Recombinant Enterococcus faecalis Polyribonucleotide nucleotidyltransferase (pnp), partial

What is Polyribonucleotide nucleotidyltransferase (PNPase) in Enterococcus faecalis?

Polyribonucleotide nucleotidyltransferase (PNPase, EC 2.7.7.8) in E. faecalis is an enzyme involved in RNA metabolism with both 3'-5' exoribonuclease activity and template-independent polymerase activity. This bifunctional enzyme plays crucial roles in RNA turnover, quality control mechanisms, and post-transcriptional regulation. In E. faecalis, a gram-positive bacterium that inhabits diverse environments including the human intestine, PNPase contributes to environmental adaptation and potentially to virulence mechanisms. The recombinant partial protein is derived from E. faecalis strain ATCC 700802/V583, which belongs to a high-risk clonal complex particularly well-adapted to hospital environments .

What are the optimal storage conditions for recombinant E. faecalis PNPase?

For optimal stability and activity retention, recombinant E. faecalis PNPase requires specific storage conditions based on its formulation:

Repeated freeze-thaw cycles significantly decrease protein activity and should be avoided. After reconstitution, working aliquots can be stored at 4°C for up to one week. For long-term storage, adding glycerol to a final concentration of 50% and storing at -20°C to -80°C is recommended . When planning long-term experiments, researchers should consider these stability limitations and prepare appropriately sized aliquots to minimize freeze-thaw cycles.

How should recombinant E. faecalis PNPase be reconstituted for experimental use?

For optimal reconstitution of lyophilized recombinant E. faecalis PNPase:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration between 5-50% for enhanced stability

• Prepare small working aliquots to avoid repeated freeze-thaw cycles

• For immediate use, store working aliquots at 4°C (stable for up to one week)

The reconstitution buffer can be modified according to specific experimental requirements, but significant deviations from neutral pH or physiological salt concentrations may affect protein activity . When designing reconstitution protocols, consider the downstream applications and potential buffer incompatibilities.

How can E. faecalis PNPase activity be assayed in vitro?

Experimental Protocol for PNPase Activity Assay:

• Degradation Assay:

  • Prepare reaction mixture containing:

    • 20 mM Tris-HCl (pH 7.5)

    • 5 mM MgCl₂

    • 1 mM DTT

    • 0.1 mM EDTA

    • 10% glycerol

    • 0.5-1 μg recombinant PNPase

    • 5'-labeled RNA substrate (0.5-1 pmol)

  • Incubate at 37°C for 5-30 minutes

  • Analyze reaction products by denaturing PAGE and autoradiography

• Polymerization Assay:

  • Prepare reaction mixture containing:

    • 20 mM Tris-HCl (pH 7.5)

    • 5 mM MgCl₂

    • 1 mM DTT

    • 0.1 mM EDTA

    • 10% glycerol

    • 0.5-1 μg recombinant PNPase

    • 3'-labeled RNA substrate (0.5-1 pmol)

    • 1 mM ADP/GDP/CDP/UDP as substrate

  • Incubate at 37°C for 15-60 minutes

  • Analyze reaction products by denaturing PAGE and autoradiography

Both assays should include appropriate controls such as heat-inactivated enzyme and time-point samples to establish reaction kinetics. The partially purified recombinant protein may contain trace contaminants that could affect assay outcomes, so running parallel reactions with commercially available PNPase from other species as standards is recommended.

How can shuttle vectors be used to study E. faecalis PNPase function in vivo?

To study E. faecalis PNPase function in vivo, researchers can use E. coli-E. faecalis shuttle vectors such as pAM401-based systems. This methodology allows for both complementation studies and protein overexpression:

• Construction of PNPase expression vector:

  • Clone the pnp gene into a shuttle vector such as pMGS100 or pMGS101 under the control of the bacA promoter

  • For protein purification purposes, use pMGS101 which contains a Strep-tag sequence for affinity purification

  • Transform the construct into E. coli for propagation and verification

• Expression in E. faecalis:

  • Transfer the verified construct into E. faecalis strains

  • For complementation studies, use a pnp deletion mutant

  • For overexpression studies, use wild-type strains

• Functional analysis options:

  • RNA decay assays comparing wild-type, deletion mutant, and complemented strains

  • Stress response assays under various environmental conditions

  • Virulence assays in infection models

This approach has been successfully used for other E. faecalis proteins and allows for cis/trans analysis of gene function . When selecting a vector system, consider compatibility with the specific E. faecalis strain being used and potential effects of antibiotics used for selection.

What methods can be used to study the role of PNPase in E. faecalis stress response?

The role of PNPase in E. faecalis stress response can be investigated using a multi-faceted approach:

For each condition, compare:

• Wild-type E. faecalis

• pnp deletion mutant

• Complemented strain (expressing recombinant PNPase)

• Overexpression strain

Additional analyses should include:

• RNA stability assays using rifampicin chase experiments

• qRT-PCR to measure expression of stress response genes

• RNA-seq to identify global effects on transcriptome

• Protein-RNA interaction studies using RNA immunoprecipitation

E. faecalis is known for its ability to tolerate diverse environmental conditions, making it an excellent model for studying stress response mechanisms . The stress conditions should be carefully optimized for each experimental system to ensure reproducible results.

How does E. faecalis PNPase contribute to virulence and pathogenicity?

While direct evidence for E. faecalis PNPase in virulence is limited in the provided search results, research on related bacterial species suggests several potential mechanisms:

• Post-transcriptional regulation of virulence factors:
  PNPase likely regulates the stability of mRNAs encoding virulence factors. This can be investigated by:

  • Comparing virulence gene expression profiles between wild-type and pnp mutant strains

  • Analyzing direct binding of PNPase to virulence factor mRNAs using CLIP-seq

  • Measuring half-lives of virulence factor transcripts in the presence/absence of PNPase

• Stress adaptation during infection:
  E. faecalis encounters various stresses during infection, and PNPase may facilitate adaptation:

  • Within the gastrointestinal tract (acid stress, bile stress)

  • During endocarditis development (shear stress, immune response)

  • In urinary tract infections (osmotic stress)

• Interaction with prophage elements:
  E. faecalis V583 harbors multiple prophage elements that contribute to pathogenicity. PNPase may regulate prophage gene expression or activation:

  • In E. faecalis V583, prophages contribute to adhesion to human platelets, a key step in infective endocarditis development

  • Fluoroquinolone antibiotics can trigger prophage induction, potentially releasing virulence factors

Experimental approaches should include infection models comparing wild-type and pnp mutant strains, focusing on:

• Colonization efficiency in various niches

• Persistence during antibiotic treatment

• Biofilm formation capacity

• Adhesion to host tissues and platelets

What is the relationship between PNPase function and antibiotic resistance in E. faecalis?

The relationship between PNPase and antibiotic resistance in E. faecalis represents an important research area with clinical implications:

• Post-transcriptional regulation of resistance genes:

  • PNPase may regulate the stability and expression of antibiotic resistance genes

  • RNA processing could affect expression of efflux pumps, cell wall modification enzymes, and other resistance determinants

• Stress response coordination:

  • Antibiotic exposure triggers bacterial stress responses

  • PNPase regulation of stress-responsive transcripts may indirectly affect resistance development

• Biofilm formation and persistence:

  • E. faecalis forms biofilms that contribute to antibiotic tolerance

  • PNPase may regulate genes involved in biofilm matrix production and cellular aggregation

• Prophage-mediated resistance transfer:

  • Fluoroquinolones can induce prophage elements in E. faecalis

  • PNPase might influence prophage excision, potentially affecting horizontal gene transfer of resistance elements

Research approach:
Compare wild-type and pnp mutant strains for:

• MIC values for various antibiotic classes

• Time-kill kinetics under antibiotic pressure

• Development of resistance during serial passage

• Expression of known resistance genes

• Biofilm formation capacity under antibiotic stress

E. faecalis is a significant nosocomial pathogen partially due to its widespread antibiotic resistance, including vancomycin resistance . Understanding PNPase's role could provide insights into resistance mechanisms and potential intervention strategies.

How does the structure of E. faecalis PNPase compare to homologs in other bacterial species?

Structural analysis of E. faecalis PNPase reveals important similarities and differences compared to homologs in other bacteria:

Structural features comparison:

Experimental approaches for structural analysis:

• Homology modeling:

  • Using existing PNPase structures as templates

  • Identifying conserved functional domains and species-specific regions

• Biochemical characterization:

  • Limited proteolysis to identify domain boundaries

  • Circular dichroism for secondary structure analysis

  • Thermal stability assays comparing full-length vs. partial constructs

• Domain-specific functional analysis:

  • Construction of truncated variants lacking specific domains

  • Site-directed mutagenesis of catalytic residues

  • RNA binding assays with different substrate types

Based on the partial nature of the recombinant protein described in the search results , careful consideration of which domains are present in the construct is essential for experimental design and interpretation of results. The UNIPROT entry (Q82ZJ2) can provide further details on the complete protein sequence and domain organization.

What are common issues in working with recombinant E. faecalis PNPase and how can they be resolved?

Researchers commonly encounter several challenges when working with recombinant E. faecalis PNPase:

Quality control recommendations:

• Verify protein purity by SDS-PAGE (should be >85% as specified in the product information)

• Confirm identity by western blot using anti-PNPase antibodies

• Perform activity assays before experimental use

• Monitor stability over time under working conditions

• Include appropriate controls in all experiments

For the partial recombinant protein, understanding which domains are present and functional is crucial. If certain domains are missing, protein activity may be compromised or altered compared to the full-length native protein.

How can researchers differentiate between the exonuclease and polymerase activities of PNPase in experimental settings?

Distinguishing between the dual activities of PNPase requires careful experimental design:

1. Buffer optimization:
Different buffer conditions can favor one activity over the other:

• Exonuclease activity: Higher phosphate concentrations (5-10 mM)

• Polymerase activity: Higher ADP concentrations (1-2 mM)

2. Substrate design:

• For exonuclease assays: Use 5'-labeled RNA substrates with structured 3' ends

• For polymerase assays: Use 3'-labeled RNA with accessible 3' ends

3. Selective inhibition:

• Citrate preferentially inhibits polymerase activity

• High Mg²⁺ concentrations (>10 mM) favor exonuclease activity

4. Site-directed mutagenesis:
Create variants with mutations in:

• Phosphorolytic active site (to eliminate exonuclease activity)

• Polymerization active site (to eliminate polymerase activity)

5. Assay readouts:

• Exonuclease activity: Measure release of nucleotides using TLC or HPLC

• Polymerase activity: Detect elongation of RNA substrates by PAGE

Understanding these dual activities is crucial for interpreting PNPase's role in bacterial RNA metabolism and regulatory networks. When working with the partial recombinant protein, researchers should determine which catalytic sites are present in the construct before designing activity assays.

How should researchers validate a PNPase knockout or complemented strain of E. faecalis?

Proper validation of PNPase knockout and complemented strains is essential for reliable research:

For PNPase knockout validation:

• Genetic confirmation:

  • PCR verification using primers flanking the deletion site

  • Sequencing across the deletion junction

  • Southern blot analysis to confirm single integration events

• Expression analysis:

  • RT-PCR to confirm absence of pnp transcript

  • Western blot to verify absence of PNPase protein

  • RNA-seq to identify potential polar effects on nearby genes

• Phenotypic characterization:

  • Growth curves under standard conditions

  • Stress response profiling

  • RNA decay assays using model substrates

For complementation strain validation:

• Expression verification:

  • RT-PCR to confirm pnp transcript levels

  • Western blot to verify PNPase protein production

  • Activity assays to confirm functional enzyme

• Genetic stability:

  • Plasmid maintenance under non-selective conditions

  • Copy number determination

  • Sequencing to confirm absence of mutations

• Phenotype restoration:

  • Comparison to wild-type in all relevant assays

  • Dose-dependency analysis if using inducible promoters

  • Growth characteristics under various conditions

When creating E. faecalis mutants, researchers can utilize approaches similar to those described for other E. faecalis genes, such as homologous recombination techniques . E. coli-E. faecalis shuttle vectors like pAM401-based systems can be used for complementation studies . The choice of vector system should consider factors like copy number, promoter strength, and stability in the absence of selection.

How can RNA-sequencing be optimized to study the impact of PNPase on the E. faecalis transcriptome?

RNA-seq experimental design for studying PNPase function requires specific considerations:

Sample preparation protocol:

• Strain preparation:

  • Wild-type E. faecalis

  • pnp knockout mutant

  • Complemented strain

  • PNPase overexpression strain

• Growth conditions:

  • Standard laboratory conditions (BHI, 37°C)

  • Relevant stress conditions (acid, oxidative, antibiotic)

  • Growth phase considerations (exponential vs. stationary)

• RNA extraction optimizations:

  • Rapid sample processing to minimize RNA degradation

  • DNase treatment to remove genomic DNA

  • rRNA depletion to enrich for mRNA and sRNAs

  • Size selection to capture small RNAs (<200 nt)

• Library preparation considerations:

  • Directional libraries to distinguish sense and antisense transcripts

  • 5'-end capture methods to identify processing sites

  • 3'-end sequencing to detect tailing and degradation intermediates

Data analysis pipeline:

• Quality control and preprocessing

• Mapping to E. faecalis genome (strain V583 recommended as reference)

• Differential expression analysis (DESeq2 or similar tools)

• RNA stability calculations from time-course data

• Motif discovery in differentially regulated transcripts

• Pathway enrichment analysis

Validation experiments:

• Northern blotting for selected targets

• qRT-PCR validation of expression changes

• RNA stability assays using rifampicin chase

• RNA immunoprecipitation to identify direct PNPase targets

This approach allows comprehensive characterization of PNPase's impact on RNA processing, degradation, and gene expression in E. faecalis .

What approaches can be used to identify RNA substrates directly bound by E. faecalis PNPase?

Several complementary techniques can identify direct RNA targets of E. faecalis PNPase:

1. RNA Immunoprecipitation (RIP):

• Cross-link RNA-protein complexes in vivo

• Lyse cells and immunoprecipitate PNPase with specific antibodies

• Extract and analyze bound RNAs by sequencing or microarray

2. CLIP-seq (Cross-Linking Immunoprecipitation followed by sequencing):

• UV cross-linking to capture direct RNA-protein interactions

• Immunoprecipitation of PNPase

• Partial RNase digestion to identify binding sites

• Library preparation and high-throughput sequencing

• Bioinformatic analysis to identify binding motifs

3. In vitro binding assays:

• Gel shift assays with purified recombinant PNPase and candidate RNAs

• Filter binding assays to determine binding affinities

• Competition assays to identify high-affinity substrates

4. Gradient fractionation:

• Separate cellular components by density

• Analyze co-fractionation of PNPase and specific RNAs

• Compare wild-type and catalytically inactive mutants

5. Structural analysis of complexes:

• Hydrogen-deuterium exchange mass spectrometry

• Chemical probing of RNA structure in the presence/absence of PNPase

• Cryo-EM of PNPase-RNA complexes

When using the partial recombinant protein , researchers should verify that RNA-binding domains are present in the construct. Comparison with full-length native protein may be necessary to validate binding results.

How can researchers investigate the role of PNPase in E. faecalis biofilm formation and virulence?

Investigation of PNPase's role in biofilm formation and virulence requires a multi-faceted approach:

Biofilm formation assays:

• Static biofilm models:

  • Microtiter plate crystal violet assay

  • Confocal microscopy with fluorescent strains

  • Biomass and viability quantification

• Flow cell biofilm models:

  • Real-time visualization of biofilm development

  • Analysis of structural parameters (thickness, roughness)

  • Response to antimicrobial challenges

• Mixed-species biofilms:

  • Co-culture with other oral or intestinal bacteria

  • Competition and cooperation dynamics

  • Species distribution analysis by FISH or qPCR

Virulence assays:

• Adhesion to relevant tissues:

  • Human platelets (for endocarditis models)

  • Intestinal epithelial cells

  • Urinary tract epithelial cells

• Resistance to host defenses:

  • Survival in human serum

  • Resistance to phagocytosis

  • Survival within macrophages

• Animal infection models:

  • Caenorhabditis elegans killing assay

  • Galleria mellonella infection model

  • Mouse models of bacteremia or endocarditis

Molecular mechanisms investigation:

• Transcriptome analysis:

  • RNA-seq of biofilm vs. planktonic cells

  • Comparison of wild-type vs. pnp mutant

  • Identification of PNPase-regulated virulence factors

• Regulatory network analysis:

  • ChIP-seq of relevant transcription factors

  • Two-component system phosphorylation assays

  • quorum sensing molecule quantification

E. faecalis prophages contribute to adhesion to human platelets, a critical step in endocarditis development . Investigating potential interactions between PNPase and prophage regulation could reveal novel aspects of virulence control.