Recombinant Corynebacterium glutamicum DNA replication and repair protein recF (recF)

What is the function of RecF protein in Corynebacterium glutamicum?

RecF in C. glutamicum is a critical component of the DNA replication and repair machinery, primarily involved in the RecFOR pathway of homologous recombination. It functions in coordination with RecO and RecR proteins to facilitate loading of RecA onto single-stranded DNA (ssDNA) at gaps, promoting strand invasion during homologous recombination . Unlike in E. coli where multiple redundant pathways exist, the RecFOR pathway appears to play a more prominent role in C. glutamicum's DNA damage response system, making it particularly significant for genomic stability in this industrial microorganism .

How does RecF in C. glutamicum differ from its homologs in other bacteria?

The RecF protein in C. glutamicum shares core functional domains with homologs in other bacteria but exhibits distinct structural features adapted to C. glutamicum's high G+C content genome. Unlike E. coli RecF, C. glutamicum RecF demonstrates enhanced binding affinity to GC-rich DNA sequences, which aligns with the organism's genomic composition. The protein contains conserved Walker A and B motifs for ATP binding and hydrolysis, but shows variations in the DNA binding domain that may contribute to its specificity in recognizing damaged DNA sites in C. glutamicum . These species-specific adaptations should be considered when designing recombination-based genome editing tools for C. glutamicum.

What genomic editing tools can be used to manipulate recF in C. glutamicum?

Several genomic manipulation technologies have been developed for C. glutamicum that can be applied to recF studies:

• RecET-based recombination systems: Heterologous recombination proteins like RecT and RecET show high efficiency in C. glutamicum and can be used with 50-1000 bp homology arms for recF editing .

• CRISPR/Cpf1 system: This system with a PAM sequence 5′-NYTV-3′ and 21 bp spacer has shown high recombination efficiency. It can be combined with RecET for simultaneous mutations of up to 4 genes, potentially including recF .

• CRISPR/Cas9 systems: Modified to reduce toxicity in C. glutamicum through lower expression levels, weak promoters, and low-copy-number plasmids. These modifications are essential when targeting recF, as uncontrolled DSBs in DNA repair genes can be lethal .

For optimal results when manipulating recF, the CRISPR/Cpf1 system combined with RecET has demonstrated superior efficiency, allowing for precise editing while minimizing off-target effects that could confound the analysis of RecF function.

How is recF expression regulated in C. glutamicum?

RecF expression in C. glutamicum is tightly regulated as part of the SOS response system. Upon DNA damage, LexA repressor auto-cleavage occurs, allowing transcription of recF and other SOS genes. Unlike in E. coli, C. glutamicum shows some constitutive expression of recF under normal conditions, suggesting its involvement in routine DNA maintenance beyond damage response.

The gene is typically co-regulated with other DNA repair components, with its expression increased during exponential growth phase when DNA replication is most active. Specific regulatory elements identified in the recF promoter region include:

These regulatory mechanisms should be carefully considered when designing recF overexpression systems for recombination-based applications in C. glutamicum .

How does RecF interact with other components of the DNA repair machinery in C. glutamicum?

RecF operates within a complex network of protein interactions in C. glutamicum's DNA repair system. Key interactions include:

• RecF-RecO-RecR complex formation: These three proteins form a functional complex that facilitates RecA loading onto ssDNA. In C. glutamicum, RecF binds to DNA ends while RecO recognizes SSB-coated ssDNA, and RecR serves as a mediator between RecF and RecO.

• Interaction with single-strand binding protein (SSB): RecF competes with SSB for binding to ssDNA. The balance between these interactions determines repair pathway choice.

• Connection to the replisome: RecF interacts with the DnaE1 subunit of DNA polymerase III at stalled replication forks, as indicated by protein co-immunoprecipitation studies.

• NucS-mediated repair pathway: Recent research has identified potential interactions between RecF and the NucS protein, which is involved in post-replicative mismatch repair in C. glutamicum .

Understanding these interactions is critical when interpreting phenotypes of recF mutants, as defects may reflect disruption of multiple repair pathways rather than loss of a single RecF function.

What methodologies are most effective for studying RecF-mediated DNA repair mechanisms in C. glutamicum?

Several methodologies have proven effective for investigating RecF functions in C. glutamicum:

• In vivo genetic approaches:

  • Construction of recF deletion and point mutants using CRISPR/Cpf1 with RecET systems

  • Complementation studies with wild-type and mutant recF alleles

  • Synthetic lethality screens to identify genetic interactions

• In vitro biochemical assays:

  • Purification of recombinant RecF protein with His-tag or other affinity tags

  • DNA binding assays using gel shift and fluorescence anisotropy techniques

  • ATPase activity assays to measure RecF enzymatic function

  • Reconstitution of RecFOR-mediated RecA loading onto ssDNA

• Advanced imaging techniques:

  • Fluorescently tagged RecF to track localization during DNA damage response

  • Super-resolution microscopy to visualize RecF foci at DNA repair sites

• Next-generation sequencing applications:

  • ChIP-seq to identify RecF binding sites genome-wide

  • RNA-seq to measure transcriptional changes in recF mutants

  • Mutation accumulation sequencing to characterize repair defects

When designing these experiments, researchers should consider the inherent challenges of working with C. glutamicum, including its thick cell wall which may require modified protocols for protein extraction and imaging .

How can I construct a recF knockout strain in C. glutamicum and what phenotypes should I expect?

To construct a recF knockout in C. glutamicum, the following methodological approach is recommended:

• Design strategy: Use CRISPR/Cpf1 combined with the RecET system, which has shown the highest efficiency for gene deletion in C. glutamicum .

• Homology arm design: Optimal results are achieved with 1000 bp homology arms flanking the recF gene, though 50 bp can work with reduced efficiency .

• Selection markers: A two-step selection process using positive (kanamycin resistance) and negative (sacB) selection markers facilitates the identification of clean deletions.

• Verification methods:

  • PCR verification with primers flanking the deletion site

  • Whole-genome sequencing to confirm deletion and check for secondary mutations

  • RT-qPCR to confirm absence of recF transcript

Expected phenotypes of a recF knockout strain include:

When designing complementation studies, use controlled expression systems like P_tac with adjustable IPTG concentrations to avoid toxicity from RecF overexpression .

What are the optimal conditions for expressing and purifying recombinant RecF protein from C. glutamicum?

For successful expression and purification of recombinant RecF from C. glutamicum:

• Expression system selection:

  • Heterologous expression in E. coli BL21(DE3) typically yields higher protein amounts

  • For more native conformation, expression in C. glutamicum with a strong inducible promoter (P_tac) is preferable

• Affinity tag selection:

  • N-terminal His₆-tag shows less interference with function than C-terminal tags

  • TEV protease cleavage site inclusion allows tag removal for functional studies

• Optimal expression conditions:

  • Temperature: 20°C for E. coli, 25°C for C. glutamicum

  • Induction: 0.1-0.5 mM IPTG for E. coli, 0.1 mM IPTG for C. glutamicum

  • Duration: 16-18 hours for highest yield with maintained solubility

• Purification protocol:

  • Lysis buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 0.1 mM EDTA, 1 mM DTT

  • Nickel affinity chromatography followed by size exclusion chromatography

  • Maintain 1 mM ATP in all buffers to stabilize protein structure

• Quality control assessments:

  • Dynamic light scattering to confirm monodispersity

  • Circular dichroism to verify proper folding

  • ATPase activity assay to confirm functionality

Typical yield from 1L culture is 2-3 mg of >95% pure RecF protein. Storage at -80°C in small aliquots with 20% glycerol maintains activity for up to 6 months .

How can I interpret contradictory results from recF mutation studies in C. glutamicum?

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

• Mutation type analysis:

  • Point mutations may affect specific functions while preserving others

  • Complete deletions eliminate all functions and may trigger compensatory mechanisms

  • Compare results with the mutational spectrum observed in adaptive laboratory evolution studies

• Genetic background considerations:

  • Secondary mutations may exist in laboratory strains

  • The prophage status of the strain can affect recombination phenotypes (compare ATCC 13032 vs. MB001)

  • Verify genotype using whole-genome sequencing to identify potential suppressor mutations

• Environmental variables:

  • RecF function may be condition-dependent (minimal vs. rich media)

  • Mg²⁺ concentration significantly affects DNA repair pathways in C. glutamicum

  • Temperature affects the stability of secondary structures in damaged DNA

• Pathway redundancy:

  • Consider potential compensation by alternative repair pathways

  • Analyze possible upregulation of other repair genes (RecET, DnaE1, NucS) in recF mutants

• Methodological differences:

  • Assay sensitivity can vary (direct repair measurement vs. survival assays)

  • Growth phase during analysis affects repair capacity

  • Construct multiple mutant alleles and test in different genetic backgrounds

When reporting contradictory results, present all data systematically in a table comparing methodologies, strain backgrounds, and environmental conditions to identify variables that may explain differences.

What statistical approaches are most appropriate for analyzing RecF-dependent recombination frequency data?

When analyzing RecF-dependent recombination frequency data in C. glutamicum:

• Appropriate transformation of data:

  • Recombination frequencies typically follow a log-normal distribution

  • Log-transformation of raw data before statistical analysis improves normality

  • Verification of normality using Shapiro-Wilk test is essential

• Statistical tests for comparing recombination frequencies:

  • Student's t-test (for comparing two conditions)

  • One-way ANOVA with Tukey's post-hoc test (for multiple comparisons)

  • Two-way ANOVA when analyzing interaction effects (e.g., recF mutation × DNA damage)

• Effect size calculation:

  • Cohen's d for comparing means between two groups

  • η² (eta squared) for ANOVA designs to quantify proportion of variance explained

• Modeling approaches for complex datasets:

  • Linear mixed-effects models when incorporating multiple variables

  • Bayesian hierarchical modeling for integrating prior knowledge

  • Time-series analysis for measuring repair kinetics

• Replication considerations:

  • Minimum of three biological replicates with technical triplicates

  • Power analysis to determine sample size for detecting expected effect sizes

  • Fluctuation analysis (Luria-Delbrück) for measuring mutation rates in recF strains

Example data presentation format:

Include clear descriptions of the statistical methods used and justification for the chosen approaches in your methods section .

How might genetic mutator approaches utilizing RecF manipulation advance strain development in C. glutamicum?

The development of genetic mutators through RecF manipulation presents promising avenues for C. glutamicum strain development:

• Controlled mutagenesis systems:

  • Creating conditional recF mutants with temperature-sensitive or inducible expression can provide tunable mutation rates

  • Integration with other DNA repair modifications (e.g., NucS impairment) could generate a binary genetic mutator system capable of increasing mutation rates up to 2000-fold

  • Targeted mutagenesis through RecF domain-specific mutations can bias mutation patterns toward specific genomic regions

• Applications in adaptive laboratory evolution:

  • RecF-modified strains can accelerate adaptation to challenging conditions

  • Sequential rounds of mutation and selection can improve stress tolerance similar to approaches used for the UBw and UBm evolved strains

  • Controlled mutator strains can be used to rapidly generate diversity in metabolic pathways for novel bioproduction capabilities

• Integration with genome editing technologies:

  • Combining RecF manipulation with CRISPR/Cpf1 or Cas9 systems can create highly efficient recombination-based genome editing platforms

  • Development of multiplex editing approaches using RecF-enhanced homologous recombination

  • Creation of genomic libraries with varying recF activity levels for phenotype screening

• Research challenges and limitations:

  • Balancing mutation rate with genomic stability

  • Developing methods to control mutagenesis spatially and temporally

  • Mitigating off-target effects that may impact industrial performance

What emerging technologies might enhance our understanding of RecF dynamics in C. glutamicum?

Several emerging technologies show promise for advancing RecF research in C. glutamicum:

• Advanced imaging technologies:

  • Single-molecule tracking of fluorescently tagged RecF to visualize real-time dynamics during DNA damage response

  • Cryo-electron microscopy to resolve the structure of RecFOR complexes in C. glutamicum

  • Expansion microscopy to visualize RecF localization patterns within the bacterial nucleoid

• Multiomics integration approaches:

  • Integration of transcriptomics, proteomics, and metabolomics data to understand system-wide effects of recF mutations

  • Chromatin immunoprecipitation sequencing (ChIP-seq) to map RecF binding sites across the genome

  • Protein-protein interaction mapping using BioID or proximity ligation assays

• CRISPR-based technologies:

  • CRISPRi for controlled gene expression modulation without genetic modification

  • Base editing for precise introduction of recF point mutations without double-strand breaks

  • Prime editing for flexibility in generating diverse recF variants

• High-throughput phenotyping:

  • Microfluidic systems for single-cell analysis of RecF function

  • Droplet-based screening of recF variant libraries

  • Automated microscopy for tracking DNA repair kinetics in living cells

• Computational approaches:

  • Molecular dynamics simulations of RecF interactions with DNA substrates

  • Machine learning algorithms to predict the impact of recF mutations on repair efficiency

  • Systems biology modeling of the entire DNA repair network in C. glutamicum

These technologies will help overcome current limitations in understanding the temporal and spatial dynamics of RecF activity in vivo and could lead to novel applications in metabolic engineering of C. glutamicum .