Recombinant Photobacterium profundum DNA mismatch repair protein MutS (mutS), partial

What is Photobacterium profundum MutS and how does it function in DNA mismatch repair?

Photobacterium profundum MutS is a DNA mismatch repair (MMR) protein that plays a critical role in post-replication repair by recognizing and binding to mismatched nucleotides or insertion/deletion loops in newly synthesized DNA. As part of the highly conserved MMR system, MutS initiates a repair cascade that safeguards genomic integrity .

The MMR process in bacteria typically follows these steps:

• MutS recognizes and binds to DNA mismatches

• MutS recruits MutL in an ATP-dependent reaction

• Together they activate downstream repair processes

• The mismatch is excised and correctly resynthesized

In P. profundum, MutS function is particularly interesting because this organism has adapted to extreme deep-sea conditions, including high hydrostatic pressure and low temperature . Understanding how MutS functions under these conditions provides insights into adaptation mechanisms of deep-sea microorganisms.

How do the structural features of MutS enable mismatch recognition?

While the specific structure of P. profundum MutS has not been fully characterized, insights from related bacterial MutS proteins suggest a conserved structural organization. MutS proteins form homodimers that adopt a theta (θ) shape when bound to mismatched DNA, with the mismatched DNA residing in the lower channel .

Co-crystal structures of MutS proteins from E. coli and T. aquaticus reveal that:

• MutS homodimers clamp around the DNA at the mismatch site

• Binding of a mismatch induces asymmetry in the two protein subunits

• Only one subunit makes direct contact with the mismatch

• Stable dimers, rather than tetramers, are the biologically functional unit

Recent single-molecule Förster resonance energy transfer studies with T. aquaticus MutS have identified three dominant DNA bending states during mismatch recognition:

• Slightly bent/unbent state (U)

• Intermediately bent state (I)

• Significantly bent state (B)

The kinetics of interconversion between these states varies for different mismatches and likely influences repair efficiency. The stability of the slightly bent/unbent state (U) appears to be particularly important for efficient repair signaling .

What experimental systems are used to study P. profundum MutS function?

Several experimental approaches have been employed to study P. profundum MutS:

Genetic approaches:

• Transposon mutagenesis to create mutS disruption strains

• Complementation analysis to verify gene mutation-growth phenotype relationships

• Growth assays under varied pressure and temperature conditions to assess phenotypes

Biochemical approaches:

• Expression of recombinant MutS protein in E. coli expression systems

• Purification using affinity chromatography (similar to methods used for T. aquaticus MutS)

• DNA binding assays to assess mismatch recognition specificity

Experimental parameters for P. profundum cultivation:

For high-pressure experiments, cultures are typically grown in heat-sealed bulbs placed in stainless steel pressure vessels at 15°C, with high-pressure/low-pressure (HP/LP) ratios calculated to assess pressure adaptation .

How does the dynamics of MutS-DNA interactions affect repair efficiency in P. profundum?

Research on MutS-DNA interactions has revealed that the dynamic behavior of these complexes may be a key determinant of repair efficiency. While specific data for P. profundum MutS is limited, studies with other bacterial MutS proteins provide valuable insights that likely apply to P. profundum as well.

The dynamics of MutS-mismatched DNA complexes include:

• Three conformational states with varying stability:

  • Strongly bent (B) state: Initial recognition complex

  • Intermediately bent (I) state: Transitional complex

  • Slightly bent/unbent (U) state: Repair signaling complex

• Conformational pathway hypothesis:

  • MutS binds DNA nonspecifically and bends it while searching for mismatches

  • Upon mismatch recognition, it forms an initial recognition complex (bent DNA)

  • It then undergoes further conformational change to the ultimate recognition complex (unbent DNA)

For efficient repair, the unbent state must be sufficiently populated. Complexes whose homologues show poor repair in vivo do not efficiently progress through the conformational pathways leading to the unbent state .

This understanding suggests that in P. profundum, MutS may have evolved specific dynamics optimized for function under high pressure conditions, potentially with altered energy barriers between conformational states.

How do environmental conditions affect MutS function in P. profundum?

P. profundum SS9 is a psychrotolerant, moderately piezophilic bacterium that grows optimally at 15°C and 28 MPa pressure. Environmental conditions significantly affect its growth and likely impact MutS function .

Pressure effects on MutS function:

• Chromosomal structure and function genes (including DNA repair) are particularly important for pressure adaptation

• In P. profundum, high pressure likely imposes specific structural constraints on DNA and DNA-binding proteins

• MutS functioning may require specific adaptations to maintain appropriate protein-DNA interactions under elevated pressure

Temperature effects on MutS function:

• Low temperature affects cell envelope biogenesis and extracellular polysaccharide production

• Ribosome assembly and function (protein synthesis) are important for both low-temperature and high-pressure growth

• MutS expression and activity may be modulated by temperature-dependent regulatory mechanisms

Experimental data demonstrating pressure adaptation in P. profundum:
Unlike pressure-sensitive bacteria like E. coli, P. profundum shows remarkable adaptations to high pressure. For example, while E. coli shows dramatically decreased swimming velocity with increasing pressure, P. profundum SS9 actually increases swimming velocity at 30 MPa and maintains motility up to 150 MPa . Similar adaptations likely exist for other cellular functions, including MutS-mediated DNA repair.

What is the molecular mechanism of MutS loading onto DNA and repair initiation?

Recent research has revealed that MutS functions as a clamp loader by positioning MutL on the DNA during mismatch repair. This mechanistic insight is particularly relevant for understanding P. profundum MutS function.

The key steps in this process are:

• MutS recognizes mismatched nucleotides forming ATP-bound sliding clamps

• These sliding clamps subsequently load MutL sliding clamps onto DNA

• MutL clamps coordinate MMR excision

• MutL sliding clamps enhance MutH endonuclease and UvrD helicase activities on the DNA

This clamp-loader function differs significantly from replication clamp-loaders. Instead of a positively charged cleft (PCC) on the MutL N-terminal domains directly binding to DNA (which is undetectable in physiological conditions), MutS sliding clamps exploit the PCC to position a MutL NTD on the DNA backbone. This likely enables diffusion-mediated wrapping of the remaining MutL domains around the DNA .

For P. profundum MutS, adaptation to high pressure may involve specific structural modifications that optimize this clamp-loading function under elevated hydrostatic pressure.

How can recombinant P. profundum MutS be expressed and purified for experimental studies?

Based on established protocols for other bacterial MutS proteins, the following methodology can be applied for recombinant P. profundum MutS:

Expression system:

• Clone the P. profundum mutS gene into an expression vector with a C-terminal His-tag

• Transform into an E. coli expression strain (e.g., BL21(DE3))

• Induce expression with IPTG under optimized conditions (temperature, time, concentration)

Purification protocol:

• Cell lysis: Sonication or French press in a buffer containing:

  • 20 mM Tris-HCl, pH 8.0

  • 250 mM NaCl

  • 0.1 mM EDTA

  • 1 mM DTT

  • Protease inhibitor cocktail

• Affinity chromatography:

  • Ni-NTA agarose column

  • Wash with increasing imidazole concentrations

  • Elute with 250-300 mM imidazole

• Further purification:

  • Ion exchange chromatography

  • Size exclusion chromatography

• Storage:

  • 20 mM Tris-HCl, pH 8.0

  • 250 mM NaCl

  • 0.1 mM EDTA

  • 1 mM DTT

  • 50% Glycerol

  • Store at -80°C

Assessing protein quality:

• SDS-PAGE (>95% purity)

• Western blot analysis

• Mass spectrometry

• Circular dichroism for secondary structure assessment

• Functional assays (DNA binding, ATPase activity)

How can researchers design experiments to evaluate MutS function under high pressure conditions?

Designing experiments to study MutS function under high pressure requires specialized equipment and careful experimental planning:

High-pressure microscopic chamber approach:

• Prepare recombinant MutS protein and fluorescently labeled mismatched DNA substrates

• Utilize a high-pressure microscopic chamber capable of maintaining pressures up to 150 MPa

• Employ single-molecule fluorescence techniques to directly observe MutS-DNA interactions

• Monitor changes in binding kinetics, conformational dynamics, and repair efficiency at various pressures

Experimental design diagram for high-pressure MutS activity assay:

Controlled Variables:

• Temperature (15°C)

• MutS protein concentration

• DNA substrate sequence and concentration

• Buffer composition

• Incubation time

• Detection method

Data collection and analysis:

• Collect quantitative data on MutS binding efficiency at each pressure level

• Calculate means and standard deviations

• Perform statistical analysis (ANOVA followed by post-hoc tests)

• Plot binding efficiency versus pressure

• Analyze kinetic parameters at different pressures

What genetic approaches can be used to study MutS function in P. profundum?

Several genetic approaches have been successfully employed to study gene function in P. profundum SS9, which can be applied to investigate MutS:

Transposon mutagenesis:
P. profundum SS9 mutant libraries have been created using mini-Tn10 and mini-Tn5 transposable elements. These approaches can be used to disrupt the mutS gene:

• Mini-Tn10 mutagenesis (though it shows some insertion bias)

• Mini-Tn5 mutagenesis (less biased)

Targeted gene deletion:

• Create in-frame deletion constructs using PCR-based approaches

• Introduce these constructs via conjugation or electroporation

• Select for double crossover events to generate clean deletions

• Verify deletions by PCR and sequencing

Complementation analysis:
To verify gene mutation-growth phenotype relationships:

• Clone the wild-type mutS gene into an appropriate expression vector

• Introduce the construct into the mutS mutant strain

• Assess restoration of phenotype under various conditions

Phenotypic characterization:

• Growth assays under varied pressure (0.1-90 MPa) and temperature (2-20°C) conditions

• HP/LP growth ratio determination

• Mutation rate measurement using fluctuation tests

• DNA damage sensitivity assays

Genomic analysis:

• Genome resequencing to identify suppressor mutations in adapted strains

• Transcriptome analysis using RNA-seq to identify genes co-regulated with mutS

• ChIP-seq to identify MutS binding sites in vivo

How does P. profundum MutS compare to MutS proteins from other bacteria?

Comparative analysis of MutS proteins provides insights into unique adaptations in P. profundum:

Structural and functional comparisons:

Evolutionary adaptations:
P. profundum MutS likely contains specific adaptations that enable:

• Protein stability under high hydrostatic pressure

• Appropriate DNA binding dynamics at low temperature

• Coordination with other MMR proteins adapted to deep-sea conditions

Functional conservation:
Despite environmental adaptations, core MutS functions are conserved across species:

• Recognition of mismatched nucleotides

• ATP-dependent conformational changes

• Interaction with MutL to initiate repair

• Strand discrimination and excision coordination

What controls should be included in experiments involving P. profundum MutS?

Proper experimental design for P. profundum MutS studies requires appropriate controls:

Genetic studies:

• Wild-type P. profundum SS9 strain (positive control)

• Known mutator strain (e.g., mutS deletion) as a reference point

• Complemented mutS mutant strain to verify phenotype restoration

• Empty vector control for complementation studies

Biochemical studies:

• Heat-inactivated MutS protein (negative control)

• MutS protein with point mutations in key functional domains:

  • Mismatch binding domain mutants

  • ATPase domain mutants (e.g., Walker A/B motifs)

• Homoduplex DNA (no mismatch) control

• Different types of mismatches and insertion/deletion loops to assess specificity

Environmental parameter controls:

• Standard pressure (0.1 MPa) and optimal temperature (15°C) as baseline conditions

• Pressure gradients (e.g., 0.1, 10, 30, 60, 90 MPa)

• Temperature gradients (e.g., 4, 10, 15, 20, 25°C)

• Combined pressure/temperature matrices

How can researchers address experimental challenges in studying deep-sea bacterial proteins?

Working with proteins from deep-sea bacteria presents unique challenges:

Challenge 1: Maintaining physiologically relevant conditions

• Solution: Use high-pressure chambers for growth and biochemical assays

• Methodology: Sealed culture vessels in pressure reactors; high-pressure spectroscopic and microscopic chambers for in vitro studies

Challenge 2: Protein stability at atmospheric pressure

• Solution: Rapid processing and analysis of samples; use of stabilizing agents

• Methodology: Immediate flash-freezing; addition of osmolytes or pressure-mimicking agents to buffers

Challenge 3: Heterologous expression issues

• Solution: Codon optimization; expression under cold-shock conditions

• Methodology: Design synthetic genes with optimized codons; use cold-inducible promoters and low-temperature expression protocols

Challenge 4: Assessing function under native conditions

• Solution: In situ and ex situ approaches combined with computational modeling

• Methodology: Compare protein behavior at different pressures; use molecular dynamics simulations to predict pressure effects on protein structure

How can researchers interpret contradictory data from MutS functional studies?

When faced with contradictory results in MutS studies, consider these analytical approaches:

• Examine experimental conditions

  • Pressure effects: Results obtained at atmospheric pressure may not reflect native function

  • Temperature effects: Low-temperature adaptations may cause unexpected behavior

  • Buffer composition: Ionic strength and specific ions can significantly affect protein-DNA interactions

• Consider biological context

  • Genetic background effects: Suppressor mutations may mask phenotypes

  • Pathway redundancy: Alternative repair pathways may compensate for MutS deficiency

  • Pleiotropic effects: MutS may have additional roles beyond mismatch repair

• Methodological reconciliation

  • Compare in vitro versus in vivo results systematically

  • Utilize multiple complementary techniques to address the same question

  • Develop assays that more closely mimic physiological conditions

• Data integration approach:

  • Compile results into a comprehensive data table

  • Identify variables that may explain discrepancies

  • Develop testable hypotheses to resolve contradictions

  • Consider computational modeling to integrate diverse datasets

For example, if MutS protein shows different mismatch binding specificities in different studies, consider:

• DNA substrate differences (length, sequence context, mismatch type)

• Protein preparation methods (tags, purification approach)

• Reaction conditions (salt, pH, temperature, pressure)

• Detection methods (sensitivity, time resolution)

What emerging techniques might advance P. profundum MutS research?

Several cutting-edge approaches show promise for deepening our understanding of P. profundum MutS:

• Cryo-electron microscopy under pressure

  • Apply emerging high-pressure cryo-EM techniques to visualize MutS-DNA complexes under native pressure conditions

  • Capture different conformational states during the repair process

• Single-molecule approaches at high pressure

  • Develop high-pressure microscopy chambers compatible with single-molecule FRET

  • Monitor real-time conformational dynamics of MutS-DNA interactions at varied pressures

• In situ gene editing

  • Apply CRISPR-Cas9 techniques optimized for P. profundum

  • Create precise mutations in the mutS gene to assess structure-function relationships

• Integrative structural biology

  • Combine X-ray crystallography, NMR, SAXS, and computational modeling

  • Generate comprehensive structural models of P. profundum MutS under different conditions

• Deep-sea bioprospecting

  • Comparative analysis of MutS proteins from various deep-sea bacteria

  • Identify convergent adaptations to high-pressure environments

These approaches will help answer key remaining questions about how deep-sea bacteria maintain genomic integrity under extreme conditions and may reveal novel molecular mechanisms of pressure adaptation.

How might insights from P. profundum MutS research be applied in broader contexts?

Understanding P. profundum MutS has potential applications in several fields:

• Biotechnology applications

  • Development of pressure-stable enzymes for industrial processes

  • Design of DNA repair systems functioning under extreme conditions

  • Engineering of pressure-resistant microorganisms for bioremediation

• Fundamental understanding of protein-DNA interactions

  • Insights into how pressure affects protein-DNA binding dynamics

  • New models for conformational changes during mismatch recognition

  • Principles of protein adaptation to extreme environments

• Evolutionary biology

  • Mechanisms of adaptation to deep-sea environments

  • Rates of evolution in extreme environments

  • Genome stability under extreme conditions

• Astrobiology

  • Models for potential life in high-pressure extraterrestrial environments

  • Understanding limits of DNA repair systems under extreme conditions

  • Insights for detecting biosignatures in extreme environments