Recombinant Campylobacter jejuni subsp. jejuni serotype O:23/36 Protein CrcB homolog (crcB)

What is the predicted function of CrcB homolog in Campylobacter jejuni?

The CrcB homolog in C. jejuni is annotated as a putative fluoride ion transporter based on sequence homology with characterized CrcB proteins from other bacterial species . This membrane protein likely contributes to fluoride resistance by facilitating the export of fluoride ions from the bacterial cytoplasm, thereby preventing inhibition of enzymes sensitive to this halide.

To experimentally validate this function, researchers should:

• Generate C. jejuni knockout strains lacking the crcB gene

• Compare growth kinetics between wild-type and ΔcrcB strains in media with varying fluoride concentrations

• Perform fluoride uptake/efflux assays using fluoride-selective electrodes with membrane vesicles prepared from both strains

• Conduct complementation studies with recombinant CrcB to confirm restoration of fluoride resistance

What is the optimal expression system for producing functional recombinant CrcB homolog?

Based on published research, E. coli has been successfully used as an expression host for CrcB homolog with an N-terminal histidine tag . For membrane proteins like CrcB, consider the following methodological approach:

For C. jejuni membrane proteins with multiple transmembrane domains, E. coli C41/C43 strains often provide better results due to their adaptation to membrane protein overexpression. The addition of 0.5-1% glucose during growth can help reduce leaky expression and toxicity before induction.

What purification protocol yields highly pure and active CrcB homolog protein?

For CrcB homolog purification, a multi-step approach is recommended based on published protocols for similar membrane proteins :

• Solubilization: After cell lysis, solubilize membranes in buffer containing an appropriate detergent (e.g., n-dodecyl-β-D-maltoside at 1% w/v, or LDAO at 1.5% w/v)

• IMAC Purification: Apply solubilized material to Ni-NTA resin, wash with buffer containing 20-50 mM imidazole and 0.1% detergent, then elute with 250-500 mM imidazole

• Size Exclusion Chromatography: For highest purity (>90%), perform gel filtration in buffer with detergent concentration just above CMC

• Quality Control: Assess purity by SDS-PAGE and western blotting; verify protein identity by mass spectrometry

• Storage: Store in Tris/PBS-based buffer containing 6% trehalose at pH 8.0 with 5-50% glycerol to prevent freeze-thaw damage

For long-term stability, aliquot and store at -80°C, avoiding repeated freeze-thaw cycles which significantly reduce protein activity.

How can I assess fluoride transport activity of purified recombinant CrcB homolog?

To directly measure fluoride transport by recombinant CrcB, researchers should implement a reconstitution system using the following methodological approach:

• Proteoliposome Preparation:

  • Mix purified CrcB (protein:lipid ratio of 1:100-1:200) with E. coli polar lipids or synthetic lipid mixtures

  • Remove detergent using Bio-Beads or dialysis

  • Confirm vesicle formation by dynamic light scattering

• Transport Assays:

  • Option A: Load proteoliposomes with potassium phosphate buffer (pH 7.0)

  • Option B: Include a pH-sensitive fluorescent dye inside vesicles

  • Add NaF to external medium (0.1-10 mM range)

  • Monitor either fluoride uptake using a fluoride-selective electrode or pH changes using fluorescence spectroscopy

• Controls and Validation:

  • Empty liposomes (negative control)

  • Proteoliposomes with known fluoride transporters (positive control)

  • Addition of protonophores to assess energy dependence

  • Varying buffer pH to determine optimal transport conditions

This approach provides quantitative data on transport kinetics, including Km and Vmax values that can be compared with other characterized fluoride transporters.

What methods can detect interactions between CrcB homolog and other C. jejuni proteins?

To identify potential protein-protein interactions involving CrcB homolog in C. jejuni, implement the following complementary approaches:

• Co-immunoprecipitation:

  • Generate C. jejuni strains expressing epitope-tagged CrcB

  • Solubilize membranes with mild detergents (digitonin or CHAPS)

  • Perform pull-down assays with anti-tag antibodies

  • Identify interacting partners by mass spectrometry

• Bacterial Two-Hybrid System:

  • Clone crcB and candidate genes into appropriate vectors

  • Transform into reporter strains and assess interaction strength

  • Validate using protein fragments to map interaction domains

• Crosslinking Studies:

  • Treat intact C. jejuni cells with membrane-permeable crosslinkers

  • Isolate CrcB-containing complexes by affinity purification

  • Identify crosslinked proteins by tandem mass spectrometry

These methods can reveal whether CrcB functions independently or as part of a larger complex, potentially connecting fluoride transport to other cellular processes in C. jejuni.

What is the optimal strategy for generating crcB gene knockouts in C. jejuni?

Creating precise crcB gene knockouts in C. jejuni requires careful methodological considerations due to the organism's transformation efficiency and homologous recombination properties. Based on techniques described for similar genetic manipulations in C. jejuni , implement the following protocol:

• Knockout Construct Design:

  • Amplify ~400 bp upstream and downstream flanking regions of crcB

  • Join these fragments to an antibiotic resistance cassette (e.g., chloramphenicol or kanamycin resistance)

  • Create the construct using overlapping PCR as described in the literature

• Transformation Protocol:

  • Prepare electrocompetent C. jejuni cells from mid-log phase cultures

  • Transform knockout construct using electroporation at 2,500 V

  • Plate on selective media containing the appropriate antibiotic

  • Incubate at 42°C under microaerobic conditions

• Verification of Knockout:

  • Design PCR primer sets for junction verification where:

    • Forward primer anneals outside the antibiotic cassette

    • Reverse primer anneals inside the antibiotic cassette

  • This approach ensures amplification only occurs when the antibiotic marker is correctly inserted into the genomic locus

  • Confirm loss of crcB expression by RT-PCR or Western blotting

• Phenotypic Validation:

  • Test knockout strains for altered sensitivity to fluoride

  • Assess changes in membrane permeability

  • Evaluate colonization ability in appropriate models

This methodology has proven successful for generating precise gene knockouts in C. jejuni as demonstrated in similar genetic manipulation studies .

How can I determine the expression pattern of crcB in different C. jejuni growth conditions?

To comprehensively analyze crcB expression patterns across different environmental conditions, implement a multi-faceted approach:

• Quantitative RT-PCR Analysis:

  • Design primers specific to crcB with amplicon size of 100-150 bp

  • Grow C. jejuni under various conditions (different temperatures, pH values, growth phases, nutrient limitations)

  • Extract total RNA and synthesize cDNA

  • Perform qRT-PCR using appropriate reference genes (16S rRNA, rpoA)

  • Calculate relative expression using the 2^(-ΔΔCT) method

• Reporter Gene Assays:

  • Create transcriptional fusion between crcB promoter and a reporter gene (gfp or luxCDABE)

  • Integrate construct into C. jejuni chromosome

  • Monitor reporter activity under different environmental conditions

• Proteomics Approach:

  • Grow C. jejuni under different conditions

  • Perform membrane fractionation

  • Quantify CrcB protein levels using targeted proteomics (MRM-MS)

  • Compare expression levels relative to other membrane proteins

• Data Integration:

  • Correlate expression levels with physiological parameters

  • Identify potential transcriptional regulators through promoter analysis

  • Compare with expression of other fluoride resistance genes

This methodological approach will reveal environmental factors that influence crcB expression and provide insights into its regulation network in C. jejuni.

How does CrcB homolog contribute to C. jejuni pathogenesis and host colonization?

While direct evidence linking CrcB to C. jejuni pathogenesis is limited, researchers can investigate this relationship through a systematic experimental approach:

• Colonization Studies:

  • Compare colonization efficiency of wild-type and ΔcrcB strains in animal models

  • Measure bacterial loads in different intestinal segments

  • Assess competition between wild-type and mutant strains in co-infection models

• Virulence Factor Expression:

  • Evaluate whether CrcB deficiency affects expression of known virulence factors

  • Perform RNA-seq comparing transcriptomes of wild-type and ΔcrcB strains

  • This approach could reveal connections to pathways like those involved in post-infection irritable bowel syndrome described in the literature

• Host Response Analysis:

  • Examine whether ΔcrcB strains elicit different inflammatory responses

  • Measure cytokine production in cell culture models

  • Assess epithelial barrier integrity following infection

  • Investigate potential connections to DNA damage mechanisms identified in other C. jejuni studies

• Fluoride Concentration in Gastrointestinal Environment:

  • Determine fluoride levels in different intestinal compartments

  • Assess whether fluoride stress affects C. jejuni colonization ability

  • Test whether dietary fluoride supplementation affects infection outcomes

This methodological framework will help determine whether CrcB-mediated fluoride resistance contributes to C. jejuni survival during infection and colonization processes.

What bioinformatic approaches can identify CrcB homolog evolutionary relationships across bacterial species?

For comprehensive evolutionary analysis of CrcB homologs, implement the following multi-step bioinformatic workflow:

• Sequence Collection and Alignment:

  • Retrieve CrcB sequences from diverse bacterial genomes using BLAST

  • Perform multiple sequence alignment using MUSCLE or MAFFT with parameters optimized for membrane proteins

  • Refine alignments manually focusing on transmembrane regions

• Phylogenetic Analysis:

  • Construct phylogenetic trees using maximum likelihood (RAxML or IQ-TREE)

  • Implement appropriate substitution models (LG+F+G or LG+I+G)

  • Assess node support through 1000 bootstrap replicates

  • Root trees using distantly related sequences or midpoint rooting

• Structural Conservation Mapping:

  • Generate structural predictions using AlphaFold2 or RoseTTAFold

  • Map conservation scores onto predicted structures

  • Identify conserved residues likely essential for function

• Horizontal Gene Transfer Detection:

  • Calculate GC content and codon usage bias

  • Perform reconciliation analysis between gene and species trees

  • Use methods like GARD to detect recombination events

  • This approach can expand on horizontal gene transfer mechanisms described for C. jejuni

• Data Visualization and Integration:

  • Create interactive phylogenetic trees with associated metadata

  • Generate structural conservation heatmaps

  • Correlate evolutionary patterns with ecological niches and pathogenicity

This comprehensive approach will reveal evolutionary patterns of CrcB across bacterial species and help identify functionally important regions conserved through selective pressure.

How can recombinant CrcB homolog be used to develop detection methods for C. jejuni contamination?

The development of CrcB-based detection systems for C. jejuni requires a methodological approach focused on specificity and sensitivity:

• Antibody Development Pipeline:

  • Immunize animals with purified recombinant CrcB homolog

  • Screen hybridoma clones for antibodies with high specificity

  • Validate antibody cross-reactivity against other Campylobacter species

  • Optimize antibody performance in various detection formats

• ELISA-Based Detection System:

  • Develop sandwich ELISA using anti-CrcB antibodies

  • Establish detection limits using spiked food samples

  • Validate against conventional culture-based methods

  • Implement signal amplification for enhanced sensitivity

• Biosensor Development:

  • Immobilize anti-CrcB antibodies on biosensor surfaces

  • Optimize sample preparation protocols for food matrices

  • Determine detection limits and dynamic range

  • Validate specificity against non-target bacteria

• Field Testing and Validation:

  • Conduct blind testing with naturally contaminated samples

  • Compare performance against standard detection methods

  • Determine false positive/negative rates

  • Assess robustness in different environmental conditions

This methodological framework transforms basic research on CrcB homolog into practical applications for food safety and clinical diagnostics.

What role might CrcB homolog play in antimicrobial resistance mechanisms in C. jejuni?

To investigate potential connections between CrcB function and antimicrobial resistance, implement the following research methodology:

• Susceptibility Testing:

  • Determine MICs of various antibiotics for wild-type and ΔcrcB strains

  • Focus on antibiotics whose efficacy might be affected by ion transport

  • Perform time-kill assays to assess killing kinetics

• Gene Expression Analysis:

  • Examine whether antibiotic exposure alters crcB expression

  • Use qRT-PCR to measure expression changes upon treatment

  • Perform RNA-seq to identify co-regulated genes

• Membrane Permeability Studies:

  • Assess changes in membrane potential using fluorescent dyes

  • Measure uptake of fluorescently labeled antibiotics

  • Determine whether CrcB affects proton motive force

• Combination Therapy Evaluation:

  • Test whether fluoride enhances antibiotic efficacy

  • Determine optimal concentration ratios

  • Evaluate synergistic effects using checkerboard assays

This research framework will reveal whether CrcB-mediated fluoride resistance intersects with antimicrobial resistance mechanisms, potentially leading to novel therapeutic approaches against C. jejuni infections.

Which amino acid residues in CrcB homolog are essential for fluoride transport function?

To identify critical residues for CrcB function, implement a systematic mutagenesis and functional analysis approach:

Methodological approach:

• Generate single-point mutations using site-directed mutagenesis

• Express and purify each mutant protein

• Reconstitute into liposomes for transport assays

• Measure fluoride transport kinetics for each variant

• Assess protein stability using thermal shift assays

• Validate structural integrity using circular dichroism

This systematic analysis will create a functional map of the CrcB protein and identify residues that could be targeted for inhibitor development.

How does the membrane environment affect CrcB homolog structure and function?

To comprehensively assess lipid-protein interactions affecting CrcB function, implement the following methodological approach:

• Lipid Dependency Testing:

  • Reconstitute purified CrcB in liposomes with varying lipid compositions:

    • Different phospholipid headgroups (PC, PE, PG, PS)

    • Varying acyl chain lengths and saturation

    • Presence/absence of bacterial-specific lipids

  • Measure transport activity in each lipid environment

  • Determine lipid preferences for optimal function

• Structural Analysis in Different Environments:

  • Perform hydrogen-deuterium exchange mass spectrometry

  • Measure conformational changes in different detergents and lipids

  • Apply molecular dynamics simulations to model protein-lipid interactions

• Lipid Binding Sites Identification:

  • Use photoactivatable lipid probes to map binding interfaces

  • Perform native mass spectrometry to identify co-purifying lipids

  • Locate conserved lipid-binding motifs through computational analysis

This methodology will reveal how membrane composition affects CrcB structure and function, providing insights into environmental adaptation mechanisms in C. jejuni.

How can structural information about CrcB homolog inform the development of specific inhibitors?

To leverage structural information for inhibitor development, implement the following structure-based drug design workflow:

• Structure Determination:

  • Generate high-resolution structural model using:

    • X-ray crystallography of purified CrcB

    • Cryo-EM of CrcB in nanodiscs

    • Computational modeling with AlphaFold2 validated by experimental data

• Binding Site Identification:

  • Perform computational pocket analysis

  • Identify conserved cavities across CrcB homologs

  • Focus on regions critical for fluoride coordination

• Virtual Screening Campaign:

  • Prepare libraries of small molecules with drug-like properties

  • Perform molecular docking against identified binding sites

  • Score compounds based on predicted binding energy and interactions

• Lead Validation and Optimization:

  • Test top virtual hits in functional assays

  • Measure inhibition of fluoride transport

  • Determine structure-activity relationships

  • Optimize potency and selectivity through medicinal chemistry

This structure-based approach could identify novel inhibitors targeting CrcB as potential antimicrobial agents against C. jejuni.

What genomic approaches can determine the prevalence of functional crcB genes across C. jejuni clinical isolates?

To comprehensively analyze crcB gene distribution and variation across clinical isolates, implement this genomic epidemiology workflow:

• Strain Collection and Sequencing:

  • Collect diverse C. jejuni clinical isolates

  • Perform whole-genome sequencing (Illumina paired-end)

  • Assemble genomes using SPAdes or similar tools

  • Annotate genomes with Prokka or PGAP

• crcB Identification and Analysis:

  • Use BLAST or HMMER to identify crcB homologs

  • Align sequences to identify variants

  • Classify variants based on predicted functional impact

  • This approach can build upon methods used in C. jejuni genotyping studies

• Correlation with Clinical Data:

  • Associate crcB variants with:

    • Patient outcomes

    • Antimicrobial resistance profiles

    • Geographic distribution

    • Source attribution (poultry, cattle, environmental)

• Transmission Analysis:

  • Construct phylogenetic trees based on core genome SNPs

  • Map crcB variants onto phylogeny

  • Identify evidence of horizontal gene transfer events

  • This can extend understanding of horizontal gene transfer documented in C. jejuni

This comprehensive genomic approach will reveal the distribution and clinical significance of crcB variants across C. jejuni populations, potentially identifying markers for virulence or treatment outcomes.

How might CrcB homolog function interact with the gut microbiome during C. jejuni infection?

To investigate interactions between CrcB function and the gut microbiome, implement this multi-faceted experimental approach:

• In vivo Colonization Studies:

  • Compare gut microbiome changes during infection with wild-type versus ΔcrcB C. jejuni

  • Use 16S rRNA sequencing and metatranscriptomics

  • Analyze shifts in microbial community structure and function

  • This builds upon microbiome analysis methods described in C. jejuni studies

• Metabolomic Analysis:

  • Profile metabolite changes in gut environment during infection

  • Identify metabolites affected by CrcB function

  • Focus on fluoride-sensitive pathways and their metabolic products

• Microbial Interaction Assays:

  • Perform co-culture experiments with gut commensals

  • Assess competitive fitness of wild-type versus ΔcrcB strains

  • Measure horizontal gene transfer frequencies in mixed communities

  • Determine whether CrcB affects colonization resistance mechanisms

• Host Response Integration:

  • Correlate microbiome changes with host inflammatory markers

  • Assess whether CrcB affects intestinal barrier integrity

  • Investigate connections to post-infection sequelae like IBS

This comprehensive approach will reveal how CrcB-mediated processes in C. jejuni influence interactions with the gut microbiome and consequent host responses.

What role might CrcB homolog play in environmental survival and transmission of C. jejuni?

To elucidate CrcB's role in environmental persistence, implement this environmental microbiology research approach:

• Survival Assays Under Environmental Stresses:

  • Compare wild-type and ΔcrcB strain survival under:

    • Water environments with varying fluoride concentrations

    • Food matrix conditions (poultry, milk, produce)

    • Disinfection treatments (chlorine, acid, heat)

    • Biofilm formation capacity

• Transcriptional Response Analysis:

  • Perform RNA-seq under environmental stress conditions

  • Identify co-regulated genes in response to fluoride exposure

  • Map regulatory networks controlling crcB expression

• Transmission Model Studies:

  • Track bacterial persistence on surfaces and fomites

  • Assess transfer efficiency during simulated contamination events

  • Determine whether CrcB affects viability in viable-but-non-culturable state

• Field Studies:

  • Screen environmental isolates for crcB variants

  • Correlate crcB sequence types with isolation sources

  • Assess fluoride levels in environmental reservoirs