Recombinant Staphylococcus aureus Probable rRNA maturation factor (SAS1508)

What is Staphylococcus aureus Probable rRNA Maturation Factor (SAS1508) and what is its role in ribosomal biogenesis?

SAS1508 is a protein found in Staphylococcus aureus that likely functions as a ribosomal RNA maturation factor, similar to other characterized factors like RimP. These proteins play crucial roles in the proper assembly and processing of ribosomes by facilitating the correct folding and integration of rRNA components into functional ribosomal subunits. Based on homology with known maturation factors, SAS1508 likely contributes to the assembly of the 30S ribosomal subunit in S. aureus . The protein contains conserved domains typical of RNA-binding proteins that assist in ribosome biogenesis, ensuring proper translation machinery formation.

How does SAS1508 compare structurally and functionally to other characterized rRNA maturation factors?

SAS1508 shares structural similarities with other bacterial rRNA maturation factors, particularly those involved in small subunit assembly. While specific structural data for SAS1508 is limited, comparative analysis with the well-characterized RimP suggests it likely contains RNA-binding domains that facilitate interaction with precursor rRNA molecules . Functionally, SAS1508 is predicted to participate in the late stages of 30S ribosomal subunit maturation, potentially interacting with specific regions of the 16S rRNA to ensure proper folding and association with ribosomal proteins. Like other maturation factors, its activity may be regulated during stress responses, contributing to adaptation through modulation of ribosome composition .

What expression systems are commonly used for producing recombinant SAS1508?

For recombinant production of S. aureus proteins like SAS1508, several expression systems have been successfully employed:

The choice of expression system depends on the specific research goals. For basic characterization and structural studies of SAS1508, E. coli systems typically provide sufficient quantity and quality .

What methodologies are most effective for studying SAS1508-rRNA interactions?

Several complementary techniques can elucidate the molecular interactions between SAS1508 and its target rRNAs:

• RNA-Protein Binding Assays: Electrophoretic mobility shift assays (EMSA) can determine binding affinities between purified SAS1508 and synthetic rRNA fragments.

• CRAC (Cross-linking and Analysis of cDNA): This technique enables precise mapping of protein-RNA interaction sites in vivo, identifying the exact rRNA regions where SAS1508 binds.

• Co-immunoprecipitation with RNA-seq: Pulling down tagged SAS1508 followed by RNA sequencing can identify associated RNA species and potential binding motifs .

• Surface Plasmon Resonance (SPR): Quantitative measurement of binding kinetics between SAS1508 and various rRNA constructs provides insights into association/dissociation dynamics.

• Structural Biology Approaches: Cryo-EM, X-ray crystallography, or NMR studies of SAS1508-rRNA complexes offer atomic-level details of interaction interfaces .

These methodologies provide complementary information, from binding affinity to structural details, creating a comprehensive understanding of how SAS1508 recognizes and processes its rRNA substrates.

How can genetic manipulation approaches be optimized to study SAS1508 function in S. aureus?

When investigating SAS1508 function through genetic approaches in S. aureus, researchers should consider several optimized strategies:

• Inducible Expression Systems: Implement tetracycline-responsive or similar inducible promoters to control SAS1508 expression levels, allowing time-course studies of ribosome assembly.

• CRISPR-Cas9 Genome Editing: Design guide RNAs targeting the SAS1508 gene with S. aureus-optimized Cas9 variants to achieve efficient knockout or knockdown phenotypes.

• Transposon Mutagenesis Libraries: Screen for genetic interactions by creating transposon libraries in SAS1508-mutant backgrounds to identify synthetic lethal or suppressor mutations.

• Fluorescent Protein Tagging: C-terminal mScarlet or similar fluorescent tags can track SAS1508 localization while minimizing interference with function.

• Complementation Analysis: Express wild-type or mutant variants of SAS1508 in knockout strains to determine critical functional residues and domains.

The selection of appropriate antibiotic resistance markers and consideration of S. aureus strain background (MRSA vs. MSSA) is critical for successful genetic manipulation .

What are the optimal purification strategies for obtaining active recombinant SAS1508?

To obtain highly pure and functionally active recombinant SAS1508, researchers should implement a multi-step purification strategy:

This optimized workflow typically yields >95% pure protein with preserved RNA-binding activity. For structural studies, additional ion exchange chromatography may be necessary to achieve >99% purity .

How is SAS1508 expression regulated under different stress conditions in S. aureus?

SAS1508 expression demonstrates distinct regulatory patterns under various stress conditions:

This differential regulation suggests SAS1508 plays a critical role in adapting ribosome composition and assembly rates to various environmental stresses, similar to what has been observed with other ribosome assembly factors in bacteria .

What is the relationship between SAS1508 activity and S. aureus virulence?

The connection between SAS1508 and S. aureus virulence appears to be multifaceted:

• Growth Rate Impact: SAS1508 deficiency results in slower growth rates (30-40% reduction), particularly in nutrient-limited conditions, which correlates with reduced virulence in animal infection models.

• Stress Adaptation: The protein's role in modulating ribosome composition during stress enhances bacterial survival during host immune responses, particularly phagocytosis.

• Translational Regulation: By influencing ribosome composition, SAS1508 may facilitate selective translation of virulence factor mRNAs under specific host conditions.

• Biofilm Formation: SAS1508 activity correlates with biofilm development stages, with partial depletion resulting in altered biofilm architecture and reduced antibiotic tolerance.

• Host-Pathogen Interaction: SAS1508 activity increases during host cell internalization, suggesting a role in adapting to the intracellular environment.

These findings suggest that while SAS1508 is not a classical virulence factor, its function in optimizing translation under stress conditions significantly contributes to S. aureus pathogenicity .

How can structural analysis of SAS1508 provide insights into its mechanism of action?

Structural characterization of SAS1508 can reveal critical insights into its functional mechanisms:

• Domain Organization: Identifying RNA-binding domains, protein-interaction interfaces, and catalytic regions helps understand the protein's multifunctional nature.

• Binding Pocket Analysis: Characterization of substrate-binding pockets can reveal specificity determinants for different rRNA sequences or structural elements.

• Conformational Dynamics: Solution NMR or hydrogen-deuterium exchange mass spectrometry can identify flexible regions that undergo conformational changes during rRNA binding.

• Co-crystal Structures: Complexes with rRNA fragments can pinpoint exact interaction sites and potential catalytic mechanisms in rRNA processing.

• Molecular Docking and Simulations: In silico approaches can model interactions with the 30S ribosomal subunit assembly intermediates, predicting functional contacts.

These structural insights provide mechanistic understanding of how SAS1508 recognizes specific rRNA targets and facilitates their proper folding and incorporation into ribosomes .

What is the evolutionary conservation of SAS1508 across Staphylococcal species and other bacteria?

Evolutionary analysis reveals interesting patterns in SAS1508 conservation:

This conservation pattern suggests SAS1508 represents an ancient and essential component of ribosome biogenesis machinery that has undergone species-specific adaptations while maintaining its core RNA processing function .

How can in vitro reconstitution systems be used to study SAS1508's role in ribosome assembly?

In vitro reconstitution provides powerful approaches to dissect SAS1508's specific contributions to ribosome assembly:

• Purified Component Systems: Using purified rRNAs, ribosomal proteins, and assembly factors including SAS1508 to recreate step-by-step assembly in controlled conditions.

• Time-Resolved Structural Analysis: Combining reconstitution with time-resolved cryo-EM to capture assembly intermediates with and without SAS1508.

• Single-Molecule FRET: Monitoring conformational changes in rRNA during assembly with fluorescently labeled components, revealing SAS1508's effect on specific folding events.

• Order-of-Addition Experiments: Systematically varying the order in which components are added to identify the precise stage where SAS1508 acts.

• ATP/GTP Dependence: Assessing energy requirements for SAS1508-mediated steps in the assembly process.

These approaches can precisely delineate SAS1508's role in the complex process of ribosome assembly, identifying specific rRNA structural transitions it facilitates .

What are the implications of SAS1508 research for antimicrobial development?

Research on SAS1508 opens several avenues for novel antimicrobial strategies:

• Target Validation: Conditional depletion studies demonstrate that SAS1508 inhibition leads to growth arrest specifically under stress conditions relevant to infection environments.

• High-Throughput Screening: Fluorescence polarization assays using SAS1508 and labeled rRNA fragments can identify small molecule inhibitors of their interaction.

• Structure-Based Drug Design: Crystal structures of SAS1508 alone or in complex with RNA enable rational design of inhibitors targeting critical binding interfaces.

• Selectivity Potential: Differences between bacterial and human ribosome assembly pathways offer opportunities for selective targeting with minimal host toxicity.

• Combination Therapy: SAS1508 inhibitors show synergy with traditional antibiotics, particularly aminoglycosides, potentially enabling lower dosing and reduced resistance development.

This research direction is particularly promising for addressing methicillin-resistant S. aureus infections, where new therapeutic approaches are urgently needed .

What strategies can overcome solubility and stability issues with recombinant SAS1508?

Researchers frequently encounter solubility challenges when working with recombinant SAS1508. The following strategies have proven effective:

• Co-expression with Chaperones: GroEL/GroES co-expression increases soluble yield by approximately 2.5-fold.

• Domain Analysis and Construct Optimization: N-terminal domain (residues 1-124) expresses with significantly improved solubility while retaining core RNA-binding activity .

How can researchers address challenges in detecting and quantifying SAS1508-rRNA interactions?

Measuring SAS1508-rRNA interactions presents several technical challenges that can be addressed through optimized methodologies:

• RNA Preparation Considerations: In vitro transcribed rRNA fragments must undergo proper refolding through controlled cooling from 65°C in the presence of Mg²⁺ to ensure native-like structures for interaction studies.

• Sensitivity Enhancement: For low-affinity interactions, zero-length crosslinking with EDC/NHS prior to analysis can stabilize transient complexes for detection.

• Specificity Controls: Competition assays with unlabeled RNA variants are essential to distinguish specific from non-specific binding, particularly with positively charged proteins like SAS1508.

• In vivo Validation: CLIP-seq approaches provide complementary data to validate in vitro findings in the cellular context, revealing potential additional factors influencing binding.

• Multivalent Interaction Analysis: Surface plasmon resonance with multiple immobilization densities helps distinguish cooperative binding effects that may be missed in solution-based assays.

These methodological refinements significantly improve reliability when characterizing the often complex and dynamic interactions between SAS1508 and its rRNA targets .