Recombinant Shewanella sp. Protein CrcB homolog (crcB)

What is the CrcB homolog protein in Shewanella species and what is its primary function?

The CrcB homolog protein in Shewanella species is a membrane protein that plays a crucial role in fluoride ion channel formation and fluoride resistance. The protein typically consists of 124 amino acids in its full-length form, as seen in Shewanella pealeana . CrcB functions primarily as a fluoride ion channel, facilitating the export of toxic fluoride ions from the bacterial cell, thereby contributing to the microorganism's ability to survive in environments with elevated fluoride concentrations.

In the broader context, Shewanella species are widely distributed gram-negative bacteria inhabiting both freshwater and marine environments, with some species gaining attention for their ability to survive in redox-stratified environments . The CrcB homolog is part of the genomic repertoire that enables Shewanella to adapt to various environmental conditions.

How does the structure of CrcB homolog protein relate to its function in Shewanella species?

The CrcB homolog protein in Shewanella species typically features transmembrane domains that assemble to form fluoride-selective ion channels. While the specific structural details of Shewanella CrcB homologs are not fully elucidated in the provided search results, research on CrcB proteins suggests they form dimeric structures with each monomer containing three transmembrane segments.

The structural arrangement creates a narrow pore that selectively permits fluoride ion transport across the cell membrane. This structural adaptation is particularly important for Shewanella species that inhabit diverse ecological niches , as it enhances their survival capability in environments containing toxic levels of fluoride. The protein's structure-function relationship is essential for understanding how Shewanella species adapt to challenging environmental conditions.

What is known about the genomic context of the crcB gene in different Shewanella species?

The genomic context of the crcB gene varies among different Shewanella species, reflecting their diverse ecological adaptations. While specific information about crcB genomic organization is limited in the search results, we can draw insights from whole-genome comparison studies of Shewanella.

In Shewanella pealeana, the crcB gene encodes a 124-amino acid protein . Genomic analyses of various Shewanella species have revealed considerable genome plasticity, with horizontal gene transfer contributing significantly to their genetic diversity. For instance, in sponge-associated Shewanella species like OPT22 and KCTC 22492, genomic islands contain genes that enhance ecological fitness and symbiotic lifestyle .

The crcB gene likely exists within genomic regions that reflect the specific environmental adaptations of each Shewanella species. Further comparative genomic analyses would be valuable to determine whether crcB is part of the core genome or the accessory genome in Shewanella species.

How does the expression of recombinant CrcB homolog protein differ between laboratory cultures and natural environmental conditions of Shewanella sp.?

In natural environments, Shewanella species encounter variable conditions that modulate crcB expression. For example, Shewanella oneidensis MR-1, found in diverse ecological niches, has developed various physiological mechanisms to respond to environmental stressors . The expression of crcB is likely regulated by environmental fluoride concentrations, pH fluctuations, and potentially redox conditions.

Research methodologies to investigate these differences include:

• Transcriptomic analysis comparing wild-type Shewanella under various environmental conditions

• Reporter gene constructs fused to the native crcB promoter to monitor expression triggers

• Quantitative proteomics to measure actual protein levels in different conditions

These approaches would help elucidate the complex regulatory mechanisms governing natural CrcB expression compared to recombinant systems.

What are the challenges in producing functionally active recombinant CrcB homolog protein from antibiotic-resistant Shewanella strains?

Producing functionally active recombinant CrcB homolog protein from antibiotic-resistant Shewanella strains presents several significant challenges, particularly with the emergence of carbapenem-resistant isolates . These challenges include:

• Expression system compatibility: The increasing prevalence of antibiotic resistance genes in Shewanella, particularly β-lactamases (including blaOXA-55-like, blaOXA-48-like, and blaOXA-54) , complicates the selection of appropriate antibiotic selection markers for expression vectors.

• Membrane protein folding: As a membrane protein, CrcB requires proper folding and insertion into membranes to maintain functionality. Antibiotic resistance mechanisms may alter membrane composition or permeability, affecting proper folding.

• Post-translational modifications: Antibiotic-resistant strains may exhibit altered post-translational modification pathways that affect CrcB functionality.

• Protein purification interference: Resistance mechanisms, including multiple efflux pump systems identified in carbapenem-resistant S. algae , may interfere with conventional protein purification strategies.

Methodological solutions include:

• Using alternative selection markers unaffected by the strain's resistance profile

• Employing membrane mimetics during purification

• Validating functionality through fluoride transport assays

• Considering heterologous expression in non-Shewanella hosts

How does horizontal gene transfer influence the genetic diversity of crcB homologs across Shewanella species in marine environments?

Horizontal gene transfer (HGT) significantly contributes to the genetic diversity of crcB homologs across Shewanella species in marine environments. This process is particularly evident in the context of genomic islands (GIs), which facilitate the dissemination of genes that enhance ecological fitness.

In sponge-associated Shewanella species, GIs have been identified as critical vehicles for the spread of symbiosis-related genes . Although the search results don't specifically mention crcB in this context, the general mechanism likely applies to this gene as well. For example, genomic island analysis of Shewanella sp. OPT22 revealed gene clusters encoding T4SS components, suggesting that similar mechanisms could facilitate crcB transfer .

Methodological approaches to investigate HGT of crcB include:

• Phylogenetic incongruence analysis: Comparing crcB gene trees with species trees to identify potential HGT events

• Analysis of GC content and codon usage: Detecting regions with atypical patterns suggesting foreign origin

• Identification of mobile genetic elements: Examining the genomic context of crcB for transposases, integrases, or other mobility-associated genes

• Comparative genomics: Analyzing synteny and conservation of crcB across Shewanella species and related genera

Understanding these HGT patterns could provide insights into how fluoride resistance spreads among marine bacteria and how crcB homologs evolve under different selective pressures.

What are the optimal conditions for expressing and purifying functional recombinant CrcB homolog protein from Shewanella species?

The optimal conditions for expressing and purifying functional recombinant CrcB homolog protein from Shewanella species involve careful consideration of the protein's membrane-associated nature and fluoride transport function. Based on protein purification principles and the limited information in the search results about the specific CrcB homolog , the following methodological approach is recommended:

Expression system optimization:

• Host selection: E. coli C41(DE3) or C43(DE3) strains are preferable for membrane protein expression

• Vector design: Incorporate a strong inducible promoter (e.g., T7) and a fusion tag (His-tag or MBP) to facilitate purification

• Induction parameters: Low temperature (16-20°C) induction with reduced IPTG concentration (0.1-0.5 mM) to prevent inclusion body formation

Purification protocol:

• Cell lysis: Gentle disruption using sonication or French press in a buffer containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, and appropriate protease inhibitors

• Membrane preparation: Ultracentrifugation to isolate membrane fractions

• Solubilization: Use of mild detergents (DDM, LMNG, or C12E8) at concentrations just above their CMC

• Affinity chromatography: IMAC purification using the His-tag

• Size exclusion chromatography: Final purification step to obtain homogeneous protein

Functional validation:

• Fluoride binding assays: Using fluorescent probes or isothermal titration calorimetry

• Reconstitution into liposomes: To verify fluoride transport activity

• Circular dichroism: To confirm proper secondary structure formation

This methodological approach maximizes the likelihood of obtaining functionally active CrcB homolog protein suitable for structural and functional studies.

What techniques are most effective for studying the interaction between CrcB homolog protein and environmental fluoride in Shewanella species?

Studying the interaction between CrcB homolog protein and environmental fluoride in Shewanella species requires a multi-faceted approach combining molecular, biophysical, and physiological techniques:

Molecular and genetic approaches:

• Gene knockout studies: Creating ΔcrcB Shewanella mutants to assess fluoride sensitivity

• Site-directed mutagenesis: Identifying key residues involved in fluoride binding and transport

• Complementation assays: Reintroducing wild-type or mutant crcB to assess functional restoration

Biophysical techniques:

• Fluoride electrode measurements: Real-time quantification of fluoride transport in whole cells or proteoliposomes

• Fluorescence spectroscopy: Using fluoride-sensitive probes to monitor transport kinetics

• Isothermal titration calorimetry (ITC): Determining thermodynamic parameters of fluoride binding

• Structural analysis: X-ray crystallography or cryo-EM to visualize fluoride binding sites

Physiological approaches:

• Growth inhibition assays: Measuring MIC (minimum inhibitory concentration) of fluoride in various Shewanella strains

• Adaptation studies: Investigating how Shewanella acclimates to different fluoride concentrations

• Environmental transcriptomics: Analyzing crcB expression patterns in response to fluoride exposure

Data analysis methodology:

• Kinetic modeling: Determining transport rates and Km values for fluoride

• Comparative analysis: Assessing functional differences between CrcB homologs from different Shewanella species

• Integration with genomic data: Correlating CrcB sequence variations with functional differences

This comprehensive approach allows researchers to understand the molecular mechanisms of CrcB-mediated fluoride resistance and its ecological significance in Shewanella adaptation to fluoride-rich environments.

How can researchers effectively analyze the role of CrcB homolog in Shewanella's resistance to environmental stressors?

To effectively analyze the role of CrcB homolog in Shewanella's resistance to environmental stressors, researchers should implement a systematic methodology that integrates genomic, transcriptomic, proteomic, and phenotypic approaches:

Genomic and comparative analysis:

• Genome mining: Identify crcB homologs and associated genes across Shewanella species

• Synteny analysis: Examine genomic context for co-localized stress response genes

• Phylogenetic analysis: Compare crcB sequences from Shewanella strains isolated from different stress conditions

Transcriptomic approach:

• RNA-seq analysis: Compare expression profiles of wild-type and ΔcrcB mutants under various stressors

• qRT-PCR validation: Confirm expression changes of crcB and related genes

• Promoter analysis: Identify regulatory elements responding to specific stressors

Functional characterization:

• Stress tolerance assays: Measure survival and growth of wild-type, knockout, and complemented strains under various conditions:

  • Fluoride stress (primary function)

  • Heavy metal exposure (relevant to Shewanella's ability to reduce metals)

  • pH fluctuations

  • Redox stress

  • Osmotic stress

Protein interaction studies:

• Co-immunoprecipitation: Identify proteins interacting with CrcB under stress conditions

• Bacterial two-hybrid assays: Confirm direct protein-protein interactions

• Proteomics: Compare global protein expression between wild-type and mutant strains

Environmental relevance:

• Microcosm experiments: Test CrcB contribution to fitness in simulated natural environments

• Field sampling: Correlate crcB sequence variants with environmental parameters

• Multi-species competition assays: Assess competitive advantage conferred by CrcB

This methodological framework would provide a comprehensive understanding of how CrcB contributes to Shewanella's remarkable adaptability to diverse ecological niches, similar to what has been observed for other Shewanella proteins involved in environmental adaptation .

How can research on Shewanella CrcB homolog contribute to our understanding of fluoride resistance mechanisms in pathogenic bacteria?

Research on Shewanella CrcB homolog can significantly advance our understanding of fluoride resistance mechanisms in pathogenic bacteria through comparative analyses and functional characterization. This knowledge has important implications given the emergence of Shewanella species as opportunistic pathogens .

Translational research approaches:

• Comparative genomics: Analyze CrcB sequence conservation between Shewanella and pathogenic bacteria to identify critical functional domains

• Heterologous expression: Express Shewanella CrcB in fluoride-sensitive pathogens to assess functional conservation

• Structure-function analysis: Use Shewanella CrcB as a model to predict resistance mechanisms in pathogens

Clinical relevance:
The increasing incidence of Shewanella infections (128 cases over 10 years in one hospital alone) highlights the importance of understanding resistance mechanisms. Shewanella algae and S. putrefaciens have been implicated in hepatobiliary, skin, soft tissue, and respiratory infections, often with significant antibiotic resistance .

Methodological approach for clinical translation:

• Isolate collection: Compare CrcB sequences from clinical and environmental Shewanella isolates

• Fluoride susceptibility testing: Correlate CrcB variants with fluoride tolerance levels

• Expression analysis: Measure crcB upregulation in response to fluoride-containing antiseptics

Future research directions:

• Development of fluoride-based antimicrobial strategies targeting CrcB function

• Investigation of cross-resistance between fluoride resistance and antibiotic resistance

• Exploration of CrcB inhibitors as potential adjuvants for antimicrobial therapy

This research path would leverage Shewanella CrcB as a model to understand fluoride resistance in the context of emerging infectious diseases.

What potential applications exist for recombinant CrcB homolog protein in environmental bioremediation of fluoride contamination?

Recombinant CrcB homolog protein from Shewanella species holds significant potential for environmental bioremediation of fluoride contamination, building upon Shewanella's established capabilities in environmental detoxification processes .

Bioremediation engineering approaches:

• Whole-cell bioremediation: Engineering Shewanella strains with enhanced CrcB expression for fluoride bioaccumulation

• Immobilized enzyme systems: Using purified recombinant CrcB in membrane-based filtration systems

• Synthetic biology circuits: Creating feedback-regulated systems that modulate CrcB expression based on environmental fluoride concentrations

Methodological framework for development:

Integration with Shewanella's other bioremediation capabilities:
Shewanella species have demonstrated abilities to transform various environmental contaminants, including heavy metals like uranium and chromium through reduction processes . A comprehensive bioremediation system could leverage CrcB's fluoride-binding capabilities alongside Shewanella's metal reduction pathways to address mixed contamination scenarios.

Potential challenges and solutions:

• Challenge: Maintaining cell viability in highly contaminated environments
  Solution: Encapsulation technologies to protect engineered cells

• Challenge: Competition with indigenous microbiota
  Solution: Co-culture approaches or cell-free systems using purified CrcB

• Challenge: Disposal of fluoride-laden biomass
  Solution: Development of regeneration protocols for sustainable operation

This application would build upon Shewanella's natural capacity to thrive in contaminated environments while utilizing the specific fluoride-binding properties of CrcB.

How might structural studies of CrcB homolog contribute to the development of new fluoride transport inhibitors for potential therapeutic applications?

Structural studies of CrcB homolog from Shewanella species could significantly contribute to the development of novel fluoride transport inhibitors with potential therapeutic applications, particularly against emerging Shewanella infections and other pathogens utilizing similar fluoride resistance mechanisms.

Structural characterization methodologies:

• X-ray crystallography: Determination of high-resolution structure of CrcB in different conformational states

• Cryo-electron microscopy: Visualization of CrcB in native membrane environment

• NMR spectroscopy: Analysis of dynamic properties and fluoride binding mechanisms

• Molecular dynamics simulations: Identification of potential binding sites for inhibitor design

Structure-based drug design approach:

Therapeutic relevance:
Shewanella infections are emerging with concerning antibiotic resistance profiles, particularly to carbapenems . In a study of 128 patients with Shewanella infections, imipenem-resistant strains were detected in 23.4% of isolates , highlighting the need for alternative therapeutic approaches. CrcB inhibitors could represent a novel class of antimicrobials targeting a mechanism not addressed by conventional antibiotics.

Challenges and considerations:

• Selectivity: Designing inhibitors that target bacterial CrcB without affecting mammalian fluoride channels

• Membrane penetration: Ensuring inhibitors can access the membrane-embedded target

• Resistance development: Assessing potential for resistance evolution through CrcB mutations

This research direction would leverage structural biology to address the growing concern of antibiotic resistance in Shewanella species while potentially creating a broadly applicable platform for targeting fluoride transport in diverse pathogens.

What are the most promising future research directions for understanding the evolution and functional diversity of CrcB homologs across the Shewanella genus?

The most promising future research directions for understanding CrcB homologs across the Shewanella genus should focus on integrating evolutionary genomics with functional characterization. Given Shewanella's remarkable genomic plasticity and ecological adaptability , several key research avenues emerge:

Evolutionary genomics approach:

• Pan-genome analysis: Comprehensive examination of crcB distribution across all sequenced Shewanella species

• Selection pressure analysis: Investigation of evolutionary forces shaping crcB sequence diversity

• Horizontal gene transfer mapping: Identification of crcB acquisition events, particularly in genomic islands

• Ancestral sequence reconstruction: Inferring the evolutionary trajectory of CrcB function

Functional diversity characterization:

• Systematic mutagenesis: Creation of CrcB variant libraries to map structure-function relationships

• Environmental adaptation studies: Correlation of CrcB sequence variations with ecological niches

• Protein engineering: Development of CrcB variants with enhanced or altered functions

Methodological integration:
Combining high-throughput sequencing, protein characterization, and environmental sampling would provide a comprehensive understanding of CrcB evolution and function. This would build upon existing knowledge of Shewanella's genomic adaptations, such as those observed in sponge-associated strains with specialized genomic repertoires .