Recombinant Staphylococcus aureus UPF0060 membrane protein SAR2425 (SAR2425)

What is the SAR2425 protein and what are its key structural features?

SAR2425 is a UPF0060 family membrane protein found in Staphylococcus aureus (strain MRSA252). It consists of 108 amino acids with a sequence of mLYPIFIFILAGLCEIGGGYLIWLWLREGQSSLVGLIGGVILmLYGVIATFQSFPSFGRVYAAYGGVFIIMSLIFAMVVDKQMPDKYDVIGAIICIVGVLVmLLPSRA . The protein contains multiple hydrophobic regions consistent with its role as a membrane protein, which suggests it spans the bacterial membrane multiple times. This structural arrangement is common in transmembrane proteins that facilitate molecular transport or signal transduction across the cell membrane.

How does SAR2425 relate to other membrane proteins in S. aureus?

SAR2425 belongs to the UPF0060 protein family, while sharing potential functional similarities with other S. aureus membrane proteins such as SA2056, which is part of the RND (Resistance-Nodulation-cell Division) family of transmembrane efflux transporters . Research on related S. aureus membrane proteins indicates potential roles in peptidoglycan synthesis and cell wall formation. For example, SA2056 has been shown to interact with FemB (involved in peptidoglycan interpeptide formation) and essential penicillin binding proteins (PBPs) such as PBP1 and PBP2 . Given these patterns, SAR2425 may participate in similar cellular processes, though direct experimental validation is required to confirm these functional relationships.

What expression systems are optimal for producing recombinant SAR2425?

For recombinant expression of SAR2425, E. coli expression systems have been successfully employed . When designing expression experiments, researchers should consider the following methodological approach:

• Select an appropriate E. coli strain optimized for membrane protein expression (e.g., C41(DE3), C43(DE3), or Lemo21(DE3))

• Design constructs with affinity tags (His-tag is commonly used) to facilitate purification

• Optimize expression conditions, including temperature (typically lowered to 16-25°C), induction time, and inducer concentration

• Include appropriate detergents during cell lysis and purification steps to maintain protein stability

Expression yields may vary based on these parameters, necessitating optimization for each research context. Alternative expression systems such as cell-free systems or yeast expression platforms may be considered if E. coli systems prove challenging for obtaining functional protein.

How should researchers design experiments to investigate potential interactions between SAR2425 and cell wall synthesis machinery?

Based on findings with similar S. aureus membrane proteins, SAR2425 may interact with components of the cell wall synthesis machinery. To investigate these potential interactions, researchers should implement a multi-method approach:

• Begin with bacterial two-hybrid (B2H) system assays, which have successfully demonstrated interactions between related membrane proteins and cell wall synthesis components

• Confirm primary findings using complementary techniques such as co-immunoprecipitation with specific antibodies against SAR2425

• Employ microscopy techniques (FRET or super-resolution microscopy) to visualize potential co-localization in vivo

• Design control experiments using known non-interacting proteins to validate specificity

The experimental design should follow established protocols for causal inference, including appropriate controls, randomization where applicable, and defined measurement parameters . For example, when using B2H systems, include both positive controls (known interacting pairs) and negative controls (non-interacting proteins) to establish baseline interaction signals.

What are the critical controls needed when studying the function of SAR2425 through deletion mutants?

When investigating SAR2425 function through genetic deletion approaches, a comprehensive set of controls is essential for valid interpretation:

• Generate a clean deletion mutant (Δsar2425) using precise genetic tools that do not disrupt adjacent genes

• Create a complementation strain by reintroducing the sar2425 gene (often on a plasmid) to confirm phenotypic changes are directly attributable to the gene deletion

• Measure growth parameters across multiple conditions to detect subtle phenotypes

• Examine cell morphology, antibiotic susceptibility profiles, and cell wall characteristics

• Consider creating double mutants with genes in related pathways to identify potential functional redundancy

Research on related proteins suggests that single deletion mutants may not always display obvious phenotypes due to functional redundancy in bacterial systems. For instance, deletion of sa2056 did not produce notable phenotypes in growth, antibiotic resistance, or cell morphology, even when combined with femB inactivation . Therefore, researchers should design experiments that can detect subtle phenotypic changes or that challenge cells under various stress conditions.

How can researchers determine the membrane topology of SAR2425?

Determining membrane protein topology requires specialized experimental approaches:

For optimal results, researchers should employ multiple complementary approaches. Begin with computational predictions of the SAR2425 sequence to identify potential transmembrane regions, then validate these predictions experimentally using at least two of the methods listed above. The amino acid sequence provided in the available data suggests multiple hydrophobic regions that likely form transmembrane helices .

What techniques should be employed to study potential protein-protein interactions involving SAR2425?

To systematically investigate SAR2425 interactions with other proteins, researchers should implement a multi-tiered approach:

• Initial screening using bacterial two-hybrid systems, which have successfully identified interactions between related membrane proteins and components of peptidoglycan synthesis machinery

• Validation of promising interactions through pull-down assays using recombinant His-tagged SAR2425 as bait

• In vivo confirmation using techniques such as:

  • FRET/BRET to detect proximal protein pairs

  • Cross-linking followed by mass spectrometry to identify interaction partners

  • Co-localization studies using fluorescence microscopy

When designing these experiments, researchers should follow the principles of valid experimental design, including appropriate randomization, controls, and measurement techniques . Given the membrane localization of SAR2425, special attention should be paid to maintaining native protein conformation during interaction studies, possibly requiring the use of mild detergents or membrane mimetics.

How can researchers investigate the potential role of SAR2425 in antibiotic resistance mechanisms?

Given that SAR2425 is found in MRSA252 (a methicillin-resistant strain) and potentially belongs to a family of membrane transporters, investigating its role in antibiotic resistance requires:

• Comparative antibiotic susceptibility testing between wild-type and Δsar2425 mutants across a panel of antibiotics at various concentrations

• Transport assays to determine if SAR2425 facilitates efflux of antibiotics

• Expression analysis to assess whether sar2425 expression changes in response to antibiotic exposure

• Structural studies to identify potential antibiotic binding sites

Research should adopt a systematic experimental design approach, ensuring proper controls and measurement methods . The experimental design should include:

• Multiple biological and technical replicates

• Appropriate statistical analysis of MIC (Minimum Inhibitory Concentration) values

• Time-course studies to detect transient resistance phenotypes

• Combinatorial antibiotic approaches to identify specific resistance mechanisms

Studies on related membrane proteins suggest potential roles in antibiotic resistance mechanisms, although deletion mutants may not always display obvious resistance phenotypes due to functional redundancy .

What methodological approaches can be used to study the evolutionary conservation of SAR2425 across Staphylococcal species?

To investigate evolutionary conservation patterns of SAR2425:

• Perform comprehensive sequence alignments of UPF0060 family proteins across Staphylococcal species and more distant bacterial taxa

• Construct phylogenetic trees to visualize evolutionary relationships

• Calculate selection pressures (dN/dS ratios) across different protein domains

• Conduct structural modeling to identify conserved functional motifs

Analysis should focus on identifying:

• Core conserved residues that may be essential for function

• Variable regions that might reflect species-specific adaptations

• Patterns of selection pressure across different protein domains

• Correlation between conservation patterns and functional domains

This evolutionary perspective can provide valuable insights into potential functions and important structural features of SAR2425, guiding experimental design for functional studies.

How should researchers approach data inconsistencies when characterizing SAR2425 function?

When encountering contradictory data regarding SAR2425 function, implement the following analytical framework:

• Systematically review experimental conditions, including:

  • Expression systems and purification methods

  • Buffer conditions and detergents used

  • Experimental temperature and pH

  • Presence of potential cofactors

• Consider alternative hypotheses:

  • Multifunctional nature of the protein

  • Context-dependent activity

  • Strain-specific differences in protein function

  • Post-translational modifications affecting activity

• Design reconciliation experiments:

  • Direct comparative studies under identical conditions

  • Sequential parameter variation to identify critical variables

  • Collaboration with laboratories reporting different results

This approach follows principles of experimental design for causal inference and variable control . Documentation of all experimental parameters is crucial for resolving apparent contradictions in experimental outcomes.

What statistical approaches are most appropriate for analyzing SAR2425 interaction data from high-throughput screens?

For high-throughput interaction screening data analysis:

When implementing these methods, researchers should:

• Establish clear threshold criteria before data collection

• Include appropriate positive and negative controls

• Validate high-confidence interactions with secondary methods

• Consider biological context when interpreting statistical significance

This approach aligns with best practices in experimental research methodology, emphasizing the importance of statistical rigor in data interpretation .

How can emerging structural biology techniques be applied to better understand SAR2425 function?

Recent advances in structural biology offer new opportunities for SAR2425 characterization:

These approaches align with current trends in experimental research design for complex biological systems , enabling researchers to connect structural features with functional hypotheses.

What are the most promising research directions for understanding the physiological role of SAR2425 in S. aureus?

Based on current knowledge and research trends, several high-priority research directions emerge:

• Investigation of potential roles in antibiotic resistance mechanisms:

  • Systematic phenotypic analysis of deletion mutants against diverse antibiotics

  • Expression profiling under antibiotic stress conditions

  • Transport assays to determine substrate specificity

• Exploration of potential roles in cell wall synthesis and integrity:

  • Detailed interaction studies with peptidoglycan synthesis machinery

  • Cell wall composition analysis in mutant strains

  • Microscopy studies of cell morphology and division

• Systems biology approaches to contextualize function:

  • Transcriptomic analysis of Δsar2425 mutants

  • Metabolomic profiling to identify affected pathways

  • Network analysis to position SAR2425 in cellular processes

• Host-pathogen interaction studies:

  • Impact of SAR2425 on virulence in infection models

  • Potential recognition by host immune system

  • Role in bacterial survival within host environments

These research directions should be pursued using rigorous experimental designs that control variables and enable strong causal inferences , with appropriate statistical approaches for data analysis and interpretation.