Recombinant Campylobacter jejuni subsp. jejuni serotype O:2 Protein CrcB homolog (crcB)

What is the basic structure and function of CrcB homolog in Campylobacter jejuni?

CrcB homolog in C. jejuni functions primarily as a membrane protein involved in ion transport, particularly fluoride ion efflux. While detailed structural information specific to C. jejuni CrcB is limited, homology with other bacterial CrcB proteins suggests multiple transmembrane domains forming ion transport channels.

Methodological approach:
To characterize CrcB structure-function relationships, researchers should employ:

• Sequence alignment with known CrcB proteins from other species

• Transmembrane topology prediction using algorithms like TMHMM and Phobius

• Membrane protein expression systems with epitope tags for localization studies

• Ion transport assays using fluoride-sensitive probes

• Site-directed mutagenesis of predicted functional residues

The protein appears to be co-regulated in specific modules with other genes involved in carbohydrate metabolic processes and transferase activity, suggesting integration in broader metabolic networks .

How is the crcB gene regulated in C. jejuni?

Regulation of crcB in C. jejuni likely responds to environmental conditions, particularly pH and oxidative stress.

Methodological approach:
To investigate crcB regulation:

• Perform RT-qPCR analysis under varying environmental conditions

• Create transcriptional fusions with reporter genes (e.g., lacZ, GFP)

• Conduct promoter mapping through 5' RACE

• Identify potential transcription factor binding sites through ChIP-seq

Regulatory studies should consider that C. jejuni competence (which may correlate with crcB expression) significantly increases from pH 6.5 to 7.5, with no competence observed below pH 5 . Additionally, aerobic conditions abolish competence development but not DNA uptake in already competent cells .

What experimental approaches are most effective for generating recombinant CrcB protein?

Methodological approach:
For recombinant CrcB expression and purification:

• Expression system selection:

  • E. coli strains specialized for membrane proteins (C41/C43)

  • Homologous expression in C. jejuni for authentic processing

  • Cell-free systems for toxic proteins

• Construct optimization:

  • Codon optimization for expression host

  • Fusion tags (His, MBP, SUMO) to enhance solubility

  • Signal sequence modification if necessary

• Expression conditions:

  • Lower temperature induction (16-20°C)

  • Reduced inducer concentration

  • Membrane-mimetic environments

• Purification strategy:

  • Detergent screening (DDM, LMNG, etc.)

  • Two-step chromatography (affinity and size exclusion)

  • Quality assessment by SDS-PAGE and Western blotting

For C. jejuni genetic manipulation, researchers can adapt the overlapping PCR protocol described for marker strain construction, involving amplification and joining of three DNA fragments: 1) resistance gene, 2) upstream flanking region, and 3) downstream flanking region of the target gene .

How does CrcB contribute to horizontal gene transfer in C. jejuni?

C. jejuni exhibits remarkable genetic plasticity through horizontal gene transfer (HGT), with potential involvement of membrane proteins like CrcB.

Methodological approach:
To investigate CrcB's role in HGT:

• Generate crcB deletion and overexpression strains

• Conduct recombination assays using marker strains with distinct antibiotic resistance genes

• Quantify natural transformation efficiency using the protocol described in search results

• Test under various environmental conditions, particularly with chicken cecal content which increases recombination efficiency approximately 10-fold compared to standard media

The significant reduction in recombination efficiency with DNase I treatment (99.92% decrease) suggests transformation primarily occurs through extracellular DNA uptake . Researchers should investigate whether CrcB affects membrane properties related to DNA binding and uptake.

What role does CrcB play in C. jejuni survival under environmental stress?

Methodological approach:
To assess CrcB's contribution to stress responses:

• Compare wild-type and crcB mutant strains under:

  • pH stress (4.0-8.0)

  • Oxidative stress (H₂O₂, paraquat)

  • Osmotic stress (high salt)

  • Temperature fluctuations

  • Bile salt exposure

• Quantitative assessment methods:

  • Survival curve analysis

  • Growth rate determination

  • Membrane integrity assays

  • Biofilm formation capacity

Environmental adaptation is particularly significant given that certain hybrid C. coli strains containing C. jejuni genetic material (possibly including genes like crcB) were preferentially isolated from egg shells, a dry and harmful environment for Campylobacter . This suggests potential selection for strains with enhanced stress resistance capabilities.

How does pH affect CrcB function and natural transformation in C. jejuni?

pH significantly impacts C. jejuni competence development and potentially CrcB function.

Methodological approach:
To investigate pH effects:

• Utilize single-cell DNA uptake assays to monitor competence development at different pH values

• Examine crcB expression levels across pH range using RT-qPCR

• Assess membrane potential changes with pH-sensitive fluorescent probes

• Measure ion transport activity of CrcB at varying pH

The increased competence at pH 7.5 has significant implications as this pH is typical of the poultry intestine, suggesting extensive genetic exchange may occur in this host environment .

How does CrcB homolog in C. jejuni compare to homologs in other bacterial species?

Methodological approach:
For comparative analysis:

• Conduct comprehensive phylogenetic analysis of CrcB homologs across bacterial species

• Perform functional complementation studies:

  • Express C. jejuni CrcB in other bacterial species with crcB mutations

  • Express heterologous CrcB proteins in C. jejuni crcB mutants

• Identify conserved and variable regions through sequence alignment

• Compare function under standardized conditions

The CrcB homolog in Mycobacterium tuberculosis (Rv3069) is associated with carbohydrate metabolic processes and growth on cholesterol , which may indicate similar metabolic roles in C. jejuni. This comparative approach can reveal functional conservation and species-specific adaptations.

What is the molecular mechanism by which CrcB influences DNA uptake during natural transformation?

Methodological approach:
To elucidate molecular mechanisms:

• Conduct structural studies of CrcB using:

  • Cryo-electron microscopy

  • Solid-state NMR for membrane proteins

  • Molecular dynamics simulations

• Examine protein-DNA interactions:

  • DNA binding assays with purified CrcB

  • Crosslinking studies followed by mass spectrometry

  • Single-molecule FRET to track conformational changes during DNA binding

• Investigate protein-protein interactions:

  • Co-immunoprecipitation with known competence proteins

  • Bacterial two-hybrid screening

  • Proximity labeling approaches (BioID, APEX)

• Create a competence interactome map using proteomics approaches

Recent research demonstrates that C. jejuni undergoes extensive genetic exchange that may enhance its adaptive potential . Understanding CrcB's specific role in this process could provide insights into C. jejuni's remarkable genetic plasticity.

How can single-cell assays be optimized to study CrcB's role in natural transformation?

Methodological approach:
For single-cell analysis:

• Adapt the single cell-based uptake assay described for monitoring competence development :

  • Incorporate fluorescently labeled DNA

  • Use microfluidic chambers for controlled environment

  • Employ time-lapse microscopy for real-time observation

• Design dual-reporter systems:

  • Fluorescent protein fusions to track CrcB localization

  • Separate fluorophores to monitor competence development

  • Correlation analysis between CrcB localization and DNA uptake

• Control environmental parameters:

  • pH gradients (particularly between 6.5-7.5)

  • Oxygen tension (aerobic vs. microaerobic)

  • Presence of host factors (chicken cecal extract)

The assay development should consider that aerobic conditions abolish competence development but not DNA uptake in already competent C. jejuni cells , suggesting complex regulation of the transformation process.

What systems biology approaches can integrate CrcB function into global C. jejuni adaptation networks?

Methodological approach:
For systems-level analysis:

• Multi-omics integration:

  • Transcriptomics: RNA-seq of wild-type vs. crcB mutants

  • Proteomics: Quantitative analysis of protein expression changes

  • Metabolomics: Profiling of metabolic shifts

  • Fluxomics: 13C metabolic flux analysis

• Network reconstruction:

  • Protein-protein interaction mapping

  • Genetic interaction screening through Tn-seq

  • Regulatory network inference

  • Metabolic pathway modeling

• Computational approaches:

  • Machine learning for pattern identification

  • Network analysis to identify critical nodes

  • Constraint-based modeling for phenotype prediction

This integrated approach could reveal how CrcB contributes to C. jejuni's ability to switch hosts and/or survive in challenging environments through extensive genetic exchange .

How does CrcB interact with the bacterial membrane to potentially facilitate DNA uptake?

Methodological approach:
To investigate membrane interactions:

• Advanced membrane studies:

  • Lipid nanodisc reconstitution of purified CrcB

  • Super-resolution microscopy for localization patterns

  • Atomic force microscopy for topographical analysis

  • Membrane fluidity measurements with fluorescent probes

• Protein dynamics:

  • Hydrogen-deuterium exchange mass spectrometry

  • Single-molecule tracking in live cells

  • FRET analysis of conformational changes

• Functional correlation:

  • Electrophysiology to measure ion flux

  • Membrane permeability assays

  • DNA binding capacity of membrane fractions

Understanding CrcB's membrane interactions could explain how it potentially contributes to competence for natural transformation, which has been shown to be affected by environmental factors like pH and oxygen levels .

What contradictory findings exist in the literature regarding CrcB function, and how can these be resolved?

Methodological approach:
To address contradictory findings:

• Meta-analysis of existing literature:

  • Systematic review of experimental conditions

  • Identification of methodological differences

  • Strain variation assessment

• Standardized experimental design:

  • Consistent growth conditions and media

  • Defined genetic backgrounds

  • Multiple assay systems for functional validation

• Advanced genetic approaches:

  • CRISPR-Cas9 precise genomic editing

  • Allelic series creation to test structure-function relationships

  • Inducible expression systems for dose-dependent studies

• Multi-laboratory validation studies with standardized protocols

The extensive genetic exchange capabilities of Campylobacter might explain some contradictory findings, as genome plasticity could lead to strain-specific functional differences . Careful consideration of strain backgrounds and environmental conditions is essential for reproducible results.