Recombinant Pseudomonas syringae pv. tomato Error-prone DNA polymerase (dnaE2), partial

What is DnaE2 and how does it differ from other DNA polymerases in P. syringae?

DnaE2 is an error-prone DNA polymerase found in several bacterial species including Pseudomonas syringae. Unlike the primary replicative polymerase DnaE1, DnaE2 lacks robust proofreading capability, resulting in higher mutation rates during DNA synthesis. Sequence analysis and evolutionary studies have revealed that DnaE2 is part of a distinct DNA polymerase family that contributes to adaptive mutagenesis under stress conditions. In myxobacteria, both dnaE1 and dnaE2 genes are highly conserved, but they serve different functions in genome maintenance and evolution . While DnaE1 is essential for normal replication fidelity, DnaE2 appears to play a specialized role in stress-induced mutagenesis, particularly in response to DNA-damaging conditions.

How does the error rate of DnaE2 compare to other DNA polymerases?

DnaE2 exhibits significantly higher error rates compared to primary replicative polymerases. Studies in myxobacteria have quantified this difference, showing that deletion of the dnaE2 gene reduces mutation rates approximately 7.6-fold compared to wild-type strains. The following table illustrates the dramatic difference in mutation rates between wild-type, dnaE2-deletion, and dnaE2-overexpression strains:

This data clearly demonstrates that DnaE2 significantly influences the basal mutation rate of bacterial genomes . Notably, overexpression of DnaE2 increases mutation rates approximately 3-fold compared to wild-type, while its deletion reduces mutation rates by nearly an order of magnitude.

What methods are most effective for creating recombinant error-prone DnaE2 constructs?

Creating recombinant error-prone DnaE2 constructs requires careful molecular cloning and expression strategies. The most effective approach involves:

• Gene isolation: Amplify the dnaE2 gene from P. syringae pv. tomato genomic DNA using high-fidelity PCR.

• Vector construction: Clone the gene into an appropriate expression vector with a controllable promoter. Research has demonstrated success using vectors like pSWU19 where the dnaE2 gene can be placed under the control of a constitutive or inducible promoter .

• Modification strategies: To enhance error-prone characteristics, introduce specific mutations in the polymerase domain or exonuclease domain. For instance, research on organelle DNA polymerases has shown that combining exonuclease deficiency with polymerization domain substitutions can increase error rates up to 140-fold compared to wild-type enzymes .

• Expression system: Transform the construct into an appropriate host strain. For studies involving P. syringae, electroporation has been proven effective for introducing recombinant DNA .

The methodology used by researchers for complementation and overexpression of dnaE2 provides a valuable template. They cloned the dnaE2 gene with its native promoter (500 bp upstream sequence) using specific primer pairs and ligated the fragment into a suitable vector. Additionally, they created fusion constructs with strong promoters like the pilA promoter to achieve overexpression .

How can recombineering techniques facilitate genetic manipulation of dnaE2 in P. syringae?

Recombineering (recombination-mediated genetic engineering) offers powerful tools for precise genetic manipulation of dnaE2 in P. syringae. This method leverages homologous recombination proteins derived from bacteriophages to promote efficient recombination between genomic loci and linear DNA fragments.

For manipulating dnaE2 in P. syringae, researchers have successfully employed a system based on RecTE homologs identified in P. syringae pv. syringae B728a . The process involves:

• Expression of recombinase proteins: The P. syringae RecT homolog is sufficient to promote recombination with single-stranded DNA oligonucleotides, while efficient recombination of double-stranded DNA requires expression of both RecT and RecE homologs .

• Substrate design: Design linear DNA fragments (either oligonucleotides or PCR products) with 40-50 bp homology arms flanking the region of interest in the dnaE2 gene.

• Transformation: Introduce the linear DNA into competent P. syringae cells expressing the recombinase proteins via electroporation.

• Selection: Apply appropriate selection methods to identify successful recombinants.

This RecA-independent recombination system has shown significant utility for targeted gene disruptions in the P. syringae chromosome . The recombinase binds to 3' single-stranded DNA ends exposed by exonuclease activity, forming a protein-DNA filament that protects the substrate DNA and promotes annealing with the homologous genomic sequence.

How is dnaE2 expression regulated in P. syringae under various environmental conditions?

The regulation of dnaE2 expression in P. syringae represents a sophisticated response system to environmental stressors. Based on studies in related bacteria, dnaE2 expression appears to be tightly controlled and specifically induced under stress conditions:

• DNA damage response: DnaE2 is part of the SOS response system, with expression significantly upregulated following UV exposure or treatment with DNA-damaging agents like H₂O₂ and MMS (methyl methanesulfonate) .

• Growth phase dependency: Research in myxobacteria has shown that dnaE2 expression levels vary depending on growth phase and nutritional status .

• Transcriptional control: The dnaE2 gene contains SOS boxes in its promoter region, allowing LexA-regulated expression in response to DNA damage.

Experimental data from myxobacteria demonstrate that under nutritional conditions (at 24 hours of incubation in CTT medium), dnaE2 overexpression reaches approximately 10 times higher levels than dnaE1. Similarly, under developmental conditions (at 6 hours of incubation on TPM), dnaE2 expression shows significant upregulation . The precise mechanisms controlling dnaE2 expression in P. syringae likely involve multiple regulatory elements responding to specific environmental cues, particularly those associated with plant-pathogen interactions and oxidative stress encountered during infection.

What role does DnaE2 play in the evolution of virulence and host specificity in P. syringae pv. tomato?

DnaE2 functions as a critical contributor to adaptive evolution in P. syringae pv. tomato, particularly in the context of host-pathogen interactions. Several lines of evidence support its significant role:

• Adaptive mutagenesis: As an error-prone polymerase, DnaE2 increases genetic diversity under stress conditions, potentially accelerating adaptation to new host environments or immune responses.

• Pathogen evolution: Studies have shown that DnaE2 contributes to bacterial pathogenicity and the emergence of drug resistance . By analogy, in P. syringae, DnaE2-mediated mutations likely contribute to evolution of virulence factors.

• Host range determination: The unique phylogenetic positioning of PtoDC3000 among P. syringae strains suggests that error-prone replication may have contributed to its unusually broad host range compared to typical tomato-specific strains .

• Effector diversification: Recombination and mutation events, potentially facilitated by error-prone polymerases like DnaE2, have played significant roles in the reassortment of type III secreted (T3S) effectors between strains . This is exemplified by the evolutionary history of the avrPto1 effector, which shows evidence of horizontal gene transfer and selective maintenance or loss depending on host resistance factors.

The phylogenetic analysis of P. syringae strains has revealed that PtoDC3000 belongs to a mixed group of almost identical P. syringae pv. maculicola and P. syringae pv. tomato isolates that can infect multiple plant species, including tomato, cauliflower, and Arabidopsis thaliana . This contrasts with typical P. syringae pv. tomato strains that form a distinct phylogenetic clade and exclusively infect tomato. The genetic plasticity provided by DnaE2 likely contributes to this phylogenetic and host range diversity.

How can DnaE2 be engineered to create controlled mutagenesis systems for evolutionary studies?

Engineering DnaE2 for controlled mutagenesis provides a powerful tool for evolutionary studies. Several approaches have proven effective:

• Inducible expression systems: Placing dnaE2 under the control of inducible promoters allows temporal control of mutagenesis. The pilA promoter has been successfully used for high-level expression in Myxococcus xanthus, resulting in approximately 500-1500 times higher expression than control strains .

• Modulation of error rates: Specific mutations in the polymerase domain can tune error rates. Research on organelle DNA polymerases demonstrated that combining exonuclease deficiency with polymerization domain substitutions increased error rates 140-fold relative to wild-type enzymes .

• Targeting mechanisms: Fusion of DnaE2 with DNA-binding domains can direct mutagenesis to specific genomic regions of interest.

• Conditional activity: Engineering temperature-sensitive variants allows temporal control of mutagenic activity.

An optimized controlled mutagenesis system would ideally include:

• A tightly regulated inducible promoter

• Ability to modulate mutation rate through protein engineering

• Targeting capability to focus mutations on genes of interest

• Reversibility to halt mutagenesis when desired

These engineered systems can accelerate experimental evolution studies, allowing researchers to observe adaptation processes in real-time and identify genetic changes associated with specific phenotypes, such as host range expansion or virulence enhancement in P. syringae.

What insights does the mutation spectrum of DnaE2 provide about its biochemical mechanism?

Analysis of the mutation spectrum generated by DnaE2 reveals crucial insights into its biochemical mechanism and functional properties:

• Mutation types: DnaE2 predominantly generates single base substitutions rather than insertions or deletions, suggesting it maintains proper base stacking and template alignment despite reduced fidelity .

• Mispair preferences: Studies of error-prone DNA polymerases have shown frequent A:A (template:dNMP) mispairings, indicating specific deficiencies in nucleotide discrimination . In the case of P. syringae and related bacteria, DnaE2 shows a distinct mutation signature different from other error-prone polymerases like DinB1.

• Sequence context effects: DnaE2-mediated mutations often exhibit sequence context preferences, suggesting interactions between the polymerase and specific DNA sequence motifs affect error rates.

• Rifampicin resistance mutations: Analysis of rpoB mutations incorporated by DnaE2 during oxidative stress revealed that unlike DinB1, DnaE2 conferred rifampicin resistance by mutating diverse rpoB codons, including Ser447(TCG>TTG), Ser438(TCG>TTG), His442(CAC>TAC), His442(CAC>GAC), and Asn435(AAC>AAG) .

• Multiple mutation events: Error-prone organelle DNA polymerases have been observed to introduce mutations at multiple locations, ranging from two to seven sites in half of mutant genes studied . This suggests that once engaged in error-prone synthesis, these polymerases may continue to introduce multiple errors during a single replication event.

These characteristics collectively suggest that DnaE2 possesses unique biochemical properties that facilitate its specialized role in stress-induced mutagenesis, particularly during plant-microbe interactions or environmental stress responses.

How conserved is DnaE2 across different Pseudomonas species and what does this tell us about its evolutionary history?

Comparative genomic analysis reveals important patterns in DnaE2 conservation across Pseudomonas species:

• Universal presence: All sequenced myxobacterial genomes possess two dnaE genes (dnaE1 and dnaE2), both highly conserved across species . This pattern likely extends to many Pseudomonas species, suggesting fundamental importance.

• Selective pressure: Evolutionary analysis using dN/dS ratios (nonsynonymous to synonymous substitution rates) indicates different selective pressures on dnaE1 and dnaE2. While dnaE1 is under strong purifying selection to maintain replication fidelity, dnaE2 shows evidence of more relaxed selection or potentially positive selection in certain domains .

• Domain conservation: Using the ConSurf algorithm to analyze conservation patterns reveals that catalytic domains are generally more conserved than other regions. The conservation scale ranges from the most variable positions (grade 1) to the most conserved positions (grade 9) .

• Phylogenetic distribution: The presence of dnaE2 across diverse bacterial phyla suggests an ancient origin, potentially acquired through horizontal gene transfer early in bacterial evolution.

• Functional specialization: Despite sequence similarities, DnaE2 has evolved specialized functions distinct from the primary replicative polymerase DnaE1, particularly in stress response and adaptive mutagenesis.

The conservation pattern of DnaE2 across Pseudomonas species suggests it provides a selective advantage in environmental adaptation, particularly for species that encounter diverse stressors or host immune responses, such as plant pathogens like P. syringae.

What role has recombination played in the evolution of P. syringae pv. tomato strains and how might DnaE2 contribute to this process?

Recombination has been a driving force in P. syringae pv. tomato evolution, with error-prone polymerases like DnaE2 potentially enhancing this process:

• Phylogenetic evidence: Multilocus sequence typing (MLST) analysis has revealed that recombination contributed more than mutation to the variation between P. syringae isolates . This finding suggests horizontal gene transfer and recombination are primary drivers of evolutionary change in these bacteria.

• Effector repertoire evolution: Recombination has played a crucial role in the reassortment of type III secreted (T3S) effectors between strains . The distribution of effectors like avrPto1 shows evidence of acquisition through horizontal gene transfer followed by selective maintenance or loss depending on host resistance factors.

• Strain diversity: The distinct phylogenetic positioning of PtoDC3000 compared to typical P. syringae pv. tomato strains highlights how recombination has created diverse lineages with different host ranges and virulence characteristics .

• DnaE2's contribution: Error-prone polymerases like DnaE2 may facilitate recombination through several mechanisms:

  • Increasing mutation rates around recombination breakpoints

  • Generating sequence diversity that serves as substrate for natural selection

  • Participating in error-prone DNA synthesis during recombinational repair

• Host adaptation: The loss of avrPto1 in some strains through recombination illustrates how this process enables adaptation to hosts with resistance genes. This exemplifies how recombination allows rapid adaptation to selective pressures .

Population genetic tests have indicated that recombination contributed more significantly than mutation to variation between isolates . This finding underscores the importance of recombination in the evolution of P. syringae pv. tomato and suggests that error-prone polymerases like DnaE2 may play an auxiliary role in generating genetic diversity that fuels this evolutionary process.

What are the key challenges in working with recombinant error-prone DNA polymerases and how can they be addressed?

Working with recombinant error-prone DNA polymerases like DnaE2 presents several technical challenges that researchers must overcome:

• Expression and purification difficulties:

  • Challenge: Error-prone polymerases often express poorly or form inclusion bodies.

  • Solution: Optimize expression conditions using lower temperatures (16-18°C), specialized host strains, or fusion tags that enhance solubility (MBP, SUMO, etc.).

• Activity assessment:

  • Challenge: Quantifying error rates accurately requires sensitive assays.

  • Solution: Implement positive selection methods like the phage lambda cI repressor gene system, which allows direct measurement of mutation frequencies .

• Controlling mutagenic activity:

  • Challenge: High error rates can lead to lethal mutations in expression hosts.

  • Solution: Use tightly regulated inducible promoters and consider expression in strains with enhanced capacity to handle protein toxicity.

• Specificity and targeting:

  • Challenge: Directing mutagenic activity to specific genomic regions.

  • Solution: Develop fusion constructs with DNA-binding domains or leverage recombineering techniques to target specific loci .

• Reproducibility issues:

  • Challenge: Variable mutation spectra between experiments.

  • Solution: Standardize reaction conditions and develop robust statistical frameworks for analyzing mutation patterns.

• Competition with endogenous polymerases:

  • Challenge: Distinguishing activity of recombinant polymerase from host enzymes.

  • Solution: Create knockout strains lacking endogenous error-prone polymerases or use distinctive mutation signatures as markers.

Researchers have successfully addressed many of these challenges. For instance, studies on error-prone organelle DNA polymerases developed a novel positive selection method involving replication of the phage lambda cI repressor gene, allowing precise quantification of error rates . Additionally, recombineering techniques using RecTE from P. syringae have provided efficient tools for targeted genetic manipulation .

How can conflicting data on DnaE2 function be reconciled, and what experimental approaches might resolve existing controversies?

Conflicting data regarding DnaE2 function can be reconciled through several targeted experimental approaches:

• Standardization of experimental systems:

  • Issue: Different bacterial strains, growth conditions, and assay methods yield variable results.

  • Resolution: Establish standardized protocols across laboratories, including defined media, growth phases, and consistent mutation detection assays.

• Direct biochemical characterization:

  • Issue: Indirect genetic evidence can lead to misattribution of functions.

  • Resolution: Perform comprehensive in vitro characterization of purified DnaE2, including determination of kinetic parameters, fidelity measurements, and structural studies.

• Genome-wide mutation mapping:

  • Issue: Targeted assays may miss global effects or context dependencies.

  • Resolution: Implement whole-genome sequencing of wildtype, ΔdnaE2, and dnaE2-overexpression strains under various stress conditions to generate comprehensive mutation landscapes.

• System-level analysis:

  • Issue: Focusing solely on DnaE2 may miss interactions with other cellular components.

  • Resolution: Employ proteomics and genetic interaction screens to identify DnaE2 binding partners and functional networks.

• Host-pathogen interaction studies:

  • Issue: Laboratory conditions may not reflect the selective pressures during plant infection.

  • Resolution: Compare mutation rates and spectra during actual plant infections versus laboratory growth to identify context-dependent functions.

• Temporal analysis:

  • Issue: Most studies provide snapshot views of DnaE2 function.

  • Resolution: Implement time-course experiments with inducible systems to track the dynamics of DnaE2-mediated mutagenesis.

A particularly promising approach combines mutation accumulation experiments with whole-genome sequencing. For example, researchers could grow parallel lineages of wild-type and ΔdnaE2 P. syringae strains for hundreds of generations under various stress conditions, followed by whole-genome sequencing to identify differences in mutation patterns, rates, and hotspots. This approach would provide a comprehensive view of DnaE2's contribution to genome evolution in different environmental contexts.