Recombinant Mycoplasma synoviae ATP synthase subunit b (atpF)

What is ATP synthase subunit b (atpF) in Mycoplasma synoviae?

ATP synthase subunit b (atpF) in Mycoplasma synoviae is a membrane protein component of the F0F1-ATP synthase complex, which plays a critical role in cellular energy production. This protein is encoded by the atpF gene and spans 193 amino acids in its full-length form . Unlike atpF in many other bacteria, the Mycoplasma synoviae atpF has unique characteristics as it contains an N-terminal lipid modification that contributes to its membrane anchoring mechanism . The protein functions as part of the stator that connects the F1 catalytic domain to the F0 membrane domain of ATP synthase, enabling the enzyme to harness the proton gradient for ATP synthesis.

How does atpF in Mycoplasma species differ from other bacterial homologs?

The atpF protein in Mycoplasma species exhibits a distinctive characteristic not found in most bacteria – it functions as a lipoprotein. Based on experimental verification in the related species Mycoplasma pneumoniae, the atpF protein is anchored to the cytoplasmic membrane by both a transmembrane domain and an N-terminal lipid modification . This dual anchoring mechanism appears to be unique to mycoplasmas, as among all sequenced bacterial atpF genes, only those from Mycoplasma species (including M. gallisepticum and M. genitalium) code for a conserved lipoprotein consensus sequence . This structural adaptation may relate to the unique membrane biology of mycoplasmas, which lack a cell wall and rely heavily on membrane proteins for structural integrity and host interactions.

What are the key structural features of Mycoplasma synoviae atpF?

Mycoplasma synoviae atpF has several key structural features that define its function:

The protein contains hydrophobic and hydrophilic regions that allow it to function as both a membrane anchor and a structural component linking the membrane-embedded F0 sector with the catalytic F1 sector of the ATP synthase complex . The first 20-30 amino acids likely contain the signal sequence and lipobox motif that directs the protein for lipid modification, similar to what has been experimentally verified in M. pneumoniae .

How does the lipoprotein nature of atpF affect its function in mycoplasmas?

The lipoprotein nature of atpF in mycoplasmas provides enhanced membrane anchoring through dual mechanisms: the N-terminal lipid modification and the transmembrane domain . This arrangement likely confers greater stability to the ATP synthase complex in the absence of a cell wall, which is characteristic of mycoplasmas. The lipid modification may also influence the protein's orientation in the membrane and its interactions with other components of the ATP synthase complex.

Experimentally, the lipoprotein nature of atpF in M. pneumoniae (a related species) has been confirmed through metabolic labeling with [14C]palmitic acid and by demonstrating the sensitivity of its processing to globomycin, a specific inhibitor of signal peptidase II that processes prolipoproteins . This dual anchoring mechanism may be an adaptation that evolved in mycoplasmas to compensate for their reduced genome size and simplified cellular structure while maintaining essential energy production functions.

What expression systems are recommended for producing recombinant Mycoplasma synoviae atpF?

E. coli is the preferred expression system for recombinant Mycoplasma synoviae atpF protein production . When selecting an expression system, researchers should consider the following methodological approach:

• Vector selection: Choose vectors with strong, inducible promoters (e.g., T7) and appropriate fusion tags (e.g., His-tag) for purification.

• Host strain selection: BL21(DE3) or derivatives are recommended for their reduced protease activity.

• Codon optimization: Consider codon optimization for E. coli expression as Mycoplasma has different codon usage patterns.

• Growth conditions: Optimize temperature (often lowered to 18-25°C after induction), induction timing, and inducer concentration to maximize soluble protein yield.

The His-tag fusion approach has been successfully implemented for Mycoplasma synoviae atpF, with the tag positioned at the N-terminus of the protein . This arrangement allows for efficient purification while minimizing interference with the protein's functional domains.

What are the recommended storage conditions for maintaining recombinant atpF protein stability?

To maintain the stability and activity of recombinant Mycoplasma synoviae atpF protein, the following storage conditions are recommended:

• Short-term storage: Store working aliquots at 4°C for up to one week .

• Long-term storage: Store at -20°C or -80°C in buffer containing 6% trehalose at pH 8.0 .

• Avoid repeated freeze-thaw cycles as they can lead to protein denaturation and aggregation .

• For optimal stability, reconstitute lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Addition of 5-50% glycerol (final concentration) is recommended when preparing aliquots for long-term storage .

Prior to use, centrifuge the vial briefly to ensure all contents are at the bottom. Proper aliquoting is necessary for multiple use scenarios to prevent protein degradation from repeated freeze-thaw cycles .

How can recombinant atpF be used in studies of Mycoplasma synoviae pathogenesis?

Recombinant Mycoplasma synoviae atpF can serve as a valuable tool in pathogenesis studies through several methodological approaches:

• Host-pathogen interaction studies: Purified recombinant atpF can be used to study direct interactions with host cell components. This is particularly relevant given that Mycoplasma synoviae is a significant cause of respiratory disease and synovitis among chickens with adverse economic impacts on broiler breeding .

• Gene expression analysis: Researchers can monitor atpF expression changes during infection using techniques like qRT-PCR or RNA-Seq, as has been done with other Mycoplasma synoviae membrane proteins . Studies have shown that lipid-associated membrane proteins (LAMPs) of M. synoviae, a category that includes atpF, undergo expression changes when the pathogen is exposed to host cells .

• Immunological studies: Recombinant atpF can be used to raise antibodies or evaluate host immune responses, providing insights into the immunogenicity of this protein during infection.

• Virulence factor characterization: Since membrane proteins in Mycoplasma species often serve as virulence factors, recombinant atpF can be used in assays to determine its contribution to adhesion, invasion, or other virulence-associated processes .

What role might atpF play in the membrane biology of Mycoplasma synoviae?

ATP synthase subunit b (atpF) likely plays multiple roles in Mycoplasma synoviae membrane biology beyond its canonical function in energy production:

• Membrane organization: As a lipoprotein with a transmembrane domain, atpF may contribute to the structural organization of the mycoplasma membrane, which is particularly important given their lack of a cell wall .

• Protein-protein interactions: AtpF may interact with other membrane proteins to form functional complexes involved in various cellular processes. Studies of M. synoviae LAMPs have identified protein-protein interaction networks that are altered during host cell infection .

• Adaptation to host environment: Transcriptomic analysis has shown that expression of membrane proteins, potentially including atpF, changes when M. synoviae interacts with host cells, suggesting roles in adaptation to the host environment .

• Energy homeostasis: Beyond its structural role in ATP synthase, atpF may participate in regulating the activity of the enzyme complex in response to changing environmental conditions, which is critical for a pathogen that must adapt to different host niches.

What are common challenges in expressing and purifying recombinant Mycoplasma proteins?

Researchers working with recombinant Mycoplasma synoviae atpF often encounter several challenges:

The recombinant expression of membrane proteins like atpF often requires optimization at multiple levels, from gene design to growth conditions and purification strategies. For example, the Mycoplasma synoviae atpF protein described in the search results was successfully expressed as an N-terminal His-tagged fusion protein in E. coli, with the product maintained as a lyophilized powder for stability .

How can the lipoprotein nature of atpF be verified experimentally?

To verify the lipoprotein nature of Mycoplasma synoviae atpF, researchers can employ the following methodological approaches:

• Metabolic labeling: Incorporate radiolabeled fatty acids (e.g., [14C]palmitic acid) into growing cultures and detect their association with the purified atpF protein, as demonstrated for M. pneumoniae atpF .

• Inhibitor studies: Use globomycin, a specific inhibitor of signal peptidase II (which processes prolipoproteins), to interfere with the processing of the prolipoprotein form of atpF and observe the accumulation of the unprocessed form .

• Mass spectrometry: Perform LC-MS/MS analysis of purified atpF to detect lipid modifications at the N-terminus, similar to approaches used for identifying other M. synoviae LAMPs .

• Bioinformatic prediction: Analyze the protein sequence using specialized tools like LipoP-1.0 to identify potential lipoprotein signal sequences and lipoboxes, complementing experimental approaches with in silico validation .

• Triton X-114 phase partitioning: Exploit the amphiphilic nature of lipoproteins by using Triton X-114 extraction, which has been successfully employed to extract LAMPs from M. synoviae for subsequent identification via LC-MS/MS .

How might atpF be exploited as a target for novel antimicrobial strategies?

ATP synthase subunit b (atpF) presents several opportunities as a target for novel antimicrobial strategies against Mycoplasma synoviae:

• Inhibitor development: The unique lipoprotein characteristics of mycoplasma atpF, absent in host ATP synthases, could be exploited to design specific inhibitors that disrupt its function or assembly without affecting host enzymes .

• Vaccine development: As a membrane-associated protein with potential surface exposure, recombinant atpF could serve as an antigen in subunit vaccine formulations. LAMPs of M. synoviae have been identified as promising vaccine candidates due to their accessibility to the host immune system .

• Diagnostic applications: The unique sequence and structural features of M. synoviae atpF could be utilized in developing specific diagnostic tests for detecting this pathogen in clinical samples, addressing the current limitations in diagnostic tools mentioned in the research .

• Drug delivery systems: Knowledge of atpF's membrane topology could inform the design of drug delivery systems that specifically target Mycoplasma membranes while sparing host cells.

What genomic diversity exists in atpF across different Mycoplasma synoviae strains?

Understanding genomic diversity in atpF across different Mycoplasma synoviae strains is crucial for developing broadly effective interventions. Current research indicates:

• The M. synoviae genome ranges from 0.766-0.848 Mb in size, with a median total length of 0.804397 Mb and a GC content of 27.90-28.40% (median: 28.25%) .

• M. synoviae harbors 635-686 protein-coding genes (median protein count: 655), suggesting potential strain-to-strain variation in proteins like atpF .

• Comparative genomic analyses would be valuable to assess conservation of atpF sequence, structure, and lipid modification sites across different isolates, particularly from various geographical regions and host species.

• Such diversity studies could identify conserved regions suitable as targets for broad-spectrum interventions versus variable regions that might explain strain-specific differences in virulence or host adaptation.

• RNA-Seq approaches, as used in studies of other M. synoviae LAMPs, could reveal strain-specific differences in atpF expression during infection, potentially correlating with variations in pathogenicity .