Recombinant Mycoplasma gallisepticum ATP synthase subunit alpha (atpA), partial

What is the structural organization of ATP synthase in Mycoplasma gallisepticum?

ATP synthase in Mycoplasma gallisepticum follows the typical F1F0 ATPase structure found in other bacteria. The F1 component contains five subunits (α, β, γ, δ, and ε) arranged in a specific stoichiometry, with the catalytic sites primarily located on the β subunits at the interface with adjacent α subunits. Despite mycoplasmas' tendency toward genome reduction, they have maintained complete operons encoding all eight subunits of the F1F0 ATPase .

In mycoplasmas, ATP synthase primarily functions in ATP hydrolysis and maintenance of the electrochemical gradient rather than ATP generation. Interestingly, mycoplasmas have evolved unique F1-like ATPase clusters not found in other bacteria, categorized as Type 2 and Type 3 ATPases, which have undergone rapid sequence evolution while maintaining structural conservation .

How do the extra copies of atpA genes found in mycoplasmas differ from standard F1F0 atpA?

Many mycoplasma genomes contain extra copies of atpA and atpD (encoding α and β subunits) outside the traditional F1F0 ATPase operon. These additional copies form part of specialized F1-like ATPase clusters containing proteins with predicted structures similar to the α, β, γ, and ε subunits of F1 ATPases, along with other unique proteins not found in typical ATP synthases .

These specialized atpA genes show evidence of more rapid sequence evolution than the standard F1F0 atpA, while still maintaining structural features necessary for function. The presence of these additional copies is particularly noteworthy given mycoplasmas' tendency toward genome reduction and elimination of redundancy, suggesting they serve important specialized functions .

What is known about the evolutionary history of ATP synthase in mycoplasmas?

Phylogenomic studies have revealed fascinating evolutionary patterns in mycoplasma ATP synthase genes. While the typical F1F0 ATPase genes show standard vertical inheritance consistent with species evolution, the additional atpA copies in specialized F1-like ATPase clusters show evidence of horizontal gene transfer (HGT) between mycoplasma species .

Researchers have identified two types of these specialized clusters (Type 2 and Type 3), with Type 3 ATPase clusters showing evidence of spreading across different phylogenetic groups through horizontal gene transfer between mycoplasmas sharing the same host. This suggests an unexpected level of genetic exchange in these organisms despite their reduced genomes .

How does the unique twin-motor structure discovered in M. mobile relate to other mycoplasma ATP synthases?

Researchers studying Mycoplasma mobile have discovered a remarkable modification of ATP synthase that forms its gliding machinery. This molecular motor consists of a chain of two ATP synthase-like molecules housed within a complex cylindrical structure - a configuration never seen before in biological systems .

This discovery suggests that ATP synthase in mycoplasmas has undergone unexpected evolutionary modifications, potentially leading to novel functional adaptations. Based on genetic information, researchers had hypothesized that M. mobile's molecular motor evolved from ATP synthase, but the discovery of this twin-motor structure was surprising . While this specific arrangement has been confirmed in M. mobile, similar specialized configurations may exist in other mycoplasma species including M. gallisepticum, representing a fascinating example of protein repurposing during evolution.

What role does ATP synthase play in mycoplasma pathogenicity?

ATP synthase may contribute to mycoplasma pathogenicity through several mechanisms. Beyond its fundamental role in energy metabolism, specialized F1-like ATPases in mycoplasmas appear to have additional functions potentially related to host interaction .

In M. gallisepticum specifically, a cluster of genes encoding an F1-likeX0 ATPase has been identified as a putative partner of the mycoplasma-specific MIB-MIP system involved in the cleavage of host immunoglobulins . This suggests a direct role in immune evasion strategies. The horizontal transfer of specialized ATPase clusters between mycoplasmas that share the same host further supports their potential role in host adaptation and pathogenicity .

What expression systems are optimal for producing recombinant M. gallisepticum atpA?

For successful expression of recombinant M. gallisepticum atpA, researchers should consider several expression systems with their respective advantages:

E. coli-based expression systems:

• BL21(DE3) and derivatives offer high expression levels and established protocols

• Critical consideration: Mycoplasmas use UGA to code for tryptophan rather than as a stop codon, requiring codon-optimization

• Low induction temperatures (16-20°C) and fusion tags (His6, MBP, SUMO) can improve solubility

• C41(DE3) and C43(DE3) strains may be beneficial if standard strains show toxicity

Alternative expression systems:

• Baculovirus expression in insect cells for complex proteins requiring eukaryotic folding machinery

• Cell-free expression systems for rapid screening or toxic proteins

• Native expression in related mycoplasma species using genetic tools like the RecET-like systems described for M. gallisepticum

Optimization strategies should include testing multiple construct designs, induction conditions, and buffer formulations, with quality assessment via SDS-PAGE, Western blotting, and activity assays to determine the most suitable system for specific research goals.

How can researchers assess ATP synthase activity in M. gallisepticum?

Multiple complementary approaches can be used to assess ATP synthase activity in M. gallisepticum:

ATP hydrolysis assays:

• EnzChek Phosphate Assay: Measures inorganic phosphate (Pi) release by monitoring absorbance at 360 nm

• Experimental protocol: Incubate membrane-enriched fractions (60 μg protein) with 80 μM ATP at room temperature, measure absorbance every 90 seconds for 40-50 minutes, with a control lacking membrane fraction to account for ATP self-hydrolysis

• Statistical analysis: Apply t-student test at 0.05 significance level to compare Pi released from different samples

Proton translocation assays:

• ACMA fluorescence quenching to monitor proton pumping in vesicles

• pH electrode measurements for direct detection of pH changes

Specificity controls:

• Include inhibitors specific to F-type ATPases (oligomycin, DCCD)

• Compare activity in wild-type versus atpA-modified strains

• Test activity across a range of pH values and ion concentrations

Each method provides different insights into ATP synthase function, with combinations of approaches yielding the most comprehensive characterization.

What structural changes occur in ATP synthase under acidic conditions?

Recent research on ATP synthase function under acidic conditions has revealed important insights that may be applicable to understanding mycoplasma ATP synthases during infection of acidic host environments:

A study examining ATP synthase at acidic pH identified four distinct conformational states, with three representing different stages in the enzyme's reaction cycle, including two unique states not previously described . These conformational changes reflect how the enzyme adapts its mechanism under hypoxic conditions that lead to acidification.

For M. gallisepticum researchers, these findings suggest that:

• ATP synthase may adopt different conformations and catalytic modes depending on the pH of the environment

• These adaptations may be particularly relevant during infection, as pathogens often encounter acidic microenvironments

• Structural plasticity in the coupling between F1 and F0 components may allow for functional adaptation under stress conditions

Understanding these pH-dependent structural changes could inform both basic research into ATP synthase mechanism and applied research targeting this enzyme in pathogens.

How can site-directed mutagenesis be applied to study M. gallisepticum atpA function?

Site-directed mutagenesis offers a powerful approach to dissect the function of M. gallisepticum atpA through systematic modification of specific amino acid residues:

Key targets for mutagenesis:

• Catalytic residues involved in nucleotide binding and hydrolysis

• Interface residues mediating interactions with other ATP synthase subunits

• Unique mycoplasma-specific residues that may confer specialized functions

• pH-sensing residues that might influence activity under different conditions

Technical implementation:

• PCR-based mutagenesis methods for introducing specific mutations

• RecET-like systems shown effective for genome modification in M. gallisepticum

• Expression of mutant proteins followed by comparative functional analysis

Expected outcomes and interpretations:

This systematic mutagenesis approach provides insights into both general mechanisms of ATP synthase function and specialized adaptations in M. gallisepticum, helping to identify residues critical for enzyme activity or species-specific functions.

What is the relationship between F1-like ATPases and the MIB-MIP system in M. gallisepticum?

Research suggests a fascinating connection between specialized F1-like ATPases and the mycoplasma immunoglobulin binding-immunoglobulin protease (MIB-MIP) system in M. gallisepticum:

A cluster of genes encoding an F1-likeX0 ATPase in M. gallisepticum has been identified as a putative partner of the MIB-MIP system, which is involved in cleaving host immunoglobulins . This suggests a direct role for this specialized ATPase in immune evasion mechanisms.

The gene GCW_RS03665, which is part of this F1-likeX0 ATPase cluster, has been targeted in genetic modification studies using RecET-like systems . While the exact functional relationship remains to be fully characterized, this association points to a potential role for specialized ATP synthase-derived complexes in host-pathogen interactions beyond energy metabolism.

This connection represents an exciting area for further research, potentially revealing how mycoplasmas have repurposed ATP synthase components for specialized functions in pathogenicity.

What approaches can be used to study horizontal gene transfer of atpA in mycoplasma species?

Investigating horizontal gene transfer (HGT) of atpA genes in mycoplasmas requires multiple genomic approaches:

Comparative genomics:

• Whole genome alignment of multiple mycoplasma species to identify syntenic regions containing atpA genes

• Analysis of operon organization and genomic context across species

• Identification of mobile genetic elements or insertion sequences associated with specialized ATPase clusters

Phylogenetic analysis:

• Construction of phylogenetic trees using atpA sequences compared to species trees

• Identification of incongruences suggesting horizontal gene transfer

• Sequence analysis to detect selection pressure and recent transfer events

From search results, we know that researchers have identified evidence for HGT of Type 3 ATPase clusters between mycoplasmas that infect the same host . This suggests that despite their reduced genomes, mycoplasmas actively exchange genes related to specialized F1-like ATPases, potentially contributing to host adaptation.

These genomic approaches can reveal the evolutionary history and ongoing dynamics of atpA gene exchange in mycoplasma populations, providing insights into both evolutionary mechanisms and potential functional adaptations.

What controls should be included when performing ATP hydrolysis assays with M. gallisepticum membrane fractions?

Rigorous ATP hydrolysis assays with M. gallisepticum membrane fractions require comprehensive controls to ensure reliable results:

Essential controls:

• ATP self-hydrolysis control: Reaction mixture without membrane fraction to account for non-enzymatic ATP hydrolysis

• Negative control: Heat-inactivated membrane fractions to confirm enzymatic nature of activity

• Positive control: Known ATPase-containing samples with established activity rates

• Inhibitor controls: Specific F-type ATPase inhibitors (oligomycin, DCCD) to confirm activity source

Statistical considerations:

• Perform biological replicates with independent membrane preparations

• Include technical replicates for each sample

• Apply appropriate statistical tests (e.g., t-student test at 0.05 significance level) to compare samples

Data presentation:

• Convert absorbance measurements to Pi released using standard curves (0.01-0.12 mM KH₂PO₄)

• Plot time-course data showing Pi release rates

• Include error bars representing standard deviation or standard error

Implementing these controls ensures that observed ATPase activity can be confidently attributed to the target enzyme complex and provides a solid foundation for comparative studies between wild-type and modified strains.

How can researchers engineer M. gallisepticum strains with modifications to atpA?

Genetic modification of atpA in M. gallisepticum can be achieved through several approaches:

RecET-like systems:

• RecET-like systems from Spiroplasma phoeniceum (Spho) or Bacillus subtilis (Bsu) have been successfully used in M. gallisepticum

• These systems enhance homologous recombination efficiency

• Two expression options are available: strong pSynMyco promoter or weaker p438 promoter

Targeting constructs:

• Design DNA templates with ~1 kb homology arms flanking the target site

• Include selectable markers such as chloramphenicol acetyl transferase (cat) gene

• Consider four template forms: linear or circular dsDNA, and linear or circular ssDNA

Target selection:

• For atpA studies, researchers can target specific regions of the gene or the entire coding sequence

• Previous work has successfully targeted a gene (GCW_RS03665) that is part of an F1-likeX0 ATPase cluster in M. gallisepticum

Verification methods:

• PCR screening to identify successful recombinants

• Sequence verification of modified regions

• Functional assays to confirm phenotypic effects

This genetic engineering approach enables various modifications including gene knockout, site-directed mutations, or insertion of tags for protein detection, providing powerful tools for investigating atpA function in M. gallisepticum.

What methods can be used to purify and characterize the complete ATP synthase complex from M. gallisepticum?

Purification and characterization of the complete ATP synthase complex from M. gallisepticum requires specialized approaches:

Membrane isolation:

• Gentle cell lysis to preserve membrane integrity

• Differential centrifugation to isolate membrane fractions

• Enrichment for membrane proteins while minimizing contamination

Solubilization strategies:

• Mild detergents (n-dodecyl-β-D-maltoside, digitonin) to maintain complex integrity

• Optimization of detergent:protein ratio to prevent dissociation

• Inclusion of stabilizing agents (ATP/ADP, Mg²⁺, glycerol)

Purification workflow:

• Ion exchange chromatography for initial enrichment

• Affinity approaches if working with tagged components

• Size exclusion chromatography for final purification and characterization of intact complexes

Structural characterization:

Functional validation:

• ATP hydrolysis assays using established protocols

• Proton pumping assays in reconstituted systems

• pH-dependent activity profiling based on recent findings about ATP synthase conformational changes at different pH values

These methods allow isolation and comprehensive characterization of intact ATP synthase complexes, enabling structural studies and detailed functional analysis of both standard F1F0 ATPase and specialized F1-like ATPases from M. gallisepticum.

How can researchers distinguish between activities of standard F1F0 ATPase versus specialized F1-like ATPases in M. gallisepticum?

Distinguishing between the activities of standard F1F0 ATPase and specialized F1-like ATPases in M. gallisepticum requires strategic experimental approaches:

Genetic approaches:

• Generate strains with deletions or mutations in specific ATPase clusters

• Create constructs where different ATPase complexes are tagged for selective purification

• Utilize the genomic manipulation tools demonstrated effective in M. gallisepticum

Biochemical differentiation:

• Exploit potential differences in inhibitor sensitivity between standard and specialized ATPases

• Investigate pH-dependence profiles, as different ATPase types may show optimal activity under different conditions

• Fractionate membranes to separate different complexes based on density or composition

Proteomic analysis:

• Use mass spectrometry to identify proteins co-purifying with different ATPase complexes

• Employ antibodies specific to unique subunits in specialized complexes

• Apply cross-linking approaches to capture interaction partners specific to each complex type

Functional distinctions:

• Test for specialized activities beyond ATP hydrolysis (e.g., connection to MIB-MIP system )

• Examine response to physiological stressors that might differentially affect standard versus specialized complexes

• Compare activities in different growth phases or media conditions

What considerations are important when designing primers for atpA amplification and cloning from M. gallisepticum?

Designing effective primers for amplification and cloning of atpA from M. gallisepticum requires attention to several critical factors:

Genetic code considerations:

• Mycoplasmas use the UGA codon to encode tryptophan rather than as a stop codon

• When cloning into standard expression systems, codon optimization may be necessary

• If designing primers for site-directed mutagenesis, account for this alternative genetic code

Sequence specificity:

• Design primers specific to the target atpA copy (standard F1F0 versus specialized F1-like ATPase)

• Account for the presence of multiple atpA copies in mycoplasma genomes

• Check for off-target binding to other regions of the M. gallisepticum genome

Technical parameters:

• Optimal melting temperature (Tm) between 55-65°C with minimal difference between forward and reverse primers

• GC content between 40-60%

• Avoid secondary structures, primer-dimers, and 3' complementarity

• Include appropriate restriction sites with additional bases for enzyme binding

Cloning strategy elements:

• Add sequences for fusion tags if required (His, FLAG, Strep)

• Consider including protease cleavage sites if tags need to be removed

• For Gibson Assembly or similar methods, include appropriate overlaps

Validation approach:

• Before large-scale cloning, verify PCR products by sequencing

• Test primers with different PCR conditions to optimize specificity and yield

• Consider designing alternative primer sets for critical constructs

These considerations ensure successful amplification, cloning, and expression of the target atpA gene, providing the foundation for subsequent functional and structural studies of this important ATP synthase component.