Recombinant Enterococcus faecalis V-type ATP synthase alpha chain (atpA), partial

What is the V-type ATP synthase alpha chain (atpA) in Enterococcus faecalis?

The V-type ATP synthase alpha chain (atpA) is a crucial component of bacterial ATP synthase that functions in ATP synthesis coupled to proton transport in Enterococcus faecalis. Unlike the more common F-type ATP synthases found in most bacteria, E. faecalis possesses V-type ATP synthases, which are typically associated with vacuolar membranes in eukaryotes. The atpA gene encodes the α subunit of this ATP synthase complex, representing approximately 73.5% of the coding region of this gene with a G+C content of approximately 43% ± 2%, which aligns with the average G+C content for the total enterococcal genome . The protein participates in energy metabolism and has been implicated in stress response mechanisms, particularly in response to bile acids and oxygen fluctuations.

How does the atpA gene differ between Enterococcus species?

The atpA gene sequences demonstrate significantly higher discriminatory power than 16S rRNA genes for differentiating closely related Enterococcus species. While members of the E. faecium species group (E. faecium, E. hirae, E. durans, E. villorum, E. mundtii, and E. ratti) show >99% similarity in 16S rRNA gene sequences, their atpA gene sequences exhibit at most 89.9% similarity . This makes atpA gene sequencing a more reliable tool for species identification within the Enterococcus genus. Even closely related species like E. haemoperoxidus and E. moraviensis, which share 99.4% 16S rRNA gene sequence similarity, display only 92% atpA gene sequence similarity . This difference highlights the utility of atpA as a phylogenetic marker with superior resolution for enterococcal species differentiation.

What functional role does V-type ATP synthase play in Enterococcus faecalis stress response?

V-type ATP synthase in E. faecalis plays a critical role in stress response, particularly to bile acids. Proteomic analyses have demonstrated that various subunits of V-type ATPase are significantly up-expressed when E. faecalis is exposed to bile acids such as deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) . This up-regulation, along with increased expression of ABC-transporters, multi-drug transporters, and proteins related to cell wall biogenesis, contributes to bile acid resistance mechanisms. The V-type ATPase likely functions to maintain internal pH homeostasis and energy balance during exposure to these membrane-disrupting compounds, enabling E. faecalis to survive in the bile-rich environment of the human gastrointestinal tract and gallbladder.

How can recombinant atpA be utilized for phylogenetic analysis of emerging Enterococcus strains?

For phylogenetic analysis of emerging Enterococcus strains, recombinant atpA can be employed following a multi-step methodological approach. First, amplify the atpA gene fragment (approximately 1,102-bp) using PCR with consensus primers designed from conserved regions of the gene. Following sequencing, conduct comparative analysis of the atpA sequences using bioinformatic tools to construct phylogenetic trees. The regression curve between atpA and 16S rRNA gene sequences reveals a logarithmic relationship that best fits a polynomial regression of the second degree, with a significant correlation (R = 0.7) . When analyzing emerging strains, focus on species-specific sequence variations, as intraspecies atpA sequence similarities typically exceed 98.5% for most Enterococcus species. The higher discriminatory power of atpA compared to 16S rRNA (maximum 92% similarity between different Enterococcus species for atpA) enables more accurate placement of novel strains within the enterococcal phylogeny, especially for closely related species that are difficult to differentiate using traditional 16S rRNA sequencing.

What are the molecular mechanisms underlying V-type ATP synthase regulation during bile acid exposure?

The molecular mechanisms regulating V-type ATP synthase expression during bile acid exposure involve complex transcriptional and post-transcriptional processes. Experimental evidence indicates that bile acids like DCA and CDCA at concentrations of 0.05% trigger significant proteomic adaptations in E. faecalis. These adaptations include up-regulation of V-type ATPase subunits as part of a "general bile acid response" . The regulatory pathway likely begins with membrane stress detection, followed by activation of stress-responsive transcription factors. Analysis of enterococcal RNA-protein interactions through Grad-seq has revealed potential involvement of RNA-binding proteins and regulatory RNAs in post-transcriptional control of gene expression during stress response . The 6S RNA-RNA polymerase complex identified in E. faecalis suggests conservation of 6S RNA-mediated global control of transcription, which may influence V-type ATPase expression under stress conditions . The precise signaling cascade requires further elucidation, but likely integrates multiple regulatory elements including two-component systems, alternative sigma factors, and small regulatory RNAs that collectively modulate V-type ATPase expression to maintain cellular homeostasis during bile acid stress.

How do post-translational modifications affect the activity of V-type ATP synthase alpha chain in Enterococcus faecalis?

Post-translational modifications (PTMs) of the V-type ATP synthase alpha chain significantly influence its enzymatic activity, protein-protein interactions, and stress response functionality. Mass spectrometry-based proteomic analyses have revealed that under bile acid stress conditions, E. faecalis exhibits altered protein expression patterns affecting posttranslational modifications and protein turnover . Using data-independent acquisition mass spectrometry (DIA-MS), researchers can identify specific PTMs such as phosphorylation, acetylation, or glycosylation that may occur on the atpA protein. These modifications likely serve as regulatory switches that fine-tune ATP synthase activity in response to environmental stressors. Methodologically, researchers should employ enrichment techniques specific for the PTM of interest, followed by high-resolution LC-MS/MS analysis. Comparing PTM profiles between normal and stress conditions (such as bile acid exposure or oxygen limitation) can reveal modification sites that correlate with altered enzymatic activity. Functional validation of identified PTM sites through site-directed mutagenesis of recombinant atpA (substituting modifiable residues with non-modifiable variants) can confirm their regulatory importance and provide insights into the molecular mechanisms underlying V-type ATP synthase regulation in enterococcal stress response.

What are the optimal conditions for expressing recombinant E. faecalis atpA protein?

For optimal expression of recombinant E. faecalis atpA protein, a systematic approach combining appropriate expression systems and purification strategies is essential. Begin by designing a codon-optimized synthetic gene construct for the expression host (typically E. coli), incorporating a suitable affinity tag (His6 or GST) to facilitate purification. The expression vector should contain a strong inducible promoter (T7 or tac) and ribosome binding site optimized for high-level expression. Initial small-scale expression trials should test multiple conditions:

• Induction temperature: Compare expression at 16°C, 25°C, and 37°C

• Inducer concentration: Test IPTG concentrations ranging from 0.1-1.0 mM

• Induction time: Evaluate 4-hour, 8-hour, and overnight induction periods

• Expression hosts: Compare BL21(DE3), C41(DE3), and Rosetta(DE3) strains
  Optimal conditions typically involve lower temperatures (16-25°C) and extended induction periods (16-20 hours) to enhance protein solubility. For purification, use a two-step process involving initial IMAC (immobilized metal affinity chromatography) followed by size exclusion chromatography. Buffer composition is critical, with 20 mM Tris-HCl, pH 7.5, 150 mM KCl, 1 mM MgCl₂, and 1 mM DTT providing stability similar to conditions used in successful enterococcal protein studies . Verify protein purity by SDS-PAGE and confirm activity through ATP hydrolysis assays before proceeding to structural or functional studies.

How can gradient centrifugation techniques be applied to study V-type ATP synthase complexes?

Gradient centrifugation represents a powerful technique for studying V-type ATP synthase complexes in their native state. Based on successful applications with enterococcal proteins, the following methodological approach is recommended:

• Sample preparation: Harvest E. faecalis cells in mid-logarithmic phase and resuspend in lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM KCl, 1 mM MgCl₂, 1 mM DTT, 1 mM PMSF, and 0.2% Triton X-100 . Include protease inhibitors to prevent degradation and DNase I to reduce viscosity.

• Cell lysis: Use mechanical disruption with glass beads (0.1 mm) through repeated vortexing cycles (30 seconds vortexing followed by 15 seconds cooling on ice, repeated 10 times) . This gentle lysis method helps preserve protein complexes.

• Gradient preparation: Create a linear 10%-40% (w/v) glycerol gradient in ultracentrifuge tubes using a gradient maker. The 10% solution contains 20 mM Tris-HCl, pH 7.5, 150 mM KCl, 1 mM MgCl₂, 1 mM DTT, 1 mM PMSF, 0.2% Triton X-100, and 10% glycerol. The 40% solution contains the same components but with 40% glycerol .

• Sample loading and centrifugation: Carefully layer 200 μl of cleared lysate onto the gradient and centrifuge at 100,000 g (approximately 23,700 rpm) for 17 hours at 4°C .

• Fractionation and analysis: Collect approximately 20 fractions (590 μl each) manually from the top of the gradient . Analyze fractions for protein content by SDS-PAGE and immunoblotting with anti-atpA antibodies. Assess ATP synthase activity in each fraction using enzyme activity assays.
  This technique separates cellular components based on their sedimentation properties, allowing identification of intact V-type ATP synthase complexes and their interaction partners. The approach has been successfully applied to study RNA-protein complexes in E. faecalis V583 and can be adapted for specific analysis of ATP synthase complexes.

What proteomic approaches are most effective for studying V-type ATP synthase regulation in response to environmental stressors?

Data-independent acquisition mass spectrometry (DIA-MS) represents the most effective proteomic approach for studying V-type ATP synthase regulation in response to environmental stressors like bile acids or oxygen limitation. This methodology offers several advantages:

• Comprehensive quantification: DIA-MS enables quantitative analysis of every detectable protein in a sample, providing high reliability in quantitative results compared to earlier techniques .

• Experimental design: For optimal results, compare E. faecalis cultures grown under control conditions versus stress conditions (e.g., 0.05% bile acids or microaerophilic conditions). Include biological triplicates for statistical validity.

• Sample preparation: After stress exposure (typically 18 hours for long-term adaptation studies), harvest cells and extract proteins using mechanical disruption with glass beads in appropriate buffer (20 mM Tris-HCl, pH 7.5, 150 mM KCl, 1 mM MgCl₂) .

• Protein digestion and peptide preparation: Perform in-solution digestion with trypsin, followed by C18 clean-up and fractionation if needed.

• DIA-MS analysis: Analyze samples using high-resolution mass spectrometers (e.g., Q-Exactive or Orbitrap instruments) in DIA mode, with spectral libraries built from data-dependent acquisition runs.

• Data analysis: Use specialized software like Spectronaut or DIA-NN for quantification, focusing on V-type ATP synthase subunits and associated proteins. Apply appropriate statistical tests (e.g., ANOVA with post-hoc corrections) to identify significantly altered proteins.
  This approach successfully identified up-expression of V-type ATPase subunits in E. faecalis exposed to bile acids, demonstrating their essential role in stress response . The method can be extended to examine various environmental conditions, providing comprehensive insights into V-type ATP synthase regulation mechanisms.

How should researchers interpret conflicting data on V-type ATP synthase function in different Enterococcus strains?

When confronted with conflicting data on V-type ATP synthase function across different Enterococcus strains, researchers should implement a systematic analytical framework:

• Strain characterization verification: First, confirm accurate taxonomic identification of all strains using both 16S rRNA and atpA gene sequencing. Given that atpA provides superior resolution for closely related enterococcal species , misidentification could explain functional discrepancies.

• Genomic context analysis: Examine the complete operon structure of V-type ATP synthase genes in each strain. Variations in regulatory elements or operon organization may explain differential expression patterns. Sequence the full atpA gene and surrounding regions to identify strain-specific polymorphisms or insertions/deletions that might affect function.

• Experimental condition standardization: Analyze methodological differences between studies, including growth conditions, stress parameters, and analytical techniques. Standardize key experimental variables including growth phase, media composition, and stress exposure protocols across comparative studies.

• Multi-omics integration: Combine proteomic data with transcriptomic and metabolomic analyses to build a comprehensive picture of V-type ATP synthase regulation. This approach can reveal strain-specific differences in post-transcriptional regulation or metabolic context that influence ATP synthase function.

• Functional validation: Perform direct enzymatic activity measurements under identical conditions across strains. ATP hydrolysis and synthesis assays, coupled with membrane potential measurements, can quantify functional differences objectively.
  Conflicting data often reflects biological reality rather than experimental error, as V-type ATP synthase regulation likely evolved differently across enterococcal lineages to adapt to specific ecological niches. By systematically addressing these analytical dimensions, researchers can distinguish genuine strain-specific functional differences from methodological artifacts.

What bioinformatic tools are recommended for analyzing atpA sequence data from clinical isolates?

For comprehensive analysis of atpA sequence data from clinical isolates, I recommend the following bioinformatic workflow:

• Sequence quality assessment and preprocessing:

  • FastQC for initial quality assessment

  • Trimmomatic or Cutadapt for adapter removal and quality filtering

  • Ensure minimum Q30 scores and read depths of >20x for reliable variant calling

• Sequence alignment and phylogenetic analysis:

  • MUSCLE or MAFFT for multiple sequence alignment of atpA sequences

  • ModelTest-NG to determine optimal evolutionary models

  • RAxML-NG or IQ-TREE for maximum likelihood phylogenetic tree construction

  • MrBayes for Bayesian phylogenetic inference when evolutionary relationships are uncertain

• Sequence variation analysis:

  • DnaSP for calculating nucleotide diversity, haplotype diversity, and tests of selection

  • MEGA for calculating evolutionary distances and identifying conserved regions

  • PAML for detecting sites under positive selection

• Strain typing and identification:

  • Construct a local BLAST database of reference atpA sequences for rapid identification

  • Set threshold of >98.5% sequence similarity for intraspecies identification based on established atpA variation patterns

  • Employ phylogenetic placement algorithms like pplacer for situating novel isolates within reference trees

• Functional prediction:

  • PROVEAN or SIFT for predicting functional impact of amino acid substitutions

  • Phyre2 or SWISS-MODEL for homology modeling of variant proteins

  • ConSurf for identifying functionally important conserved residues
    When analyzing clinical isolates, special attention should be paid to mutations that might confer increased stress tolerance, as evidenced by the up-regulation of V-type ATPase subunits during bile acid stress . Comparison of atpA sequences from clinical isolates with environmental strains can reveal adaptation signatures related to healthcare settings.

What are the future research directions for Enterococcus faecalis V-type ATP synthase studies?

Future research on Enterococcus faecalis V-type ATP synthase should focus on several promising directions that build upon recent advances in understanding its structure, function, and regulation:

• Structure-function relationships: Determine high-resolution structures of the complete V-type ATP synthase complex from E. faecalis using cryo-electron microscopy. This will provide insights into unique structural features that distinguish it from F-type ATP synthases and identify potential antimicrobial target sites.

• Regulatory networks: Expand upon the Grad-seq analysis of E. faecalis to elucidate the complete transcriptional and post-transcriptional regulatory networks controlling V-type ATP synthase expression under various stress conditions. This should include identification of small regulatory RNAs and RNA-binding proteins that modulate atpA expression.

• Host-pathogen interactions: Investigate how V-type ATP synthase functions during in vivo infection and colonization using animal models. Particularly important is understanding its role during transition between different host environments with varying oxygen levels and bile acid concentrations.

• Antimicrobial development: Given the essential nature of V-type ATP synthase and its upregulation during stress responses , develop targeted inhibitors that specifically disrupt its function. The structural differences between bacterial V-type and human F-type ATP synthases offer potential for selective targeting.

• Systems biology integration: Combine multi-omics approaches (transcriptomics, proteomics, metabolomics) to build comprehensive models of energy metabolism in E. faecalis, with V-type ATP synthase as a central component. This will provide a systems-level understanding of how energy production is coordinated with stress responses and virulence.

• Evolutionary adaptations: Expand comparative analyses of atpA across diverse enterococcal species and strains to understand how sequence variations contribute to niche adaptation and pathogenicity. This builds upon established atpA phylogenetic frameworks to connect sequence diversity with functional adaptation. These research directions will not only advance our fundamental understanding of E. faecalis biology but also potentially lead to new therapeutic approaches for combating enterococcal infections.