Recombinant Macrococcus caseolyticus ATP synthase subunit beta (atpD)

What is Macrococcus caseolyticus and what is its significance in microbiology?

Macrococcus caseolyticus is a Gram-positive, coagulase-negative bacterial species belonging to the family Staphylococcaceae. The genus Macrococcus was first described in 1998 when it was differentiated from Staphylococcus . M. caseolyticus has been isolated from various sources including dairy products, animal skin, and meat. While generally considered less pathogenic than Staphylococcus species, M. caseolyticus has been documented in animal infections including bovine mastitis .

The species has gained significant research interest as a potential reservoir of antibiotic resistance genes, notably the methicillin resistance determinants mecB and mecD, which could potentially transfer to more pathogenic staphylococci . Genome analysis has revealed that these resistance determinants are present in diverse bacterial populations and are widely spread geographically .

What is the function of ATP synthase subunit beta (atpD) in bacterial metabolism?

ATP synthase subunit beta (atpD) is a critical component of the F1 portion of the F1F0-ATP synthase complex, which is responsible for ATP production during oxidative phosphorylation. The beta subunit contains the catalytic sites for ATP synthesis and hydrolysis, making it essential for energy metabolism in bacteria.

This protein plays a fundamental role in energy conservation by coupling the proton motive force to ATP synthesis. The atpD gene is considered a housekeeping gene due to its essential function and has been used as a phylogenetic marker for bacterial classification and identification within the Staphylococcaceae family, which includes both Macrococcus and Staphylococcus genera.

Why do researchers express recombinant ATP synthase components rather than purify them from native sources?

Researchers express recombinant ATP synthase components for several methodological advantages:

• Controlled expression: Recombinant systems allow precise control over protein expression levels

• Scalability: Higher yields can be achieved compared to native purification

• Genetic manipulation: Site-directed mutagenesis can be performed to study structure-function relationships

• Addition of tags: Affinity tags can be incorporated for easier purification and detection

• Homogeneity: Pure protein preparations can be obtained without contamination from other ATP synthase subunits

For M. caseolyticus specifically, recombinant expression is practical since this bacterium is not as routinely cultured as model organisms like E. coli, and specialized growth conditions might be required for optimal native protein production.

How is the atpD gene used in phylogenetic analysis of Macrococcus species?

The atpD gene serves as an excellent phylogenetic marker due to several characteristics:

• It is a housekeeping gene with essential function, resulting in evolutionary conservation

• It contains both conserved regions (useful for primer design) and variable regions (useful for species discrimination)

• It is typically present as a single copy in bacterial genomes, avoiding paralog complications

• It evolves at a moderate rate, allowing resolution of both recent and ancient divergences

Studies of methicillin-resistant Macrococcus isolates have used genetic markers to understand the epidemiology and evolutionary relationships between strains . Similar approaches with atpD can help track the spread of specific lineages and potentially correlate genetic traits with antibiotic resistance patterns or virulence factors.

What expression systems are typically used for recombinant M. caseolyticus proteins?

Based on successful expression of other M. caseolyticus proteins, the following systems have proven effective:

For M. caseolyticus OleT MC expression, researchers have successfully used E. coli BL21(DE3) with the pNN33 plasmid, achieving sufficient expression for both in vivo activity and in vitro enzyme characterization .

How might antibiotic resistance mechanisms in M. caseolyticus interact with ATP synthase function?

M. caseolyticus harbors important antibiotic resistance determinants, particularly mecB and mecD genes that confer methicillin resistance . These resistance mechanisms could potentially affect ATP synthase function through:

• Increased energy demand to power antibiotic efflux pumps, creating selective pressure for more efficient ATP synthase variants

• Alterations in membrane potential due to resistance mechanisms, indirectly affecting ATP synthase efficiency

• Co-regulation of energy metabolism genes with resistance determinants

• Adaptations in ATP synthase to function optimally under antibiotic stress conditions

Genome analysis has revealed diverse methicillin-resistant M. caseolyticus populations with both mecB and mecD genes . Understanding how these resistance determinants interact with energy metabolism could provide insights into bacterial adaptation and potential targets for overcoming resistance.

What challenges exist in expressing functional recombinant ATP synthase subunits?

Expressing functional recombinant ATP synthase subunits presents several challenges:

• Maintaining proper folding and conformation, crucial for catalytic function

• Ensuring proper assembly with other subunits if studying the entire complex

• Potential toxicity to host cells when overexpressed

• Challenges in solubility, as membrane-associated proteins can form inclusion bodies

• Post-translational modifications that may differ between the native organism and expression host

Similar challenges have been observed with other M. caseolyticus proteins. For example, researchers working with OleT MC encountered electron transfer limitations that were overcome by introducing a two-component redox system (CamA/CamB) . This approach increased terminal alkene production in recombinant E. coli from 21.92 mg/L to higher levels, demonstrating the importance of addressing cofactor requirements in heterologous expression systems .

How can researchers differentiate between ATP synthase activity and other ATPases when characterizing recombinant atpD?

Differentiating ATP synthase activity from other ATPases requires specific experimental approaches:

These approaches can provide conclusive evidence that the observed activity is specifically from the ATP synthase complex rather than contaminating ATPases.

What insights can comparative genomics provide about atpD evolution in Macrococcus species compared to Staphylococcus?

Comparative genomics studies of Macrococcus and Staphylococcus have revealed important insights about gene evolution and horizontal gene transfer between these genera:

• Chromosomal resistance islands containing mecD have been identified in M. caseolyticus

• Novel macrolide resistance genes mef(D) and msr(F) have been found on mobile genetic elements in both Macrococcus and Staphylococcus species

• Similar genetic elements have been detected in M. canis, M. caseolyticus, and S. aureus, suggesting potential for inter-genus horizontal gene transfer

While these studies focus on antibiotic resistance genes, similar comparative approaches can be applied to atpD to understand its evolution. Of particular interest would be whether atpD shows evidence of selection pressure in antibiotic-resistant strains, potentially indicating adaptation of energy metabolism to support resistance mechanisms.

What are the optimal conditions for expressing recombinant M. caseolyticus atpD?

Based on successful expression of other M. caseolyticus proteins, the following conditions are recommended:

This approach has been successful for expressing the OleT MC enzyme from M. caseolyticus, which showed good activity in both in vitro and in vivo settings .

What purification strategies maintain native conformation of recombinant atpD?

To maintain the native conformation of atpD during purification:

• Cell lysis: Use gentle methods like enzymatic lysis or controlled sonication

• Buffer composition:

  • pH 7.0-8.0 (physiological range)

  • 20-50 mM Tris-HCl or phosphate buffer

  • 100-300 mM NaCl for ionic strength

  • 10% glycerol as stabilizer

  • 1-5 mM MgCl2 (cofactor for ATP binding)

  • 1-2 mM DTT or β-mercaptoethanol as reducing agent

• Chromatography sequence:

  • IMAC (immobilized metal affinity chromatography) for His-tagged protein

  • Ion exchange chromatography for further purification

  • Size exclusion chromatography as final polishing step

• Activity preservation:

  • Add 0.1-0.5 mM ATP or non-hydrolyzable ATP analogs to stabilize conformation

  • Perform all steps at 4°C

  • Include protease inhibitors to prevent degradation

What functional assays can assess recombinant atpD activity?

Several assays can be employed to assess the functionality of recombinant atpD:

For comprehensive characterization, a combination of these assays should be employed to assess both the structural integrity and catalytic function of the recombinant protein.

How can researchers troubleshoot low expression yields of recombinant atpD?

When facing low expression yields, researchers can implement the following troubleshooting strategies:

• Genetic optimization:

  • Codon optimization for E. coli expression

  • Use of different promoters (T7, tac, or arabinose-inducible)

  • Inclusion of ribosome binding site optimization

• Host strain selection:

  • Test specialized strains (C41/C43 for membrane proteins)

  • Use Rosetta strains providing rare tRNAs

  • Consider BL21(DE3)pLysS to reduce basal expression

• Expression conditions:

  • Lower induction temperature (16°C)

  • Reduce IPTG concentration (0.1 mM)

  • Extended expression time (overnight)

• Protein engineering:

  • N- or C-terminal fusion partners (MBP, SUMO, TrxA)

  • Truncation constructs removing problematic domains

  • Addition of stabilizing mutations

Researchers working with the OleT MC enzyme from M. caseolyticus found that addressing electron transfer limitations by co-expressing a two-component redox system (CamA/CamB) significantly improved functional protein activity , highlighting the importance of considering cofactor requirements.

What approaches can identify interaction partners of atpD in multiprotein complexes?

To identify and characterize interaction partners of atpD in the ATP synthase complex:

These techniques can provide complementary information about the assembly, stoichiometry, and dynamics of the ATP synthase complex, particularly how the beta subunit interacts with other components.

How can recombinant atpD be used as a target for antimicrobial development?

ATP synthase represents a promising antimicrobial target due to its essential role in bacterial metabolism:

• Target validation approaches:

  • Conditional knockout studies to confirm essentiality

  • Determination of structure-activity relationships

  • Screening for subunit-specific inhibitors

• Advantages as an antimicrobial target:

  • Essential for bacterial survival

  • Structurally distinct from mammalian counterparts

  • Located in bacterial membrane, accessible to drugs

  • Well-characterized biochemistry

• Drug discovery strategies:

  • High-throughput screening against recombinant atpD

  • Structure-based drug design targeting catalytic sites

  • Fragment-based lead discovery

  • Repurposing of known ATP synthase inhibitors

Understanding the unique structural features of M. caseolyticus atpD could enable the development of species-specific inhibitors with potential applications in treating resistant infections.

What biotechnological applications exist for engineered ATP synthase components?

Engineered ATP synthase components have several biotechnological applications:

These applications could leverage the highly evolved catalytic properties of ATP synthase components while engineering them for specific technological purposes.

How might comparative studies between Macrococcus and Staphylococcus ATP synthases inform evolution of antibiotic resistance?

Comparative studies of ATP synthases could provide insights into antibiotic resistance evolution:

• Energy cost analysis:

  • Determine if resistant strains show adaptations in ATP synthase efficiency

  • Quantify energetic burden of resistance mechanisms

• Co-evolutionary patterns:

  • Identify if ATP synthase mutations correlate with acquisition of resistance genes

  • Determine if compensatory mutations in energy metabolism accompany resistance

• Transfer potential:

  • Assess if ATP synthase genes show evidence of horizontal gene transfer alongside resistance determinants

  • Evaluate if mobile genetic elements carrying resistance genes also impact energy metabolism

Given that chromosomal resistance islands in M. caseolyticus can carry methicillin resistance genes like mecD , understanding how these genetic elements affect core metabolic functions could provide valuable insights into the evolution and spread of antibiotic resistance.

What recent advances have improved recombinant expression of challenging bacterial proteins?

Recent technological advances improving recombinant protein expression include:

These technologies could be applied to improve the expression of functional M. caseolyticus atpD, particularly if traditional approaches yield insufficient protein quantities or activity.

How do genomic approaches inform our understanding of ATP synthase evolution in Macrococcus species?

Genomic approaches provide several insights into ATP synthase evolution:

• Whole genome sequencing has revealed the genetic context of Macrococcus species, including mobile genetic elements that can carry resistance genes between species

• Comparative genomics shows that chromosomal resistance islands in M. caseolyticus and M. canis can integrate into specific genomic locations and carry various resistance determinants

• Analysis of methicillin-resistant M. caseolyticus has demonstrated both local and distant spread of related isolates, suggesting the potential for widespread distribution of genetic elements

• Phylogenetic analysis indicates that highly related isolates may carry different resistance genes (mecB or mecD), suggesting dynamic gene acquisition and loss

Similar approaches applied specifically to ATP synthase genes could reveal patterns of conservation, selection, and potential adaptation to different ecological niches or resistance states.

What is known about the role of ATP synthase in antibiotic tolerance and persistence?

ATP synthase plays important roles in antibiotic tolerance and persistence:

• Energy modulation: Reduced ATP synthase activity can induce a low-energy state associated with antibiotic tolerance

• Persister cell formation: Metabolic dormancy mediated by altered ATP synthase function contributes to persister cell development

• Membrane potential: ATP synthase activity affects proton motive force, which influences uptake of certain antibiotics

• Stress response: Energy conservation through ATP synthase regulation helps bacteria survive antibiotic stress

These aspects are particularly relevant for M. caseolyticus given its role as a reservoir of resistance genes that can potentially transfer to more pathogenic staphylococci . Understanding how ATP synthase function intersects with resistance mechanisms could provide new targets for combating antibiotic resistance.