Recombinant Geobacter sulfurreducens Catalase-peroxidase (katG), partial

What is the role of catalase-peroxidase (katG) in Geobacter sulfurreducens?

Catalase-peroxidase (katG) in G. sulfurreducens plays a critical role in oxidative stress protection by catalyzing the decomposition of hydrogen peroxide (H₂O₂) to water and oxygen. The enzyme belongs to the cellular detoxification system and has been identified as an important component for cellular processes in G. sulfurreducens . Research indicates that katG is part of a broader oxidative stress response system that helps G. sulfurreducens survive in environments with fluctuating oxygen levels, contributing to its recently recognized aerotolerance .

Methodology for investigating katG function:

• Measure catalase activity using spectrophotometric assays that monitor H₂O₂ consumption

• Create gene deletion strains (ΔkatG) and assess their survival under oxidative stress conditions

• Monitor gene expression levels of katG under varying oxygen or peroxide exposure conditions

How does G. sulfurreducens' oxidative stress response system function?

G. sulfurreducens employs multiple systems to manage oxidative stress. The recently discovered aerotolerance of this previously classified strict anaerobe highlights the importance of these systems . Research shows that periplasmic triheme cytochromes (PpcA-E) interact with the diheme cytochrome peroxidase MacA to form a redox complex that helps mitigate oxidative stress . MacA functions as the second cytochrome c peroxidase in G. sulfurreducens and has demonstrated hydrogen peroxide reductase activity with a KM of 38.5 ± 3.7 μM H₂O₂ .

In this system:

• Reduced PpcA-E cytochromes transfer electrons to oxidized MacA

• Reduction of MacA's high-potential heme triggers a conformational change

• This displaces the axial histidine of the low-potential heme with peroxidase activity

• The activated peroxidase then reduces hydrogen peroxide to water

What genetic systems are available for working with G. sulfurreducens katG?

A well-developed genetic system exists for G. sulfurreducens that can be applied to study katG. The following methodological approaches have been established :

• Transformation protocol:

  • Electroporation of cells with foreign DNA using optimized buffer conditions

  • Cell preparation includes harvesting at 4°C, washing with electroporation buffer (1 mM HEPES [pH 7.0], 1 mM MgCl₂, and 175 mM sucrose)

  • Addition of DMSO to a final concentration of 10% improves transformation efficiency

  • Minimize shearing of cells by using large-bore pipette tips

• Vector systems:

  • Two classes of broad-host-range vectors (IncQ and pBBR1) replicate in G. sulfurreducens

  • IncQ plasmid pCD342 is suitable for expression of recombinant proteins

• Gene replacement methods:

  • Single-step gene replacement protocols allow for targeted gene disruption

  • Markerless deletion techniques have been developed for clean genetic modifications

What are the optimal conditions for expressing and purifying recombinant G. sulfurreducens katG?

Based on methodologies used for similar proteins in G. sulfurreducens, recombinant expression and purification of katG should follow these parameters:

Expression system optimization:

• Heterologous expression in E. coli has been successful for other G. sulfurreducens proteins like MacA

• Consider microaerobic conditions, which have proven successful for expressing other redox proteins from G. sulfurreducens

• C-terminal histidine tagging strategies can be employed for purification using metal affinity chromatography

Purification considerations:

• Multiple forms of the protein may be observed (as seen with PgcA, which appeared as both 57 kDa and 41 kDa forms when expressed in Shewanella)

• Verify proper incorporation of heme cofactors using pyridine hemochrome assay and mass spectrometry

• Additional purification may be required to separate differentially processed forms

Activity verification:

• Hydrogen peroxide reductase activity can be measured using ABTS²⁻ as an electron donor

• Expected KM values would be similar to MacA (~38.5 μM for H₂O₂)

How does G. sulfurreducens katG compare with catalase-peroxidases from other bacteria?

Bacterial catalase-peroxidases show considerable variation in structure and function across species. The table below compares key features of catalase-peroxidases from different bacterial sources:

Methodological considerations for comparative analysis:

• Perform phylogenetic analysis of katG sequences across bacterial species

• Compare structural models using homology modeling if crystal structure is unavailable

• Conduct kinetic parameter comparison under standardized conditions

What is the relationship between katG and electron transfer pathways in G. sulfurreducens?

G. sulfurreducens is known for its extensive electron transfer network, particularly for extracellular electron transfer. Research suggests complex interactions between oxidative stress responses and electron transfer pathways:

• Integration with periplasmic electron carriers:

  • Periplasmic triheme cytochromes (PpcA-E) can transfer electrons to cytochrome peroxidases like MacA

  • PpcA-E cytochromes are crucial for electron transfer across the periplasm

  • These same cytochromes may channel electrons to katG for peroxide reduction

• Connection to central metabolism:

  • Electron flow through central metabolic pathways may influence katG activity

  • The flux distributions through central metabolism in G. sulfurreducens (shown in ) could affect NADH availability for peroxidase functions

• Regulatory crosstalk:

  • Oxidative stress and electron transfer pathway regulation likely overlap

  • Changes in electron acceptor availability may influence katG expression and activity

Experimental approaches to investigate these relationships:

• Use cyclic voltammetry to characterize electron transfer to purified katG

• Apply metabolic flux analysis to track electron flow under oxidative stress

• Perform transcriptomic analysis comparing expression patterns under different redox conditions

How does katG contribute to G. sulfurreducens' unique cell composition and metabolism?

G. sulfurreducens has a unique cell composition compared to other bacteria, with distinctive metabolic features that may influence and be influenced by katG function:

• Metal content relationships:

  • G. sulfurreducens has significantly different trace metal content compared to E. coli, with enrichment in elements like Ni, Co, Cr, and Fe

  • These metals may serve as cofactors in metalloenzymes like katG

  • Iron content is particularly relevant as katG requires heme iron for catalytic activity

• Oxidative stress adaptation:

  • The high iron content in G. sulfurreducens (an order of magnitude higher than E. coli) may increase vulnerability to oxidative damage through Fenton chemistry

  • This vulnerability likely necessitates robust peroxide-scavenging systems including katG

• Metabolic integration:

  • KatG activity may be particularly important during shifts in metabolic states

  • The adaptive evolution demonstrated in G. sulfurreducens for new substrate utilization suggests metabolic flexibility that may extend to oxidative stress responses

Methodological approaches:

• Compare proteomics profiles between wild-type and ΔkatG strains under oxidative stress

• Measure intracellular redox potential changes in response to peroxide exposure

• Analyze metabolite profiles to identify shifts in central metabolism during oxidative stress

What are the challenges and solutions for assessing katG function in vivo?

Investigating katG function in G. sulfurreducens presents several methodological challenges:

Advanced experimental approaches:

• Create reporter fusions to monitor katG expression in real-time

• Apply genetically encoded H₂O₂ sensors to track intracellular peroxide levels

• Use microfluidic systems to create controlled oxygen gradients

• Implement metabolic modeling to predict the impact of katG activity on cellular redox state

How does G. sulfurreducens katG function change under different environmental conditions?

The function and importance of katG likely varies significantly across different growth conditions:

• Aerobic vs. anaerobic environments:

  • KatG importance increases during oxygen exposure or aerotolerant growth

  • Under strictly anaerobic conditions, its role may be minimized

• Growth phase effects:

  • In other bacteria, monofunctional catalases are typically induced during stationary phase

  • Bifunctional catalases like katG are often induced during exponential growth or upon environmental H₂O₂ detection

  • Similar patterns may exist in G. sulfurreducens

• Electron acceptor availability:

  • During growth with different electron acceptors (Fe(III), fumarate, electrodes), the importance of katG may vary

  • When growing on insoluble electron acceptors like Fe(III) oxide, oxidative stress may increase due to Fenton chemistry

• Nutrient limitation:

  • Under carbon starvation, catalase may become the primary peroxide scavenger as cells cannot provide reductants to peroxidases

  • Nitrogen limitation may also affect katG expression as part of a general stress response

Experimental protocol:

• Culture G. sulfurreducens under varied conditions (different electron acceptors, growth phases, nutrient limitations)

• Measure katG expression using qRT-PCR and protein levels via Western blotting

• Assess catalase-peroxidase activity in cell extracts from each condition

• Compare H₂O₂ sensitivity profiles across growth conditions

What are the implications of G. sulfurreducens katG for bioremediation applications?

G. sulfurreducens is important in various bioremediation applications, particularly for metal contaminants, and katG may play several roles in these processes:

• Uranium bioremediation:

  • G. sulfurreducens is involved in U(VI) reduction to less soluble U(IV)

  • Oxidative stress during this process may require katG activity for cell protection

  • U(VI) reduction occurs primarily through outer-surface c-type cytochromes, not pili

• Long-term survival in contaminated environments:

  • KatG may contribute to persistence in environments with fluctuating oxygen levels

  • This could improve bioremediation efficiency in field applications

• Biofilm formation and maintenance:

  • Oxidative stress protection may support biofilm development on electrodes or mineral surfaces

  • Robust biofilms are crucial for effective bioremediation processes

Research methodologies to explore these implications:

• Compare wild-type and ΔkatG strains in laboratory-scale bioremediation experiments

• Analyze katG expression in field samples from active bioremediation sites

• Assess the effect of oxidative stress preconditioning on subsequent bioremediation efficiency

• Develop genetic engineering approaches to enhance katG expression for improved bioremediation performance