would illuminate several key aspects of G. sulfurreducens metabolism:

• Oxygen Adaptation Mechanisms:
  Given that G. sulfurreducens can utilize oxygen as a terminal electron acceptor up to specific concentrations , structural features of moaC may reveal adaptations that protect the protein and its function during oxygen exposure.

• Electron Transfer Coordination:
  Structural insights could reveal how moaC activity is coordinated with the menaquinol oxidase system implicated in oxygen reduction , potentially through shared regulatory mechanisms or protein-protein interactions.

• Metabolic Integration:
  Since molybdenum metabolism is tightly connected to Fe-S cluster synthesis , structural studies might identify features that facilitate this integration, particularly in the context of G. sulfurreducens' ability to utilize diverse electron acceptors.

Technical Approaches:

These structural insights would be particularly valuable given the unique position of G. sulfurreducens as a facultative microaerobe rather than a strict anaerobe , potentially revealing adaptations that allow molybdenum cofactor biosynthesis to function across varying oxygen conditions.

What are the most reliable methods for assessing moaC expression levels in G. sulfurreducens under different experimental conditions?

For reliable quantification of moaC expression in G. sulfurreducens under varying experimental conditions, researchers should consider multiple complementary approaches:

Transcript-Level Methods:

• RT-qPCR:

  • Design primers specific to G. sulfurreducens moaC

  • Validate efficiency and specificity under experimental conditions

  • Select stable reference genes (validated candidates include proC, rpoD)

  • Perform proper normalization accounting for G. sulfurreducens growth characteristics

• RNA-Seq:

  • Use rRNA depletion rather than poly(A) selection

  • Include spike-in controls for absolute quantification

  • Apply G. sulfurreducens-specific bias correction algorithms

  • Minimum 20M reads per sample with 3-5 biological replicates

Protein-Level Methods:

• Western Blotting:

  • Generate moaC-specific antibodies or use epitope tagging approaches

  • Validate with recombinant protein and knockout controls

  • Use internal loading controls appropriate for G. sulfurreducens

  • Apply quantitative analysis with standard curves

• Targeted Proteomics (PRM/MRM):

  • Identify unique peptides for moaC using in silico digestion

  • Synthesize isotopically labeled peptide standards

  • Optimize extraction protocols for G. sulfurreducens membrane-associated proteins

  • Perform absolute quantification using calibration curves

Reporter Systems:

• Translational Fusions:

  • Create moaC promoter-reporter constructs (GFP, luciferase)

  • Validate in varied growth conditions relevant to G. sulfurreducens

  • Consider microaerobic/anaerobic compatible reporters

  • Use flow cytometry or plate readers for quantification

Methodological Considerations:

When interpreting results, researchers should consider that G. sulfurreducens exhibits different survival strategies depending on oxygen concentration , which may influence moaC expression patterns. Additionally, the connection between molybdenum metabolism and Fe-S cluster synthesis suggests that iron availability may impact moaC expression through regulatory mechanisms.

What are the critical considerations for designing experiments involving recombinant G. sulfurreducens moaC and oxygen exposure?

When designing experiments involving recombinant G. sulfurreducens moaC and oxygen exposure, researchers must address several critical considerations:

Oxygen Concentration Management:

• Precise Oxygen Control:

  • Use specialized bioreactors with continuous O₂ monitoring

  • Maintain oxygen below the maximum specific oxygen uptake rate of 95 mg O₂ g CDW⁻¹ h⁻¹

  • Implement gradient-based experiments to identify threshold responses

  • Include rapid sampling techniques that maintain oxygen conditions

• Experimental Design Parameters:

Protein Stability and Activity:

• Oxidative Modifications:

  • Monitor potential oxidation of critical cysteine residues

  • Assess activity changes following oxygen exposure

  • Use reducing agents during purification to maintain native state

  • Consider site-directed mutagenesis of oxygen-sensitive residues

• Enzyme Kinetic Analysis:

  • Perform kinetic assays under precisely controlled oxygen conditions

  • Compare activity parameters (kcat, Km) across oxygen gradients

  • Analyze product formation using oxygen-insensitive detection methods

  • Include time-course studies to capture potential inactivation

Experimental Controls:

• Essential Positive Controls:

  • Parallel experiments with known oxygen-tolerant and oxygen-sensitive enzymes

  • Inclusion of chemical oxygen scavengers in control reactions

  • Activity measurements under strict anaerobic conditions as baseline

• Statistical Design:

  • Minimum 5-6 biological replicates per oxygen condition

  • Power analysis to determine sample size for detecting anticipated effects

  • Factorial design to assess interaction between oxygen and other variables

  • Time-series analysis to capture dynamic responses