Recombinant Burkholderia xenovorans Protein CrcB homolog (crcB)

What is the Burkholderia xenovorans CrcB homolog and what is its functional significance?

The CrcB homolog in Burkholderia xenovorans LB400 is a putative membrane protein likely involved in fluoride ion transport and homeostasis. While specific research on the B. xenovorans version is limited, CrcB proteins generally function as fluoride channels that protect bacterial cells from fluoride toxicity. B. xenovorans LB400 is particularly notable as a non-pathogenic strain with one of the largest known bacterial genomes (9.73 Mbp) and remarkable metabolic versatility, particularly in degrading polychlorinated biphenyls (PCBs) . The significance of studying its CrcB homolog relates to understanding how this environmentally important bacterium manages ion homeostasis, which may contribute to its ability to survive in diverse ecological niches. The protein would be one of thousands encoded in the B. xenovorans genome, which features significant functional specialization across its three replicons (chromosomes) .

How is the crcB gene organized within the B. xenovorans genome?

The crcB gene in B. xenovorans would be located on one of its three replicons - either the 4.90-Mbp chromosome 1, the 3.36-Mbp chromosome 2, or the 1.42-Mbp megaplasmid . Given the genomic organization patterns observed in B. xenovorans, it's important to determine whether crcB is part of the core genome (conserved across Burkholderia species) or unique to B. xenovorans. Approximately 44% of genes are conserved between B. xenovorans LB400 and Burkholderia cepacia complex strain 383, indicating high genomic plasticity within the genus . The specific replicon location would provide insights into the evolutionary history and importance of the gene, as genes on the two smaller replicons typically experience more relaxed selective pressure compared to those on the largest replicon . Researchers should analyze the surrounding genetic context, as over 20% of the LB400 sequence was acquired through lateral gene transfer, which may influence the evolutionary history of the crcB homolog .

What expression systems are most effective for producing recombinant B. xenovorans CrcB homolog protein?

For producing recombinant B. xenovorans CrcB homolog protein, an E. coli-based expression system is typically most effective due to its ease of use and high yield potential. Based on successful expression approaches used for other B. xenovorans proteins, such as RcoM-1, a pUX-type plasmid system with a C-terminal 6×His tag has demonstrated efficacy . For membrane proteins like CrcB, consider using specialized E. coli strains (such as C41/C43(DE3) or Lemo21(DE3)) that are designed to accommodate potentially toxic membrane proteins.

The expression protocol should include:

• Induction with IPTG at lower temperatures (16-20°C)

• Extended expression periods (16-24 hours)

• Use of mild detergents for extraction (DDM or LDAO)

• Purification via nickel affinity chromatography followed by size exclusion chromatography

A dual-plasmid system, similar to that used for RcoM-1 protein studies, could be adapted for functional verification of the recombinant protein . For challenging expression cases, consider Burkholderia-specific codon optimization or fusion with solubility-enhancing partners like MBP or SUMO.

How does lateral gene transfer influence the evolution of the crcB homolog in B. xenovorans compared to other Burkholderia species?

The evolution of the crcB homolog in B. xenovorans should be examined in the context of the significant lateral gene transfer (LGT) that has shaped this organism's genome. Research shows that >20% of the B. xenovorans LB400 sequence was recently acquired via LGT , which creates an important evolutionary framework for studying any single gene. To properly investigate this question, researchers should:

• Perform comparative genomic analyses across multiple Burkholderia strains to determine conservation patterns of crcB

• Calculate the G+C content and codon usage of crcB compared to the genome average

• Evaluate the presence of mobile genetic elements or genomic islands near the crcB locus

• Construct phylogenetic trees comparing crcB sequences from B. xenovorans and related species

The high genomic plasticity within Burkholderia is evidenced by the conservation of only 44% of genes between LB400 and B. cepacia complex strain 383, and significant genome size variations even among four B. xenovorans strains (ranging from 7.4 to 9.73 Mbp) . Additionally, researchers should determine whether crcB is present on the megaplasmid, which contains 70% genes unique to LB400 and appears to be a mosaic of foreign genomic material with a G+C% approximately 1% lower than the chromosomes . If crcB exhibits characteristics of recently acquired genes, this would suggest adaptation to specific environmental niches rather than core cellular function.

What structural modifications can optimize the stability and functionality of recombinant B. xenovorans CrcB homolog for crystallography studies?

Optimizing recombinant B. xenovorans CrcB homolog for crystallography studies requires several strategic structural modifications:

• Terminal modifications:

  • N-terminal truncation to remove signal peptides or disordered regions

  • C-terminal fusion with crystallization chaperones (T4 lysozyme, BRIL, rubredoxin)

  • Strategic placement of His-tags with TEV cleavage sites

• Stability-enhancing mutations:

  • Introduce disulfide bridges at computationally predicted positions

  • Replace surface-exposed hydrophobic residues with hydrophilic alternatives

  • Perform alanine scanning to identify and mutate destabilizing residues

• Construct design table:

• Solubilization optimization:

  • Screen detergent classes systematically (maltoside, glucoside, amine oxide)

  • Test lipid cubic phase methods for membrane protein crystallization

  • Evaluate nanodiscs or amphipols as alternative stabilization platforms

Given B. xenovorans' genetic complexity with three replicons and significant functional specialization between them , expression of heterologous membrane proteins may benefit from understanding the native genetic context of crcB, particularly considering the relaxed selective pressure observed for genes on the two smaller vs. largest replicon .

How does the B. xenovorans CrcB homolog function differ in PCB-degrading environments compared to standard laboratory conditions?

The functional activity of B. xenovorans CrcB homolog likely varies significantly between PCB-degrading environments and standard laboratory conditions due to several factors:

• Regulatory influences:

  • PCB degradation induces significant transcriptional changes that may affect crcB expression

  • Stress responses triggered by aromatic compounds could alter membrane protein function

• Metabolic context:

  • B. xenovorans possesses at least eleven "central aromatic" and twenty "peripheral aromatic" pathways , creating unique metabolic states

  • Secondary metabolites from PCB degradation may directly interact with membrane transport systems

• Comparative expression profile:

• Methodological approach for investigation:

  • RNA-seq analysis comparing expression profiles across conditions

  • Fluoride-sensitive reporter assays in various growth conditions

  • Proteomic analysis of membrane fraction from cells grown in different conditions

  • Lipidomic analysis to identify membrane composition changes that might affect CrcB function

This investigation connects to broader observations about B. xenovorans adapting to its ecological niche through specialized metabolic pathways. The presence of genetic factors associated with in vivo survival and intercellular interactions relate to niche breadth rather than pathogenicity , suggesting that CrcB may play a role in environmental adaptation. Additionally, the redundancy observed in other metabolic pathways (17.6% of proteins having better LB400 paralogs than orthologs in different genomes ) raises questions about potential functional redundancies in ion transport systems.

What are the optimal protocols for assessing fluoride transport activity of purified recombinant B. xenovorans CrcB homolog?

Assessing fluoride transport activity of purified recombinant B. xenovorans CrcB homolog requires careful experimental design using these complementary approaches:

• Liposome-based fluorescence assays:

  • Reconstitute purified CrcB into liposomes with encapsulated fluoride-sensitive probes (PBFI or fluoride-selective electrodes)

  • Monitor fluoride transport kinetics upon creation of an external/internal gradient

  • Compare transport rates with known CrcB homologs as positive controls

• Electrophysiological methods:

  • Planar lipid bilayer recordings to measure single-channel conductance

  • Patch-clamp analysis of CrcB-expressing giant unilamellar vesicles

  • Determination of ion selectivity through competition experiments

• Experimental setup table:

• Control experiments:

  • Empty liposomes (negative control)

  • Known fluoride transporters (positive control)

  • CrcB with site-directed mutations at predicted pore residues

  • Varying lipid compositions to determine optimal CrcB function

For data analysis, apply Michaelis-Menten kinetics to determine Km and Vmax values for fluoride transport. This approach parallels techniques used for characterizing other B. xenovorans proteins, such as the methods used to study the RcoM-1 protein's binding characteristics , adapted for membrane protein analysis.

How can researchers effectively analyze the gene duplication patterns and potential functional redundancies of crcB in the B. xenovorans genome?

Analyzing gene duplication patterns and functional redundancies of crcB in B. xenovorans requires a systematic approach that takes advantage of the organism's unique genomic features:

• Computational sequence analysis:

  • Perform BLAST searches against the B. xenovorans genome to identify all crcB homologs

  • Calculate sequence identity, similarity, and construct phylogenetic trees

  • Analyze synteny of genomic regions containing crcB homologs

• Evolutionary analysis framework:

  • Compare with crcB homologs in other Burkholderia species to determine duplication timing

  • Calculate Ka/Ks ratios to identify selective pressures on different copies

  • Identify potential recombination or lateral gene transfer events through phylogenetic incongruence

• Functional complementation approach:

  • Generate single and combined knockout mutants of crcB homologs

  • Test growth under fluoride stress conditions

  • Rescue experiments with individual homologs to determine functional overlap

• Expression pattern analysis:

  • RNA-seq under various conditions to determine differential expression

  • Promoter-reporter fusions to visualize expression patterns

  • Proteomic analysis of membrane fractions

This approach is particularly relevant as 17.6% of proteins in B. xenovorans have a better paralog within LB400 than an ortholog in a different genome , highlighting the importance of gene duplication. For experimental design, researchers should consider the multi-replicon nature of B. xenovorans, as the observed functional specialization between the three replicons may extend to the evolution of crcB homologs. Techniques similar to those used for analyzing the RcoM paralogues in B. xenovorans (RcoM-1 and RcoM-2) could be adapted for crcB homolog analysis.

What techniques are most effective for investigating protein-protein interactions involving the CrcB homolog in B. xenovorans?

For investigating protein-protein interactions (PPIs) involving the CrcB homolog in B. xenovorans, researchers should employ multiple complementary techniques:

• In vivo approaches:

  • Bacterial two-hybrid system adapted for membrane proteins

  • Split fluorescent protein complementation assays

  • Co-immunoprecipitation with tagged CrcB followed by mass spectrometry

• In vitro methods:

  • Pull-down assays using purified His-tagged CrcB

  • Surface plasmon resonance (SPR) for quantitative binding kinetics

  • Microscale thermophoresis for detecting interactions in solution

• Comparative cross-linking strategies:

• Computational integration:

  • Protein interaction network analysis

  • Structural modeling of interaction interfaces

  • Co-evolution analysis to predict interaction partners

When analyzing CrcB interactions, consider the genomic context in B. xenovorans, including its multi-replicon structure and functional specialization between replicons . The approach should account for the unique genomic characteristics of B. xenovorans, where >20% of the genome was acquired by lateral gene transfer , potentially influencing the evolution of protein interaction networks. The dual-plasmid system approach used for studying RcoM-1 protein could be adapted to verify functional interactions in vivo.

How can researchers integrate transcriptomic, proteomic, and metabolomic data to understand the role of CrcB homolog in B. xenovorans stress response?

Integrating multi-omics data to understand the role of CrcB homolog in B. xenovorans stress response requires a systematic framework:

• Experimental design considerations:

  • Expose B. xenovorans cultures to different stressors (fluoride, pH variation, aromatic compounds)

  • Collect matched samples for transcriptomic, proteomic, and metabolomic analyses

  • Include time-course measurements to capture dynamic responses

• Multi-omics integration workflow:

  • Normalize data across platforms using appropriate statistical methods

  • Identify coordinated changes across all three data types

  • Apply network analysis to construct stress response pathways

• Statistical integration approaches:

• Visualization and interpretation:

  • Construct multi-level regulatory networks

  • Map metabolic fluxes affected by CrcB activity

  • Identify regulatory elements controlling crcB expression

This integrative approach should consider B. xenovorans' genomic complexity, including its three replicons with significant functional specialization . The analysis should examine whether stress responses involving CrcB are connected to the organism's extensive aromatic degradation pathways (at least eleven "central aromatic" and twenty "peripheral aromatic" pathways) . Additionally, researchers should determine if CrcB function correlates with the expression of other transport systems, particularly considering the significant gene redundancy observed in B. xenovorans (17.6% of proteins having better paralogs within LB400 than orthologs in different genomes) .

What bioinformatic approaches can best predict structural features and functional motifs of the B. xenovorans CrcB homolog?

Predicting structural features and functional motifs of the B. xenovorans CrcB homolog requires a multi-layered bioinformatic approach:

• Primary sequence analysis:

  • Multiple sequence alignment with diverse CrcB homologs

  • Conservation analysis to identify functionally important residues

  • Transmembrane topology prediction using consensus methods (TMHMM, TOPCONS, MEMSAT)

• Structure prediction pipeline:

  • AlphaFold2 or RoseTTAFold for ab initio structure prediction

  • Homology modeling based on available CrcB structures

  • Molecular dynamics simulations to refine models and identify flexible regions

• Functional motif prediction:

• Structural validation approach:

  • Cysteine scanning mutagenesis to validate transmembrane regions

  • Fluoride binding assays with predicted pore residue mutants

  • Evolutionary coupling analysis to validate predicted domain interactions

The bioinformatic analysis should account for B. xenovorans' genomic context, including its multi-replicon structure and significant functional specialization between replicons . Researchers should consider potential adaptations specific to B. xenovorans' ecological niche, as genetic factors related to in vivo survival are likely related to niche breadth rather than pathogenicity . Additionally, if multiple CrcB paralogs exist, analysis of gene duplication patterns should be conducted to understand potential functional divergence, considering the significant gene redundancy observed in B. xenovorans .

What are the most promising research directions for understanding the evolutionary and functional significance of CrcB homologs in environmental bacteria like B. xenovorans?

The most promising research directions for CrcB homologs in environmental bacteria like B. xenovorans include:

• Ecological context studies:

  • Investigation of CrcB function in natural environments with varying fluoride levels

  • Correlation between CrcB variants and habitat adaptation

  • Role of CrcB in microbial community interactions

• Evolutionary trajectory analysis:

  • Comparative genomics across Burkholderia species to track CrcB evolution

  • Investigation of lateral gene transfer patterns involving CrcB

  • Selective pressures on CrcB in different environmental contexts

• Functional diversity exploration:

  • Characterization of potential secondary functions beyond fluoride transport

  • Involvement in stress responses to pollutants

  • Connection to B. xenovorans' remarkable metabolic versatility

• Biotechnological applications:

  • Engineering CrcB variants for enhanced fluoride bioremediation

  • Development of biosensors for environmental fluoride detection

  • Potential applications in industrial bioprocesses requiring fluoride tolerance

These directions build upon the understanding that B. xenovorans has undergone significant genome evolution, with >20% of its sequence acquired through lateral gene transfer . The organism's ability to adapt to diverse ecological niches is reflected in its genomic plasticity, with significant differences in functional specialization between its three replicons . Understanding CrcB evolution in this context may provide insights into how membrane transporters contribute to environmental adaptation, particularly in connection with B. xenovorans' extensive aromatic degradation capabilities (eleven "central aromatic" and twenty "peripheral aromatic" pathways) . Additionally, investigating potential functional redundancies of CrcB homologs would connect to broader observations about gene duplication in B. xenovorans, where 17.6% of proteins have better paralogs within the organism than orthologs in different genomes .

What quality control metrics should be applied when evaluating recombinant B. xenovorans CrcB homolog protein preparations?

When evaluating recombinant B. xenovorans CrcB homolog protein preparations, researchers should implement comprehensive quality control metrics:

• Purity assessment protocols:

  • SDS-PAGE with Coomassie and silver staining (target >95% purity)

  • Western blot with anti-His antibodies

  • Mass spectrometry for accurate mass determination and contaminant identification

• Functional integrity evaluation:

  • Circular dichroism to assess secondary structure

  • Fluoride binding assays

  • Transport activity in reconstituted systems

• Stability assessment metrics:

• Batch consistency verification:

  • Lot-to-lot comparison using activity assays

  • MS fingerprinting

  • Detailed documentation of expression and purification parameters