Recombinant Geobacter sulfurreducens UDP-N-acetylglucosamine--N-acetylmuramyl- (pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase (murG)

What is Geobacter sulfurreducens and why is it significant for recombinant protein studies?

Geobacter sulfurreducens is an electroactive bacterium that grows on metallic minerals by transferring electrons to them, effectively "breathing" metals. In engineered systems, it can respire electrodes to produce measurable electric current. Its unique metabolism depends heavily on an extensive network of cytochromes, requiring a distinctive cell composition . As a model organism for electroactive microorganisms, G. sulfurreducens bridges the gap between biology and electrical signals, with significant impact on the global iron cycle as a ubiquitous iron reducer in soils .

The organism's unique properties include:

• High lipid content indicated by elevated C:O (1.7:1) and H:O (0.25:1) ratios

• Significantly higher iron content compared to model organisms like E. coli

• Over 100 cytochromes facilitating electron transfer to the cell exterior

• Remarkable adaptability to different growth substrates through genetic mutations

These characteristics make G. sulfurreducens an intriguing but challenging organism for recombinant protein expression, particularly for studying essential enzymes like MurG that may have adapted to its unique cellular environment.

What is the function of MurG in bacterial cell wall biosynthesis?

MurG is an essential glycosyltransferase that forms the glycosidic linkage between N-acetyl muramyl pentapeptide and N-acetyl glucosamine during peptidoglycan biosynthesis . This enzyme belongs to a major superfamily of NDP-glycosyltransferases, utilizing UDP-GlcNAc (UDP-N-acetylglucosamine) as its donor substrate . The reaction catalyzed by MurG is a crucial step in cell wall formation, directly affecting bacterial cell integrity and survival.

What methodological approaches are recommended for purifying recombinant proteins from G. sulfurreducens?

Based on successful examples with other G. sulfurreducens proteins, the following methodological approaches are recommended:

• Expression system selection:

  • E. coli expression systems have proven effective when properly modified

  • For cytochrome proteins, co-expression with the cytochrome c maturation gene cluster (ccmABCDEFGH) on a separate plasmid is essential

  • Consider untagged constructs, as N-terminal His-tags have been shown to interfere with proper maturation of some G. sulfurreducens proteins

• Optimization guidelines:

  • For cytochromes, aerobic culture conditions can yield up to 6 mg/L of fully matured protein

  • Carefully assess the impact of affinity tags on protein folding and function

  • Verify proper folding by comparing spectroscopic properties with native proteins

• Purification verification:

  • Confirm molecular weight matches the native protein

  • For cytochromes, verify proper spectral characteristics in both reduced and oxidized forms

  • Validate functional activity (e.g., metal ion reduction capability for cytochromes)

  • Consider small-angle X-ray scattering to confirm proper folding matches structural predictions

When expressing MurG specifically, researchers should consider its potential membrane association and develop appropriate solubilization and purification strategies based on protocols established for other glycosyltransferases.

How should researchers optimize E. coli expression systems for recombinant G. sulfurreducens MurG production?

When developing an E. coli-based expression system for recombinant G. sulfurreducens MurG, researchers should consider the following optimization strategies:

• Construct design considerations:

  • Evaluate both tagged and untagged versions, as affinity tags can sometimes interfere with proper folding

  • If using tags, consider C-terminal rather than N-terminal placement based on success with other G. sulfurreducens proteins

  • Design constructs with appropriate promoters that allow controlled expression levels

  • Consider codon optimization for E. coli expression

• Expression strain selection:

  • Choose E. coli strains optimized for membrane protein expression if MurG shows membrane association

  • Consider strains with enhanced disulfide bond formation capabilities if structural analysis suggests disulfide bonds

  • Evaluate strains with additional chaperones to assist protein folding

• Growth and induction parameters:

  • Test multiple induction temperatures (typically lower temperatures favor proper folding)

  • Optimize inducer concentration and induction timing

  • Consider extended expression times at lower temperatures

• Purification strategy development:

  • Design a multi-step purification process including initial capture, intermediate purification, and polishing steps

  • Evaluate different detergents for membrane protein solubilization if needed

  • Include stabilizing additives based on G. sulfurreducens' unique cellular environment, particularly metals like iron

• Functional verification methods:

  • Develop activity assays using UDP-GlcNAc as donor substrate

  • Confirm proper folding through circular dichroism and thermal stability assays

  • Validate substrate binding through isothermal titration calorimetry or surface plasmon resonance

This systematic approach will help identify optimal conditions for producing functional recombinant G. sulfurreducens MurG.

What analytical techniques should be employed to verify proper folding and function of recombinant MurG?

To ensure proper folding and function of recombinant G. sulfurreducens MurG, researchers should employ a comprehensive suite of analytical techniques:

• Structural characterization:

  • Circular dichroism (CD) spectroscopy to assess secondary structure elements

  • Fluorescence spectroscopy to evaluate tertiary structure through intrinsic tryptophan fluorescence

  • Thermal shift assays to determine protein stability and proper folding

  • Limited proteolysis to probe domain organization and folding quality

  • Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) to assess oligomeric state

• Functional characterization:

  • Enzyme activity assays measuring the formation of the glycosidic linkage

  • Substrate binding studies using isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR)

  • Kinetic analysis to determine Km, kcat, and catalytic efficiency parameters

  • Inhibition studies with known glycosyltransferase inhibitors as functional probes

• Biophysical stability assessments:

  • Differential scanning calorimetry to determine thermal stability

  • Differential scanning fluorimetry to identify stabilizing buffer conditions

  • Long-term stability studies under various storage conditions

  • Aggregation analysis using dynamic light scattering

• Structural comparison with homologs:

  • Homology modeling based on the E. coli MurG crystal structure (2.5 Å resolution)

  • Potential crystallization trials to determine the actual structure

  • Structural alignments with other bacterial MurG enzymes to identify unique features

Researchers should use E. coli MurG as a positive control in these analyses where possible, as its structure and function have been well characterized, including in complex with its donor substrate UDP-GlcNAc .

How does the high metal content of G. sulfurreducens affect experimental design for recombinant protein production?

G. sulfurreducens has significantly higher metal content compared to model organisms like E. coli, particularly iron . This characteristic necessitates specific considerations when designing experiments for recombinant protein production:

• Media composition adjustments:

  • Standard G. sulfurreducens medium may only support cell densities of ~0.1 g/L due to iron limitation

  • Supplementation with iron, copper, and zinc may be necessary to achieve higher cell densities

  • Consider potential metal precipitation issues, as observed with copper in G. sulfurreducens samples

• Expression host modifications:

  • When using E. coli as an expression host, growing it in Geobacter medium results in significant increases in Cu, Fe, Mn, and Se content compared to standard minimal media

  • Evaluate whether these metal content changes affect recombinant protein quality and yield

• Protein purification considerations:

  • Develop purification strategies that account for potential metal co-purification

  • Include appropriate metal chelators or preservatives in buffers when needed

  • Analyze metal content of purified recombinant proteins to ensure proper incorporation

• Functional assessment adjustments:

  • Test enzyme activity under varying metal concentrations to identify optimal conditions

  • Consider the impact of different metals on protein stability and oligomerization

  • Evaluate whether the recombinant MurG requires specific metals for proper folding or function

The table below summarizes key metals in G. sulfurreducens and their potential impact on recombinant protein production:

By accounting for these metal-related factors, researchers can design more effective experimental approaches for recombinant G. sulfurreducens MurG production.

How might the structure and function of G. sulfurreducens MurG differ from E. coli MurG given their distinct cellular environments?

The unique cellular environment of G. sulfurreducens likely influences the structure and function of its MurG enzyme compared to the well-characterized E. coli counterpart. Key differences may include:

• Adaptations to lipid-rich environment:

  • G. sulfurreducens has unusually high lipid content with C:O and H:O ratios of approximately 1.7:1 and 0.25:1, respectively

  • MurG interacts with membrane-bound lipid II substrate, so these lipid differences could necessitate structural adaptations in substrate-binding regions

  • Higher hydrophobicity in membrane-interaction domains might be expected

• Metal coordination capabilities:

  • Given G. sulfurreducens' high iron content and metal-reducing capabilities, its MurG might have evolved metal-binding sites not present in E. coli MurG

  • These potential metal-binding sites could influence protein stability or even catalytic activity

  • Structural elements that protect the enzyme from potential interference by high metal concentrations might be present

• Substrate specificity variations:

  • While the core catalytic mechanism would be conserved, the E. coli MurG structure suggests that variation in two key loops can create new glycosyltransferase specificities

  • G. sulfurreducens MurG might have evolved loop variations that optimize function in its unique cellular context

  • These variations could affect substrate binding affinity, catalytic efficiency, or even expand substrate range

• Regulatory interfaces:

  • Different protein-protein interaction surfaces might exist to integrate cell wall synthesis with G. sulfurreducens' extensive electron transfer machinery

  • Potential regulatory sites responding to the metal-rich environment could have evolved

  • Adaptation to the unique stress conditions experienced by G. sulfurreducens in its natural environment

Experimental approaches to investigate these potential differences would include comparative structural analysis, detailed enzyme kinetics under varying metal and lipid conditions, and chimeric enzyme studies swapping domains between G. sulfurreducens and E. coli MurG.

What research approaches can identify potential interactions between MurG activity and the extensive cytochrome network in G. sulfurreducens?

Investigating potential crosstalk between MurG-mediated cell wall synthesis and G. sulfurreducens' extensive cytochrome network requires sophisticated research approaches spanning from molecular to systems levels:

• Co-expression and localization studies:

  • Fluorescent protein tagging to track co-localization of MurG with cytochromes during growth

  • Membrane fractionation followed by proteomic analysis to identify physical proximity

  • Immunoprecipitation studies to detect direct or indirect protein-protein interactions

  • Bacterial two-hybrid screening for potential interaction partners

• Mutant phenotype analysis:

  • Creation of MurG conditional mutants to examine effects on cytochrome expression and localization

  • Analysis of cytochrome mutants for cell wall composition changes

  • Measurement of electron transfer capabilities when MurG expression is modulated

  • Growth and survival assays under various electron acceptor conditions

• Systems biology approaches:

  • Transcriptomic analysis comparing gene expression profiles under conditions where either MurG activity or cytochrome function is altered

  • Metabolomic studies to identify changes in metabolic fluxes linking cell wall synthesis and electron transfer

  • Network analysis to identify potential regulatory nodes connecting these pathways

  • Flux balance analysis incorporating both cell wall synthesis and electron transfer pathways

• Biophysical characterization:

  • Atomic force microscopy to examine cell surface properties when MurG activity is modulated

  • Electrochemical measurements of intact cells with altered MurG expression

  • In situ NMR to track metabolic fluxes between these pathways

  • Biofilm formation assays on electrodes with varying MurG expression levels

These complementary approaches would provide a comprehensive understanding of how cell wall synthesis through MurG activity might be integrated with the extensive cytochrome network that defines G. sulfurreducens' unique electron transfer capabilities.

How can adaptive laboratory evolution techniques be applied to enhance recombinant MurG production or activity?

Adaptive laboratory evolution (ALE) offers powerful approaches for enhancing recombinant MurG production and activity, as demonstrated by successful evolution of G. sulfurreducens for improved lactate metabolism . The following methodological framework can be applied:

• Selection strategy design:

  • Create selective pressure directly targeting MurG function by using cell wall synthesis inhibitors at sub-lethal concentrations

  • Design growth conditions where enhanced MurG activity provides a fitness advantage

  • Establish screening methods to identify colonies with improved MurG expression or activity

• Parallel evolution approach:

  • Conduct multiple parallel evolution experiments to identify convergent mutations, as seen in the lactate utilization evolution where five parallel cultures developed mutations in the same gene (GSU0514)

  • Apply different selection pressures across parallel cultures to explore diverse adaptation routes

  • Vary starting strains to explore different genetic backgrounds

• Genomic analysis workflow:

  • Perform whole-genome sequencing of evolved strains to identify mutations

  • Focus particularly on mutations in transcriptional regulators, as these can significantly impact gene expression

  • Validate causal mutations by introducing specific single-base-pair mutations into wild-type strains

• Molecular characterization:

  • Measure transcript abundance changes for MurG and related genes in evolved strains

  • Perform DNA-binding assays with identified transcriptional regulators to map regulatory networks

  • Characterize enzyme kinetics and stability in evolved variants

The effectiveness of this approach is demonstrated by previous work with G. sulfurreducens, where a single-base-pair mutation in a transcriptional regulator (GSU0514) led to significantly improved lactate utilization through increased expression of key metabolic enzymes like succinyl-CoA synthase . Similar mutations affecting MurG expression or activity could be identified through carefully designed ALE experiments.

How can researchers resolve conflicting data regarding metal content and distribution in G. sulfurreducens?

The scientific literature contains conflicting results regarding metal content in G. sulfurreducens, with some studies showing similar metal content to E. coli when grown on fumarate, while others report an order of magnitude higher iron content . To resolve these contradictions, researchers should implement the following methodological approaches:

• Standardized growth protocols:

  • Establish consistent growth media composition with precise metal concentrations

  • Define standard harvest points based on growth phase rather than arbitrary time points

  • Compare identical growth conditions across different electron acceptors (fumarate, Fe(III), electrode)

  • Document precise medium composition, including trace elements and potential contaminating metals

• Comprehensive analytical methods:

  • Employ multiple complementary metal analysis techniques (ICP-MS, ICP-OES, atomic absorption)

  • Develop washing protocols that remove extracellular precipitates without leaching cellular metals

  • Include appropriate certified reference materials for calibration

  • Report detailed method validation parameters including limits of detection, recovery rates, and precision metrics

• Cellular fractionation approaches:

  • Develop gentle fractionation methods to separate periplasmic, cytoplasmic, and membrane fractions

  • Analyze metal distribution across cellular compartments

  • Use multiple fractionation techniques to validate results

  • Control for potential metal redistribution during fractionation

• Statistical rigor implementation:

  • Perform adequate biological and technical replicates (minimum n=3 for each)

  • Apply appropriate statistical tests to determine significant differences

  • Report variability measures (standard deviation, confidence intervals)

  • Consider meta-analysis across multiple studies when possible

By implementing these standardized approaches, researchers can resolve contradictions and establish a consensus on the true metal content and distribution in G. sulfurreducens under various growth conditions, which is essential for understanding how this environment might affect MurG function .

What experimental designs can distinguish between direct and indirect effects of genetic mutations on MurG function?

To distinguish between direct and indirect effects of genetic mutations on MurG function, researchers should implement multi-level experimental designs that link genotype to phenotype through mechanistic understanding:

• Genetic confirmation strategies:

  • Introduce the specific mutation into wild-type strains to confirm causality, as demonstrated with lactate utilization mutations

  • Create revertants to verify that phenotype loss corresponds with mutation correction

  • Construct allelic series with varying mutations in the same gene to establish structure-function relationships

  • Implement complementation studies with wild-type genes

• Molecular mechanism determination:

  • Perform DNA-binding assays to identify if mutated transcriptional regulators directly bind to the murG promoter region

  • Measure transcript abundance changes for murG and related genes using RT-qPCR or RNA-seq

  • Use reporter gene assays to monitor promoter activity with and without mutations

  • Implement ChIP-seq to map genome-wide binding patterns of transcription factors

• Protein-level analyses:

  • Quantify MurG protein levels through western blotting or targeted proteomics

  • Assess MurG enzymatic activity directly using purified proteins from mutant and wild-type strains

  • Determine post-translational modifications that might be affected by mutations

  • Evaluate protein-protein interactions that could be altered

• Systems-level integration:

  • Perform metabolic flux analysis to determine how mutations affect cell wall precursor availability

  • Map epistatic interactions through double-mutant analysis

  • Conduct suppressor screens to identify genes that can compensate for mutations

  • Implement comparative multi-omics to build comprehensive models of mutation effects

This multi-level approach would provide robust evidence to distinguish between mutations directly affecting MurG structure or function versus those indirectly affecting MurG through regulatory networks or metabolic shifts, similar to how mutations in GSU0514 were shown to affect lactate metabolism indirectly through altered succinyl-CoA synthase expression .

How can researchers accurately measure the kinetic parameters of recombinant MurG in experimental settings that mimic G. sulfurreducens' unique cellular environment?

To accurately determine kinetic parameters of recombinant MurG under conditions that reflect G. sulfurreducens' unique cellular environment, researchers should implement the following methodological approaches:

• Membrane mimetic development:

  • Create liposome compositions that reflect G. sulfurreducens' unusual lipid content (high C:O and H:O ratios)

  • Test various membrane models including nanodiscs, liposomes, and supported bilayers

  • Incorporate appropriate membrane fluidity and charge characteristics

  • Compare activity in different membrane models to identify optimal reconstitution systems

• Metal environment reconstitution:

  • Systematically test enzyme activity across physiologically relevant concentrations of Fe, Cu, Mn, and other metals found in G. sulfurreducens

  • Develop buffer systems that maintain metal solubility while preventing precipitation

  • Include appropriate redox components to maintain metals in their native oxidation states

  • Use metal chelators as controls to determine metal dependency

• Advanced enzyme kinetics approaches:

  • Implement progress curve analysis rather than initial rate measurements for more robust parameter estimation

  • Develop continuous assays for real-time monitoring of reaction progress

  • Apply global fitting of multiple experiments to constrain parameter estimation

  • Account for potential product inhibition and substrate depletion effects

• Coupling reaction system optimization:

  • Design continuous coupled enzyme assays to monitor MurG activity in real-time

  • Validate that coupling enzymes function properly under experimental conditions

  • Ensure coupling reactions are not rate-limiting

  • Include appropriate controls to correct for background reactions

By implementing these approaches, researchers can obtain kinetic parameters that accurately reflect MurG function in its native G. sulfurreducens environment, providing insights into how this enzyme has adapted to the organism's unique cellular conditions.

How might bioelectrochemical systems benefit from fundamental research on G. sulfurreducens MurG?

Fundamental research on G. sulfurreducens MurG could advance bioelectrochemical systems through several innovative applications:

• Engineered cell-electrode interfaces:

  • Understanding how cell wall composition affects electron transfer could enable rational design of bacteria with enhanced electrode interaction

  • MurG modifications might allow controlled cell wall permeability for improved electron shuttling

  • Knowledge of how peptidoglycan structure influences cytochrome localization could inform electrode surface modifications

• Biosensor development:

  • Insights into MurG structure and regulation could enable design of whole-cell biosensors with improved signal transduction

  • Understanding the relationship between cell wall synthesis and electron transfer might allow creation of sensitive detection systems for antimicrobials targeting cell wall synthesis

  • Knowledge of how G. sulfurreducens integrates cell envelope maintenance with electron transfer could inspire new sensor architectures

• Biofilm engineering on electrodes:

  • Understanding how MurG activity influences biofilm formation and stability could enable design of more robust electroactive biofilms

  • Controlled modification of cell wall properties might enhance electron transfer in multilayer biofilms

  • Insights into cell-cell interactions mediated by the cell wall could improve biofilm conductivity

• Enhanced microbial fuel cell performance:

  • Fundamental knowledge of how cell wall properties affect electron transfer kinetics could guide optimization of power output

  • Understanding MurG regulation under various electron acceptor conditions might allow tuning expression for optimal performance

  • Insights into metal-cell wall interactions could improve electrode materials and interfaces

The comprehensive understanding of G. sulfurreducens' cell wall biosynthesis could ultimately lead to engineered strains with optimized properties for specific bioelectrochemical applications, similar to how adaptive evolution has already improved substrate utilization in this organism .

What novel approaches could resolve the structure-function relationship between cell wall composition and electron transfer in G. sulfurreducens?

Resolving the intricate relationship between cell wall composition and electron transfer in G. sulfurreducens requires innovative experimental approaches spanning multiple scales:

• Advanced imaging technologies:

  • Correlative light and electron microscopy (CLEM) to visualize cytochrome distribution relative to cell wall architecture

  • High-resolution atomic force microscopy with conductive probes to map electron transfer sites on the cell surface

  • Cryo-electron tomography to visualize the native arrangement of cell wall components and cytochromes

  • Super-resolution microscopy with specific labeling of cell wall components and electron transfer proteins

• In situ structural biology techniques:

  • Solid-state NMR to determine peptidoglycan structure in intact cells under electron transfer conditions

  • Neutron scattering to distinguish between protein and lipid components at the cell-electrode interface

  • X-ray footprinting to map solvent accessibility changes during electron transfer

  • Cross-linking mass spectrometry to identify proximity relationships between cell wall components and electron transfer proteins

• Synthetic biology approaches:

  • Creation of minimal cell wall systems with defined composition to identify essential features for electron transfer

  • Development of chimeric organisms with hybrid cell walls to isolate functional components

  • Controlled expression of MurG variants to modulate cell wall properties

  • Design of synthetic electron conduits integrated into engineered cell walls

• Real-time monitoring systems:

  • Development of fluorescent peptidoglycan probes to track cell wall synthesis during electron transfer

  • Integration of electrical measurements with live-cell microscopy

  • Implementation of microfluidic devices that allow rapid alteration of electron acceptor availability while monitoring cell wall dynamics

  • Creation of reporter systems that respond to both cell wall stress and electron transfer efficiency

These innovative approaches would provide unprecedented insights into how G. sulfurreducens' cell wall, influenced by MurG activity, facilitates its remarkable electron transfer capabilities. This knowledge could then inform rational design of enhanced bioelectrochemical systems and expand our understanding of this unique biological interface between cellular metabolism and extracellular electron acceptors.