Recombinant Photobacterium profundum Formamidopyrimidine-DNA glycosylase (mutM)

What is the basic function of formamidopyrimidine-DNA glycosylase (mutM) in Photobacterium profundum?

Formamidopyrimidine-DNA glycosylase (Fapy-DNA glycosylase) encoded by the mutM gene in P. profundum functions as a critical DNA repair enzyme that removes oxidatively damaged bases from DNA. Based on characterization of mutM in other bacterial species, this enzyme specifically excises lesions such as 8-oxo-7,8-dihydro-2'-deoxyguanine (8-OxodG) from DNA, preventing mutagenic G.C to T.A transversions . In the deep-sea bacterium P. profundum, this enzyme likely plays an essential role in maintaining genomic integrity under conditions of high pressure and low temperature that can increase oxidative stress. The enzyme recognizes and removes damaged purine residues, initiating the base excision repair pathway to restore the correct DNA sequence.

How does P. profundum mutM differ from its homologs in other bacterial species?

While the core enzymatic function of mutM-encoded Fapy-DNA glycosylase remains conserved across bacterial species, the P. profundum variant likely contains adaptations that optimize its activity under high-pressure and low-temperature conditions characteristic of the deep sea. These adaptations may include amino acid substitutions that enhance protein flexibility at low temperatures or maintain structural integrity under high pressure. Comparative genomics studies of P. profundum have revealed numerous adaptations for piezopsychrophilic growth, affecting various cellular processes including DNA replication and repair . Specific structural differences may include modifications to substrate binding domains or catalytic sites that optimize enzyme kinetics in the deep-sea environment compared to mesophilic bacterial homologs.

What physiological conditions trigger increased expression of mutM in P. profundum?

In P. profundum, mutM expression is likely upregulated under conditions that increase oxidative stress and DNA damage. Large-scale transposon mutagenesis studies have shown that both low-temperature and high-pressure adaptations in P. profundum involve numerous sensory and regulatory loci, suggesting complex signal transduction mechanisms respond to these physical parameters . The expression of DNA repair genes like mutM would logically be part of these stress responses. While specific regulators of mutM in P. profundum haven't been fully characterized in the available search results, oxidative stress and exposure to DNA-damaging agents would predictably increase its expression to protect genomic integrity under the challenging conditions of the deep-sea environment.

What are the most effective methods for cloning and expressing recombinant P. profundum mutM?

The most effective approach for cloning and expressing recombinant P. profundum mutM involves PCR amplification of the gene using carefully designed primers, followed by insertion into an appropriate expression vector. Based on successful transposon mutagenesis techniques used with P. profundum, researchers can employ a PCR method similar to that described for transposon insertion site identification . Specifically, genomic DNA can be amplified using gene-specific primers, with subsequent cloning into expression vectors containing inducible promoters. For expression, E. coli BL21(DE3) or similar strains are commonly used, with protein production optimized at lower temperatures (15-20°C) to enhance proper folding of this cold-adapted enzyme. Purification typically involves affinity chromatography using polyhistidine tags, followed by ion exchange and size exclusion chromatography to obtain pure, active enzyme.

How can researchers design effective transposon mutagenesis strategies for studying mutM function in P. profundum?

To design effective transposon mutagenesis strategies for studying mutM function in P. profundum, researchers should carefully select appropriate transposable elements and screening conditions. Based on previous successful studies with P. profundum, mini-Tn5 transposable elements demonstrate less insertion bias compared to mini-Tn10 elements and thus would be preferred for mutM functional analysis . The mutagenesis protocol should include:

• Conjugative transfer of suicide plasmids containing the transposon into P. profundum

• Selection for transposon insertion using appropriate antibiotics

• Screening mutant libraries for altered phenotypes related to DNA repair, such as sensitivity to oxidative agents or UV radiation

• Verification of insertion sites using arbitrary PCR methods as described for P. profundum previously, with primers targeting transposon ends and random genomic sequences

• Complementation analysis with wild-type mutM to confirm phenotype-genotype relationships

For pressure and temperature-related phenotypes, mutants should be screened under various pressure (0.1-50 MPa) and temperature (4-20°C) conditions to identify conditional phenotypes that might reveal specialized functions of mutM in deep-sea adaptation.

What are the optimal conditions for measuring recombinant P. profundum mutM enzymatic activity?

The optimal conditions for measuring recombinant P. profundum mutM enzymatic activity should reflect its adaptation to the deep-sea environment while providing reliable quantitative results. Based on knowledge of piezopsychrophilic adaptations, recommended assay conditions include:

Buffer Composition:

• 50 mM phosphate buffer or Tris-HCl (pH 7.5-8.0)

• 50-100 mM NaCl

• 1 mM EDTA

• 1 mM DTT or 2-mercaptoethanol

• 10% glycerol as stabilizer

Physical Parameters:

• Temperature: 4-15°C (primary) with comparative assays at 25-37°C

• Pressure: Atmospheric pressure for standard assays, with specialized high-pressure chambers (10-50 MPa) for studying pressure effects

Substrate:

• Synthetic oligonucleotides containing 8-oxoG lesions paired with cytosine

• Formamidopyrimidine-containing DNA substrates

Activity can be measured by:

• Release of modified bases from DNA substrate

• DNA nicking assays using gel electrophoresis

• Fluorescence-based methods with labeled substrates

Comparing enzymatic parameters (kcat, Km) across temperature and pressure gradients will provide insights into the enzyme's adaptation to the deep-sea environment.

How does the mutation rate in P. profundum mutM mutants compare to wild-type under various pressure conditions?

The mutation rate in P. profundum mutM mutants likely shows significant elevation compared to wild-type strains, particularly under conditions that increase oxidative stress. While specific data for P. profundum mutM is not directly provided in the search results, studies of mutM in other bacterial species have characterized it as a mutator locus that specifically leads to G.C to T.A transversions when inactivated . For P. profundum, this effect would be particularly relevant under high-pressure conditions, as studies have shown that genes for chromosomal structure and function are especially important for high-pressure growth .

Based on comparable bacterial systems, we would expect to observe:

The elevated mutation rates under high pressure would result from increased oxidative damage to DNA combined with the inability of mutM mutants to repair 8-oxoG lesions effectively. Complementation with functional mutM would be expected to restore mutation rates to near wild-type levels, confirming the specific role of this gene in preventing pressure-associated mutagenesis.

What genomic context surrounds the mutM gene in P. profundum and how does this compare to other bacterial species?

The genomic context surrounding the mutM gene in P. profundum likely includes co-regulated genes involved in DNA repair and oxidative stress response. While the specific genomic organization in P. profundum is not directly described in the search results, comparative genomics with other bacterial systems can provide insights. In many bacteria, mutM may be located in operons or gene clusters with functionally related genes, possibly including other base excision repair enzymes or oxidative stress response regulators.

Potential genomic arrangements might include:

• Association with mutY (adenine glycosylase), which works complementarily with mutM to prevent mutations from 8-oxoG

• Proximity to genes encoding oxidative stress response factors

• Possible co-regulation with genes encoding other DNA repair pathways

Comparative genomics analyses would examine conservation of gene order and operon structure across related bacterial species, with particular attention to differences between deep-sea adapted bacteria like P. profundum and their shallow-water or terrestrial counterparts. Such analysis could reveal whether the genomic context of mutM has been specifically adapted for the deep-sea environment, potentially through altered regulatory elements or gene duplications.

How do the structural features of P. profundum mutM product contribute to its function under high pressure?

The structural features of P. profundum mutM protein that contribute to its function under high pressure likely include specific adaptations that maintain catalytic activity while preventing pressure-induced denaturation. Although the search results don't provide direct structural data for P. profundum mutM, research on piezopsychrophilic adaptations suggests several probable structural modifications:

• Increased Protein Flexibility: The enzyme likely contains fewer rigid structural elements and more flexible regions that allow conformational changes under pressure without losing functional structure.

• Modified Hydrophobic Core: Piezopsychrophilic proteins often feature reduced hydrophobic cores with altered amino acid compositions that resist pressure-induced water infiltration.

• Strategic Salt Bridges and Hydrogen Bonds: Additional stabilizing interactions that maintain tertiary structure under pressure conditions where weaker interactions would typically be disrupted.

• Specialized Active Site Architecture: The catalytic pocket may be structured to maintain precise geometry for substrate binding and catalysis under pressure, possibly with increased volume to accommodate pressure-induced compression.

• Surface Charge Distribution: Altered distribution of charged residues that interact favorably with water molecules under pressure.

These structural adaptations would collectively enable the enzyme to maintain its critical DNA repair function in the deep-sea environment, removing oxidatively damaged bases efficiently even under the high-pressure conditions that characterize P. profundum's natural habitat.

How does temperature affect the activity and stability of recombinant P. profundum mutM compared to homologs from mesophilic bacteria?

Temperature significantly influences both the activity and stability of recombinant P. profundum mutM, with distinct differences compared to homologs from mesophilic bacteria. As a protein from a piezopsychrophilic organism, P. profundum mutM likely exhibits cold adaptation features that enhance catalytic efficiency at low temperatures (4-15°C) while sacrificing thermal stability at higher temperatures. While specific data for P. profundum mutM is not directly provided in the search results, large-scale studies of P. profundum adaptations have identified numerous genes conditionally required for low-temperature growth .

Based on established patterns of cold-adapted enzymes, we would expect to observe:

The molecular basis for these differences typically includes reduced numbers of stabilizing interactions (fewer salt bridges, hydrogen bonds) and increased flexibility of catalytic regions, which enhance function at low temperatures but reduce thermal stability. These adaptations would be particularly important for maintaining DNA repair capacity in the cold deep-sea environment.

What is the relationship between oxidative stress, pressure, and mutM expression in P. profundum?

The relationship between oxidative stress, pressure, and mutM expression in P. profundum likely forms a critical adaptive response network. High hydrostatic pressure can increase the production of reactive oxygen species (ROS) in bacterial cells, creating elevated levels of oxidative DNA damage. Since mutM-encoded Fapy-DNA glycosylase repairs oxidative DNA damage, its expression would logically be upregulated under high-pressure conditions as part of the cell's defense mechanism against increased oxidative stress.

Studies of P. profundum have demonstrated that adaptation to both temperature and pressure involves numerous sensory and regulatory loci, suggesting sophisticated signal transduction mechanisms respond to these physical parameters . While the specific regulation of mutM in P. profundum isn't directly detailed in the search results, the following regulatory model is plausible:

• High pressure sensors (possibly membrane-associated proteins) detect pressure changes

• Signal transduction cascades activate stress response regulators

• These regulators induce expression of oxidative stress response genes, including mutM

• Increased mutM levels enhance DNA repair capacity to manage pressure-associated DNA damage

This coordinated response would enable P. profundum to maintain genomic integrity despite the challenging conditions of its deep-sea habitat, with mutM serving as a key component of the adaptive response to pressure-induced oxidative stress.

How does the substrate specificity of P. profundum mutM differ from shallow-water bacterial homologs?

The substrate specificity of P. profundum mutM likely shows adaptations optimized for its deep-sea environment compared to shallow-water bacterial homologs. While the core function of recognizing and removing oxidatively damaged bases like 8-oxoG remains conserved, several adaptations in substrate recognition and processing efficiency may exist to address the specific challenges of DNA damage under high pressure and low temperature.

Potential substrate specificity differences may include:

• Enhanced Recognition of Cold-Induced Lesions: Improved ability to recognize and excise DNA damages that occur more frequently at low temperatures, such as certain types of depurination or deamination products.

• Pressure-Damaged DNA Structures: Potentially improved recognition of unusual DNA conformations that might arise under high pressure conditions, where DNA structure can be subtly altered.

• Broader Substrate Range: Possibly expanded substrate specificity to efficiently process multiple types of damaged bases, providing a more versatile repair capability when enzyme expression or activity might be limited by environmental constraints.

• Altered Kinetics: Different kinetic parameters for various substrates, with faster processing of the most pressure-relevant DNA damages and potentially slower processing of less common lesions.

These adaptations would collectively represent an evolutionary fine-tuning of the enzyme to prioritize repair of the most problematic or frequent DNA damages encountered in the deep-sea environment, potentially at the cost of reduced efficiency for damages more common in shallow-water environments.

How can P. profundum mutM be utilized in studying evolutionary adaptations to extreme environments?

P. profundum mutM represents an excellent model for studying evolutionary adaptations to extreme environments, particularly the deep sea's combination of high pressure and low temperature. Researchers can utilize this system to investigate molecular evolution through several approaches:

• Comparative Sequence Analysis: Alignment of mutM sequences across bacterial species from different environmental niches (deep sea, shallow water, terrestrial) can reveal adaptive mutations. Positive selection analysis can identify amino acid positions under selective pressure specifically in piezopsychrophilic lineages.

• Ancestral Protein Reconstruction: Reconstructing ancestral versions of mutM and comparing their biochemical properties with modern P. profundum mutM can reveal the evolutionary trajectory of adaptation to the deep sea.

• Domain Swapping Experiments: Creating chimeric proteins combining domains from deep-sea and shallow-water homologs can identify which protein regions are most critical for pressure adaptation.

• Experimental Evolution: Laboratory evolution of model organisms expressing P. profundum mutM under various pressure regimes, followed by sequence and functional analysis, can reveal how selective pressure drives adaptation.

• Correlation with Habitat Depth: Analyzing mutM sequences from bacteria isolated across a depth gradient can reveal how progressive adaptation occurs with increasing pressure, potentially identifying key transitional mutations.

These approaches collectively provide insights into how essential DNA repair mechanisms adapt to extreme conditions, revealing fundamental principles of protein evolution under selective pressure from physical parameters like temperature and pressure.

What role might P. profundum mutM play in the bacterium's symbiotic or pathogenic interactions?

While P. profundum itself is not primarily known as a symbiont or pathogen, its mutM gene could potentially influence interactions with other organisms through several mechanisms. Drawing parallels from related bacterial systems provides insights into possible roles:

• Maintenance of Genomic Stability During Host Interaction: Similar to how lytic transglycosylase mutations in Vibrio fischeri affect host colonization and superinfection resistance , proper DNA repair through mutM function might be crucial for P. profundum during any potential interactions with deep-sea organisms.

• Response to Host-Generated Oxidative Stress: If P. profundum engages in any host associations, mutM would be crucial for countering reactive oxygen species generated as host defense mechanisms.

• Modulation of Mutation Rates Under Stress: The balance between genomic stability and adaptive mutation during host interaction could be influenced by mutM function, potentially affecting the evolution of any symbiotic relationships.

• Horizontal Gene Transfer Stability: mutM function might influence the integration and maintenance of horizontally acquired genes that could carry symbiosis factors or virulence determinants.

While not directly studied in P. profundum, these potential roles highlight how fundamental DNA repair mechanisms like mutM-mediated base excision repair could influence complex ecological interactions beyond their primary cellular function, potentially contributing to the bacterium's ecological fitness in its deep-sea habitat.

What are the implications of P. profundum mutM research for understanding DNA repair in other extremophiles?

Research on P. profundum mutM has broad implications for understanding DNA repair mechanisms across diverse extremophiles, providing insights into convergent and divergent evolutionary strategies for maintaining genomic integrity under challenging conditions:

These broader implications position P. profundum mutM research as a valuable model system for understanding fundamental principles of molecular adaptation to extreme environments across the tree of life.

What strategies can address low yield or activity when expressing recombinant P. profundum mutM in E. coli?

When facing challenges with low yield or activity of recombinant P. profundum mutM expressed in E. coli, researchers can implement several targeted strategies:

• Optimize Expression Temperature: Cold-adapted enzymes often fold incorrectly at standard expression temperatures (37°C). Lowering expression temperature to 10-20°C and extending expression time to 24-48 hours can dramatically improve folding and yield of active enzyme.

• Codon Optimization: P. profundum's GC content and codon usage differ from E. coli. Synthesizing a codon-optimized gene can significantly improve translation efficiency and protein yield.

• Solubility Enhancement:

  • Use fusion partners like thioredoxin, SUMO, or MBP that enhance solubility

  • Add osmolytes (0.5-1M sorbitol, 5-10% glycerol) to expression media

  • Include low concentrations (1-5 mM) of chemical chaperones like arginine or proline

• Expression Host Selection: Consider Arctic Express or other cold-adapted E. coli strains that express chaperones functional at lower temperatures.

• Buffer Optimization during Purification:

  • Include stabilizers like 10-20% glycerol and 100-200 mM NaCl

  • Maintain slightly alkaline pH (7.5-8.5)

  • Add 1-5 mM reducing agents (DTT or β-mercaptoethanol)

  • Consider pressure-stable preservatives like trimethylamine N-oxide (TMAO)

• Enzymatic Assay Conditions: Test activity under conditions mimicking the deep-sea environment (low temperature, high pressure when possible) rather than standard laboratory conditions.

These approaches address the fundamental challenge of expressing a cold-adapted, pressure-adapted enzyme in mesophilic hosts under atmospheric pressure, focusing on creating conditions that respect the unique structural and functional properties of P. profundum mutM.

How can researchers distinguish between direct and indirect effects when analyzing P. profundum mutM mutant phenotypes?

Distinguishing between direct and indirect effects when analyzing P. profundum mutM mutant phenotypes requires a systematic approach incorporating multiple complementary methods:

• Complementation Analysis: The gold standard approach involves reintroducing the wild-type mutM gene into the mutant strain. If the wild-type phenotype is restored, this strongly supports a direct relationship between mutM and the observed phenotype. This approach has been successfully used in P. profundum for verifying gene mutation-growth phenotype relationships .

• Controlled Expression Systems: Using inducible promoters to control mutM expression levels can establish dose-response relationships between mutM activity and phenotypic effects, helping to distinguish primary from secondary effects.

• Site-Directed Mutagenesis: Creating variants with specific mutations in catalytic residues versus structural regions can separate enzyme activity effects from potential protein-protein interaction effects.

• Temporal Analysis: Monitoring the kinetics of phenotypic changes after mutM inactivation or restoration can help distinguish immediate (likely direct) effects from delayed (possibly indirect) consequences.

• Epistasis Analysis: Examining double mutants of mutM with genes in related pathways can reveal functional relationships and pathway positions.

• Biochemical Verification: Directly measuring DNA repair activity and oxidative damage levels in mutM mutants versus wild-type under various conditions provides mechanistic evidence for direct effects.

• Global Analysis Approaches: Transcriptomics, proteomics, or metabolomics comparing mutM mutants to wild-type can reveal the scope of cellular changes and help distinguish primary from secondary effects.

These comprehensive approaches collectively provide strong evidence for causal relationships between mutM function and observed phenotypes, overcoming the challenge of pleiotropy that often complicates interpretation of mutant phenotypes in bacteria adapted to extreme environments.

What are the most significant open questions in P. profundum mutM research?

Despite progress in understanding P. profundum mutM, several significant open questions remain that represent important directions for future research:

• Pressure-Temperature Adaptation Mechanisms: How exactly do structural modifications in P. profundum mutM confer adaptation to both high pressure and low temperature simultaneously? The molecular details of this dual adaptation remain incompletely understood and represent a frontier in protein biophysics.

• Regulatory Networks: How is mutM expression regulated in response to environmental signals in P. profundum? The complete signal transduction pathways linking pressure/temperature sensing to DNA repair gene expression remain to be fully elucidated, despite evidence for their importance in adaptation .

• Functional Redundancy: What other DNA repair pathways complement or overlap with mutM function in P. profundum, and how is this redundancy orchestrated under different stress conditions? The relative importance of various DNA repair mechanisms in maintaining genomic integrity in the deep sea remains unclear.

• Evolutionary History: What was the evolutionary trajectory that led to the current form of P. profundum mutM? Understanding whether adaptations arose through gradual modification or more rapid selection events would provide insights into molecular evolution under extreme conditions.

• Ecological Significance: How does mutM function contribute to P. profundum's ecological fitness and population dynamics in its natural deep-sea habitat? The connection between molecular function and ecosystem role remains poorly characterized.

These open questions highlight that while significant progress has been made in understanding the basic function and adaptation of P. profundum mutM, much remains to be discovered about how this important DNA repair enzyme supports life in one of Earth's most extreme environments.

How might future methodological advances enhance our understanding of P. profundum mutM?

Future methodological advances across multiple fields promise to significantly enhance our understanding of P. profundum mutM through several transformative approaches:

• High-Pressure Structural Biology: Emerging techniques for protein structure determination under high pressure, including specialized NMR methods and high-pressure crystallography chambers, will reveal how the enzyme's structure responds dynamically to pressure changes.

• Single-Molecule Enzymology: Advanced single-molecule techniques adapted for high-pressure conditions will enable direct observation of mutM catalysis under native-like conditions, revealing mechanistic details obscured in bulk assays.

• In Situ Gene Editing: CRISPR-Cas technologies adapted for deep-sea microorganisms will enable precise genetic manipulation of P. profundum in its natural environment, allowing direct testing of mutM function without laboratory cultivation biases.

• Deep-Sea Metatranscriptomics: Improved sampling and sequencing technologies will enable analysis of mutM expression patterns in natural deep-sea communities, revealing its ecological importance and regulation under truly natural conditions.

• Computational Approaches: Advanced molecular dynamics simulations incorporating pressure effects will provide atomic-level insights into mutM's pressure adaptations, while machine learning approaches may identify subtle sequence patterns associated with pressure adaptation across diverse homologs.

• Synthetic Biology: Cell-free systems engineered to function under high pressure will enable rapid prototyping and testing of mutM variants without the complications of whole-cell physiology.

These methodological advances will collectively bridge the gap between molecular mechanisms and ecological functions, providing a comprehensive understanding of how P. profundum mutM contributes to life's adaptation to extreme environments, with potential applications ranging from biotechnology to astrobiology.

What broader implications does P. profundum mutM research have for understanding genome stability mechanisms across diverse environments?

Research on P. profundum mutM extends far beyond this specific enzyme, offering profound insights into fundamental principles of genome stability across diverse environments:

• Universal vs. Environment-Specific Mechanisms: By revealing which aspects of DNA repair are conserved across environments and which are specialized, P. profundum mutM research helps delineate the core universal requirements for genome stability versus environment-specific adaptations. This fundamental distinction informs our understanding of the evolutionary constraints on DNA repair systems.

• Adaptation Rate Limitations: Understanding how essential DNA repair enzymes like mutM adapt to extreme conditions provides insights into potential rate-limiting steps in colonization of new environments. The necessity for functional DNA repair may represent a critical bottleneck in adaptation to novel niches.

• Biophysical Constraints and Solutions: Research on pressure-adapted mutM reveals fundamental biophysical principles governing protein function under extreme conditions, with implications for understanding adaptation to other physical extremes like temperature, radiation, or desiccation.

• Evolutionary Plasticity of Essential Systems: The study of adaptations in core housekeeping functions like DNA repair reveals the surprising evolutionary plasticity of seemingly constrained systems, informing broader evolutionary theory about the modification of essential cellular processes.

• Biomedical Applications: Insights from pressure-adapted DNA repair enzymes may inform the development of enhanced DNA repair systems for biomedical applications, potentially addressing conditions where oxidative DNA damage plays a pathological role.

• Extremophile Biotechnology: Understanding how DNA repair functions in extreme environments enables development of biotechnological applications involving extremophilic organisms or their molecular components, including applications in bioremediation, biosensing, and sustainable industrial processes.