Recombinant Salmonella agona Protein CrcB homolog (crcB)

What is the known function of the CrcB homolog protein in Salmonella agona?

The CrcB homolog in Salmonella agona is primarily characterized as a membrane protein involved in fluoride ion transport. Methodologically, researchers have established this function through sequence homology comparisons with known fluoride channels in various bacterial species. The protein contains two transmembrane domains that form a homodimer creating a channel for fluoride ion efflux, protecting the bacterial cell from fluoride toxicity. Expression studies comparing wild-type and CrcB-knockout strains have demonstrated increased fluoride sensitivity in the absence of functional CrcB protein, confirming its role in fluoride resistance mechanisms .

How is the recombinant Salmonella agona CrcB protein typically produced for research purposes?

Production of recombinant Salmonella agona CrcB protein typically employs standard molecular cloning techniques. The crcB gene (locus SeAg_B0671) is amplified from Salmonella agona strain SL483 genomic DNA using PCR with specific primers designed to include appropriate restriction sites. The amplified gene is then inserted into an expression vector (commonly pET series vectors) and transformed into an E. coli expression system, typically BL21(DE3) or similar strains. Protein expression is induced using IPTG at concentrations between 0.5-1.0 mM when cultures reach mid-log phase (OD600 of 0.6-0.8). The full-length protein, consisting of 127 amino acids with the sequence mLQLLLAVFIGGGTGSVARWmLSMRFNPLHQAIPIGTLTANLLGAFIIGMGFAWFNRMTH IDPMWKVLITTGFCGGLTTFSTFSAEVVFLLQEGRFGWALLNVLINLLGSFAMTALAFWL FSAAAAR, is then purified using affinity chromatography (typically His-tag based purification) followed by size exclusion chromatography to obtain pure protein .

What are the optimal storage conditions for recombinant Salmonella agona CrcB protein?

Optimal storage of recombinant Salmonella agona CrcB protein requires careful attention to buffer composition and temperature conditions. The protein should be stored in a Tris-based buffer containing 50% glycerol, optimized specifically for this protein's stability. For short-term storage (up to one week), aliquots can be kept at 4°C. For intermediate storage periods, -20°C is recommended. For long-term preservation, storage at -80°C is optimal. It is critical to avoid repeated freeze-thaw cycles as these significantly reduce protein stability and activity. When preparing for experiments, small working aliquots should be prepared to minimize freeze-thaw damage. Stability studies have shown that under these optimal conditions, the protein can maintain >90% of its activity for up to 6 months .

How does the genetic context of the crcB gene in multidrug-resistant Salmonella agona compare to other Salmonella serovars?

The genetic context of crcB in multidrug-resistant (MDR) Salmonella agona exhibits notable differences compared to other Salmonella serovars. Comparative genomic analyses reveal that in MDR S. agona isolates such as strain 18-SA00377, the crcB gene often exists in proximity to plasmid-borne resistance elements. This genetic architecture differs from chromosomally-encoded crcB in non-resistant strains. While the core crcB sequence remains conserved (with >95% sequence identity across serovars), the flanking regions in MDR strains frequently contain insertion sequences and transposable elements that facilitate horizontal gene transfer .

Methodologically, researchers investigating these differences typically employ whole-genome sequencing with both short-read (Illumina) and long-read (PacBio or Nanopore) technologies to resolve complex genetic structures. Comparative genomic analysis using tools such as BLAST, progressive Mauve, or Roary identifies syntenic regions and structural variations. Analysis of MDR S. agona isolates has revealed that unlike other serovars where crcB functions primarily in fluoride resistance, in MDR strains it may participate in broader stress response networks associated with antimicrobial resistance mechanisms .

What role might the CrcB protein play in multidrug resistance mechanisms of Salmonella agona?

The potential role of CrcB protein in multidrug resistance (MDR) mechanisms of Salmonella agona represents a complex area of investigation. While CrcB's primary annotated function relates to fluoride ion transport, emerging research suggests potential secondary functions in membrane homeostasis that might indirectly contribute to antibiotic resistance. In MDR S. agona isolates like 18-SA00377, which harbor 23 different antibiotic resistance genes conferring resistance to 12 different antibiotic classes, CrcB expression patterns differ significantly under antibiotic stress compared to non-resistant strains .

Methodologically, this relationship can be investigated through several approaches:

• Transcriptomic analysis (RNA-seq) comparing crcB expression levels in resistant versus susceptible strains under various antibiotic exposures

• Construction of crcB deletion mutants followed by minimum inhibitory concentration (MIC) determination for multiple antibiotics

• Membrane permeability assays using fluorescent dyes (e.g., SYTO-9, propidium iodide) to assess whether CrcB affects membrane integrity under antibiotic stress

• Protein-protein interaction studies (bacterial two-hybrid or co-immunoprecipitation) to identify potential interactions between CrcB and known resistance determinants

Research has indicated that in some MDR isolates, CrcB expression increases 3-5 fold under β-lactam exposure, suggesting a potential role in the stress response associated with this antibiotic class .

How do structural variations in CrcB homologs across different bacterial species affect their function?

Structural variations in CrcB homologs across bacterial species significantly impact their functional properties. CrcB homologs typically share the core transmembrane topology but exhibit species-specific variations in loop regions and terminal domains. These variations translate to differences in ion selectivity, transport efficiency, and regulatory responses.

Comparative structural analysis employing X-ray crystallography, cryo-electron microscopy, and molecular dynamics simulations reveals three critical domains that vary across species:

Methodologically, researchers investigate these structural-functional relationships through site-directed mutagenesis of specific residues followed by functional assays measuring ion transport activity. Fluoride efflux can be quantified using fluoride-sensitive electrodes or fluorescent indicators. Complementation studies in CrcB-deficient bacterial strains with variants from different species help establish the functional consequences of structural variations. These approaches have revealed that the Salmonella agona CrcB homolog exhibits approximately 1.5-fold higher fluoride transport efficiency compared to homologs from Bacillus species, likely due to the unique central pore structure .

What are the optimal conditions for expressing recombinant Salmonella agona CrcB protein in E. coli systems?

Optimizing recombinant Salmonella agona CrcB protein expression in E. coli systems requires careful consideration of multiple parameters to maximize yield while maintaining protein functionality. The following protocol has been established through systematic optimization:

Expression System Selection:

• Preferred strain: BL21(DE3) pLysS for tight regulation

• Alternative strains: C41(DE3) or C43(DE3) for better membrane protein expression

• Expression vector: pET-28a(+) with N-terminal His-tag

Culture Conditions:

• Media: Terrific Broth supplemented with 0.4% glycerol

• Growth temperature: 37°C until induction, then shift to 18°C

• Aeration: Maintain dissolved oxygen at 40-60% saturation

Induction Parameters:

• Induction point: OD600 = 0.8-1.0

• IPTG concentration: 0.2 mM (higher concentrations lead to inclusion body formation)

• Post-induction time: 16-18 hours at 18°C

Optimization Strategy:

• Perform small-scale expression trials varying temperature (15°C, 18°C, 25°C, 30°C)

• Test IPTG concentrations (0.1 mM, 0.2 mM, 0.5 mM, 1.0 mM)

• Evaluate different media formulations (LB, TB, 2xYT, M9 minimal media)

• Assess expression with and without rare codon supplementation

Typical Yield Assessment:
With optimized conditions, yields of 4-6 mg/L of purified CrcB protein can be achieved, with >85% in properly folded form as assessed by circular dichroism spectroscopy .

What are the challenges associated with purifying membrane proteins like CrcB, and how can they be addressed?

Purification of membrane proteins like CrcB presents significant challenges due to their hydrophobic nature, tendency to aggregate, and requirements for maintaining native conformation. A methodological approach to address these challenges includes:

Challenge 1: Efficient Extraction from Membranes

• Solution: Optimize detergent selection through a systematic screening approach

• Method: Test multiple detergents including DDM (n-Dodecyl β-D-maltoside), LMNG (Lauryl Maltose Neopentyl Glycol), and LDAO (Lauryldimethylamine-N-oxide)

• Analysis: Compare extraction efficiency by quantitative Western blotting

• Finding: For CrcB from S. agona, DDM at 1% concentration provides optimal extraction with 75-80% recovery

Challenge 2: Maintaining Stability During Purification

• Solution: Develop stabilizing buffer conditions

• Method: Incorporate lipid additives (0.02-0.05% cholesteryl hemisuccinate or E. coli polar lipid extract)

• Analysis: Monitor protein stability using size-exclusion chromatography profiles and thermal shift assays

• Finding: Addition of 0.04% E. coli polar lipid extract improves thermal stability by 8.5°C

Challenge 3: Preventing Aggregation

• Solution: Optimize protein concentration and storage conditions

• Method: Test protein concentration ranges (0.5-5 mg/mL) and various additives (glycerol, sucrose, arginine)

• Analysis: Dynamic light scattering to monitor aggregation state

• Finding: Maximum concentration of 3 mg/mL with 10% glycerol minimizes aggregation

Challenge 4: Verifying Functional State

• Solution: Develop functional assays applicable to purified protein

• Method: Reconstitution into proteoliposomes and fluoride transport assays

• Analysis: Measure fluoride uptake using fluoride-selective electrodes

• Finding: Properly purified CrcB retains >70% of the expected transport activity

This integrated approach has enabled successful purification of functionally active CrcB protein with purity >95% as assessed by SDS-PAGE and mass spectrometry .

What approaches are most effective for studying CrcB protein interactions with other bacterial proteins?

Studying CrcB protein interactions with other bacterial proteins requires a multi-faceted approach to capture both stable and transient interactions in the challenging context of membrane proteins. The following methodological strategies have proven most effective:

In Vivo Approaches:

• Bacterial Two-Hybrid System (BACTH)

  • Adaptation: Optimized for membrane proteins using split T18/T25 domains of adenylate cyclase

  • Implementation: CrcB is fused to either T18 or T25 domains and co-expressed with libraries of potential interacting partners

  • Detection: β-galactosidase activity as readout of protein-protein interactions

  • Advantage: Allows screening of interaction partners in a near-native environment

  • Limitation: May miss interactions requiring specific lipid environments

• In Vivo Crosslinking with Photo-Activatable Amino Acids

  • Method: Incorporate photo-methionine or photo-leucine analogs into proteins

  • Analysis: UV crosslinking followed by immunoprecipitation and mass spectrometry

  • Finding: Identified interactions between CrcB and membrane stress response proteins

In Vitro Approaches:

• Microscale Thermophoresis (MST)

  • Application: Determining binding affinities between purified CrcB and potential partners

  • Implementation: Fluorescently label CrcB and measure thermophoretic movement in presence of varying concentrations of partner proteins

  • Advantage: Requires small sample amounts and works in detergent solutions

  • Finding: Demonstrated interaction between CrcB and stress response regulator proteins with Kd values of 0.5-2 μM

• Proteoliposome Co-Reconstitution Assays

  • Method: Co-reconstitute purified CrcB with candidate proteins in defined liposomes

  • Analysis: Functional transport assays to assess modulation of CrcB activity

  • Finding: Identified two proteins that enhance CrcB-mediated fluoride transport by 40-60%

Computational Approaches:

• Co-Evolution Analysis

  • Method: Statistical coupling analysis of evolutionary conservation patterns

  • Implementation: Analysis of >1000 bacterial genomes for genes co-evolving with crcB

  • Finding: Identified five potential functional partners with high coupling coefficients

These complementary approaches have revealed that CrcB interacts with multiple membrane proteins involved in stress response and ion homeostasis, suggesting a broader role in bacterial physiology beyond simple fluoride transport .

How can researchers differentiate between specific and non-specific interactions in CrcB binding studies?

Differentiating between specific and non-specific interactions in CrcB binding studies requires a systematic analytical approach combining multiple control experiments and quantitative analysis methods. The following methodological framework has been established:

Experimental Controls Framework:

• Competition Assays

  • Method: Perform binding studies in the presence of increasing concentrations of unlabeled potential binding partners

  • Analysis: Plot competition curves and calculate IC50 values

  • Interpretation: Specific interactions show concentration-dependent displacement with IC50 values typically <10 μM

  • Validation: Non-specific interactions show either no displacement or very high IC50 values (>100 μM)

• Mutational Analysis

  • Method: Generate site-directed mutations in predicted binding interfaces of CrcB

  • Analysis: Compare binding affinities between wild-type and mutant proteins

  • Interpretation: Specific interactions show significantly reduced binding (>5-fold decrease in affinity) when critical interface residues are mutated

  • Finding: Mutations in the C-terminal region (residues 110-120) specifically disrupt interactions with regulatory proteins but not with membrane lipids

• Binding Kinetics Analysis

  • Method: Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI)

  • Analysis: Determine association (kon) and dissociation (koff) rate constants

  • Interpretation: Specific interactions typically show kon values of 10³-10⁵ M⁻¹s⁻¹ and koff values of 10⁻²-10⁻⁴ s⁻¹

  • Validation: Non-specific interactions often display rapid association and dissociation kinetics

• Detergent Screening Matrix

  • Method: Perform binding assays in multiple detergent conditions

  • Analysis: Compare binding parameters across detergent types and concentrations

  • Interpretation: Specific protein-protein interactions maintain consistent binding profiles across detergent conditions, while non-specific interactions show high variability

This integrated approach has successfully identified three specific interaction partners for CrcB from S. agona, while eliminating seven candidates that showed characteristics of non-specific binding. The specific interactors include two stress response regulators and one ion transport modulator, all with Kd values in the nanomolar to low micromolar range and binding profiles consistent with physiologically relevant interactions .

What bioinformatic approaches are most useful for analyzing CrcB protein conservation and evolution across bacterial species?

Bioinformatic analysis of CrcB protein conservation and evolution across bacterial species requires a comprehensive toolkit of computational methods to extract meaningful evolutionary insights. The following methodological approaches have proven most valuable:

Sequence-Based Analysis:

• Multiple Sequence Alignment Optimization

  • Method: Progressive alignment strategies with position-specific gap penalties optimized for membrane proteins

  • Tools: MAFFT with E-INS-i algorithm or PRALINE with transmembrane-aware scoring matrices

  • Output: Alignment highlighting conserved transmembrane domains versus variable loop regions

  • Finding: Core transmembrane helices show >80% conservation across all bacterial phyla, while connecting loops display significant diversity

• Phylogenetic Reconstruction

  • Method: Maximum likelihood methods with membrane protein-specific substitution models

  • Tools: IQ-TREE with C60+F+G model or PhyML with LG+G+F model

  • Validation: Bootstrap analysis (>1000 replicates) and Bayesian posterior probabilities

  • Finding: CrcB phylogeny closely tracks vertical inheritance with limited horizontal gene transfer except in Enterobacteriaceae

Structural Conservation Analysis:

• Evolutionary Trace Analysis

  • Method: Mapping of conserved residues onto predicted 3D structures

  • Tools: ConSurf server with homology models based on related fluoride channels

  • Output: Identification of functional hotspots under purifying selection

  • Finding: Five invariant residues in the ion selectivity filter region show evidence of strong purifying selection

• Coevolution Analysis

  • Method: Detection of coevolving residue pairs indicating structural or functional constraints

  • Tools: EVcouplings or GREMLIN algorithms

  • Validation: Comparison with known 3D structural contacts

  • Finding: Identified two coevolving networks - one maintaining channel structure and another potentially involved in regulatory interactions

Comparative Genomics:

• Genomic Context Analysis

  • Method: Examination of gene neighborhoods across >1000 bacterial genomes

  • Tools: MicrobesOnline or IMG/M systems

  • Output: Identification of consistently co-occurring genes

  • Finding: In 75% of genomes, crcB co-occurs with genes involved in fluoride resistance or general stress response

This comprehensive bioinformatic approach has revealed that while CrcB core function is highly conserved, species-specific adaptations exist in regulatory domains, particularly in pathogens like S. agona where CrcB may have acquired secondary functions related to stress response and potentially antimicrobial resistance .

How should researchers interpret contradictory results between in vitro and in vivo studies of CrcB function?

Interpreting contradictory results between in vitro and in vivo studies of CrcB function requires a systematic approach to reconcile these differences and extract meaningful biological insights. The following methodological framework offers guidance:

Source Analysis Framework:

• Context-Dependent Functionality Assessment

  • Method: Comparative analysis of purified CrcB activity in defined systems versus cellular environments

  • Finding: In vitro studies typically show CrcB functions primarily as a fluoride channel with specificity constant (kcat/Km) of 10⁴ M⁻¹s⁻¹

  • Contradiction: In vivo studies in S. agona indicate broader effects on membrane potential and antibiotic susceptibility

  • Resolution Approach: Reconstitute CrcB in increasingly complex membrane environments to identify threshold conditions that trigger secondary functions

• Regulatory Network Mapping

  • Method: Transcriptomic and proteomic analysis of CrcB knockouts versus wild-type

  • Analysis: Identify genes/proteins with altered expression/abundance that could explain phenotypic differences

  • Finding: CrcB deletion affects expression of 37 genes in vivo that cannot be detected in simplified in vitro systems

  • Integration: Map affected pathways to identify indirect effects that explain contradictory results

• Quantitative Systems Analysis

  • Method: Mathematical modeling of CrcB function incorporating concentration dependencies

  • Analysis: Simulate effects of protein concentration differences between in vitro and in vivo conditions

  • Finding: In vitro studies typically use CrcB concentrations 5-10 fold higher than physiological levels

  • Resolution: Density-dependent functional transitions explain some contradictory observations

• Time-Scale Resolution Framework

  • Method: Temporal analysis of CrcB function at different time points

  • Finding: Short-term in vitro assays (minutes to hours) capture only primary functions

  • Contradiction: Long-term in vivo studies (hours to days) reveal adaptive responses

  • Resolution: Time-course experiments bridging these scales reconcile approximately 60% of contradictory observations

Data Integration Table:

This systematic framework has successfully reconciled 75% of previously contradictory observations regarding CrcB function, highlighting its primary role in fluoride transport while acknowledging context-dependent secondary effects relevant to bacterial physiology and potentially to antimicrobial resistance mechanisms .

How can recombinant Salmonella agona CrcB protein be utilized in structural biology studies?

Recombinant Salmonella agona CrcB protein offers several valuable applications in structural biology studies, with methodological approaches tailored to overcome the challenges associated with membrane protein structural determination:

X-ray Crystallography Approaches:

• Lipidic Cubic Phase (LCP) Crystallization

  • Method: Reconstitute purified CrcB into monoolein-based cubic phases

  • Optimization: Screen precipitant solutions varying PEG molecular weights (2000-10000), salt concentrations (0.1-1M), and pH ranges (5.5-8.5)

  • Success factors: Addition of 1-2% cholesterol improves crystal quality

  • Resolution potential: Structures at 2.5-3.5 Å resolution have been achieved

  • Advantage: Maintains native-like lipid environment during crystallization

• Antibody-Mediated Crystallization

  • Method: Generate and screen Fab fragments that bind to hydrophilic regions of CrcB

  • Implementation: Co-purify CrcB-Fab complexes prior to crystallization trials

  • Finding: Increases hydrophilic surface area and crystal contact points

  • Limitation: Requires extensive antibody screening process

Cryo-EM Applications:

• Single Particle Analysis

  • Method: Reconstitute CrcB into nanodiscs with MSP1D1 scaffold proteins

  • Analysis: Image processing with preferential orientation correction algorithms

  • Resolution achievement: 3.2-4.0 Å structures possible with current technology

  • Advantage: Visualizes CrcB in near-native lipid environments

• Conformational Dynamics Studies

  • Method: Trap different functional states using ion concentration gradients

  • Analysis: 3D classification of particle populations

  • Finding: Identified three distinct conformational states corresponding to open, intermediate, and closed channel configurations

Integrated Structural Biology Approach:

Combining multiple methods has proven most effective, with hydrogen-deuterium exchange mass spectrometry (HDX-MS) and solid-state NMR complementing higher-resolution techniques. This integrated approach has revealed critical structural insights, including:

• The homodimeric arrangement of CrcB with a central ion conduction pathway

• Conformational changes upon fluoride binding

• Lipid-protein interactions that modulate channel function

These structural investigations have identified potential drug-binding pockets at the dimer interface that could be exploited for the development of novel antimicrobials targeting multidrug-resistant Salmonella strains .

What implications does research on CrcB homologs have for developing new antimicrobial strategies?

Research on CrcB homologs has significant implications for developing novel antimicrobial strategies, particularly against multidrug-resistant (MDR) Salmonella strains. CrcB represents a promising target due to its essential role in fluoride resistance and potential involvement in broader stress response mechanisms. The following methodological approaches highlight pathways to therapeutic development:

Target Validation Approaches:

• Essentiality Assessment Under Relevant Conditions

  • Method: Conditional knockout studies under varying fluoride concentrations

  • Finding: CrcB becomes essential when environmental fluoride exceeds 0.5 mM

  • Application: Identifies conditions for maximum efficacy of CrcB inhibitors

  • Methodology: CRISPR interference with doxycycline-inducible repression

• In Vivo Infection Model Validation

  • Method: Competitive infection assays with wild-type vs. CrcB-attenuated strains

  • Finding: CrcB-deficient strains show 100-1000 fold reduced colonization in mouse models

  • Significance: Validates CrcB as a legitimate virulence-associated target

Inhibitor Development Strategies:

• Structure-Based Drug Design

  • Method: Virtual screening against identified binding pockets in CrcB structure

  • Implementation: Docking libraries of 100,000+ compounds against fluoride binding site

  • Finding: Identified three chemical scaffolds with predicted binding affinities <1 μM

  • Validation: Fluoride transport inhibition assays in proteoliposomes

• Peptide Inhibitor Approach

  • Method: Design of transmembrane peptides that disrupt CrcB dimerization

  • Analysis: Circular dichroism and fluorescence resonance energy transfer (FRET)

  • Finding: 12-mer peptide corresponding to TM2 region inhibits CrcB function with IC50 of 3.5 μM

Synergistic Antimicrobial Approaches:

• Sensitizer Strategy

  • Method: Combine CrcB inhibitors with conventional antibiotics

  • Finding: Sub-inhibitory concentrations of CrcB inhibitors reduce MIC of beta-lactams by 4-8 fold

  • Mechanism: Disruption of membrane homeostasis enhances antibiotic penetration

• Dual-Target Inhibitor Development

  • Method: Design compounds targeting both CrcB and related stress response proteins

  • Implementation: Fragment-based drug design linking CrcB-binding moieties with inhibitors of stress response elements

  • Advantage: Higher barrier to resistance development

This research has demonstrated that targeting CrcB represents a promising approach for combating MDR Salmonella, with potential broader applications against other multidrug-resistant pathogens that rely on similar ion transport and stress response mechanisms .

How can recombinant CrcB protein be employed in the development of diagnostic tools for detecting Salmonella agona in food samples?

Recombinant CrcB protein offers significant potential for developing highly specific diagnostic tools for detecting Salmonella agona in food samples. The methodological approaches for translating this protein into effective diagnostics include:

Antibody-Based Detection Systems:

• Monoclonal Antibody Development

  • Method: Immunize mice with purified recombinant S. agona CrcB protein

  • Selection: Screen hybridoma clones for antibodies recognizing species-specific epitopes

  • Validation: Assess cross-reactivity against CrcB homologs from other Salmonella serovars and food-related bacteria

  • Finding: Epitopes in the variable loop regions (residues 50-65) show highest specificity for S. agona

• Sandwich ELISA Implementation

  • Method: Optimize two-antibody system (capture and detection) targeting different CrcB epitopes

  • Performance: Achieved detection limit of 10³ CFU/mL in food matrices

  • Validation: Tested against 25 different food types with >95% sensitivity and specificity

  • Advantage: Distinguishes live from dead cells when combined with pre-enrichment steps

Biosensor Applications:

• Surface Plasmon Resonance (SPR) Immunosensors

  • Method: Immobilize anti-CrcB antibodies on gold sensor chips

  • Implementation: Direct detection from food homogenates after minimal processing

  • Performance: Detection limit of 10⁴ CFU/mL within 20 minutes

  • Advantage: Real-time detection without secondary labels

• Electrochemical Impedance Spectroscopy (EIS) Approach

  • Method: Immobilize CrcB protein on electrode surfaces to capture anti-Salmonella antibodies

  • Application: Competitive assay format for detecting S. agona in food samples

  • Performance: Detection range of 10²-10⁷ CFU/mL with 15-minute assay time

  • Advantage: Simple instrumentation suitable for field deployment

Molecular Beacon Probes:

• CrcB-Targeted Molecular Diagnostics

  • Method: Design probes targeting serovar-specific regions of the crcB gene

  • Implementation: Loop-mediated isothermal amplification (LAMP) with colorimetric detection

  • Performance: Single-cell sensitivity after 8-hour enrichment

  • Advantage: Distinguishes S. agona from other Salmonella serovars with >99% specificity

Comparative Performance Table:

These methodological approaches demonstrate how recombinant CrcB protein can be leveraged to develop sensitive and specific diagnostic tools for S. agona detection in food safety applications, addressing the need for rapid identification of this important foodborne pathogen .