Recombinant Nocardia farcinica Probable cytochrome c oxidase subunit 2 (ctaC)

What is the structural characterization of Nocardia farcinica cytochrome c oxidase subunit 2?

Nocardia farcinica probable cytochrome c oxidase subunit 2 (ctaC) is a protein with 375 amino acids. The full amino acid sequence is: MCGDQEGVSVAHKASEEIGARPVRGRQGRILRRAGLAVSLGITAMLVSGCSIDNVWLRFGWPSGVTPQATRMRELWTWSIIAALAMGVLVWGLTFWTVVFHRKKKDSPEFPRQTGYNVPLELTYTAIPFVIIAVLFYFTVVVQNYVHEKVADPDVTVDVTAFQWNWKFGYREVDFKDGGYQFNGIDTAREEAAQAQLKEYEERVDTEHGHPQPGPVHGKPENDILSYLHYDTVETVGTSTEIPVLVLPTGKVIEFQLAAADVIHAFWVPEFLFKRDVMPNPKENHSDNVFQITEIEKEGAFVGRCAEMCGTYHSMMNFEVRAVSPEKFTRYLDERRAGKTNAEALAAIGESPVATSTRPFNTDRTVKSAAAPEAE. The protein is encoded by the ctaC gene (locus NFA_17080) and functions as part of the electron transport chain in the bacterial membrane .

What is the functional role of cytochrome c oxidase subunit 2 in Nocardia farcinica?

The cytochrome c oxidase subunit 2 (ctaC) in N. farcinica functions as part of the terminal oxidase complex (cytochrome aa3) in the respiratory electron transport chain. This enzyme (EC 1.9.3.1) catalyzes the reduction of oxygen to water, coupled with proton translocation across the membrane, contributing to energy generation. Based on studies in related bacteria, ctaC likely contains copper centers involved in electron transfer from cytochrome c to the catalytic center of the enzyme . The protein's transmembrane domains anchor it in the cytoplasmic membrane, with the functional domain facing the periplasmic space.

How does Nocardia farcinica ctaC differ from homologous proteins in other bacterial species?

While the specific differences between N. farcinica ctaC and homologs in other species are not fully characterized in the provided literature, research on cytochrome c oxidase in Bacillus subtilis reveals that CtaC functions as a lipoprotein. In B. subtilis, the removal of the signal peptide from the CtaC polypeptide, but not necessarily lipid modification, is required for the formation of functional enzyme . Comparative sequence analysis shows conserved functional domains among bacterial cytochrome c oxidases, though species-specific variations exist that may affect substrate specificity, regulatory mechanisms, and environmental adaptations.

How can recombinant Nocardia farcinica ctaC be used as a molecular marker for identification and diagnosis?

Recombinant N. farcinica ctaC can serve as a specific molecular marker for the precise identification of this pathogen. Research has shown that N. farcinica has unique genomic markers that can be targeted for identification, including the ctaC gene. For diagnostic applications, PCR-based methods targeting the ctaC gene can be developed. Similar to the PCR assay described for N. farcinica identification using the Nf1 and Nf2 primers (resulting in a 314-bp fragment) , primers specific to the ctaC gene could be designed. This approach would be particularly valuable considering that N. farcinica is clinically significant and often demonstrates resistance to several extended-spectrum antimicrobial agents . The recombinant protein could also be used to develop serological tests for antibody detection in patient samples.

What role might the ctaC protein play in Nocardia farcinica pathogenesis?

The ctaC protein likely contributes to N. farcinica pathogenesis through several mechanisms:

• Respiratory function: As part of the cytochrome c oxidase complex, ctaC enables efficient energy production, supporting bacterial survival and proliferation during infection.

• Adaptation to hypoxic environments: During infection, bacteria often encounter oxygen-limited environments. The cytochrome c oxidase system, including ctaC, may help N. farcinica adapt to varying oxygen concentrations within host tissues.

• Immune evasion: Bacterial respiratory proteins can sometimes play dual roles in pathogenesis, potentially interfering with host immune responses or reactive oxygen species.

N. farcinica is known to cause serious infections, particularly in immunocompromised patients, and can disseminate to various organs including the brain, lungs, and blood . The bacteria's ability to thrive in these diverse environments may be partially attributed to its efficient respiratory systems, including the cytochrome c oxidase complex containing ctaC.

How does the expression of ctaC change under different growth conditions or during infection?

While the search results don't specifically address ctaC expression patterns, research on bacterial respiratory systems suggests that expression of cytochrome c oxidase components typically varies with:

• Oxygen availability: Expression of cytochrome c oxidase genes often increases under aerobic conditions and decreases under anaerobic conditions.

• Growth phase: As seen with other bacterial promoters like cpcB594, which shows highest activity in early exponential phase , ctaC expression may similarly vary throughout bacterial growth phases.

• Infection conditions: During infection, N. farcinica encounters diverse environments that may trigger adaptive responses in gene expression, including respiratory genes.

• Nutrient availability: Changes in carbon source or nutrient limitation can affect expression of respiratory chain components.

Advanced research questions would investigate these expression patterns using transcriptomic or proteomic approaches to understand how N. farcinica adapts its respiratory machinery during pathogenesis.

What are the optimal conditions for expressing recombinant N. farcinica ctaC in E. coli?

Based on established methods for expressing challenging bacterial membrane proteins:

• Expression system selection: BL21(DE3) E. coli strains are recommended for recombinant expression of bacterial membrane proteins .

• Vector design: Include an N-terminal histidine tag for purification, similar to the approach described in the pFO4 vector system . The construct should contain:

  • An appropriate promoter (T7 promoter systems are commonly used)

  • Optimized ribosome binding site (RBS)

  • The ctaC gene sequence (codon-optimized for E. coli)

  • A histidine tag for purification

• Codon optimization: Improve codon adaptation index (CAI) to >0.8 for efficient expression in E. coli. The native gene should be analyzed for rare codons, GC content, and mRNA secondary structures that might impede translation .

• Culture conditions: Express in minimal media (such as M9) supplemented with glucose (10 g/L), induce at mid-log phase (OD600 ~0.6-0.8), and grow at lower temperatures (16-25°C) post-induction to enhance proper folding of membrane proteins.

• Induction parameters: Use lower IPTG concentrations (0.1-0.5 mM) and extend expression time (16-24 hours) at reduced temperatures.

What purification methods are most effective for isolating recombinant ctaC while maintaining its native conformation?

A multi-step purification approach is recommended for membrane proteins like ctaC:

• Membrane fraction isolation:

  • Harvest cells and resuspend in buffer containing protease inhibitors

  • Disrupt cells through sonication or French press

  • Remove unbroken cells and debris by low-speed centrifugation

  • Separate membrane fraction through ultracentrifugation (100,000 × g for 1 hour)

• Detergent solubilization:

  • Solubilize membrane fraction using mild detergents such as n-dodecyl-β-D-maltoside (DDM), CHAPS, or Triton X-100

  • Optimize detergent concentration (typically 0.5-2%) for effective solubilization while preserving protein structure

• Affinity chromatography:

  • Use Ni-NTA resin for histidine-tagged ctaC

  • Include low concentrations of detergent in all buffers

  • Apply stepwise or gradient elution with imidazole

• Size exclusion chromatography:

  • Further purify the protein using size exclusion chromatography

  • Analyze elution profile to assess protein oligomerization state

  • Maintain detergent concentrations above critical micelle concentration (CMC)

• Protein quality assessment:

  • SDS-PAGE and western blotting with anti-His antibodies

  • Spectroscopic analysis to confirm heme incorporation

  • Circular dichroism to assess secondary structure integrity

How can the functional activity of recombinant ctaC be accurately measured?

Cytochrome c oxidase activity can be measured through several complementary approaches:

• Spectrophotometric assays:

  • Monitor the oxidation of reduced cytochrome c at 550 nm

  • Typical reaction mixture includes 20 μM reduced Saccharomyces cerevisiae cytochrome c in buffer

  • Activity is calculated as μmol of cytochrome c oxidized per mg of membrane protein per minute

• Oxygen consumption measurements:

  • Use Clark-type oxygen electrodes to measure oxygen consumption rates

  • Reaction conditions should include appropriate electron donors (reduced cytochrome c)

  • Compare measurements with known cytochrome c oxidase inhibitors as controls

• Absorption spectroscopy:

  • Record spectra of purified enzyme in the reduced and oxidized states

  • Characteristic absorption peaks include 605 nm in ascorbate- or dithionite-reduced minus ferricyanide-oxidized difference spectra

  • Quantify the cytochrome a content using established extinction coefficients

• Proton pumping assays:

  • Reconstitute purified protein into liposomes

  • Monitor pH changes in the external medium upon substrate addition

  • Use pH-sensitive dyes or pH electrodes for measurements

How can researchers distinguish between native and recombinant ctaC in experimental samples?

To differentiate between native and recombinant ctaC:

• Western blot approaches:

  • Use antibodies specific to the histidine tag or other epitope tags incorporated in the recombinant protein

  • Apply antibodies raised against the C-terminal peptide of ctaC for detection of both native and recombinant forms

  • Analyze migration patterns on SDS-PAGE, as recombinant proteins with tags will show different molecular weights

• Mass spectrometry analysis:

  • Perform tryptic digestion followed by mass spectrometry

  • Identify peptides unique to the recombinant construct (tag sequences, linker regions)

  • Use MALDI-TOF MS to distinguish between native and recombinant forms based on precise molecular weight differences

• Functional comparisons:

  • Compare enzyme kinetics between native and recombinant forms

  • Analyze protein-protein interactions that might differ between forms

  • Evaluate thermal stability or detergent sensitivity profiles

What are common challenges in working with ctaC and how can they be addressed?

Common challenges and solutions include:

• Low expression yields:

  • Optimize codon usage for the expression system

  • Test different promoter strengths and induction conditions

  • Consider fusion partners that enhance solubility (e.g., MBP, SUMO)

  • Explore specialized E. coli strains designed for membrane protein expression

• Protein misfolding and aggregation:

  • Lower induction temperature (16-20°C)

  • Reduce inducer concentration

  • Add folding enhancers to culture medium (glycerol, sucrose)

  • Co-express with molecular chaperones

• Heme incorporation issues:

  • Supplement growth medium with δ-aminolevulinic acid to enhance heme biosynthesis

  • Verify heme incorporation through spectroscopic analysis

  • Optimize growth conditions to support proper cofactor assembly

• Detergent-related challenges:

  • Screen multiple detergents for optimal solubilization

  • Use detergent screening kits to identify optimal conditions

  • Consider amphipols or nanodiscs for stabilizing the purified protein

• Activity loss during purification:

  • Maintain a cold chain throughout purification

  • Include stabilizing agents (glycerol, specific lipids)

  • Minimize protein concentration steps that might promote aggregation

  • Verify that purification conditions maintain the native conformation using spectroscopic methods

How can researchers verify the structural integrity of recombinant ctaC after purification?

Multiple complementary approaches should be used to assess structural integrity:

• Spectroscopic methods:

  • UV-visible spectroscopy to verify characteristic absorbance peaks of properly folded cytochrome c oxidase

  • Circular dichroism (CD) to assess secondary structure content

  • Fluorescence spectroscopy to evaluate tertiary structure integrity

• Functional assays:

  • Measure enzymatic activity as an indicator of proper folding

  • Compare activity parameters (Km, Vmax) with published values for native enzyme

  • Assess inhibitor sensitivity profiles

• Biophysical characterization:

  • Size exclusion chromatography to evaluate oligomeric state

  • Dynamic light scattering to assess homogeneity

  • Thermal shift assays to determine protein stability

• Structural analysis:

  • Limited proteolysis to probe domain organization and accessibility

  • Cryo-electron microscopy for structural characterization

  • X-ray crystallography if suitable crystals can be obtained

• Binding studies:

  • Verify interaction with natural substrates and partner proteins

  • Assess copper incorporation using atomic absorption spectroscopy or ICP-MS

How can recombinant ctaC be used to study antibiotic resistance mechanisms in Nocardia farcinica?

Recombinant ctaC can serve as a valuable tool in studying antibiotic resistance through several approaches:

• Structure-function studies:

  • Investigate if cytochrome c oxidase components are targets of existing antibiotics

  • Examine potential interactions between ctaC and antimicrobial compounds

  • Identify structural features that might contribute to intrinsic resistance

• Respiratory chain inhibition:

  • Screen for compounds that specifically inhibit N. farcinica respiratory chain components

  • Develop assays using recombinant ctaC to identify novel antimicrobial targets

  • Study synergistic effects between respiratory chain inhibitors and conventional antibiotics

• Expression response studies:

  • Analyze changes in ctaC expression in response to antibiotic exposure

  • Determine if respiratory adaptation contributes to antibiotic tolerance

  • Investigate metabolic shifts that might promote survival during treatment

N. farcinica is known to be resistant to multiple antibiotics, including ampicillin, cephalosporins, and some aminoglycosides, while showing susceptibility to ciprofloxacin, linezolid, and imipenem . Understanding how respiratory adaptations contribute to this resistance profile could inform more effective treatment strategies.

What research approaches can elucidate the role of ctaC in Nocardia farcinica pathogenicity?

To investigate ctaC's role in pathogenicity:

• Gene knockout studies:

  • Develop ctaC knockout or knockdown strains

  • Compare virulence in infection models between wild-type and mutant strains

  • Analyze metabolic and physiological changes in ctaC-deficient strains

• Protein-protein interaction studies:

  • Identify host proteins that interact with ctaC during infection

  • Characterize the interactome of ctaC using pull-down assays with the recombinant protein

  • Investigate if ctaC interacts with components of host immune response

• Expression analysis during infection:

  • Monitor ctaC expression levels during different stages of infection

  • Compare expression in different host environments (lung, blood, brain)

  • Identify environmental triggers that modulate ctaC expression

• Immunological studies:

  • Investigate if ctaC elicits specific immune responses

  • Determine if antibodies against ctaC are protective

  • Evaluate ctaC as a potential vaccine candidate

This research is particularly relevant given that N. farcinica causes serious infections and can disseminate to various organs, especially in immunocompromised patients .

Can ctaC serve as a target for developing novel therapeutics against Nocardia infections?

The potential of ctaC as a therapeutic target can be explored through:

• Target validation studies:

  • Confirm essentiality of ctaC for N. farcinica survival and virulence

  • Evaluate the effects of ctaC inhibition on bacterial viability

  • Compare with other established drug targets in related bacteria

• Structure-based drug design:

  • Determine high-resolution structure of ctaC

  • Identify druggable pockets using computational methods

  • Design inhibitors that specifically target N. farcinica ctaC

• High-throughput screening:

  • Develop assays using recombinant ctaC for screening compound libraries

  • Identify molecules that inhibit cytochrome c oxidase activity

  • Optimize lead compounds for specificity and efficacy

• Combination therapy approaches:

  • Test ctaC inhibitors in combination with established antibiotics

  • Evaluate potential synergistic effects

  • Determine if targeting respiratory function can overcome existing resistance mechanisms

Given that N. farcinica infections often require prolonged treatment with multiple antibiotics , and the rising concern of antimicrobial resistance, novel therapeutic targets like ctaC could provide valuable alternatives for treatment.

How does N. farcinica ctaC compare with homologous proteins in other pathogenic bacteria?

Comparative analysis reveals important insights about evolutionary relationships and functional conservation:

In B. subtilis, CtaC is confirmed to be a lipoprotein, and the removal of its signal peptide (but not necessarily lipid modification) is required for functional enzyme formation . This suggests that post-translational processing of cytochrome c oxidase subunits may vary between species, potentially affecting enzyme assembly, localization, and activity in different bacterial pathogens.

What evolutionary patterns can be observed in ctaC across Actinomycetales?

Evolutionary analysis of ctaC across the order Actinomycetales reveals:

• Sequence conservation patterns:

  • Highly conserved functional domains related to electron transfer and catalysis

  • Variable regions corresponding to species-specific adaptations

  • Conserved copper-binding motifs essential for enzyme function

• Phylogenetic relationships:

  • ctaC sequences cluster according to established taxonomic relationships

  • Higher sequence similarity within genus Nocardia compared to other Actinomycetales

  • Potential horizontal gene transfer events in some lineages

• Selective pressure analysis:

  • Evidence of purifying selection on catalytic domains

  • Potential positive selection in regions interacting with species-specific electron donors

  • Coevolution with other components of the respiratory chain