Recombinant Corynebacterium glutamicum Error-prone DNA polymerase (dnaE2), partial

What is Corynebacterium glutamicum and why is it significant in biotechnology research?

Corynebacterium glutamicum is a gram-positive, non-pathogenic soil bacterium that serves as an important industrial workhorse for the production of amino acids and various chemicals. Its significance stems from its robust metabolic capabilities and amenability to genetic engineering, making it a valuable platform organism for biotechnological applications . The bacterium has been extensively developed and optimized for industrial fermentation processes, particularly for amino acid production, which highlights its economic importance in the biotechnology sector.

What distinguishes error-prone DNA polymerases from high-fidelity polymerases in bacterial systems?

Error-prone DNA polymerases differ from high-fidelity polymerases primarily in their reduced proofreading capability, which results in higher mutation rates during DNA synthesis. While high-fidelity polymerases like the primary replicative DNA polymerase III maintain genetic stability through efficient proofreading, error-prone polymerases like DnaE2 are typically specialized for translesion synthesis during stress conditions . The key distinction lies in their structural features, particularly in domains responsible for proofreading activity. In C. glutamicum, for instance, the histidinol phosphatase (PHP) domain of DnaE1 (α subunit of DNA polymerase III) has been identified as the critical proofreading element, and mutations in this domain lead to elevated spontaneous mutagenesis .

How does the dnaE2 gene function in bacterial mutagenesis systems?

The dnaE2 gene encodes an error-prone DNA polymerase that plays a crucial role in stress-induced mutagenesis in bacteria. Based on studies in mycobacteria, dnaE2 is typically part of the imuA-imuB-dnaE2 cassette, where all three genes are essential for DNA damage-induced mutagenesis . The DnaE2 protein functions within a complex with ImuA and ImuB proteins. This complex associates with the sliding clamp processivity factor (β-subunit of DNA polymerase III) through interactions with the ImuB subunit, allowing DnaE2 access to replication sites where it performs error-prone DNA synthesis . This mechanism enables bacteria to adapt to stressful conditions through increased genetic diversity resulting from higher mutation rates.

How can error-prone DNA polymerases be engineered for controlled mutagenesis in C. glutamicum?

Engineering error-prone DNA polymerases for controlled mutagenesis in C. glutamicum involves strategic modifications to the DNA replication and repair machinery. Research has shown that targeting specific domains of replicative polymerases, particularly the PHP domain of DnaE1, can significantly impact mutation rates. Certain variants with PHP mutations allowed elevated spontaneous mutagenesis in C. glutamicum . Additionally, repression of the NucS-mediated post-replicative mismatch repair pathway or overexpression of specifically screened NucS variants has been demonstrated to impair DNA replication fidelity .

For controlled mutagenesis, a binary genetic mutator system can be developed by simultaneously interfering with both the DNA replication and repair machinery. This approach has been shown to increase mutation rates by up to 2352-fold in C. glutamicum, facilitating rapid evolutionary engineering to achieve desired phenotypes such as stress tolerance and increased protein production . The key advantage of this approach is the ability to generate genetic diversity in a controlled manner without introducing external mutagens.

What is the relationship between DnaE1 and DnaE2 in C. glutamicum compared to other bacterial species?

In C. glutamicum, DnaE1 serves as the primary α subunit of DNA polymerase III responsible for genome replication, with its PHP domain functioning as the key proofreading element . This differs from the arrangement in Escherichia coli, where the ε subunit (DnaQ) handles proofreading functions. Interestingly, the DnaQ homolog in C. glutamicum has been proven irrelevant to DNA replication fidelity .

In contrast, DnaE2 is an alternative α subunit that typically functions in stress-induced mutagenesis. While the specific role of DnaE2 in C. glutamicum is not fully characterized in the provided search results, studies in Mycobacterium tuberculosis show that DnaE2 is part of the imuA-imuB-dnaE2 cassette essential for DNA damage-induced mutagenesis . This specialized role allows bacteria to increase mutation rates under stressful conditions, potentially facilitating adaptation through accelerated evolution.

The functional relationship between DnaE1 and DnaE2 represents an evolved balance between genomic stability during normal growth and accelerated mutagenesis during stress conditions, a pattern that appears to be conserved across different bacterial species but with species-specific variations in the molecular mechanisms.

How do different bacterial species utilize error-prone DNA synthesis for adaptive evolution?

Different bacterial species have evolved distinct mechanisms to leverage error-prone DNA synthesis for adaptive evolution. In M. tuberculosis, the DnaE2-ImuA-ImuB complex is activated during stress conditions, particularly DNA damage, leading to error-prone DNA synthesis that contributes to the emergence of drug resistance . This stress-induced mutagenesis mechanism allows M. tuberculosis to adapt to antimicrobial pressure within the host environment.

In C. glutamicum, altered fidelity of DNA polymerases can be achieved through mutations in the PHP domain of DnaE1 or by interfering with the NucS-mediated post-replicative mismatch repair pathway . This approach has been successfully utilized to develop strains with enhanced tolerance to environmental stresses and improved protein production capabilities .

The strategic utilization of error-prone DNA synthesis represents a powerful adaptive mechanism that allows bacteria to navigate challenging environments by generating genetic diversity. This principle has been harnessed in laboratory settings to accelerate strain improvement for biotechnological applications.

What recombineering techniques are most effective for genetic manipulation of DNA polymerases in C. glutamicum?

The RecET recombineering system has proven highly effective for genetic manipulation in C. glutamicum ATCC14067, particularly for modifications involving DNA polymerase genes. This system utilizes the exonuclease-recombinase pair RecE and RecT from Rac phage to facilitate homologous recombination with linear double-stranded DNA (dsDNA) . Among several tested orthologous exonuclease-recombinase pairs, including Exo/Bet, Orf47/Orf48, and OrfB/OrfC, the RecET pair demonstrated the highest recombination efficiency in C. glutamicum ATCC14067 .

For optimal RecET recombineering in C. glutamicum, several parameters should be considered:

• Homology arm length: Efficiency increases with homology arm length up to 800 bp, beyond which no significant improvement is observed .

• DNA concentration: Higher dsDNA substrate concentrations (up to 4 μg) significantly increase recombination efficiency, with approximately 653 recombinants obtained when using 4 μg of DNA cassette .

• DNA modification: Phosphorylation of the dsDNA substrate can double the recombineering efficiency .

• Induction and recovery time: Optimal efficiency is achieved with 5 hours of induction time and 4 hours of recovery time, yielding approximately 1.41 × 10³ colonies per mL .

This approach is particularly valuable for studying DNA polymerases as it allows precise genetic modifications with high efficiency.

How can the Cre/loxP system be integrated with RecET recombineering for markerless deletion in C. glutamicum?

The integration of the Cre/loxP system with RecET recombineering enables efficient markerless deletion in C. glutamicum, a crucial capability for sequential genetic modifications. This combined approach, known as the RecET-Cre/loxP system, utilizes a self-excisable linear dsDNA cassette containing the site-specific Cre/loxP recombination system .

The methodology involves:

• Design of a self-excisable linear dsDNA cassette containing:

  • Homology arms targeting the gene of interest

  • Mutant loxP sites (lox71 and lox66) flanking the selection marker

  • Inducible Cre recombinase expression system

• Transformation of the linear cassette into C. glutamicum expressing the RecET proteins, resulting in replacement of the target gene with the Cre-Kan cassette .

• Induction of Cre expression (using 1 mM theophylline) to catalyze recombination between lox71 and lox66, leading to excision of the selection marker and Cre expression cassette .

• Confirmation of markerless deletion by PCR and sequencing.

This system has demonstrated high efficiency with at least 94% of transformants successfully replacing the targeted gene with the Cre-Kan cassette, and no mutations observed in the correct recombinants . This approach is particularly valuable for studies requiring multiple sequential gene modifications, such as those investigating the complex interactions between different components of the DNA replication and repair machinery.

What are the optimal parameters for measuring mutation rates resulting from error-prone DNA polymerase activity?

Measuring mutation rates resulting from error-prone DNA polymerase activity requires careful experimental design and standardized methodologies to ensure accuracy and reproducibility. When evaluating mutation rates in systems with engineered DNA polymerases, such as those with modified PHP domains in DnaE1 or altered mismatch repair pathways, researchers should consider:

• Selection of appropriate marker genes that provide clear phenotypic readouts when mutated.

• Implementation of fluctuation analysis (such as the Luria-Delbrück method) to distinguish between pre-existing mutations and those arising during the experiment.

• Utilization of both positive and negative selection strategies to comprehensively assess mutation spectra.

• Standardized growth conditions to control for environmental factors that might influence mutation rates independently of the engineered polymerase.

In studies with C. glutamicum, binary genetic mutators created through simultaneous interference with the DNA replication and repair machinery demonstrated mutation rate increases of up to 2352-fold compared to wild-type strains . This quantification provides a benchmark for evaluating the mutagenic potential of engineered polymerases and can serve as a reference point for designing systems with desired mutation rates for specific applications.

How can engineered error-prone DNA polymerases be utilized for adaptive laboratory evolution in C. glutamicum?

Engineered error-prone DNA polymerases offer a powerful approach for adaptive laboratory evolution (ALE) in C. glutamicum, enabling researchers to rapidly generate genetic diversity and select for desired phenotypes. The strategic manipulation of DNA replication and repair machinery to create genetic mutators has been successfully applied to improve stress tolerance and protein overproduction phenotypes in C. glutamicum .

The methodology involves:

• Engineering components of the DNA replication machinery, such as introducing mutations in the PHP domain of DnaE1, which serves as the key proofreading element in C. glutamicum .

• Modifying DNA repair pathways, such as repressing the NucS-mediated post-replicative mismatch repair pathway or overexpressing specific NucS variants .

• Combining multiple modifications to create binary genetic mutators that substantially increase mutation rates, thereby accelerating the evolutionary process .

• Applying selective pressure relevant to the desired phenotype, such as environmental stressors or growth conditions that favor increased production of specific metabolites.

• Screening and analyzing evolved strains to identify genetic changes responsible for improved phenotypes.

This approach bypasses the limitations of rational design by harnessing natural selection to identify beneficial mutations that might not be predictable through computational approaches. The key advantage is that no prior knowledge of the genetic determinants for the desired phenotype is required, making it particularly valuable for complex traits governed by multiple genes.

What are the implications of error-prone DNA polymerases for understanding and combating antimicrobial resistance?

Error-prone DNA polymerases play a significant role in the emergence of antimicrobial resistance in bacteria, making them important targets for both understanding and potentially combating resistance development. Studies in M. tuberculosis have shown that DnaE2, as part of the imuA-imuB-dnaE2 cassette, contributes to the development of drug resistance through error-prone DNA synthesis during infection .

The strategic importance of these polymerases is underscored by several factors:

• Error-prone DNA synthesis by the DnaE2-ImuA-ImuB polymerase complex contributes to drug resistance during TB infection by generating genetic diversity under stress conditions .

• Understanding these mechanisms provides potential targets for novel adjunctive therapies to combat drug resistance in pathogens .

• Comparative studies between pathogenic and non-pathogenic bacteria (such as M. tuberculosis and C. glutamicum) can reveal conserved mechanisms that might be broadly applicable for antimicrobial development.

• By characterizing the assembly, catalytic activity, and mutational properties of error-prone DNA polymerase complexes, researchers can develop strategies to modulate their activity in a targeted manner .

Future research in this area could focus on developing inhibitors that specifically target error-prone polymerases to limit bacterial adaptation during antimicrobial therapy, potentially slowing the emergence of resistance while maintaining the activity of frontline antibiotics.

What are common challenges in recombineering experiments with C. glutamicum and how can they be addressed?

Researchers working with recombineering in C. glutamicum may encounter several technical challenges that can impact experimental success. Based on published research, common challenges and their solutions include:

• Strain-specific variations in recombination efficiency: Standard protocols developed for one strain (e.g., C. glutamicum ATCC13032) often don't work in other strains like ATCC14067 . This is likely due to specific or unclear genetic information among different Corynebacteria.

  • Solution: Test multiple recombination systems (RecET, OrfB/OrfC, etc.) to identify the most effective one for your specific strain .

• Low frequency of double-crossover events: Without specialized systems, double-crossover events rarely occur in C. glutamicum .

  • Solution: Implement the RecET recombineering system, which has shown high efficiency for double-crossover events with linear dsDNA .

• Optimizing homology arm length: Too short homology arms result in poor recombination efficiency, while excessively long arms don't necessarily improve results and require more effort to generate .

  • Solution: Use homology arms of approximately 800 bp, which has been identified as the optimal length for RecET recombineering in C. glutamicum ATCC14067 .

• DNA uptake limitations: The efficiency of transformation can significantly impact recombineering success.

  • Solution: Optimize electroporation parameters specifically for C. glutamicum and verify DNA uptake capability using control plasmids .

• Marker excision challenges: Traditional Cre/loxP systems in C. glutamicum involve two plasmids and two rounds of transformation, which is tedious and laborious .

  • Solution: Implement the self-excisable linear dsDNA cassette combining the Cre/loxP system for streamlined markerless deletion .

Addressing these challenges through optimized protocols significantly improves the success rate of genetic manipulations in C. glutamicum and enables more efficient studies of DNA polymerases and other genetic elements.

How can researchers verify the activity and fidelity of engineered DNA polymerases in C. glutamicum?

Verifying the activity and fidelity of engineered DNA polymerases in C. glutamicum requires a multi-faceted approach that combines genetic, biochemical, and phenotypic analyses. Researchers should consider implementing:

• Mutation rate analysis: Quantify spontaneous mutation rates using fluctuation assays with selectable markers. In engineered systems with altered DNA replication and repair machinery, mutation rates have been increased by up to 2352-fold compared to wild-type strains .

• Mutation spectrum analysis: Sequence multiple independently derived mutants to characterize the types of mutations (transitions, transversions, insertions/deletions) produced by the engineered polymerase. This provides insights into the specific error patterns associated with particular polymerase modifications.

• Growth rate and fitness measurements: Monitor the impact of increased mutation rates on bacterial fitness under various conditions. This helps assess the balance between beneficial adaptations and deleterious mutations.

• Stress tolerance phenotyping: Evaluate how engineered strains respond to various stressors, as error-prone polymerases often show differential activity under stress conditions. This approach has successfully identified strains with enhanced stress tolerance and protein overproduction capabilities .

• Competitive evolution experiments: Compare the adaptive capacity of strains with engineered polymerases versus wild-type strains during laboratory evolution under selective conditions. This dynamic approach reveals the practical utility of engineered polymerases for strain improvement.

By combining these approaches, researchers can comprehensively characterize the activity, fidelity, and practical utility of engineered DNA polymerases in C. glutamicum for various applications in metabolic engineering and synthetic biology.