Recombinant Lactobacillus plantarum MutS2 protein (mutS2), partial

What is MutS2 protein and how does it differ from other MutS family proteins?

MutS2 belongs to a subfamily of bacterial MutS proteins that is distinct from the well-characterized MutS1 family involved in mismatch repair (MMR). While MutS1 proteins recognize DNA replication errors during their participation in MMR, MutS2 proteins serve different functions that vary between bacterial species. In Bacillus subtilis, MutS2 has been demonstrated to promote homologous recombination, which contrasts with its role in Helicobacter pylori and Thermus thermophilus where it suppresses recombination . MutS2 proteins contain an ABC ATPase domain and a C-terminal small MutS-related (Smr) domain with potential endonuclease activity, but they lack the mismatch binding domain and β-clamp interaction site found in MutS1 proteins .

What is the domain architecture of L. plantarum MutS2 and how does each domain contribute to function?

Based on structural and functional analyses of MutS2 proteins from other bacterial species, L. plantarum MutS2 likely contains several key domains:

• N-terminal region - Important for proper protein function; in B. subtilis, the N-terminal portion complements the function of the C-terminal Smr domain

• ABC ATPase domain - Provides energy for mechanical activities such as ribosome splitting

• KOW domain - Involved in ribosome interaction, particularly with collided ribosomes

• C-terminal Smr domain - Contains endonuclease activity, necessary but not sufficient for protein function

In B. subtilis, studies have shown that the C-terminal Smr domain requires the N-terminal portion of MutS2 for proper function in vivo. When expressed alone, the Smr domain was unable to complement the mitomycin C sensitivity phenotype of a mutS2 deletion strain . The KOW and SMR domains work together in binding to collided ribosomes, while the ABC ATPase domain provides the mechanical force for ribosome splitting .

How does MutS2 contribute to homologous recombination in bacteria?

The role of MutS2 in homologous recombination appears to be species-dependent. In B. subtilis, several lines of evidence suggest that MutS2 promotes homologous recombination:

• Deletion of mutS2 renders B. subtilis sensitive to mitomycin C (MMC), which requires homologous recombination for repair

• The Δmuts2 strain shows decreased transformation efficiency with both plasmid and chromosomal DNA

• Genetic analysis revealed that MMC sensitivity in the Δmuts2 strain was dependent on recombination (recA) and not on nucleotide excision repair

• The mutS2 deletion is additive with a recU deletion (Holliday junction endonuclease), suggesting they may have redundant functions in homologous recombination

This promotion of recombination in B. subtilis contrasts with the antirecombination activity observed in H. pylori and T. thermophilus, where MutS2 proteins suppress homologous recombination through their endonuclease activity toward recombination intermediates, including Holliday junctions . If L. plantarum MutS2 functions similarly to B. subtilis MutS2, it might be involved in processing recombination intermediates and facilitating DNA repair through homologous recombination.

What is the role of MutS2 in ribosome quality control?

Recent research has uncovered a fascinating role for B. subtilis MutS2 in ribosome quality control. According to Cerullo et al. (2022), MutS2 is recruited to collided ribosomes that have stalled during translation . The study revealed several key aspects of this function:

• MutS2 binds to collided ribosomes through its SMR and KOW domains

• It forms a dimer when bound to collided ribosomes, with its ATPase domains contacting the lead ribosome

• MutS2 uses its ABC ATPase activity to split ribosomes into subunits

• This splitting targets the nascent peptide for degradation through the ribosome quality control pathway

• Unlike the related protein SmrB in E. coli, B. subtilis MutS2 does not appear to cleave mRNA during this process

This function represents a distinct role for MutS2 beyond DNA maintenance and recombination. The ability to recognize and resolve stalled translation complexes suggests MutS2 plays a broader role in cellular quality control than previously appreciated.

How might MutS2 function in L. plantarum stress response?

While direct evidence for MutS2's role in L. plantarum stress response is limited, we can infer potential functions based on related findings:

In L. plantarum, the LuxS enzyme has been linked with acid stress response and increased adherence capabilities of cells as a bacterial survival mechanism . LuxS is responsible for synthesizing the bacterial interspecies signaling molecule autoinducer-2 (AI-2) and has been characterized as part of stress response pathways .

If MutS2 in L. plantarum functions similarly to other bacterial MutS2 proteins in maintaining genome integrity, it might be involved in responding to DNA-damaging stressors. The sensitivity of B. subtilis ΔmutS2 strains to mitomycin C suggests MutS2 plays a role in DNA damage response pathways . L. plantarum MutS2 might similarly function in responding to environmental stressors that cause DNA damage, such as oxidative stress, radiation, or antimicrobial compounds.

What are the optimal conditions for expressing recombinant L. plantarum MutS2?

Based on general principles for bacterial protein expression and insights from the literature, optimal conditions for expressing functional L. plantarum MutS2 would include:

Western blot analysis can be used to monitor protein expression levels, as demonstrated in B. subtilis MutS2 studies where antiserum raised against purified MutS2 was used to track protein abundance in vivo .

How can CRISPR/Cas9 genome editing be utilized to study MutS2 function?

CRISPR/Cas9 genome editing provides powerful tools for studying MutS2 function. A single plasmid-based CRISPR/Cas9 system was described for B. subtilis that allows for efficient markerless genetic manipulation . Similar approaches could be adapted for L. plantarum to:

• Generate precise deletion mutants of mutS2 or specific domains

• Introduce point mutations in functional motifs (ATP binding sites, catalytic residues)

• Create reporter fusions to study localization and expression patterns

• Engineer conditional expression systems for functional studies

The advantage of CRISPR/Cas9 over traditional methods is the ability to make markerless, precise genomic modifications without leaving antibiotic resistance markers that might affect phenotypic analysis . This is particularly valuable for studying MutS2, which has multiple domains with potentially distinct functions.

What biochemical assays can measure MutS2 activity in vitro?

Several biochemical assays can be used to characterize different aspects of MutS2 function:

DNA Binding and Processing:

• Electrophoretic mobility shift assays (EMSA) to measure binding to various DNA structures, particularly recombination intermediates like Holliday junctions

• Nuclease assays using labeled DNA substrates to assess potential Smr domain-mediated cleavage activity

• Branch migration and strand exchange assays to evaluate effects on recombination intermediates

ATPase Activity:

• Malachite green assays to quantify ATP hydrolysis rates

• Analysis of ATPase stimulation by different DNA substrates or ribosomal components

Ribosome Interaction:

• Ribosome binding assays using purified components

• Ribosome splitting assays measuring separation of subunits

• Sucrose gradient centrifugation to analyze ribosome profiles

Structural Analysis:

• Limited proteolysis to identify flexible regions and domain boundaries

• Circular dichroism to assess secondary structure composition

• Analytical ultracentrifugation to determine oligomeric state

In B. subtilis studies, complementation assays were used to determine functional domains of MutS2 important for mitomycin C tolerance, revealing that the C-terminal Smr domain is necessary but not sufficient for MutS2 function .

How should contradictory findings about MutS2 function across species be interpreted?

The contradictory findings regarding MutS2 function across bacterial species present an interesting scientific puzzle that requires careful interpretation:

• Evolutionary Divergence: MutS2 proteins may have evolved different functions in different bacterial lineages in response to specific environmental pressures and genomic requirements. For example, the promotion of homologous recombination by B. subtilis MutS2 contrasts with the suppression of recombination by T. thermophilus MutS2, which may reflect adaptations to different ecological niches .

• Multifunctionality: Rather than having contradictory functions, MutS2 proteins may be multifunctional, with different activities being emphasized or repressed in different species or under different conditions. The recent finding that B. subtilis MutS2 participates in ribosome quality control in addition to DNA metabolism illustrates this multifunctionality .

• Context Dependency: MutS2 function may depend on genomic context, including the presence or absence of other DNA repair and recombination systems. The genetic interaction observed between mutS2 and recU in B. subtilis suggests redundancy or cooperation between these systems .

To resolve these contradictions for L. plantarum MutS2, researchers should consider:

• Comparing MutS2 sequences across species to identify key residues associated with functional differences

• Testing L. plantarum MutS2 function under various conditions to identify context-dependent activities

• Examining genetic interactions with other recombination and repair genes

• Considering the ecological context of L. plantarum and how it might influence MutS2 function

What bioinformatic approaches can identify potential MutS2 interaction partners?

Several bioinformatic approaches can help identify potential interaction partners for L. plantarum MutS2:

Genomic Context Analysis:

• Examine gene neighborhood conservation across bacterial species

• Identify consistently co-localized genes that may encode interacting proteins

• Analyze operon structures that might suggest functional relationships

Co-evolution Analysis:

• Apply direct coupling analysis to identify co-evolving residues between proteins

• Use the mirror-tree method to find proteins with similar evolutionary histories

• Perform mutual information analysis of sequence alignments

Expression Correlation:

• Analyze transcriptomic datasets for co-regulated genes

• Identify proteins with similar expression patterns across conditions

• Perform cluster analysis of expression profiles

Domain-Based Predictions:

• Identify proteins with complementary domains known to interact with MutS2 components

• Analyze domain architecture similarities between potential partners

• Search for proteins with domains known to interact with ABC ATPases or SMR domains

In B. subtilis, MutS2 protein abundance was found to be stable throughout exponential growth and into the stationary phase, providing clues about its regulation and potential functional contexts . Studies in L. plantarum have identified stress-responsive proteins like LuxS that might function in related pathways to MutS2 .

How can structural modeling inform MutS2 functional studies?

Structural modeling provides valuable insights into MutS2 function through several approaches:

The cryo-EM structure of B. subtilis MutS2 revealed that it binds to collided ribosomes as a dimer, with its ATPase domains contacting the lead ribosome and its SMR and KOW domains interacting with the collided ribosomes . This structural information provides a framework for understanding how MutS2 might function in L. plantarum.

How does MutS2 contribute to bacterial adaptation to environmental stressors?

MutS2's role in bacterial adaptation to environmental stressors represents an emerging area of research with several intriguing possibilities:

• DNA Damage Response: In B. subtilis, MutS2 promotes tolerance to the DNA-damaging agent mitomycin C through its role in homologous recombination . This suggests MutS2 contributes to genomic stability under genotoxic stress conditions.

• Translation Quality Control: The recently discovered role of B. subtilis MutS2 in splitting stalled ribosomes indicates it helps cells cope with translation stress . By resolving collided ribosomes, MutS2 may prevent the accumulation of potentially toxic incomplete protein products.

• Stress-Induced Recombination: If L. plantarum MutS2 promotes recombination like its B. subtilis counterpart, it might facilitate adaptive genomic changes under stress conditions, potentially accelerating evolution in challenging environments.

• Cross-Talk with Stress Response Pathways: In L. plantarum, proteins involved in stress response like LuxS have been linked to acid stress response and increased adherence capabilities . MutS2 might interact with these pathways to coordinate cellular responses to stress.

Understanding how MutS2 contributes to stress adaptation in L. plantarum would require:

• Comparing wild-type and mutS2-deficient strains under various stress conditions

• Analyzing changes in gene expression and protein interaction networks during stress

• Measuring mutation rates and recombination frequencies under stress conditions

• Investigating potential connections to known stress response pathways

What is the significance of the dual functionality of MutS2 in DNA metabolism and ribosome quality control?

The discovery that B. subtilis MutS2 functions in both DNA metabolism (promoting homologous recombination) and ribosome quality control (splitting stalled ribosomes) represents a fascinating case of functional duality with several important implications:

• Resource Efficiency: By utilizing one protein for multiple cellular quality control processes, bacteria can achieve economy in their genome size and protein synthesis requirements.

• Coordinated Responses: The dual functionality may allow for coordinated responses to certain stressors that affect both DNA integrity and translation fidelity, such as nutrient limitation or antibiotic exposure.

• Evolutionary Innovation: This functional duality may represent an evolutionary innovation that allows bacteria like B. subtilis to efficiently manage quality control across different cellular processes.

• Mechanistic Similarities: The ability of MutS2 to recognize and process both DNA structures (recombination intermediates) and RNA-protein complexes (stalled ribosomes) suggests interesting mechanistic similarities between these seemingly disparate processes.

If L. plantarum MutS2 shares this dual functionality, it would suggest a conserved strategy for cellular quality control across different Gram-positive bacteria. Understanding the relative importance of these functions in L. plantarum would provide insights into how this bacterium maintains cellular homeostasis in its natural environments, including fermented foods and the gastrointestinal tract.

How can our understanding of MutS2 function inform biotechnological applications of L. plantarum?

Lactobacillus plantarum is widely used in food fermentation and as a probiotic, making insights into MutS2 function potentially valuable for biotechnological applications:

• Strain Improvement: Understanding MutS2's role in recombination could inform strategies for genetic manipulation of L. plantarum strains for improved technological properties. If MutS2 promotes recombination as in B. subtilis, modulating its expression might enhance transformation efficiency for strain engineering .

• Stress Resistance: Knowledge of how MutS2 contributes to stress tolerance could help develop more robust L. plantarum strains for industrial applications. The apparent connection between MutS2 and stress response mechanisms suggests it might be a target for enhancing survival under processing conditions .

• Protein Production Systems: The newly discovered role of MutS2 in ribosome quality control suggests it might influence protein expression efficiency . Optimizing MutS2 function could potentially enhance recombinant protein production in L. plantarum-based expression systems.

• Genomic Stability: For applications requiring stable maintenance of introduced genetic elements, understanding MutS2's impact on genomic stability would be valuable. The contrasting roles of MutS2 in different bacteria (promoting versus suppressing recombination) highlight the importance of species-specific characterization .

• Interspecies Communication: If L. plantarum MutS2 interacts with signaling pathways as suggested by connections to LuxS in stress response, it might influence the bacterium's interactions with other microbes in food fermentations or the gut microbiome .