Recombinant Renibacterium salmoninarum Acyl carrier protein (acpP)

What is Renibacterium salmoninarum and why is it significant in research?

Renibacterium salmoninarum is the causative agent of bacterial kidney disease (BKD), a significant pathogen affecting salmon (Oncorhynchus spp.) populations throughout the northern hemisphere. This gram-positive bacterium presents substantial research importance due to its impact on both wild salmon ecosystems and aquaculture operations. R. salmoninarum infections are particularly concerning as they affect young salmon, potentially compromising population sustainability in marine, aquatic, and terrestrial ecosystems that depend on healthy salmon populations .

The research significance extends beyond ecological concerns to practical applications in aquaculture and hatchery management. Early detection of R. salmoninarum allows managers to implement targeted antibiotic treatments and containment measures before widespread outbreaks occur. The bacterium's unique pathogenicity mechanisms and host interactions make it an important model for studying fish pathogens and developing novel detection and treatment approaches .

What detection methods are available for Renibacterium salmoninarum in research settings?

Several detection methods with varying sensitivity and application contexts are available for R. salmoninarum research:

The isothermal recombinase polymerase amplification (RPA) coupled with CRISPR-Cas12a represents one of the newest methodological advances, offering sensitivity comparable to qPCR but with significantly reduced time requirements and potential field application. This method has demonstrated a limit of detection of approximately 20-40 copies/μL within 10 minutes of Cas activity, making it particularly valuable for time-sensitive applications .

For research requiring quantitative assessment, real-time PCR methods remain valuable, especially when bacterial load correlation with pathology is being investigated. The choice of detection method should align with specific research questions, considering factors such as required sensitivity, time constraints, field vs. laboratory application, and whether quantification is necessary .

What samples are optimal for studying Renibacterium salmoninarum in laboratory conditions?

The optimal sample selection for R. salmoninarum research depends on the specific research objective:

For clinical diagnosis and pathogenesis studies:

• Kidney tissue (primary site of infection, unfixed)

• Spleen tissue (unfixed)

• These tissues can be analyzed individually or pooled (maximum 3 samples) for higher throughput

For epidemiological and environmental monitoring:

• Water samples from hatcheries or natural habitats

• Environmental DNA (eDNA) extracted from water samples

• These samples are particularly valuable for detecting subclinical infections and monitoring pathogen prevalence

For aquaculture quality control and vertical transmission studies:

• Embryonated eggs (minimum volume: 60mL, stored dry without transport medium)

• Coelomic fluid (collected in tubes or dry pots without transport medium)

• These samples allow monitoring of vertical transmission routes and early intervention

Sample processing method significantly impacts detection sensitivity. For water samples, concentration techniques such as filtration followed by DNA extraction protocols optimized for environmental samples improve detection rates. For tissue samples, homogenization in appropriate buffers followed by nucleic acid extraction yields optimal results for molecular detection methods .

How can recombinant techniques be applied to study Renibacterium salmoninarum acpP?

Recombinant techniques offer powerful approaches to studying R. salmoninarum acpP, enabling detailed functional and structural characterization:

Expression System Selection:
The choice of expression system is critical for successful recombinant acpP production. E. coli expression systems using vectors such as pET28a have been successfully employed for expressing diverse bacterial acyltransferases and associated proteins. For R. salmoninarum acpP, codon optimization may be necessary due to potential differences in codon usage between R. salmoninarum and E. coli .

Purification Strategy:
A methodological approach for purification typically involves:

• Affinity chromatography using His-tag or other fusion tags

• Size exclusion chromatography for further purification

• Verification of protein integrity using SDS-PAGE and Western blotting

• Activity assessment using in vitro assays

Functional Characterization:
To assess acpP functionality, researchers can:

• Perform in vitro reconstitution assays with purified fatty acid synthesis enzymes

• Analyze acpP-dependent lipid production using mass spectrometry

• Study protein-protein interactions with predicted partner enzymes using pull-down assays or surface plasmon resonance

• Conduct complementation studies in acpP-deficient bacterial strains

Structural Studies:
X-ray crystallography or NMR spectroscopy can provide valuable insights into the structural basis of acpP function, particularly the binding pocket accommodating the phosphopantetheine prosthetic group and interactions with partner enzymes .

What host-pathogen interactions involve Renibacterium salmoninarum and potentially acpP?

Host-pathogen interactions between Renibacterium salmoninarum and its salmonid hosts involve complex immune responses that could potentially implicate acpP, though specific involvement of acpP in these interactions remains an area requiring further research.

Macrophage Response:
Fish macrophages play a central role in the host response to R. salmoninarum infection. Methodological approaches to study these interactions include:

• Isolation of pronephros macrophages using Percoll density gradient centrifugation

• In vitro infection models using isolated macrophages

• RNA extraction and reverse transcription for gene expression analysis

• Quantification of immune response genes following infection

Potential acpP Involvement:
While direct evidence of acpP's role in host-pathogen interactions is limited, bacterial acyl carrier proteins often contribute to pathogenesis through:

• Involvement in the synthesis of cell envelope components that interact with host immune receptors

• Production of lipid-based virulence factors

• Potential moonlighting functions beyond primary metabolic roles

Research Methodology for Investigating acpP in Host Interactions:

• Generate recombinant acpP and assess its immunostimulatory properties on fish immune cells

• Develop acpP-deficient R. salmoninarum strains (if possible) to assess virulence attenuation

• Perform transcriptomic analysis to determine if acpP expression changes during host infection

• Use fluorescently labeled acpP to track localization during infection processes

How does Renibacterium salmoninarum acpP compare to similar proteins in other bacterial species?

Comparative analysis of acpP across bacterial species provides valuable insights into functional conservation and specialization. While specific comparative data for R. salmoninarum acpP is limited in the current literature, methodological approaches to such comparison can be outlined:

Phylogenetic Analysis Framework:
The evolutionary relationships between acpP homologs can be studied by:

• Identifying homologs through BLAST searches against protein databases

• Performing multiple sequence alignment using tools like MUSCLE or CLUSTALW

• Constructing phylogenetic trees using maximum likelihood or Bayesian methods

• Analyzing conservation of key functional residues across species

Functional Domain Comparison:
Acyl carrier proteins typically contain a conserved serine residue that serves as the attachment site for the phosphopantetheine prosthetic group. Comparative analysis would examine conservation of:

• The phosphopantetheine attachment site

• Residues involved in protein-protein interactions

• Surface charges that facilitate interactions with partner enzymes

Methodological Approach to Functional Comparison:

• Heterologous expression of acpP homologs from different species

• Biochemical characterization of substrate preferences

• Cross-complementation studies in acpP-deficient strains

• Structural comparison through homology modeling or experimental structure determination

What are the challenges in isolating and characterizing acpP from Renibacterium salmoninarum?

Researchers face several technical challenges when isolating and characterizing acpP from R. salmoninarum:

Cultivation Challenges:
R. salmoninarum is a slow-growing, fastidious organism requiring specialized media and extended incubation periods (weeks rather than days). This complicates efforts to obtain sufficient biomass for native protein purification. Methodological approaches to address this include:

• Optimization of growth media with supplements that enhance growth rate

• Scaled-up cultivation systems with careful monitoring of growth parameters

• Alternative approaches using recombinant expression in heterologous hosts

Protein Stability Issues:
Acyl carrier proteins are relatively small (~9-10 kDa) and can present stability challenges during purification. The phosphopantetheine prosthetic group critical for function can be labile under certain conditions. Strategies to address this include:

• Use of protease inhibitors throughout the purification process

• Optimization of buffer compositions to enhance stability

• Careful temperature control during handling and storage

• Rapid purification protocols to minimize degradation time

Functional Characterization Complexity:
AcpP functions as part of a multi-enzyme system, requiring partner enzymes for complete functional characterization. Research approaches include:

• Reconstitution of minimal synthetic systems with key partner enzymes

• Development of specialized assays to monitor acpP-dependent reactions

• Use of mass spectrometry to identify acpP-bound intermediates

• Isothermal titration calorimetry to characterize protein-protein interactions

Post-translational Modification Requirements:
Proper functioning of acpP requires post-translational modification by a phosphopantetheinyl transferase to convert the apo-form to the holo-form. Ensuring this modification occurs correctly in recombinant systems requires:

• Co-expression with appropriate phosphopantetheinyl transferase

• Verification of modification status using mass spectrometry

• Careful design of purification strategies that preserve the modified form

How can isothermal amplification techniques be optimized for Renibacterium salmoninarum research?

Isothermal amplification techniques offer significant advantages for R. salmoninarum research, particularly for field applications and rapid diagnostics. The optimization of these methods involves several critical considerations:

RPA-CRISPR/Cas12a System Optimization:
The recombinase polymerase amplification (RPA) coupled with CRISPR-Cas12a has shown promising results for R. salmoninarum detection, achieving a sensitivity of approximately 20-40 copies/μL within 10 minutes. Methodological optimization strategies include:

• Target sequence selection: Identifying highly conserved regions of the R. salmoninarum genome that maximize specificity while maintaining sensitivity. The design should account for sequence variability among different strains .

• Primer and crRNA design considerations:

  • Primer length (typically 30-35 nucleotides)

  • GC content (optimally 40-60%)

  • Secondary structure minimization

  • Positioning of the protospacer adjacent motif (PAM) site for efficient Cas12a recognition

• Reaction condition optimization:

  • Temperature (typically 37-42°C)

  • Magnesium concentration

  • Enzyme concentration ratios

  • Incubation time optimization

Assay Validation Framework:
For reliable research applications, validation should include:

• Specificity testing against related bacteria (a panel of at least 10 co-occurring or closely related bacteria is recommended)

• Sensitivity assessment using serial dilutions of quantified DNA

• Environmental sample testing with comparison to established methods (e.g., qPCR)

• Reproducibility assessment across different operators and equipment

Field Application Considerations:
A key advantage of isothermal methods is potential field application. Optimization for field use includes:

• Lyophilization of reagents for ambient temperature stability

• Simplified sample processing workflows

• Integration with portable fluorescence detection systems

• Development of internal controls to verify assay performance

What are the latest methodological advances in studying acyl carrier proteins in bacterial pathogens?

Recent methodological advances have expanded researchers' capabilities to study acyl carrier proteins in bacterial pathogens, including approaches potentially applicable to R. salmoninarum acpP research:

Activity-Based Protein Profiling (ABPP):
This chemical biology approach uses probes that covalently bind to active site residues of enzymes. For acyl carrier proteins:

• Phosphopantetheine analogs with clickable handles allow visualization of active acpP in complex mixtures

• These probes can distinguish between apo and holo forms of the protein

• ABPP can be applied in living cells to monitor acpP activation dynamics

Structural Biology Advances:
Beyond traditional X-ray crystallography, newer approaches include:

• Cryo-electron microscopy for visualizing acpP in complex with partner enzymes

• Nuclear magnetic resonance for studying the dynamic aspects of acpP function

• Hydrogen-deuterium exchange mass spectrometry to map protein-protein interaction surfaces

Systems Biology Integration:
Multi-omics approaches provide comprehensive insights:

• Transcriptomics to identify co-expressed genes in the acpP pathway

• Proteomics to identify interaction partners through proximity labeling

• Metabolomics to track the flux of substrates through acpP-dependent pathways

• Integration of these datasets through computational modeling

Genetic Tool Development:
Advanced genetic manipulation techniques applicable to studying acpP include:

• CRISPR-Cas9 gene editing for precise modification of acpP and associated genes

• Inducible expression systems for controlled production of acpP variants

• Fluorescent protein fusions for tracking acpP localization

• Synthetic biology approaches to reconstitute minimal acpP-dependent systems

How can contradictory data in Renibacterium salmoninarum research be analyzed and reconciled?

Researchers frequently encounter seemingly contradictory data when studying R. salmoninarum, particularly regarding detection methods, virulence factors, and host interactions. A systematic approach to analyzing and reconciling such contradictions includes:

Sources of Variation Identification:
Several factors can contribute to apparently contradictory findings:

• Methodological differences:

  • Detection limit variations between techniques (e.g., RPA-CRISPR/Cas12a vs. qPCR)

  • Sample processing variations affecting nucleic acid recovery

  • Different reference standards for quantification

• Biological variables:

  • Strain differences in R. salmoninarum isolates

  • Host species and population variations

  • Environmental conditions affecting bacterial physiology

  • Infection stage when samples were collected

Analytical Framework for Contradictory Data:
A structured approach to reconciling contradictions includes:

• Meta-analysis methodology:

  • Systematic review of methodological details across studies

  • Statistical analysis accounting for inter-study heterogeneity

  • Identification of moderator variables explaining discrepancies

• Experimental validation:

  • Side-by-side comparison of detection methods using identical samples

  • Multi-laboratory validation with standardized protocols

  • Analysis of diverse R. salmoninarum strains under controlled conditions

Case Study: Reconciling Detection Method Discrepancies:
RPA-CRISPR/Cas12a assays detected R. salmoninarum in 70% of samples previously identified as positive by qPCR. Analysis of the discrepancies revealed:

• A detection threshold effect (reliable detection >10 copies/μL, stochastic detection below)

• Variation in sampling locations within the same environments

• Differences in inhibitor tolerance between the two methods

These findings highlight the importance of considering detection thresholds and sampling strategies when interpreting apparently contradictory results .

What experimental design considerations are crucial when studying recombinant acpP function?

Robust experimental design is critical for meaningful research on recombinant R. salmoninarum acpP function. Key considerations include:

Control Selection and Validation:
The choice of appropriate controls significantly impacts experimental reliability:

• Positive controls:

  • Well-characterized acpP homologs from model organisms (e.g., E. coli)

  • Synthetic substrates with known reaction kinetics

  • Previously validated reaction conditions from related systems

• Negative controls:

  • Inactive acpP variants (site-directed mutagenesis of key residues)

  • Reactions lacking essential cofactors

  • Heat-denatured enzyme preparations

Parameter Optimization Matrix:
A systematic approach to optimization includes:

Reproducibility Considerations:
To ensure reliable results:

• Perform biological replicates across multiple protein preparations

• Include technical replicates for each experimental condition

• Establish statistical power through preliminary studies

• Document detailed protocols including reagent sources and lot numbers

Functional Context Reconstitution:
Since acpP functions as part of a multi-enzyme system, experimental design should consider:

• Minimal reconstituted systems with partner proteins

• Complementation assays in appropriate model organisms

• Comparison of in vitro activity with predicted in vivo function

• Assessment of substrate specificity across physiologically relevant options

What RNA extraction protocols are most effective for Renibacterium salmoninarum research?

Effective RNA extraction is critical for gene expression studies involving R. salmoninarum. The methodological approach must account for the gram-positive cell wall and potential sample complexities:

Protocol Selection Based on Sample Type:

For pure bacterial cultures:

• Harvest cells during appropriate growth phase

• Treat with lysozyme to disrupt the peptidoglycan layer

• Extract using commercial reagents like RNAzol B

• Include DNase I treatment to remove genomic DNA contamination

• Assess RNA quality by measuring optical density at 260/280 nm (optimal ratio: 1.8-2.0)

For infected fish tissues:

• Homogenize tissues (e.g., pronephros, spleen) in RNAzol B or equivalent reagent

• Extract following manufacturer's instructions with modifications for high-lipid content

• Treat with DNase I (Amplification Grade)

• Verify RNA integrity using bioanalyzer or gel electrophoresis

• Standardize input RNA amount (e.g., 5 μg total RNA per reverse transcription reaction)

Critical Quality Control Measures:

• Include no-RT controls to detect genomic DNA contamination

• Use random primers (e.g., 300 ng per reaction) for reverse transcription to ensure comprehensive transcriptome coverage

• Validate extraction efficiency using spiked-in control RNA

• Normalize to constant starting RNA amount rather than cell count

Optimization Considerations:

• For environmental samples, modify protocols to account for potential inhibitors

• When analyzing gene expression during infection, consider differential extraction efficiency between host and pathogen RNA

• For low-biomass samples, include carrier RNA to improve recovery

• Store extracted RNA at -80°C with RNase inhibitors to prevent degradation

How can CRISPR-Cas systems be utilized for Renibacterium salmoninarum detection and research?

CRISPR-Cas systems offer powerful tools for both detection and functional studies of R. salmoninarum:

Detection Applications:

The RPA-CRISPR/AsCas12a detection system demonstrates significant potential for R. salmoninarum research and monitoring:

• Methodological approach:

  • Target DNA amplification using isothermal RPA

  • AsCas12a-mediated recognition of target sequences

  • Trans-cleavage of reporter molecules upon target recognition

  • Fluorescence or colorimetric readout of positive detection

• Performance characteristics:

  • Specificity: 100% (0/10 co-occurring or related bacteria detected)

  • Sensitivity: 0.0128 pg/μL DNA (approximately 20-40 copies/μL)

  • Detection time: 10 minutes after Cas activity initiation

  • Field application potential: High due to isothermal (37°C) reaction conditions

• Optimization strategies:

  • Guide RNA design to target conserved genomic regions

  • Buffer composition adjustment for maximum enzyme activity

  • Reporter molecule selection based on detection context

  • Sample processing protocols to minimize inhibitors

Research Applications Beyond Detection:

CRISPR-Cas systems can also be applied to functional studies of R. salmoninarum:

• Gene function analysis:

  • Targeted gene knockdown/knockout to study virulence factors

  • CRISPRi for inducible gene repression without genome editing

  • Transient expression of Cas9 and guide RNAs via conjugation or transformation

  • Screening approaches to identify essential genes

• Transcriptional regulation studies:

  • dCas9-based systems for modulating gene expression

  • CRISPR activation (CRISPRa) to upregulate gene expression

  • Multiplexed targeting to study gene interaction networks

  • Integration with reporter systems to monitor expression changes

What are the optimal conditions for expressing recombinant proteins from Renibacterium salmoninarum?

Successful expression of recombinant proteins from R. salmoninarum requires careful optimization of multiple parameters:

Expression System Selection:

Based on published work with other bacterial proteins, the pET28a expression system in E. coli represents a practical starting point for most R. salmoninarum proteins, including acpP .

Expression Condition Optimization:

• Temperature considerations:

  • Standard induction at 37°C for 3-4 hours

  • Reduced temperature (16-20°C) for overnight expression to improve folding

  • Assessment of different temperatures based on protein solubility analysis

• Induction parameters:

  • IPTG concentration typically 0.1-1.0 mM

  • Induction timing at mid-log phase (OD600 = 0.6-0.8)

  • Consideration of auto-induction media for gradual protein expression

• Media composition:

  • Rich media (LB, TB) for maximum biomass

  • Minimal media for isotope labeling in structural studies

  • Supplementation with specific cofactors if required for folding or activity

Post-expression Analysis:

• SDS-PAGE analysis of soluble and insoluble fractions

• Western blotting to confirm identity of expressed protein

• Activity assays to verify functional integrity

• Assessment of post-translational modifications where relevant

How can environmental DNA techniques be applied to Renibacterium salmoninarum research?

Environmental DNA (eDNA) approaches offer powerful tools for R. salmoninarum research, particularly for ecological studies and early detection applications:

Sample Collection and Processing Methodology:

• Water sample collection:

  • Sample volume optimization (typically 1-5 liters)

  • Filtration through 0.22-0.45 μm filters

  • Preservation methods (ethanol, buffer solutions, or immediate processing)

  • Field collection protocols to minimize contamination

• DNA extraction optimization:

  • Commercial kits modified for environmental samples

  • Inclusion of additional purification steps to remove inhibitors

  • Addition of carrier DNA for low-abundance samples

  • Internal extraction controls to assess efficiency

Detection and Quantification Approaches:

• Conventional methods:

  • qPCR targeting R. salmoninarum-specific sequences

  • Digital PCR for absolute quantification

  • Nested PCR for improved sensitivity in complex samples

• Advanced approaches:

  • RPA-CRISPR/Cas12a showing limit of detection comparable to qPCR

  • Metabarcoding for simultaneous detection of multiple pathogens

  • Nanopore sequencing for real-time field detection

  • Droplet digital PCR for absolute quantification without standard curves

Research Applications:

Environmental DNA techniques enable several research approaches:

• Tracking pathogen distribution in natural waterways

• Monitoring hatchery water systems for early detection

• Studying transmission dynamics between wild and farmed populations

• Evaluating effectiveness of disease management interventions

Data Interpretation Framework:

Proper interpretation of eDNA results requires consideration of:

• Detection limits (>10 copies/μL for reliable detection, stochastic detection below)

• Correlation between eDNA concentration and infection prevalence

• Persistence of DNA after bacterial death

• Potential for environmental inhibitors affecting detection reliability

The RPA-CRISPR/Cas12a assay successfully detected R. salmoninarum eDNA in 70% of water samples previously identified as positive by qPCR, demonstrating its utility while highlighting the need for understanding detection thresholds and environmental variables affecting performance .