Recombinant Erwinia tasmaniensis Protein CrcB homolog (crcB)

What is the Protein CrcB homolog from Erwinia tasmaniensis?

Protein CrcB homolog is a membrane protein expressed by Erwinia tasmaniensis (strain DSM 17950 / Et1/99), a bacterium belonging to the Erwiniaceae family. The protein is encoded by the crcB gene (locus tag: ETA_25880) and has been assigned UniProt accession number B2VHL9. The complete amino acid sequence consists of 125 residues (1-125 expression region), with the following sequence: MIKPLLAVMIGGCAGCVIRWLLAVRLNAWFPNLPPGTLLVNLVGGLIIGATVAWFARYPGIDPNWKLLITTGLCGGMTTFSTFSLEVVTLLQAGNYLWAVISVLTHVTGSLLMTIAGFWLVSLLF . This protein appears to be a transmembrane protein based on its hydrophobic amino acid composition and structural motifs.

How does the CrcB homolog differ from other proteins in Erwinia species?

While the search results don't provide direct comparative data for CrcB across Erwinia species, phylogenetic analyses of Erwinia tasmaniensis show it has close relationships with several other species in the Erwiniaceae family. E. tasmaniensis shows 99.21% 16S rRNA gene sequence similarity with other Erwinia species and is closely related to several members including E. phyllosphaerae, E. amylovora, and E. uzenensis . Unlike many Erwinia species that are primarily known as phytopathogens, E. tasmaniensis may include non-phytopathogenic strains that potentially promote plant growth (PGPR), suggesting its proteins, including CrcB homolog, might have unique functional characteristics compared to pathogenic strains .

What is the predicted secondary and tertiary structure of the CrcB homolog?

Based on the amino acid sequence analysis, the CrcB homolog is predominantly hydrophobic with multiple predicted transmembrane domains. The sequence MIKPLLAVMIGGCAGCVIRWLLAVRLNAWFPNLPPGTLLVNLVGGLIIGATVAWFARYPGIDPNWKLLITTGLCGGMTTFSTFSLEVVTLLQAGNYLWAVISVLTHVTGSLLMTIAGFWLVSLLF suggests a protein with several membrane-spanning regions . While specific structural studies of this protein are not detailed in the search results, the high proportion of hydrophobic residues (L, I, V, F) and the presence of glycine residues at potential turns indicate a multi-pass membrane protein architecture. Researchers working with this protein would typically employ prediction tools such as TMHMM, PSIPRED, or I-TASSER to generate theoretical models before conducting experimental structure determination.

What are the optimal expression systems for recombinant production of E. tasmaniensis CrcB homolog?

While the search results don't specifically address expression systems for CrcB homolog, we can draw insights from successful expression of other E. tasmaniensis proteins. Based on the recombinant production of E. tasmaniensis Ribulokinase, yeast expression systems have proven effective for recombinant proteins from this organism. For membrane proteins like CrcB, researchers should consider:

• Bacterial expression systems (E. coli) with specific strains optimized for membrane protein expression (C41, C43, or Lemo21)

• Yeast systems (Pichia pastoris or Saccharomyces cerevisiae) which can provide appropriate post-translational modifications

• Insect cell expression systems (Sf9, Sf21) for complex membrane proteins requiring eukaryotic processing

The choice of expression system should be guided by the intended application, required yield, and preservation of native structure and function.

What purification strategies are most effective for isolating CrcB homolog while maintaining protein integrity?

For membrane proteins like CrcB homolog, effective purification typically involves:

• Affinity chromatography: The addition of affinity tags (similar to the His6 tag used for other E. tasmaniensis recombinant proteins) enables efficient initial purification

• Detergent solubilization: Careful selection of detergents (DDM, LMNG, or digitonin) for extraction from membranes without denaturation

• Size exclusion chromatography: For separating properly folded protein from aggregates

• Ion exchange chromatography: For further purification based on charge properties

Researchers should monitor protein integrity at each step using techniques such as SDS-PAGE (aiming for >85% purity as achieved with other E. tasmaniensis recombinant proteins) and functional assays. For long-term storage, the purified protein can be maintained at -20°C, or -80°C for extended storage, potentially with 50% glycerol as a cryoprotectant .

What is the known or predicted function of the CrcB homolog in E. tasmaniensis?

The CrcB homolog in E. tasmaniensis is primarily predicted to function as a membrane protein involved in ion transport, specifically fluoride ion export. While the search results don't explicitly detail its function, CrcB family proteins are generally known to protect cells against fluoride toxicity by exporting fluoride ions from the cytoplasm. Given that E. tasmaniensis is found in plant environments where it may encounter various ions and toxins, the CrcB homolog likely plays a role in maintaining ion homeostasis and contributing to the bacterium's survival in diverse ecological niches .

Additionally, considering that some E. tasmaniensis strains appear to be non-phytopathogenic and potentially plant-growth promoting , the CrcB homolog might indirectly contribute to the bacterium's beneficial interactions with plants by supporting bacterial survival in the plant microenvironment.

How can researchers design assays to measure CrcB homolog activity in vitro?

To measure the activity of CrcB homolog in vitro, researchers can implement several approaches:

• Fluoride ion transport assays:

  • Reconstitute purified CrcB in liposomes loaded with a fluoride-sensitive fluorescent dye

  • Monitor fluorescence changes upon addition of external fluoride

  • Quantify transport rates under various conditions (pH, temperature, inhibitors)

• Patch clamp electrophysiology:

  • Express CrcB in model membrane systems (oocytes, giant liposomes)

  • Measure ion conductance across membranes

  • Determine ion selectivity through competition experiments

• Isothermal titration calorimetry (ITC):

  • Measure binding affinities of potential substrates to purified CrcB

  • Determine thermodynamic parameters of binding events

• Fluoride resistance complementation assays:

  • Express E. tasmaniensis CrcB in crcB-knockout bacterial strains

  • Assess restoration of fluoride resistance in complemented strains

  • Compare growth curves in media containing various fluoride concentrations

How does the E. tasmaniensis CrcB homolog compare to CrcB proteins in other bacterial species?

A comparative analysis of the E. tasmaniensis CrcB homolog with those from other bacterial species would typically involve:

• Sequence alignment analysis showing conservation of key functional domains and motifs

• Phylogenetic analysis to determine evolutionary relationships

• Structural comparisons if 3D structures are available

While the search results don't provide direct comparison data, researchers could expect the E. tasmaniensis CrcB to share significant sequence similarity with CrcB proteins from closely related Erwinia species (E. amylovora, E. billingiae) and other members of the Erwiniaceae family . The core transmembrane domains responsible for ion transport function would likely show higher conservation than peripheral regions. Functional differences might correlate with the non-pathogenic nature of some E. tasmaniensis strains compared to pathogenic relatives.

What insights can be gained by studying CrcB homologs across the Erwinia genus?

Studying CrcB homologs across the Erwinia genus can provide valuable insights into:

• Evolution of ion transport mechanisms in plant-associated bacteria

• Adaptations related to different ecological niches (pathogenic vs. non-pathogenic lifestyles)

• Conservation of essential functions across divergent species

The Erwinia genus encompasses both phytopathogenic bacteria and potentially plant-friendly strains that can promote plant growth . Comparing CrcB homologs between these functionally diverse strains could reveal whether differences in ion transport contribute to pathogenicity or plant-growth-promoting abilities.

Additionally, since some Erwinia species have been shown to carry antimicrobial resistance genes, including chromosomally-encoded beta-lactamases , studying conserved membrane proteins could potentially reveal novel targets for antimicrobial development specific to plant pathogens.

How can researchers effectively use fluorescence techniques to study CrcB localization and dynamics?

To study CrcB localization and dynamics using fluorescence techniques, researchers can implement:

• Fluorescent protein fusions:

  • Generate N- or C-terminal fusions with GFP or other fluorescent proteins

  • Express in native host or heterologous systems

  • Verify protein functionality is maintained after fusion

  • Use confocal microscopy to visualize subcellular localization

• Fluorescence Recovery After Photobleaching (FRAP):

  • Apply to fluorescently tagged CrcB to measure protein mobility within membranes

  • Calculate diffusion coefficients under different conditions

  • Assess factors affecting protein dynamics (temperature, membrane composition)

• Förster Resonance Energy Transfer (FRET):

  • Create donor-acceptor pairs with potential interaction partners

  • Measure energy transfer as evidence of protein-protein interactions

  • Map interaction domains through mutational analysis

• Super-resolution microscopy:

  • Apply STORM or PALM techniques to visualize nanoscale organization

  • Track single molecules to reveal transient interactions

  • Correlate localization patterns with cellular functions

When designing these experiments, researchers should consider the transmembrane nature of CrcB and optimize protocols accordingly, including careful selection of membrane-compatible fixation methods and appropriate controls to distinguish specific from non-specific signals.

What are the most reliable methods for studying protein-protein interactions involving the CrcB homolog?

For studying protein-protein interactions involving membrane proteins like CrcB homolog, researchers should consider:

• Co-immunoprecipitation with membrane solubilization:

  • Use mild detergents to solubilize membranes

  • Employ antibodies against CrcB or potential interaction partners

  • Confirm interactions by western blotting or mass spectrometry

• Proximity labeling techniques:

  • BioID or APEX2 fusions to CrcB

  • In vivo labeling of proximal proteins

  • Mass spectrometry identification of labeled proteins

  • Validation through reverse proximity labeling

• Split-protein complementation assays:

  • BiFC (Bimolecular Fluorescence Complementation)

  • Split luciferase assays

  • Optimization for membrane protein topology

• Membrane-specific yeast two-hybrid systems:

  • MYTH (Membrane Yeast Two-Hybrid)

  • Split-ubiquitin systems designed for membrane proteins

• Crosslinking mass spectrometry:

  • In vivo or in vitro crosslinking using DSS, formaldehyde, or photo-activatable crosslinkers

  • Digestion and mass spectrometry to identify crosslinked peptides

  • Computational modeling of interaction interfaces

Each method has specific advantages and limitations for membrane proteins, and researchers should consider using multiple complementary approaches to build a robust interaction network.

How can structural studies of CrcB homolog contribute to understanding fluoride transport mechanisms?

Structural studies of the CrcB homolog can provide critical insights into fluoride transport mechanisms through:

• Determination of high-resolution structures:

  • X-ray crystallography of purified protein in detergent micelles or lipidic cubic phase

  • Cryo-electron microscopy for membrane protein complexes

  • Solution NMR for smaller domains or fragments

• Identification of key structural elements:

  • Fluoride binding sites through co-crystallization with fluoride or anomalous diffraction

  • Channel/pore architecture and dimensions

  • Conformational changes associated with transport cycle

• Structure-guided functional analyses:

  • Site-directed mutagenesis of predicted key residues

  • Functional assays to correlate structural features with transport activity

  • Computational simulations of ion permeation pathways

While no structural data for E. tasmaniensis CrcB homolog is provided in the search results, researchers can draw insights from structural studies of other membrane proteins from Erwinia species. For example, the NMR structure determination approach used for the RcsB DNA binding motif from E. amylovora demonstrates successful structural characterization of a protein from a related organism.

What role might the CrcB homolog play in bacterial stress responses and environmental adaptation?

The CrcB homolog likely plays significant roles in bacterial stress responses and environmental adaptation through:

• Protection against fluoride toxicity:

  • Fluoride is naturally present in soil and water

  • CrcB proteins export fluoride ions that would otherwise inhibit essential enzymes

  • This protection may be particularly important in acidic environments where fluoride entry is enhanced

• Contribution to general ion homeostasis:

  • Maintaining appropriate intracellular ion concentrations

  • Supporting membrane potential and cellular energetics

  • Potential roles in pH homeostasis

• Environmental adaptation in plant-associated niches:

  • E. tasmaniensis strains have been isolated from plant tissues, including apple and pear trees

  • Some strains may be non-phytopathogenic and potentially plant-growth promoting

  • Ion transport functions could support colonization of specific plant microenvironments

• Potential roles in symbiotic interactions:

  • Contributing to bacterial fitness during plant colonization

  • Possibly influencing plant-bacterial signaling through ion flux modulation

Research in this area would benefit from comparative transcriptomic and proteomic analyses of E. tasmaniensis under various stress conditions to determine how CrcB expression is regulated in response to environmental challenges.

What are common challenges in expressing and purifying CrcB homolog, and how can they be addressed?

Common challenges in expressing and purifying membrane proteins like CrcB homolog include:

While not specific to CrcB, the preparation of other E. tasmaniensis recombinant proteins has achieved >85% purity using affinity tags and appropriate purification protocols, suggesting similar approaches could be successful for CrcB homolog.

How can researchers overcome challenges in functional characterization of CrcB homolog?

Overcoming functional characterization challenges requires systematic approaches:

• Activity assay development:

  • Begin with established protocols for related proteins

  • Systematically optimize buffer conditions (pH, salt, temperature)

  • Consider reconstitution into artificial membranes (liposomes, nanodiscs)

  • Develop robust positive and negative controls

• Substrate specificity determination:

  • Screen multiple potential substrates (fluoride, other halides)

  • Use radioactive or fluorescently labeled substrates for increased sensitivity

  • Implement competition assays to determine relative affinities

  • Consider indirect readouts (pH changes, membrane potential)

• Addressing low signal-to-noise ratios:

  • Increase protein concentration (if stability permits)

  • Reduce background through more stringent purification

  • Enhance detection sensitivity through signal amplification

  • Develop equilibrium-based assays for slow transporters

• Confirming physiological relevance:

  • Correlate in vitro findings with in vivo phenotypes

  • Generate gene deletion mutants and complement with wild-type or mutant variants

  • Test function under physiologically relevant conditions

  • Consider heterologous expression in model organisms lacking endogenous activity

What statistical approaches are most appropriate for analyzing CrcB functional data?

When analyzing functional data from CrcB studies, researchers should consider:

• For transport kinetics:

  • Michaelis-Menten kinetic analysis for substrate concentration dependence

  • Linear or non-linear regression for determining Km and Vmax values

  • Comparison of transport models (facilitated diffusion vs. active transport)

  • Analysis of variance (ANOVA) to compare experimental conditions

• For dose-response studies:

  • Four-parameter logistic regression for IC50/EC50 determination

  • Hill equation fitting for cooperativity assessment

  • Two-way ANOVA for testing multiple variables (e.g., substrate type and concentration)

• For electrophysiology data:

  • Non-linear curve fitting for current-voltage relationships

  • Power spectral analysis for channel noise characterization

  • Statistical comparison of open probability under different conditions

• For fluorescence-based assays:

  • Baseline correction and normalization procedures

  • Time-series analysis for transport kinetics

  • Paired statistical tests for before/after comparisons

  • Correction for multiple comparisons when screening conditions

For all analyses, researchers should report appropriate statistics including sample sizes, p-values, confidence intervals, and effect sizes. Visualization should include error bars representing standard deviation or standard error of the mean as appropriate.

How can researchers effectively integrate structural and functional data to develop comprehensive models of CrcB function?

To integrate structural and functional data for developing comprehensive CrcB function models:

• Structure-function correlation:

  • Map functional data onto structural models

  • Correlate conservation patterns with functional importance

  • Use site-directed mutagenesis to test structure-based hypotheses

  • Employ molecular dynamics simulations to predict functional mechanisms

• Multi-scale modeling approach:

  • Atomic-level simulations of ion permeation and binding

  • Coarse-grained models for larger-scale conformational changes

  • Kinetic models incorporating experimental rate constants

  • Systems biology models for contextualizing function within cellular networks

• Integrative visualization:

  • Create interactive visualizations linking structural features to functional parameters

  • Develop animated models of proposed transport mechanisms

  • Use statistical coupling analysis to identify co-evolving residues

  • Implement computational docking to identify interaction partners

• Iterative model refinement:

  • Generate testable predictions from initial models

  • Design experiments to specifically address model uncertainties

  • Update models based on new experimental data

  • Quantify model accuracy and precision

By integrating structural information with functional data, researchers can develop mechanistic models that explain how specific structural features of CrcB enable its fluoride transport function and how mutations or environmental conditions might alter this activity.

What are promising research directions for understanding the physiological importance of CrcB homolog in E. tasmaniensis?

Future research on the physiological importance of CrcB homolog could explore:

• Role in plant-microbe interactions:

  • Determine if CrcB contributes to E. tasmaniensis colonization of plant tissues

  • Investigate whether CrcB function affects plant growth promotion capabilities

  • Compare CrcB function between pathogenic and non-pathogenic Erwinia strains

• Stress adaptation mechanisms:

  • Characterize CrcB expression under various environmental stresses

  • Determine if CrcB contributes to survival in fluoride-rich environments

  • Investigate potential roles in acid stress resistance

• Metabolic integration:

  • Explore connections between fluoride homeostasis and central metabolism

  • Investigate effects of CrcB deletion on metabolomic profiles

  • Determine if CrcB function affects sensitivity to metabolic inhibitors

• Evolutionary significance:

  • Compare CrcB sequences across Erwinia species with different host ranges

  • Investigate horizontal gene transfer patterns of crcB genes

  • Study crcB gene expression regulation across related species

These research directions would benefit from generating crcB knockout strains in E. tasmaniensis and performing comprehensive phenotypic analyses under various growth conditions relevant to its natural habitat.

How might CrcB homolog research contribute to broader understanding of bacterial adaptation and evolution?

CrcB homolog research could contribute to understanding bacterial adaptation and evolution through:

• Investigation of selective pressures:

  • Determine how fluoride resistance shapes bacterial adaptation to specific niches

  • Analyze CrcB sequence conservation in relation to environmental fluoride exposure

  • Study how ion transport mechanisms influence competitive fitness

• Comparative genomics approaches:

  • Examine synteny and gene neighborhood conservation around crcB

  • Identify co-evolving genes that might functionally interact with CrcB

  • Track evolutionary history of fluoride resistance mechanisms

• Exploration of functional diversification:

  • Determine if CrcB homologs have acquired additional functions in some lineages

  • Investigate potential moonlighting functions beyond fluoride transport

  • Study how subtle sequence variations influence substrate specificity

• Contribution to ecological understanding:

  • Explore how CrcB function influences microbial community structures

  • Investigate potential roles in interspecies competition

  • Determine if CrcB function affects bacterial persistence in agricultural settings

This research would provide insights into how fundamental cellular processes like ion homeostasis contribute to bacterial adaptation and evolution in diverse environments, with potential applications to understanding both beneficial and pathogenic plant-microbe interactions.