Recombinant Porphyromonas gingivalis DNA mismatch repair protein MutS (mutS), partial

What is the role of MutS in P. gingivalis DNA repair mechanisms?

MutS is a critical component of the DNA mismatch repair (MMR) system in P. gingivalis that recognizes and binds to mismatched base pairs, particularly those resulting from oxidative damage. While P. gingivalis lacks the MutM protein found in many bacteria, it does contain homologues to MutY, MutT, and MutS, suggesting a modified repair pathway for oxidative stress-induced DNA damage . MutS may participate in repairing 8-oxoG:A mismatches to 8-oxoG:C, similar to the function observed in E. coli . Preliminary microarray analysis indicates that mutS is upregulated in P. gingivalis when exposed to hydrogen peroxide, suggesting its importance in oxidative stress resistance .

Methodologically, researchers investigating MutS function should consider:

• Comparative genomic analysis with MutS proteins from other bacterial species

• Expression studies under various oxidative stress conditions

• Protein-DNA interaction assays focusing on mismatched substrates

How does P. gingivalis MutS differ structurally and functionally from other bacterial MutS proteins?

While specific structural data for P. gingivalis MutS is limited, it likely shares the core domains common to bacterial MutS proteins, including mismatch recognition, connector, and ATPase domains. The unique aspect of P. gingivalis MutS appears to be its potential role in a modified DNA repair pathway that compensates for the absence of MutM .

In E. coli, MutS is part of the methyl-directed mismatch repair system and has been shown to repair base pairs containing oxidative lesions . P. gingivalis MutS may have evolved specialized functions to address the challenges of surviving in the inflammatory environment of periodontal pockets where oxidative stress is prevalent.

To investigate these differences, researchers should:

• Perform sequence alignment analyses comparing P. gingivalis MutS with homologs

• Conduct complementation studies in E. coli mutS mutants

• Use site-directed mutagenesis to identify critical functional residues

What are the expression patterns of mutS in P. gingivalis under different environmental conditions?

The expression of mutS in P. gingivalis appears to be responsive to oxidative stress conditions. Preliminary microarray analysis has shown that mutS is upregulated in P. gingivalis FLL92 (a non-pigmented vimA-defective mutant) compared to wild-type W83 when exposed to hydrogen peroxide . This suggests that MutS plays a role in protecting the cell against oxidative stress.

*Exact fold change values not specified in available research

For comprehensive expression analysis, researchers should employ:

• RT-qPCR for targeted gene expression studies

• RNA-seq for transcriptome-wide analysis under various conditions

• Reporter gene fusions (e.g., mutS-gfp) for real-time monitoring

How can researchers effectively express and purify recombinant P. gingivalis MutS protein?

Expressing and purifying recombinant P. gingivalis MutS requires careful optimization to ensure the protein maintains its native conformation and activity. Based on general principles for recombinant protein production and the specific characteristics of P. gingivalis proteins:

Expression System Recommendations:

• E. coli BL21(DE3) or Rosetta strains (to account for rare codons)

• Expression vector with an N-terminal 6xHis-tag for purification

• Induction with 0.1-0.5 mM IPTG at lower temperatures (16-20°C)

• Addition of 5% glycerol to the culture medium to enhance solubility

Purification Protocol:

• Cell lysis under reducing conditions to preserve cysteine residues

• Immobilized metal affinity chromatography using Ni-NTA resin

• Size exclusion chromatography to remove aggregates

• Ion exchange chromatography for final polishing

• Storage in buffer containing 20% glycerol, 1mM DTT at -80°C

This approach parallels successful methodologies used for other recombinant P. gingivalis proteins, such as the recombinant Gingipain R1 (RgpA) production process, which involves plasmid vector insertion followed by expression and affinity chromatography purification .

What experimental approaches can determine the binding specificity of P. gingivalis MutS to different DNA mismatches?

Understanding the binding specificity of P. gingivalis MutS to various DNA mismatches is crucial for characterizing its role in DNA repair. Several experimental approaches can be employed:

Gel Mobility Shift Assays:
Similar to the approach used with P. gingivalis MutY, electrophoretic mobility shift assays (EMSA) can determine binding to oligonucleotides containing specific mismatches . Purified recombinant MutS is incubated with labeled DNA oligomers containing various mismatches, and the resulting complexes are analyzed by native gel electrophoresis.

*Predicted ranges based on general MutS characteristics; specific values for P. gingivalis MutS require experimental determination

Advanced Binding Analysis Techniques:

• Surface plasmon resonance (SPR) for real-time binding kinetics

• Fluorescence anisotropy for quantitative affinity measurements

• Microscale thermophoresis for binding under various buffer conditions

• DNA footprinting to identify the precise binding site

How does the oxidative stress response pathway in P. gingivalis integrate MutS function?

P. gingivalis encounters significant oxidative stress in the periodontal pocket environment, and MutS appears to be an important component of its stress response system. The integration of MutS into this pathway likely involves:

• Sensing Oxidative Damage: MutS recognizes and binds to DNA containing oxidative damage-induced mismatches, particularly 8-oxoG:A

• Coordinated Regulation: The upregulation of mutS observed during hydrogen peroxide exposure suggests coordinated transcriptional regulation with other stress response genes

• Interaction with Other Repair Systems: Despite lacking MutM, P. gingivalis may utilize MutS in conjunction with MutY to address oxidative DNA damage through alternative pathways

• Redundant Mechanisms: The similar repair activities observed in both wild-type and mutY-defective P. gingivalis strains suggest redundant repair mechanisms that may involve MutS

Researchers investigating this pathway should consider:

• Chromatin immunoprecipitation (ChIP) to identify proteins interacting with MutS

• Transcriptome analysis comparing wild-type and mutS-deficient strains under oxidative stress

• Metabolomic profiling to identify changes in cellular processes affected by MutS function

What are the optimal conditions for assessing P. gingivalis MutS ATPase activity in vitro?

MutS proteins require ATP hydrolysis for their function in DNA mismatch repair. Optimizing assay conditions for P. gingivalis MutS ATPase activity is essential for functional characterization:

Recommended Assay Conditions:

Detection Methods:

• Malachite green assay for phosphate release

• Coupled enzyme assay linking ATP hydrolysis to NADH oxidation

• Thin-layer chromatography separation of ATP and ADP

• HPLC analysis of reaction products

Comparing ATPase activity in the presence of different DNA substrates (homoduplex vs. various mismatches) can provide insights into how DNA binding affects the catalytic activity of P. gingivalis MutS.

What are the most effective strategies for creating mutS-deficient P. gingivalis mutants?

Creating mutS-deficient P. gingivalis mutants is crucial for understanding the protein's function. Based on successful approaches used for other P. gingivalis genes (such as mutY), the following strategies are recommended:

Allelic Exchange Mutagenesis:
The most established approach involves insertional inactivation using an antibiotic resistance cassette (e.g., ermF-ermAM) . This method requires:

• Amplification of regions flanking the mutS gene

• Cloning these regions into a suicide vector with the antibiotic cassette

• Introduction into P. gingivalis via electroporation

• Selection of transformants on appropriate antibiotics

• Confirmation of gene disruption by PCR and sequencing

This approach was successfully used to create a mutY-deficient mutant (FLL147) in P. gingivalis , suggesting it would be applicable for mutS as well.

CRISPR-Cas9 Approach:
While less established in P. gingivalis, CRISPR-Cas9 systems offer potential advantages:

• Greater precision in targeting specific regions of mutS

• Ability to create markerless mutations

• Potential for higher efficiency

Verification of Mutants:
Successful mutants should be verified through:

• PCR confirmation of the disrupted gene

• RT-PCR/qPCR to confirm absence of transcription

• Western blotting to confirm absence of protein (if antibodies are available)

• Phenotypic analysis (increased mutation rate, sensitivity to oxidative stress)

How can researchers quantify mutation rates in wild-type versus mutS-deficient P. gingivalis strains?

Measuring mutation rates is essential for understanding the impact of MutS deficiency on genomic stability. Based on approaches used with mutY-deficient P. gingivalis , several methodologies are recommended:

Rifampicin Resistance Assay:

• Grow multiple independent cultures of wild-type and mutS-deficient strains

• Plate appropriate dilutions on media with and without rifampicin

• Calculate mutation frequency as the ratio of rifampicin-resistant colonies to total viable cells

• Convert frequencies to rates using appropriate statistical methods (e.g., Luria-Delbrück fluctuation test)

Expected Results:
Similar to observations with mutY-deficient P. gingivalis, mutS-deficient strains would likely show increased spontaneous mutation rates .

*Predicted ranges based on general patterns observed with DNA repair mutants; specific values require experimental determination

Whole Genome Sequencing Approach:

• Culture wild-type and mutant strains for multiple generations

• Sequence genomes from different time points

• Analyze mutation accumulation rates and spectra

• Pay particular attention to mutations that would result from unrepaired mismatches

What phenotypic assays best characterize the response of mutS-deficient P. gingivalis to oxidative stress?

Given the likely role of MutS in oxidative stress resistance, comprehensive phenotypic characterization should include:

Hydrogen Peroxide Sensitivity Assay:
Similar to tests performed with mutY-deficient P. gingivalis , compare growth inhibition zones or survival rates after H₂O₂ exposure.

Expected Results:
mutS-deficient strains would likely show increased sensitivity to hydrogen peroxide compared to wild-type, similar to observations with mutY-deficient strains .

*Predicted ranges based on patterns observed with DNA repair mutants; specific values require experimental determination

Additional Phenotypic Assays:

• Survival under periodontal pocket simulation conditions

• Biofilm formation capacity

• Virulence factor expression

• Growth rate measurements under various oxygen tensions

• Long-term survival in stationary phase

How can researchers perform cross-species complementation studies with P. gingivalis mutS?

Cross-species complementation studies can provide valuable insights into the functional conservation and unique properties of P. gingivalis MutS. Based on successful complementation of E. coli mutY mutants with P. gingivalis mutY , similar approaches can be applied to mutS:

E. coli Complementation System:

• Clone the P. gingivalis mutS gene into an E. coli expression vector

• Transform into an E. coli mutS-deficient strain

• Assess restoration of mutation frequency to wild-type levels

• Compare with positive control (E. coli mutS) and negative control (empty vector)

Expected Results:
If P. gingivalis mutS can functionally complement E. coli mutS deficiency, this would indicate conservation of core functions despite potential adaptations to the periodontal environment.

*Predicted ranges based on general patterns observed with DNA repair genes; specific values require experimental determination

Optimization Considerations:

• Codon optimization may be necessary for efficient expression in E. coli

• Expression level adjustments to prevent toxicity

• Testing under various oxidative stress conditions

What protein interaction studies can reveal the partners of P. gingivalis MutS in DNA repair pathways?

Understanding the interaction network of P. gingivalis MutS is crucial for elucidating its role in DNA repair pathways:

Recommended Approaches:

• Bacterial Two-Hybrid System:

  • Create fusion constructs of P. gingivalis MutS with DNA-binding domain

  • Screen against P. gingivalis genomic library fused to activation domain

  • Identify positive interactions through reporter gene activation

• Co-Immunoprecipitation:

  • Generate antibodies against recombinant P. gingivalis MutS

  • Perform pull-down experiments from P. gingivalis lysates

  • Identify interacting partners by mass spectrometry

• Crosslinking Mass Spectrometry:

  • Use chemical crosslinkers to stabilize transient interactions

  • Digest and analyze by LC-MS/MS

  • Identify crosslinked peptides to map interaction interfaces

Potential Interaction Partners:
Based on known MutS interaction networks in other bacteria and the unique DNA repair landscape of P. gingivalis, potential partners include:

• MutL homologs

• DNA polymerase III

• Single-strand binding proteins

• MutY (given their potential functional overlap in 8-oxoG:A repair)

• Novel proteins unique to P. gingivalis repair pathways

How does temperature affect the stability and activity of recombinant P. gingivalis MutS?

Temperature stability is a critical parameter for functional studies and storage of recombinant proteins. For P. gingivalis MutS:

Temperature Stability Profile:

*Predicted ranges based on general properties of DNA repair proteins; specific values require experimental determination

Experimental Design for Temperature Stability Studies:

• Incubate purified recombinant P. gingivalis MutS at various temperatures

• At defined time points, assess:

  • Structural integrity by circular dichroism

  • DNA binding activity by EMSA

  • ATPase activity by phosphate release assay

• Determine half-life at each temperature condition

Since P. gingivalis is an anaerobic organism that colonizes the human oral cavity, its proteins may have evolved temperature stability properties optimized for this environment.

What is the relationship between P. gingivalis MutS function and periodontal disease progression?

P. gingivalis is strongly associated with periodontal disease, found in 69-79% of individuals with periodontal disease compared to only 10-25% of periodontically healthy individuals . The role of MutS in this context may be significant:

Hypothesized Relationships:

• Adaptation to Inflammatory Environment:
  MutS likely helps P. gingivalis survive the oxidative burst from host immune cells in periodontal pockets by repairing oxidative DNA damage .

• Genomic Stability During Chronic Infection:
  Maintenance of genomic integrity through MutS activity may be crucial for persistent infection over the years that characterize periodontal disease .

• Mutation Rate Modulation:
  MutS may help balance genomic stability with adaptive mutation rates, allowing P. gingivalis to evolve in response to host defenses while maintaining core virulence functions.

Research Approaches:

• Compare mutS sequence and expression between isolates from healthy subjects and those with varying severity of periodontal disease

• Assess the virulence of wild-type versus mutS-deficient P. gingivalis in animal models of periodontitis

• Examine mutation accumulation in P. gingivalis during long-term in vitro modeling of periodontal pocket conditions

How have P. gingivalis MutS functions evolved compared to related oral pathogens?

Evolutionary analysis of MutS across oral pathogens can provide insights into adaptation to specific periodontal niches:

Comparative Analysis Framework:

*Specific homology percentages require sequence comparison analysis

Evolutionary Pattern Analysis:

• Phylogenetic analysis of MutS sequences across oral pathogens

• Identification of positively selected residues that may indicate functional adaptation

• Correlation of MutS sequence variation with habitat and oxidative stress exposure

• Comparative analysis of mutS regulation across species

This evolutionary perspective can provide insights into how different oral pathogens have adapted their DNA repair mechanisms to specific niches within the oral microbiome.