Recombinant Campylobacter jejuni subsp. jejuni serotype O:6 Protein CrcB homolog (crcB)

What is the genomic context of the crcB homolog in Campylobacter jejuni subsp. jejuni serotype O:6?

The crcB homolog in C. jejuni is typically found within the chromosomal DNA rather than in plasmids. Based on comparative genomic analyses of C. jejuni strains, the gene encoding this protein exists in a relatively conserved region across different serotypes, though specific flanking genes may vary between strains. When investigating the genomic context, researchers should employ whole genome sequencing followed by comparative genomic analysis with reference strains such as NCTC 11168, 81-176, and 81116 . For precise mapping, PCR assays using locus-specific primers should be designed to amplify both the crcB gene and its flanking regions, allowing confirmation of its chromosomal position and identification of potential regulatory elements .

What experimental protocols are recommended for expressing recombinant CrcB protein from C. jejuni?

Expression of recombinant CrcB requires careful optimization due to the membrane-associated nature of this protein. An effective expression protocol involves:

• Gene amplification using high-fidelity PCR from C. jejuni serotype O:6 genomic DNA

• Cloning into an expression vector with a suitable promoter (T7 or tac promoters work well)

• Transformation into an appropriate E. coli expression strain (BL21(DE3) or similar)

• Expression induction using IPTG at concentrations between 0.1-0.5 mM

• Growth at lower temperatures (16-25°C) to enhance proper folding of membrane proteins

For purification, a combination of detergent solubilization (using mild detergents like DDM or LDAO) and affinity chromatography yields the most functional protein. Western blotting using anti-His or custom anti-CrcB antibodies should be employed to confirm expression and purification success .

How do researchers verify the functional activity of recombinant CrcB homolog protein?

Verification of CrcB functional activity requires multiple complementary approaches:

• Ion transport assays: Using fluoride-sensitive probes to measure transport activity across membranes in proteoliposomes containing reconstituted CrcB protein.

• Complementation studies: Introducing the recombinant crcB gene into crcB knockout strains to assess restoration of fluoride resistance.

• Electrophysiology: Patch-clamp techniques can be used to directly measure ion conductance through CrcB channels.

• Binding assays: Isothermal titration calorimetry (ITC) or microscale thermophoresis (MST) to measure fluoride binding constants.

A comprehensive functional verification should include at least two different methodological approaches, with appropriate controls including inactive protein mutants .

What are the best approaches for studying the membrane topology of CrcB in C. jejuni?

The membrane topology of CrcB can be investigated using:

• Computational prediction: Initial topology prediction using algorithms like TMHMM, Phobius, or TOPCONS.

• Cysteine scanning mutagenesis: Systematically replacing residues with cysteine and using membrane-impermeable sulfhydryl reagents to identify exposed regions.

• GFP fusion constructs: Creating fusion proteins with GFP at different predicted loops to determine orientation.

• Protease protection assays: Limited proteolysis of membrane preparations followed by mass spectrometry to identify protected fragments.

The comprehensive topology model should integrate results from at least three independent experimental approaches. Current models suggest CrcB typically contains multiple transmembrane domains with both N and C termini facing the cytoplasm .

How can researchers investigate horizontal genetic exchange involving the crcB homolog in C. jejuni populations?

Investigating horizontal genetic exchange of crcB requires sophisticated genetic tracking systems. One effective methodology involves:

• Construction of marker strains carrying distinct antibiotic resistance genes flanking the crcB locus

• Co-culture of different marker strains under conditions that promote DNA exchange

• Selection of recombinants on media containing multiple antibiotics

• PCR verification of recombination events using locus-specific primers

Recent studies demonstrate that the recombination efficiency in C. jejuni can reach 0.02811 ± 0.0035% of parental strains under optimized conditions . This efficiency increases approximately 10-fold when chicken cecal content is added to the growth medium, suggesting environmental factors significantly impact horizontal genetic exchange. The recombination efficiency data is summarized in the table below:

These experiments should include DNase I controls to determine the contribution of transformation versus conjugation mechanisms .

What approaches can be used to investigate the potential role of CrcB in antimicrobial resistance in C. jejuni serotype O:6?

Investigating CrcB's role in antimicrobial resistance requires multi-level analyses:

• Generation of crcB knockout mutants: Using homologous recombination to replace crcB with an antibiotic resistance cassette, confirming by PCR and sequencing .

• Comparative MIC testing: Determining minimum inhibitory concentrations of various antimicrobials against wild-type, crcB knockout, and complemented strains.

• Transcriptomic analysis: RNA-seq to identify genes differentially expressed in response to crcB deletion, especially under antimicrobial stress.

• Fluoride resistance assays: Testing growth in increasing concentrations of sodium fluoride, as CrcB proteins are known to confer fluoride resistance.

• In vivo colonization studies: Assessing the ability of crcB mutants to colonize animal models when challenged with antimicrobials.

The experimental design should include appropriate controls and multiple biological replicates to account for C. jejuni's genetic variability. Results should be validated across different strains of the same serotype to ensure generalizability .

How does the molecular structure of CrcB homolog from C. jejuni serotype O:6 differ from other bacterial CrcB proteins, and what methodologies are appropriate for structural determination?

Structural determination of CrcB homolog requires specialized approaches due to the challenges of membrane protein crystallization:

• X-ray crystallography: Requires detergent screening to identify conditions that maintain protein stability while permitting crystal formation. Lipidic cubic phase crystallization has shown success with similar membrane proteins.

• Cryo-electron microscopy: Increasingly the method of choice for membrane proteins, enabling structural determination in a more native-like lipid environment.

• NMR spectroscopy: Suitable for determining dynamic structural features, particularly for smaller domains or loops connecting transmembrane segments.

• Computational modeling: Homology modeling based on related structures, validated by experimental data from cross-linking or mutagenesis studies.

A combination of structural approaches provides the most comprehensive understanding. When comparing C. jejuni CrcB to homologs from other bacteria, particular attention should be paid to the fluoride ion coordination site and transmembrane domains, as these regions likely determine functional specificity .

What is the relationship between CrcB expression and virulence in C. jejuni serotype O:6, and how can this be experimentally determined?

The relationship between CrcB and virulence can be investigated through:

• Virulence gene expression profiling: qRT-PCR or RNA-seq comparing expression of known virulence factors between wild-type and crcB mutant strains.

• Cell invasion assays: Using human intestinal epithelial cell lines to compare invasion efficiency of wild-type versus crcB mutants.

• Animal infection models: Testing colonization and pathogenicity in appropriate animal models, such as chickens or specialized mouse models.

• Stress response assays: Measuring survival under various stress conditions (acid, bile, oxidative stress) relevant to host colonization.

• Comparative genomics: Analyzing crcB sequence variations across strains with different virulence profiles. The mosaic structure of virulence-associated plasmids in C. jejuni (such as ICDCCJ07001_pTet) suggests complex relationships between genomic elements .

Researchers should note that C. jejuni virulence is multifactorial, with significant roles played by multiple systems including LOS biosynthesis loci, polysaccharide capsular biosynthesis (CPS) loci, and flagella modification loci . Therefore, experiments should control for potential confounding factors and strain variations.

How can researchers design effective experiments to study the impact of environmental conditions on crcB expression and function in C. jejuni?

Designing experiments to study environmental regulation of crcB requires:

• Promoter reporter systems: Fusion of the crcB promoter to reporter genes like luciferase or GFP to monitor expression under different conditions.

• qRT-PCR assays: Measuring crcB transcript levels under various environmental conditions (temperature, pH, oxygen levels, nutrient availability, fluoride concentration).

• Proteomics approaches: Quantitative proteomics to measure CrcB protein levels and potential post-translational modifications under different conditions.

• In vivo expression studies: Measuring expression during colonization of different host environments (e.g., chicken cecum vs. human cell cultures).

• ChIP-seq analysis: Identifying transcription factors that bind to the crcB promoter region under different conditions.

The experimental design should incorporate accurate modeling of relevant environments, including microaerobic conditions (5% O₂, 10% CO₂, 85% N₂), temperature shifts (37°C, 42°C), and exposure to host-derived factors. Statistical analysis should account for the high genetic variability observed in C. jejuni populations .

What are the recommended genetic tools for manipulating the crcB gene in C. jejuni serotype O:6?

Genetic manipulation of crcB in C. jejuni requires specific tools optimized for this organism:

• Shuttle vectors: Use of E. coli-Campylobacter shuttle vectors like pRY112 that contain compatible origins of replication.

• Selection markers: Chloramphenicol and kanamycin resistance genes work effectively in C. jejuni.

• Transformation protocols: Natural transformation is highly efficient in C. jejuni when using biphasic media systems. Electroporation at 2,500V is an effective alternative.

• Homologous recombination: For chromosomal integration, homologous flanking regions of approximately 400bp are optimal, as demonstrated in marker insertion experiments .

• CRISPR-Cas9 systems: Recently adapted for C. jejuni, though optimization may be required for specific strains.

The recombination efficiency varies considerably between strains, so preliminary transformation efficiency tests should be conducted when working with new isolates. The biphasic culture system significantly enhances transformation efficiency compared to liquid or solid media alone .

What bioinformatic approaches are most effective for analyzing the evolutionary relationships between CrcB homologs across different C. jejuni strains and serotypes?

Effective bioinformatic analysis of CrcB evolutionary relationships includes:

• Sequence retrieval: Comprehensive collection of crcB sequences from diverse C. jejuni strains and related species through database mining (NCBI, UniProt).

• Multiple sequence alignment: Using MUSCLE or MAFFT with parameters optimized for membrane proteins.

• Phylogenetic analysis: Maximum likelihood and Bayesian inference methods to construct robust phylogenetic trees.

• Selection pressure analysis: Calculating dN/dS ratios to identify regions under purifying or diversifying selection.

• Structural mapping: Mapping sequence conservation onto predicted structural models to identify functionally important residues.

• Serotype correlation: Analysis of crcB sequence variations in context of Penner serotyping data to identify potential associations with capsule polysaccharide (CPS) structures .

The analysis should account for horizontal gene transfer events, which are common in C. jejuni populations and can complicate traditional phylogenetic inference. Recombination detection algorithms such as RDP4 or ClonalFrameML should be incorporated into the analysis pipeline .

How can researchers accurately quantify and compare CrcB protein expression levels across different experimental conditions?

Accurate quantification of CrcB expression requires:

• Western blotting: Using specific anti-CrcB antibodies with careful optimization of membrane protein extraction protocols.

• Quantitative proteomics: LC-MS/MS using techniques like SILAC, TMT, or label-free quantification to compare protein abundance.

• Flow cytometry: For cells expressing tagged CrcB variants that can be detected with fluorescent antibodies.

• Targeted selected reaction monitoring (SRM): For highly sensitive and specific quantification of CrcB peptides.

• Transcriptional analysis: qRT-PCR with validated reference genes appropriate for the experimental conditions being tested.

When comparing expression across conditions, researchers should normalize data using multiple housekeeping proteins that maintain stable expression under the tested conditions. For membrane proteins like CrcB, careful attention to extraction efficiency is crucial, as different conditions may affect membrane composition and protein extractability .

What are the key considerations for designing antigenic epitopes from CrcB for antibody production and immunological studies?

When designing antigenic epitopes from CrcB for antibody production:

• Epitope prediction: Use of algorithms that combine hydrophilicity, surface accessibility, and antigenicity scores to identify candidate epitopes.

• Topological considerations: Focus on extracellular or periplasmic loops rather than transmembrane regions, which are often poorly immunogenic and less accessible.

• Conjugation strategies: Coupling selected peptides to carrier proteins like KLH or BSA to enhance immunogenicity.

• Cross-reactivity testing: Screening against related proteins from commensal bacteria to ensure specificity.

• Validation methods: Confirming antibody specificity using wild-type and crcB knockout strains.

For optimal results, researchers should generate antibodies against multiple epitopes and pool them for applications requiring high sensitivity. Both polyclonal and monoclonal approaches should be considered, with the latter offering higher specificity but potentially lower sensitivity .

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

When faced with contradictory results between in vitro and in vivo studies:

• Environmental context assessment: Evaluate differences in experimental conditions between systems, particularly oxygen levels, pH, and presence of host factors.

• Strain variation considerations: Verify genetic stability of the strain during passage and experimentation, as C. jejuni is known for genomic plasticity.

• Methodological validation: Confirm that phenotypes observed in vitro are not artifacts of the experimental setup through multiple methodological approaches.

• Host interaction factors: Consider that host environments contain factors that may alter gene expression or protein function not replicated in vitro.

• Statistical robustness: Ensure sufficient biological replicates in both systems to account for inherent variability.

Recent studies demonstrate that chicken cecal content increases the recombination efficiency of C. jejuni approximately 10-fold compared to laboratory media, highlighting the profound impact of environmental factors on bacterial genetic processes . Similarly, factors in the host environment may significantly influence CrcB function in ways not apparent in simplified laboratory systems.

What are the most common technical challenges when working with recombinant CrcB protein, and how can they be addressed?

Common technical challenges with recombinant CrcB include:

• Low expression levels: Overcome by optimizing codon usage for the expression host, using stronger promoters, or testing different expression strains.

• Protein misfolding: Address by expressing at lower temperatures (16-25°C), adding folding enhancers (glycerol, sucrose), or using specific E. coli strains designed for membrane protein expression.

• Aggregation during purification: Minimize by carefully selecting detergents, adding stabilizing agents, and avoiding freezing-thawing cycles.

• Loss of function during reconstitution: Improve by reconstituting into lipid compositions similar to C. jejuni membranes and validating function using multiple assays.

• Difficulty obtaining crystals: Explore alternative structural determination methods such as cryo-EM or use nanobodies to stabilize the protein.

When troubleshooting, systematic variation of expression parameters is recommended, testing at least three different detergents and two expression systems before exploring more substantial modifications to the protein construct .

How can researchers effectively control for strain variation when studying CrcB function across different C. jejuni isolates?

To control for strain variation:

• Genome sequencing verification: Perform whole genome sequencing of all strains to identify potential confounding genetic differences.

• Isogenic strain construction: Generate mutations in the same parental background to minimize variation from other genetic factors.

• Complementation controls: Introduce the wild-type gene back into mutant strains to confirm phenotypes are specifically linked to crcB.

• Multi-strain validation: Test key findings across multiple independent isolates of the same serotype.

• Background normalization: When comparing different wild-type backgrounds, normalize functional data to account for inherent strain differences.

What statistical approaches are most appropriate for analyzing complex phenotypic data related to CrcB function in C. jejuni?

For complex phenotypic data analysis:

• Mixed-effects models: Account for both fixed effects (experimental conditions) and random effects (biological variation between replicates).

• Non-parametric methods: When data does not follow normal distribution, use Kruskal-Wallis or Mann-Whitney tests.

• Multivariate analysis: Principal Component Analysis (PCA) or clustering methods to identify patterns in multidimensional phenotypic data.

• Time-series analysis: For kinetic data, use repeated measures ANOVA or growth curve modeling.

• Machine learning approaches: For identifying complex patterns in large datasets, consider supervised learning algorithms.

Statistical power analysis should be performed prior to experimentation to determine appropriate sample sizes. For C. jejuni, which shows higher variability than many other bacterial models, larger sample sizes are typically required (n≥6 biological replicates) to achieve sufficient statistical power (β≥0.8) .

What emerging technologies could advance our understanding of CrcB function in C. jejuni serotype O:6?

Emerging technologies with potential to advance CrcB research include:

• Single-cell approaches: Techniques like single-cell RNA-seq and time-lapse microscopy to study heterogeneity in CrcB expression and function within bacterial populations.

• Advanced imaging techniques: Super-resolution microscopy and correlative light and electron microscopy (CLEM) to visualize CrcB localization and dynamics in living cells.

• Microfluidic systems: Devices that can create defined environmental gradients to study CrcB regulation under precisely controlled conditions.

• In situ structural determination: Techniques like cellular cryo-electron tomography to study CrcB structure in its native membrane environment.

• CRISPR interference (CRISPRi): For precise, tunable transcriptional control of crcB to study dosage effects.

These technologies will be particularly valuable for understanding the temporal and spatial aspects of CrcB function in response to changing environmental conditions, which are difficult to capture with traditional experimental approaches .

How might research on CrcB homologs in C. jejuni contribute to our broader understanding of bacterial ion transport mechanisms?

Research on CrcB in C. jejuni may contribute to broader understanding of bacterial ion transport through:

• Evolutionary insights: Comparative analysis of CrcB homologs across species can reveal conserved mechanisms and specialized adaptations in ion transport.

• Structure-function relationships: Detailed characterization of C. jejuni CrcB structure may identify novel ion coordination mechanisms applicable to other transport systems.

• Regulatory network discovery: Mapping the regulatory pathways controlling crcB expression may reveal shared control mechanisms for ion homeostasis genes.

• Environmental adaptation mechanisms: Understanding how CrcB function changes in response to environmental factors may illuminate general principles of bacterial adaptation.

• Host-microbe interaction principles: Clarifying CrcB's role during host colonization could reveal general mechanisms by which bacterial ion transport systems adapt to host environments.