Recombinant Mycoplasma agalactiae ATP synthase subunit c (atpE)

What is Mycoplasma agalactiae ATP synthase subunit c (atpE)?

Mycoplasma agalactiae ATP synthase subunit c (atpE) is an essential component of the F-type ATPase in M. agalactiae, a pathogenic bacterium responsible for contagious agalactia in small ruminants. The atpE gene encodes a small hydrophobic protein that forms part of the F0 sector of ATP synthase. According to structural information, the protein consists of 75 amino acids with a molecular weight of approximately 8 kDa (based on comparison with M. penetrans atpE which is 78 amino acids with a molecular weight of 8159.55 Da) . The protein functions as part of the proton channel in the F0 complex, which is critical for ATP synthesis in the bacterial cell.

What is the amino acid sequence of M. agalactiae ATP synthase subunit c?

The complete amino acid sequence of M. agalactiae ATP synthase subunit c is:
MEKGLIAIGIGISMISGLGVGLGQGLAAGKAAEAAVGRNPEAASKIRTMMLVGQAVAESAAIYALVISILLMFAFN

This 75-amino acid protein is highly hydrophobic, containing multiple glycine and alanine residues, which facilitate its membrane-spanning function within the ATP synthase complex. The protein's hydrophobic nature is evident from the prevalence of non-polar amino acids such as leucine, isoleucine, and valine throughout the sequence.

What conservation patterns exist for ATP synthase subunit c across Mycoplasma species?

ATP synthase subunit c shows varying degrees of conservation across Mycoplasma species. When comparing M. agalactiae atpE with its counterpart in M. penetrans, similarities in length and function are observed, though species-specific variations exist . The conservation of this protein is important for understanding evolutionary relationships between Mycoplasma species and identifying potential targets for species-specific detection methods.

Similar to other Mycoplasma proteins, atpE likely contains the typical genetic code feature where TGA encodes tryptophan rather than acting as a stop codon (as seen in standard genetic code), which has significant implications for recombinant expression in non-Mycoplasma hosts .

What is the genomic context of the atpE gene in M. agalactiae?

The atpE gene in Mycoplasma species is typically part of the ATP synthase operon. In M. agalactiae strain PG2, the gene has been identified and annotated. Though specific genomic coordinates for M. agalactiae atpE are not directly provided in the search results, comparative information from M. penetrans shows that its atpE gene spans positions 67456 to 67692 on the positive strand . The genomic organization of the ATP synthase genes in Mycoplasma species is important for understanding the regulation of energy metabolism in these minimal bacteria.

What expression systems are optimal for producing recombinant M. agalactiae ATP synthase subunit c?

For optimal expression of recombinant M. agalactiae ATP synthase subunit c, E. coli-based expression systems have proven effective for Mycoplasma proteins, though they require specific modifications:

• Codon optimization: Site-directed mutagenesis is essential to convert Mycoplasma TGA codons (which encode tryptophan in Mycoplasma) to TGG codons (the universal tryptophan codon) for proper expression in E. coli .

• Vector selection: Expression vectors such as pPRO EX HTb have been successfully used for other Mycoplasma proteins and could be applied to atpE . For improved solubility and purification, fusion tag systems like pGex-2T (GST fusion) have proven effective for Mycoplasma membrane proteins .

• Expression conditions: For membrane proteins like ATP synthase subunit c, modified growth conditions including lower temperatures (15-25°C) and reduced IPTG concentrations often improve proper folding and reduce inclusion body formation.

The transformation method used for M. agalactiae involves centrifugation of cultures at 10,000 g at 4°C, washing with sterile cold DPBS, and treating with cold 0.1 M CaCl2 before introduction of the expression vector DNA .

How can site-directed mutagenesis enhance recombinant expression of M. agalactiae proteins?

Site-directed mutagenesis is crucial when expressing Mycoplasma proteins in heterologous systems due to genetic code differences. For M. agalactiae proteins:

• TGA codon conversion: The most critical application is converting TGA codons (which encode tryptophan in Mycoplasma) to TGG codons (the standard tryptophan codon) to prevent premature termination in E. coli . For P48 protein, researchers successfully converted three TGA codons to TGG codons , while another study reported converting four TGA codons .

• Methodological approach: The mutagenesis process typically involves:

  • PCR amplification of the target gene

  • Design of mutagenic primers containing the desired nucleotide changes

  • Sequential or simultaneous mutation of all TGA codons

  • Validation of the mutated sequence by DNA sequencing before cloning into an expression vector

• Validation of functionality: Following mutagenesis, validating that the protein retains its structural and functional properties is essential. For P48, western blotting with anti-P48 serum confirmed that the recombinant protein maintained its immunogenic properties after TGA→TGG conversion .

This approach is likely necessary for atpE expression as well, though the specific number of TGA codons in M. agalactiae atpE would need to be determined from the complete gene sequence.

What purification strategies yield high-purity recombinant M. agalactiae ATP synthase subunit c?

For effective purification of recombinant M. agalactiae ATP synthase subunit c, the following strategies are recommended based on successful approaches with other Mycoplasma proteins:

• Fusion protein expression: Expression as a fusion protein, such as with glutathione-S-transferase (GST), enhances solubility and provides an initial affinity purification step .

• Affinity chromatography: For His-tagged proteins, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is effective. For GST-fusion proteins, glutathione-agarose affinity chromatography allows for specific binding .

• Protease cleavage: Following initial purification, the target protein can be released from the fusion partner using specific proteases. For example, thrombin digestion has been successfully used to separate P48 from its GST fusion partner .

• Storage conditions: For maintaining stability of the purified protein, storage in Tris-based buffer with 50% glycerol at -20°C is recommended, with aliquoting to avoid repeated freeze-thaw cycles .

Table 1: Recommended Purification Protocol for Recombinant M. agalactiae ATP Synthase Subunit c

How can recombinant M. agalactiae ATP synthase subunit c be used in diagnostic applications?

Recombinant M. agalactiae ATP synthase subunit c could serve as a valuable diagnostic antigen, based on the successful use of other M. agalactiae recombinant proteins in diagnostic applications:

• ELISA-based detection: Recombinant atpE could be immobilized on ELISA plates to detect antibodies in serum samples from potentially infected animals . Studies with recombinant P48 have demonstrated that specific antibodies are detectable as early as 3 weeks after the onset of clinical disease .

• Western blot confirmation: Recombinant atpE could be used in western blotting as a confirmatory test, similar to the application of P48 . The immunogenic nature of membrane proteins makes them good candidates for serological detection.

• Species differentiation: While P48 has shown cross-reactivity between M. agalactiae and M. bovis , investigating the species-specificity of atpE could potentially provide a more discriminating diagnostic marker. Differential testing could be established by comparing reactivity patterns across multiple antigens.

• PCR-based detection: As a complement to protein-based assays, PCR amplification of the atpE gene could be incorporated into molecular diagnostic panels, though care must be taken to distinguish it from other Mycoplasma species . Typical amplicon sizes for Mycoplasma detection range from 171-374 bp depending on the target .

What are the immunogenic properties of M. agalactiae membrane proteins?

M. agalactiae membrane proteins demonstrate significant immunogenic properties that make them valuable for both diagnostics and potential vaccine development:

• Antigenic profile: Studies have revealed 24 polypeptides in whole cell antigens (WCA) and sonicated supernatant antigen (SSA) of M. agalactiae, ranging from 20.89 to 181.97 kDa. Seven major proteins with molecular weights of 63.10, 60.25, 58.88, 47.86, 44.66, 33.88, and 28.84 kDa have been identified as particularly important .

• Immunogenic response: All major proteins have demonstrated immunogenicity when tested with polyclonal rabbit serum against M. agalactiae, with 12-14 polypeptides showing strong immunogenic reactions . For membrane protein P48 specifically, antibodies are detectable in infected animals within 3 weeks after disease onset .

• Conservation across isolates: Immunoblotting of cell lysates from various Indian isolates of M. agalactiae against anti-P48 serum resulted in a consistent single band at approximately 48 kDa across all isolates, indicating conservation of this antigen . This conservation is important for developing reliable diagnostic tools.

• Cross-reactivity considerations: While membrane proteins like P48 can be used to differentiate M. agalactiae from many other pathogenic Mycoplasma species, some cross-reactivity exists with M. bovis . This is an important consideration when developing specific diagnostic tests, though the two species tend to be host-specific.

How does DNA methylation impact gene transfer and recombinant protein production in M. agalactiae?

DNA methylation plays a significant role in horizontal gene transfer (HGT) and potentially in recombinant protein production in M. agalactiae:

• Restriction-modification systems: M. agalactiae possesses restriction-modification (RM) systems that methylate specific DNA sequences as a protective mechanism against foreign DNA, including both m6A and cytosine modifications (m4C and m5C) .

• Methodological detection: A combination of SMRT-seq and Illumina bisulphite sequencing (BS-seq) has been used to detect the full range of DNA modifications in M. agalactiae, including m6A, m4C, and m5C .

• Strain variation: Analysis of multiple strains (including reference strains 5632 and PG2) has revealed diversity in the methylome landscape within the M. agalactiae species, with different strains possessing different active RM systems and recognition motifs .

• Impact on transformation: For recombinant protein expression in Mycoplasma itself, the presence of strain-specific restriction-modification systems must be considered, as they can reduce transformation efficiency. For transformation of M. agalactiae, researchers have used specific protocols involving CaCl2 treatment and PEG-mediated transformation .

• Growth phase considerations: The methylation status can vary depending on the growth phase of M. agalactiae, with DNA being extracted at different time points (24h for exponential phase and 48h for stationary phase) to assess these differences .

What PCR conditions are optimal for amplifying M. agalactiae genes for recombinant expression?

For successful amplification of M. agalactiae genes including atpE, the following optimized PCR conditions are recommended:

• DNA extraction: Genomic DNA should be extracted from mycoplasma cells after 48 hours of growth (beginning of stationary phase) using the phenol-chloroform method . For methylation studies, extraction at 24 hours (exponential phase) may also be important .

• PCR primer design: Primers should be designed to include appropriate restriction sites for subsequent cloning. For genes containing TGA codons that will be expressed in E. coli, primers can also be designed to incorporate the TGA→TGG mutations .

• Amplicon size expectations: For atpE gene amplification, the expected amplicon would be approximately 237 nucleotides (based on the coding sequence length) . For comparison, other Mycoplasma gene targets typically produce:

  • 16S rDNA: 269 bp

  • 16S-23S rDNA ITS: 236-374 bp (variable depending on species)

  • uvrC (M. bovis specific): 171 bp

• Electrophoresis conditions: For optimal separation of PCR products, 1.5% agarose gels run at 105V for 50 minutes with 100 bp DNA molecular weight markers in both the first and last lanes are recommended .

• Verification steps: Sequencing of PCR products is essential to confirm successful amplification and, if applicable, mutation of TGA codons to TGG codons. Both 16S and 16S-23S ITS sequences are highly conserved for Mycoplasma and can serve as controls .

What validation methods ensure successful expression of functional recombinant M. agalactiae proteins?

To ensure that recombinant M. agalactiae ATP synthase subunit c is properly expressed and maintains its structural integrity, the following validation methods are recommended:

• SDS-PAGE analysis: Assess protein expression and purity through SDS-PAGE, which should show a band corresponding to the expected molecular weight of approximately 8-9 kDa for ATP synthase subunit c .

• Western blotting: Confirm the identity and immunoreactivity of the recombinant protein using specific antibodies. For novel proteins, antibodies can be raised in rabbits against synthesized peptides or the purified recombinant protein .

• Mass spectrometry: Verify the molecular weight and sequence of the purified protein using MS/MS analysis, which can also confirm the success of site-directed mutagenesis efforts.

• Functional assays: For ATP synthase components, assess functionality through ATPase activity assays or proton translocation studies in reconstituted membrane systems.

• Storage stability assessment: Evaluate the stability of the purified protein under different storage conditions, with recommended storage in Tris-based buffer with 50% glycerol at -20°C for extended periods, and working aliquots kept at 4°C for up to one week .

• Immunogenicity testing: If intended for diagnostic use, validate the recombinant protein against well-characterized positive and negative serum samples to determine sensitivity and specificity parameters .

How can recombinant M. agalactiae proteins contribute to vaccine development?

Recombinant M. agalactiae ATP synthase subunit c and other membrane proteins hold significant potential for vaccine development against contagious agalactia:

• Subunit vaccine candidates: As membrane proteins are often immunogenic, recombinant atpE could serve as a component in subunit vaccines. Research with P48 has already demonstrated strong immunogenic properties that could be leveraged for protective immunity .

• Multi-epitope approaches: The antigenic heterogeneity of M. agalactiae suggests that effective vaccines may require multiple antigenic determinants . A cocktail of recombinant proteins including atpE, P48, and other immunogenic proteins could provide broader protection.

• Adjuvant optimization: Studies combining recombinant M. agalactiae membrane proteins with various adjuvants would be valuable to determine optimal formulations for inducing protective immunity in sheep and goats.

• Cross-protection assessment: Given the cross-reactivity observed between some M. agalactiae and M. bovis proteins , research into whether recombinant proteins from one species could provide cross-protection against the other would be valuable.

• Delivery system development: Novel delivery systems such as nanoparticles or liposomes could enhance the immunogenicity of recombinant M. agalactiae proteins and improve vaccine efficacy.

What bioinformatic approaches can predict antigenic determinants in M. agalactiae proteins?

Advanced bioinformatic approaches can help identify antigenic determinants in M. agalactiae proteins including ATP synthase subunit c:

• Epitope prediction algorithms: B-cell and T-cell epitope prediction tools can identify potential antigenic regions within the atpE sequence, guiding the design of synthetic peptides for antibody production or vaccine development.

• Structural modeling: Homology modeling of M. agalactiae ATP synthase subunit c based on crystal structures from related organisms can reveal surface-exposed regions likely to be immunogenic.

• Comparative genomics: Analysis of atpE sequence conservation across multiple Mycoplasma strains can identify both conserved regions (for broad-spectrum diagnostics) and variable regions (for species-specific detection) .

• Protein-protein interaction prediction: Computational prediction of interactions between M. agalactiae proteins and host immune components could identify key antigenic determinants involved in pathogenesis.

• Reverse vaccinology: Genome-wide screening for potential vaccine candidates, including membrane proteins like ATP synthase components, can identify novel antigenic targets beyond the currently characterized proteins .