Recombinant Enterococcus faecalis DNA-directed RNA polymerase subunit beta' (rpoC), partial

What is the rpoC gene in Enterococcus faecalis and what is its function?

The rpoC gene in Enterococcus faecalis encodes the β' (beta prime) subunit of RNA polymerase (RNAP), which is an essential component of the bacterial transcription machinery. This subunit forms part of the catalytic center of RNAP and plays crucial roles in DNA binding, RNA synthesis, and interactions with regulatory factors. As part of the core enzyme, the RpoC protein works in conjunction with other subunits to enable transcription of genetic information from DNA to RNA .

In E. faecalis, mutations in the rpoC gene can lead to significant changes in gene expression patterns throughout the genome, affecting diverse cellular processes including stress responses, virulence gene expression, and antimicrobial resistance mechanisms .

How does RpoC interact with other RNA polymerase subunits?

RpoC (β' subunit) forms the core RNA polymerase enzyme along with RpoA (α subunit) and RpoB (β subunit). These core subunits associate with sigma factors to form the holoenzyme capable of promoter recognition and transcription initiation. In E. faecalis, the RpoC subunit contains conserved domains that mediate interactions with:

• RpoB: Forms extensive interactions creating the main channel for DNA binding

• Sigma factors: Contains regions for sigma factor docking, enabling promoter recognition

• Regulatory proteins: Includes surfaces for interaction with transcription factors

• Nucleic acids: Contains motifs that contact template DNA and nascent RNA

The precise arrangement of these interaction surfaces ensures proper assembly of the transcription complex and appropriate regulation of gene expression in response to environmental conditions.

How do mutations in the rpoC gene affect bacterial phenotypes?

Mutations in the rpoC gene can profoundly impact bacterial phenotypes through global alterations in gene expression. According to research, in E. faecalis, an rpoC mutation in the M7 strain confers resistance to the effects of Fst toxin, which normally causes defects in chromosome segregation, cell division, and membrane integrity .

Similar effects have been observed in other bacterial species. For instance, in Staphylococcus aureus, an rpoC(P440L) mutation is associated with the "slow VISA" (sVISA) phenotype, characterized by prolonged doubling time, increased vancomycin resistance, and altered cell wall thickness . These phenotypic changes reflect how modifications to RNA polymerase function can systemically alter cellular physiology.

What is the relationship between rpoC mutations and antimicrobial resistance?

The rpoC mutations contribute to antimicrobial resistance through several distinct mechanisms:

• Altered cell wall synthesis: In S. aureus, the rpoC(P440L) mutation causes the sVISA phenotype by augmenting cell wall peptidoglycan synthesis .

• Modified stress responses: rpoC mutations can affect the stringent response pathway, as evidenced by mutations in relQ found in revertant strains .

• Transporter expression: In E. faecalis, rpoC mutations may affect the expression of membrane transporters and efflux pumps. Research shows that certain transporters were induced in the parent strain OG1X after Fst exposure but not in the M7 mutant with altered rpoC .

• Global transcriptional changes: As a component of RNA polymerase, rpoC mutations can cause widespread alterations in gene expression, affecting multiple resistance determinants simultaneously.

These mechanisms collectively explain how single mutations in rpoC can lead to complex resistance phenotypes that may be difficult to overcome with conventional antimicrobial strategies.

What compensatory mutations arise in response to rpoC mutations?

When bacteria acquire rpoC mutations that confer resistance but also impose fitness costs, they often develop compensatory mutations to restore growth and fitness. Research has identified three main categories of compensatory mutations:

• Secondary mutations within rpoC itself: For example, in S. aureus, the F562L mutation in rpoC was found to compensate for the initial P440L mutation, reverting the strain from sVISA to hVISA phenotype .

• Mutations in peptidoglycan biosynthesis genes: These help restore cell wall homeostasis disrupted by altered transcription patterns .

• Mutations in stress response genes: Particularly those involved in the stringent response pathway, such as relQ .

This adaptive capability highlights the genetic flexibility of bacteria in maintaining both resistance and fitness—a challenge for antimicrobial therapy.

What techniques are used to identify and characterize rpoC mutations?

Several methodologies are employed to identify and characterize rpoC mutations in E. faecalis:

• Full-genome sequencing: This comprehensive approach was used to identify the rpoC mutation in the M7 mutant derivative of E. faecalis strain OG1X, revealing the genetic basis for Fst toxin resistance .

• Allelic exchange experiments: This technique confirms whether observed phenotypes are specifically due to rpoC mutations by introducing the mutation into a wild-type background or restoring the wild-type sequence in mutant strains .

• Microarray analysis: Used to examine global gene expression changes resulting from rpoC mutations, this method revealed differential expression of transporters between parent and mutant strains .

• PCR amplification and targeted sequencing: For rapid screening of specific regions within the rpoC gene.

• Phenotypic assays: Including growth rate measurements, antimicrobial susceptibility testing, and specific assays such as monitoring efflux pump activity using indicator compounds like berberine .

How can recombinant DNA techniques be applied to study rpoC function?

Modern recombinant DNA technologies offer powerful approaches for investigating rpoC function in Enterococcus:

• CRISPR-Cas9 mediated recombineering: Recent advances have enabled efficient gene editing in Enterococcus species. The use of RecT recombinase significantly improves DNA integration efficiency in both commensal and antibiotic-resistant strains .

• Single-stranded DNA recombineering: The expression of RecT in combination with CRISPR-Cas9 and guide RNAs enables highly efficient scarless gene editing in E. faecium, a technique that could be adapted for E. faecalis .

• Chromosomal insertions: E. faecium RecT expression facilitates the insertion of double-stranded DNA templates encoding antibiotic selectable markers, allowing for the generation of gene deletion mutants .

• Tagged constructs: Creating recombinant rpoC variants with epitope tags or fluorescent protein fusions for localization and interaction studies.

These techniques circumvent the limitations of traditional passive homologous recombination methods, which typically require weeks to perform and have lower efficiency .

What protocols are recommended for purifying recombinant RpoC protein?

Purification of recombinant RpoC protein from E. faecalis requires specialized protocols due to its large size and tendency to form complexes with other RNAP subunits:

• Cell lysis protocol:

  • Resuspend cells in appropriate buffer

  • Add lysozyme (50 mg/ml) and mutanolysin (2500U/ml)

  • Incubate at 37°C for 2 hours

  • Add 20% Sarkosyl and RNase (10 mg/ml)

  • Incubate at 37°C for 30 minutes

  • Add pronase (10 mg/ml) and incubate for an additional 30 minutes

• Protein extraction:

  • Perform phenol-chloroform extraction (1:1 ratio) five times

  • Perform a final chloroform extraction

  • Precipitate protein using standard methods

• Chromatography approaches:

  • Affinity chromatography using engineered tags (His, GST, etc.)

  • Ion-exchange chromatography to separate based on charge

  • Size exclusion chromatography for final purification

• Quality control:

  • SDS-PAGE to verify size and purity

  • Mass spectrometry for identity confirmation

  • Activity assays to confirm functionality

How does RpoC structure relate to its function in transcriptional regulation?

Understanding the structure-function relationship of RpoC is essential for elucidating transcriptional regulation mechanisms:

• Functional domains: The RpoC protein contains several conserved domains, including:

  • The active site for RNA synthesis

  • DNA binding regions

  • Interface surfaces for interaction with other RNAP subunits

  • Binding sites for regulatory factors

• Mutation effects: Specific mutations, such as those in S. aureus rpoC(P440L), can alter the conformation of the protein in ways that affect gene expression patterns, particularly those involved in cell wall synthesis and stress responses .

• Structural studies: Advanced techniques such as X-ray crystallography and cryo-electron microscopy would be valuable for determining how specific mutations alter RpoC structure and function in E. faecalis.

• Regulatory interactions: The structure of RpoC determines its ability to interact with regulatory proteins, including those involved in the stringent response pathway, which has been implicated in antimicrobial resistance mechanisms .

How can rpoC mutations be utilized for synthetic biology applications?

The directed modification of rpoC offers opportunities for synthetic biology applications:

• Designer transcription systems: Engineered rpoC variants could create RNA polymerases with altered promoter specificity or elongation properties.

• Growth control mechanisms: Since rpoC mutations can significantly affect growth rates (as seen in the sVISA phenotype with a 72-minute doubling time), engineered rpoC could serve as a tunable growth control element .

• Resistance mechanisms: Understanding how specific rpoC mutations confer resistance could inform the design of bacterial strains with controlled sensitivity to antimicrobials.

• Stress response engineering: Modified rpoC could alter how bacteria respond to environmental stresses, potentially creating strains with enhanced survival in specific conditions.

• Heterologous expression hosts: E. faecalis strains with engineered rpoC might serve as specialized hosts for the expression of difficult-to-produce proteins.

What specific rpoC mutations have been identified in Enterococcus species?

The following table summarizes key rpoC mutations that have been characterized in Enterococcus and related species:

What expression systems are most effective for recombinant rpoC production?

Based on research with related recombinant proteins in Enterococcus species, the following expression systems show promise for rpoC production:

• IPTG-inducible systems: Similar to the system used for RecT expression in E. faecium, an IPTG-inducible promoter with the lacI repressor provides controlled expression .

• Vector considerations:

  • pRecT-like plasmids containing appropriate antibiotic resistance markers (e.g., erythromycin resistance gene ermC)

  • Vectors with suitable copy number for the large rpoC gene

  • Compatibility with both E. coli (for cloning) and Enterococcus (for expression)

• Host strain selection:

  • Consider using modified strains with reduced protease activity

  • Strains with mutations in native rpoC may provide insights into functional complementation

  • Both commensal and clinical isolates can be used with proper optimization

How do growth conditions affect recombinant RpoC expression and activity?

Various growth parameters significantly impact the expression and functionality of recombinant RpoC:

What are common difficulties in working with rpoC mutants and how can they be addressed?

Researchers working with rpoC mutants frequently encounter several challenges:

• Genetic instability: As seen in the V6-5 strain of S. aureus, which frequently reverted to the hVISA phenotype with concomitant loss of its growth characteristics .

  • Solution: Maintain selection pressure and regularly confirm genotype by sequencing.

• Growth defects: Many rpoC mutations significantly impact growth rates.

  • Solution: Optimize media composition and growth conditions; consider using rich media supplemented with appropriate nutrients.

• Compensatory mutations: Secondary mutations often arise to compensate for fitness costs.

  • Solution: Genome sequencing to identify all mutations; use freshly constructed mutants for critical experiments.

• Pleiotropic effects: rpoC mutations affect many cellular processes simultaneously.

  • Solution: Use global approaches (transcriptomics, proteomics) to comprehensively characterize changes.

• Strain-specific effects: The impact of identical rpoC mutations may vary between strains.

  • Solution: Validate findings in multiple genetic backgrounds.

How can researchers distinguish between direct and indirect effects of rpoC mutations?

Differentiating direct from indirect effects of rpoC mutations presents a significant challenge:

• Temporal analysis: Examine gene expression changes at multiple time points after inducing expression of mutant rpoC. The research on Fst toxin in E. faecalis showed that certain transporters were induced late (60 min) after exposure, representing secondary rather than primary responses .

• Complementation studies: Introduce wild-type rpoC on a plasmid into mutant strains to determine which phenotypes are reversed. This approach confirmed the role of rpoC(P440L) in the sVISA phenotype when complementation with wild-type rpoC resolved the phenotype .

• Directed mutagenesis: Introduce specific domains or individual residue changes to map functional regions.

• In vitro transcription: Reconstitute transcription systems with purified components to directly assess the impact of RpoC variants on transcription from different promoters.

• Chromatin immunoprecipitation (ChIP): Compare DNA binding profiles of wild-type and mutant RNA polymerase to identify differentially regulated genes.