Recombinant Staphylococcus aureus Putative ribosomal protein L7Ae-like (SAR0549)

What is the Staphylococcus aureus putative ribosomal protein L7Ae-like (SAR0549)?

SAR0549 is a putative ribosomal protein from Staphylococcus aureus that belongs to the L7Ae protein family. This protein family is highly conserved across different domains of life, including archaea, bacteria, and eukaryotes. In archaea, L7Ae proteins function as RNA-binding proteins that recognize and bind to specific RNA structures called kink-turns (K-turns). Based on sequence homology and structural predictions, SAR0549 is classified as an L7Ae-like protein in S. aureus, suggesting it may have RNA-binding capabilities similar to its archaeal counterparts .

How does SAR0549 relate to other L7Ae family proteins across species?

L7Ae family proteins are found across different domains of life but show varying functional capacities. Archaeal L7Ae proteins have been extensively studied and demonstrate strong RNA-binding activities, particularly to kink-turn motifs. Research indicates that only L7Ae members from archaea retain the capacity to inhibit specific RNA processing events, such as the accumulation of M2 and NS2 viral proteins . Comparative sequence analysis reveals that SAR0549 from S. aureus shares homology with other bacterial L30 proteins but likely differs functionally from archaeal L7Ae proteins due to specific amino acid differences in key functional domains.

What are the characteristic structural features of L7Ae-like proteins?

L7Ae-like proteins typically contain:

• A conserved RNA-binding domain

• Specific residues involved in kink-turn recognition

• A core structure with >50% conservation across domains of life

Key residues that define the archaeal lineage (I88/E89/V90) appear to be critical contributors to the functional specificity of these proteins . The S. aureus SAR0549 protein would be expected to maintain the core structural elements while potentially having bacterial-specific modifications in the binding domains.

What expression systems are recommended for producing recombinant SAR0549?

For recombinant expression of S. aureus SAR0549, the following expression systems have proven effective for similar bacterial proteins:

For optimal expression, consider using a C-terminal His-tag purification strategy similar to that employed for other recombinant proteins, which facilitates single-step affinity purification while maintaining protein function .

What is the recommended purification protocol for recombinant SAR0549?

A multi-step purification protocol is recommended for obtaining high-purity recombinant SAR0549:

• Initial Lysis: Lyse bacterial cells expressing His-tagged SAR0549 in phosphate buffer containing appropriate protease inhibitors

• Affinity Chromatography: Purify using Ni-NTA resin with imidazole gradient elution

• Size Exclusion Chromatography: Remove aggregates and further purify using gel filtration

• Quality Control: Assess purity by SDS-PAGE (>95% purity recommended)

• Activity Verification: Confirm RNA-binding activity using electrophoretic mobility shift assays (EMSA)

For optimal stability, consider lyophilization from a 0.2 μm filtered solution in PBS with trehalose as a stabilizing agent, similar to protocols used for other recombinant proteins .

How can I verify the RNA-binding activity of purified recombinant SAR0549?

To verify RNA-binding activity of purified SAR0549:

• Electrophoretic Mobility Shift Assay (EMSA):

  • Incubate increasing concentrations of purified SAR0549 with labeled RNA containing putative binding motifs

  • Resolve on native polyacrylamide gels

  • Shifted bands indicate protein-RNA complex formation

• Surface Plasmon Resonance (SPR):

  • Immobilize purified SAR0549 on a sensor chip

  • Flow solutions containing target RNA sequences

  • Measure binding kinetics and determine affinity constants (KD)

  • Compare with known L7Ae proteins as controls

• RNA Pull-down Assays:

  • Use biotinylated RNA targets and streptavidin beads

  • Incubate with SAR0549

  • Analyze pulled-down proteins by Western blotting

Positive controls should include known kink-turn-containing RNAs, while negative controls should include RNAs without these structures .

What mutagenesis strategies are recommended for studying SAR0549 function?

When designing mutagenesis experiments for SAR0549, focus on:

• Conserved Residues: Target amino acids equivalent to K37, R41, I88, E89, and V90 in archaeal L7Ae, which are critical for RNA recognition

• Binding Domain Mutations: Create systematic alanine substitutions in predicted RNA-binding regions

• Chimeric Constructs: Exchange domains between SAR0549 and archaeal L7Ae to identify functional regions

A recommended experimental approach involves:

• Site-directed mutagenesis using overlap extension PCR

• Expression of wild-type and mutant proteins under identical conditions

• Functional comparison using RNA-binding assays (EMSA, SPR)

• Structural analysis of mutants using circular dichroism to ensure proper folding

Mutations should be analyzed both individually and in combinations to detect cooperative effects between residues.

How might SAR0549 contribute to S. aureus pathogenicity?

SAR0549, as an RNA-binding protein, may contribute to S. aureus pathogenicity through several potential mechanisms:

• Regulation of Virulence Factors:

  • May bind to mRNAs encoding virulence factors, influencing their expression

  • Could potentially modulate toxin production in response to environmental cues

• Stress Response Adaptation:

  • RNA-binding proteins often play roles in bacterial stress responses

  • Could regulate translation during host infection conditions (pH changes, nutrient limitation)

• Interaction with Host RNA:

  • Based on archaeal L7Ae interactions with viral transcripts , SAR0549 might target host cell RNAs

  • Could potentially disrupt host defense mechanisms by interfering with RNA processing

Research methodologies to investigate these hypotheses could include:

• Transcriptome analysis of S. aureus strains with SAR0549 deletion or overexpression

• RNA-immunoprecipitation followed by sequencing (RIP-seq) to identify target RNAs

• Infection models comparing wild-type and SAR0549 mutant strains

How does the RNA-binding specificity of SAR0549 compare to archaeal L7Ae proteins?

The RNA-binding specificity of SAR0549 likely differs from archaeal L7Ae proteins due to evolutionary divergence. Comparative analysis should focus on:

• Structural Differences in Binding Domains:

  • Archaeal L7Ae proteins have specific residues (I88/E89/V90) that define their lineage and contribute to RNA binding

  • SAR0549 may have bacterial-specific adaptations in these regions

• Target RNA Structure Recognition:

  • Archaeal L7Ae recognizes conventional kink-turns

  • SAR0549 may recognize alternative RNA structures prevalent in bacteria

Experimental approaches to investigate these differences include:

• Competitive binding assays with different RNA motifs

• Structural studies (X-ray crystallography, cryo-EM) of SAR0549-RNA complexes

• Systematic evolution of ligands by exponential enrichment (SELEX) to identify preferred binding sequences

A comparison table of binding affinities for different RNA targets would help quantify these differences:

What role might SAR0549 play in antibiotic resistance mechanisms of S. aureus?

S. aureus is known for developing antibiotic resistance, including methicillin-resistant strains (MRSA) . SAR0549 could potentially contribute to resistance mechanisms through:

• Regulation of Resistance Gene Expression:

  • RNA-binding proteins can modulate translation efficiency of resistance genes

  • SAR0549 might regulate expression of efflux pumps or cell wall modification enzymes

• Stress Response during Antibiotic Exposure:

  • May participate in ribosome protection or modification

  • Could alter translation patterns during antibiotic stress

• Biofilm Formation Support:

  • RNA regulators often influence biofilm development

  • Biofilms increase antibiotic tolerance in S. aureus infections

Research strategies to explore these hypotheses include:

• Transcriptome and proteome analysis of wild-type vs. SAR0549 mutants under antibiotic pressure

• Minimum inhibitory concentration (MIC) determinations for various antibiotics

• Biofilm formation assays with SAR0549 variants

How can SAR0549 be utilized for developing novel antimicrobial strategies?

Based on the inhibitory effects observed with archaeal L7Ae on viral processes , SAR0549 presents several potential antimicrobial development avenues:

• Target for Antimicrobial Development:

  • Small molecule inhibitors of SAR0549 could disrupt essential RNA-processing functions

  • High-throughput screening of compound libraries against purified SAR0549

• Exploitation of RNA-binding Properties:

  • Engineered SAR0549 variants could deliver antimicrobial RNA molecules

  • Design of decoy RNA targets to sequester endogenous SAR0549

• Diagnostic Applications:

  • SAR0549-specific antibodies could be used for rapid S. aureus detection

  • RNA aptamers targeting SAR0549 might distinguish between S. aureus strains

Development pipeline considerations include:

• In silico modeling of small molecule interactions with SAR0549 binding pocket

• Functional screening using RNA-binding competition assays

• Bacterial growth inhibition assays with lead compounds

• Cytotoxicity testing in mammalian cell lines

How has SAR0549 evolved within the Staphylococcus genus?

Evolutionary analysis of SAR0549 within Staphylococcus species provides insights into its functional importance:

• Sequence Conservation Analysis:

  • Compare SAR0549 orthologues across Staphylococcus species

  • Identify conserved domains versus variable regions

  • Calculate selection pressure (dN/dS ratios) across the protein sequence

• Phylogenetic Relationships:

  • Construct phylogenetic trees based on SAR0549 sequences

  • Compare with species trees to identify potential horizontal gene transfer events

  • Analyze co-evolution with interacting RNA partners

S. aureus engages in co-evolutionary processes with its human hosts, leading to adaptations that allow asymptomatic carriage in approximately 20-30% of the human population . SAR0549 may have evolved specific functions that contribute to this host-pathogen relationship.

What experimental approaches can differentiate between the functions of SAR0549 and archaeal L7Ae proteins?

To differentiate between SAR0549 and archaeal L7Ae functions:

• Cross-complementation Studies:

  • Express SAR0549 in archaeal systems lacking L7Ae

  • Express archaeal L7Ae in S. aureus with SAR0549 deletion

  • Assess phenotypic rescue and molecular function

• Domain Swap Experiments:

  • Create chimeric proteins with domains from both SAR0549 and archaeal L7Ae

  • Test RNA-binding specificity and cellular functions of chimeras

  • Identify critical domains responsible for functional differences

• RNA Target Identification:

  • Perform CLIP-seq (crosslinking immunoprecipitation followed by sequencing) with both proteins

  • Compare binding sites and RNA structural motifs

  • Analyze differences in binding preferences

Archaeal L7Ae proteins show specific inhibition of viral transcript splicing not observed with bacterial homologs . This functional divergence provides a clear experimental readout for comparative studies.

How do post-translational modifications affect SAR0549 function in S. aureus?

Post-translational modifications (PTMs) can significantly impact protein function. For SAR0549:

• Identification of PTMs:

  • Mass spectrometry analysis of native SAR0549 from S. aureus

  • Comparison with recombinantly expressed protein

  • Targeted analysis for common bacterial PTMs (phosphorylation, acetylation)

• Functional Impact Assessment:

  • Site-directed mutagenesis of modified residues

  • Comparative RNA-binding assays with modified and unmodified protein

  • In vivo phenotypic analysis of modification-deficient mutants

• Regulation of Modifications:

  • Identify environmental conditions that alter modification patterns

  • Characterize enzymes responsible for SAR0549 modifications

  • Develop antibodies specific to modified forms for quantitative analysis

A systematic approach using phosphomimetic mutations (e.g., Ser to Asp) or modification-preventing mutations (e.g., Lys to Arg) can help determine the functional significance of each modification.

What are the major challenges in structural studies of SAR0549 and how can they be addressed?

Structural studies of RNA-binding proteins like SAR0549 present several challenges:

• Protein Solubility Issues:

  • Challenge: SAR0549 may form aggregates during purification

  • Solution: Optimize buffer conditions (pH, salt concentration, additives like glycerol)

  • Alternative: Use solubility-enhancing fusion tags (MBP, SUMO, thioredoxin)

• Crystallization Difficulties:

  • Challenge: RNA-binding proteins often have flexible regions that hinder crystallization

  • Solution: Surface entropy reduction mutations in flexible loops

  • Alternative: Crystallize with RNA binding partners to stabilize conformation

• NMR Spectroscopy Limitations:

  • Challenge: Size limitations for traditional NMR approaches

  • Solution: Selective isotopic labeling strategies

  • Alternative: Combine NMR with small-angle X-ray scattering (SAXS) for hybrid modeling

• Cryo-EM Considerations:

  • Challenge: SAR0549 (~15-20 kDa) is below the typical size limit for cryo-EM

  • Solution: Study as part of larger complexes with RNA or other proteins

  • Alternative: Use recent advances in micro-ED for small protein crystallography

How can I troubleshoot expression and purification issues with recombinant SAR0549?

Common issues and solutions for recombinant SAR0549:

When optimizing purification protocols, implement a stepwise approach:

• Test small-scale expressions with various conditions

• Analyze soluble vs. insoluble fractions

• Optimize lysis and purification buffers

• Consider on-column refolding for inclusion bodies

• Validate final product by activity assays and circular dichroism

What are the best methods for identifying RNA targets of SAR0549 in vivo?

To comprehensively identify RNA targets of SAR0549 in vivo:

• CLIP-seq (Cross-linking and Immunoprecipitation followed by Sequencing):

  • UV cross-linking of RNA-protein complexes in vivo

  • Immunoprecipitation of SAR0549 with specific antibodies

  • RNA extraction, library preparation, and high-throughput sequencing

  • Bioinformatic analysis to identify binding sites and motifs

• RIP-seq (RNA Immunoprecipitation followed by Sequencing):

  • Similar to CLIP-seq but without crosslinking

  • Gentler approach that may preserve weaker interactions

  • May have higher background than CLIP-seq

• Grad-seq (Gradient Profiling by Sequencing):

  • Separate cellular complexes by density gradient centrifugation

  • Analyze co-sedimentation of SAR0549 and RNAs

  • Identify RNAs in SAR0549-containing fractions by sequencing

• Proximity-dependent RNA Labeling:

  • Fuse SAR0549 to RNA-modifying enzymes

  • Identify RNAs that become modified due to proximity

  • Allows detection of transient interactions

For all these methods, appropriate controls must include:

• Input samples before immunoprecipitation

• Non-specific antibody controls

• SAR0549 knockout strains as negative controls

• Known binding partners as positive controls

What are the key unresolved questions about SAR0549 function in S. aureus?

Despite advances in understanding L7Ae family proteins, several critical questions about SAR0549 remain unresolved:

• Precise Biological Role:

  • What are the primary RNA targets in S. aureus?

  • How does it contribute to normal cellular processes?

  • Is it essential under specific environmental conditions?

• Structural Determinants of Function:

  • How does its structure differ from archaeal counterparts?

  • What specific RNA motifs does it recognize?

  • Do structure-function relationships differ from other L7Ae family members?

• Regulatory Networks:

  • What regulates SAR0549 expression?

  • Does it interact with other RNA-binding proteins?

  • How is its activity modulated during infection?

What emerging technologies will advance our understanding of SAR0549?

Emerging technologies that will likely contribute to SAR0549 research include:

• Single-molecule Approaches:

  • Single-molecule FRET to study SAR0549-RNA binding dynamics

  • Optical tweezers to measure binding forces and kinetics

  • Super-resolution microscopy to visualize cellular localization

• Integrative Structural Biology:

  • Cryo-electron tomography for in situ structural studies

  • Integrative modeling combining multiple experimental inputs

  • AlphaFold2 and other AI approaches for structure prediction

• Systems Biology Methods:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics)

  • Network analysis of SAR0549-dependent processes

  • Mathematical modeling of regulatory networks

• Genome Engineering:

  • CRISPR-Cas9 for precise genomic modifications

  • Base editing for targeted mutagenesis without double-strand breaks

  • Conditional knockdown systems for essential genes

These technologies will help connect molecular mechanisms to physiological functions and potentially reveal therapeutic applications targeting SAR0549.

How might understanding SAR0549 contribute to addressing antibiotic resistance in S. aureus?

S. aureus remains a leading cause of antibiotic-resistant infections, with approximately 500,000 hospital patients in the United States contracting staphylococcal infections annually and up to 50,000 related deaths . Understanding SAR0549 could contribute to addressing this crisis through:

• Novel Drug Target Development:

  • If SAR0549 proves essential or important for virulence, it could be targeted by new antimicrobials

  • Structure-based drug design using resolved SAR0549 structures

  • Screening for inhibitors of SAR0549-RNA interactions

• Biomarker Applications:

  • SAR0549 expression patterns could indicate antibiotic susceptibility

  • Could provide targets for rapid diagnostic development

  • May help distinguish between resistant and susceptible strains

• Understanding Resistance Mechanisms:

  • If SAR0549 regulates resistance genes, understanding this regulation could lead to methods to disrupt resistance

  • Could reveal novel resistance pathways

  • May provide insights into evolutionary processes driving resistance