Recombinant Shewanella baltica Protein CrcB homolog (crcB)

How do CrcB proteins differ among Shewanella species?

Comparative analysis of CrcB homologs across Shewanella species reveals both conservation and species-specific variations:

These sequence variations may reflect adaptation to different ecological niches. Despite these differences, the core functional domains remain conserved, with most variations occurring in non-critical regions. The high sequence similarity (95-98% identity between closely related species) suggests functional conservation of CrcB across the Shewanella genus .

What expression systems are optimal for recombinant CrcB production?

The optimal expression system for recombinant Shewanella baltica CrcB homolog protein is Escherichia coli. According to multiple product specifications, all commercially available recombinant CrcB proteins are produced in E. coli expression systems . For successful expression, researchers should consider:

• Vector selection: Vectors containing strong inducible promoters (T7, tac) with tight regulation to control potentially toxic membrane protein expression

• E. coli strain optimization:

  • BL21(DE3) and derivatives for general expression

  • C41(DE3) or C43(DE3) for membrane proteins that may be toxic

  • Rosetta strains for proteins with rare codons

• Induction conditions:

  • Lower temperatures (16-25°C) during induction to slow folding and prevent inclusion body formation

  • Reduced inducer concentrations (0.1-0.5 mM IPTG)

  • Extended expression times (overnight)

• Affinity tag placement:

  • N-terminal His-tag as used in commercial preparations

  • Consider TEV protease cleavage sites if tag removal is required

For membrane proteins like CrcB, optimization of membrane fraction isolation and detergent solubilization are critical steps that may require empirical testing of different detergents (DDM, LMNG, or C12E8) for optimal protein extraction and stability.

What are effective purification strategies for recombinant CrcB protein?

Purification of recombinant His-tagged CrcB protein requires specialized techniques for membrane proteins. Based on specifications from commercial preparations, the following strategy is recommended :

Step 1: Cell lysis and membrane preparation

• Mechanical disruption (sonication or high-pressure homogenization)

• Differential centrifugation to isolate membrane fractions

• Washing steps to remove peripheral proteins

Step 2: Protein solubilization

• Screening of detergents for optimal solubilization

• Typical conditions: 1% detergent, 150-300 mM NaCl, pH 7.5-8.0

• Incubation at 4°C with gentle agitation for 1-2 hours

Step 3: Affinity chromatography

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

• Binding buffer containing detergent at concentrations above CMC

• Wash steps with increasing imidazole (20-40 mM)

• Elution with 250-300 mM imidazole

Step 4: Secondary purification

• Size exclusion chromatography to remove aggregates

• Buffer optimization for stability (typical buffer: Tris/PBS-based, pH 8.0)

Step 5: Storage and handling

• Concentration to 0.1-1.0 mg/mL

• Addition of 5-50% glycerol (50% recommended)

• Storage at -20°C/-80°C in aliquots

• Avoid repeated freeze-thaw cycles

The final product should be >90% pure as determined by SDS-PAGE analysis .

How can researchers assess CrcB ion transport activity in vitro?

Assessing the ion transport activity of CrcB requires specialized methodologies for membrane proteins. The following experimental approaches are recommended:

• Reconstitution in proteoliposomes:

  • Purified CrcB protein incorporated into artificial liposomes

  • Liposome composition should mimic bacterial membranes

  • Verification of proper orientation by protease protection assays

• Fluoride-selective electrode measurements:

  • Direct measurement of fluoride concentration changes

  • Assay buffer typically contains 100-150 mM KCl, 20 mM HEPES (pH 7.0-7.5)

  • Initiation of transport by establishing ion gradients

• Fluorescent probe-based assays:

  • Internal pH-sensitive fluorescent dyes to monitor pH changes during transport

  • MQAE fluorescent probe (quenched by halides) for indirect measurement

• Electrophysiological techniques:

  • Planar lipid bilayer recordings for single-channel analysis

  • Patch-clamp analysis of CrcB-expressing giant proteoliposomes

  • Solid-supported membrane electrophysiology

• Isotope flux assays:

  • Using 18F-labeled fluoride for direct transport measurement

  • Scintillation counting to quantify transport rates

Each method has advantages for specific research questions about CrcB function. For example, electrode measurements provide real-time transport kinetics, while fluorescent probes allow high-throughput screening of transport modulators.

What approaches can assess CrcB's role in Shewanella baltica's spoilage activity?

Investigating CrcB's potential role in Shewanella baltica's seafood spoilage activity requires integrated experimental approaches:

• Gene knockout and complementation studies:

  • Create precise crcB deletion mutants

  • Compare spoilage parameters between wild-type, knockout, and complemented strains

  • Assess whether CrcB expression correlates with spoilage potential

• Spoilage parameter measurements:

  • Total volatile basic nitrogen (TVB-N) production using standard methods

  • H2S production quantification (S. baltica is identified as the main H2S-producing bacterium)

  • Sensory evaluation and lightness measurements of inoculated fish samples

• Transcriptional analysis:

  • qPCR to measure crcB expression under spoilage conditions

  • RNA-Seq to identify co-regulated genes during spoilage

  • Comparison with RpoS regulon (RpoS positively affects spoilage activity)

• Environmental condition testing:

  • Low temperature (4°C) storage experiments mimicking seafood preservation

  • Varied salt concentrations reflecting different processing methods

  • Oxygen limitation studies simulating packaged seafood conditions

Research has shown that the sigma factor RpoS positively affects spoilage activity in S. baltica, with significant changes in total viable counts, sensory scores, TVB-N, and lightness of fish fillets when comparing wild-type and RpoS mutant strains . Similar approaches could be applied to investigate CrcB's specific contribution to these spoilage mechanisms.

How do genomic variations in crcB genes correlate with Shewanella baltica ecological adaptations?

Shewanella baltica shows remarkable genomic variation related to ecological adaptation, which may extend to the crcB gene. Analysis of this correlation requires:

• Comparative sequence analysis:

  • Alignment of crcB sequences from different S. baltica strains

  • Identification of SNPs and structural variations

  • Correlation of sequence variations with ecological origin

• Population genomics approach:

  • Analysis of S. baltica populations from different redox niches

  • Assessment of selection signatures on crcB genes

  • Identification of niche-specific alleles

• Ecological correlation studies:

  • Sampling across redox gradients in the Baltic Sea

  • Isolation and genomic characterization of strains from different depths

  • Experimental verification of fitness effects under different conditions

Research has shown that S. baltica populations exhibit genomic variations underlying speciation and niche specialization, particularly related to redox conditions . The Baltic Sea provides a natural laboratory with its stratified water column creating a gradient of environmental niches with varying electron acceptors and donors. S. baltica strains from different niches show contrasting gene-sharing patterns , and analysis of the crcB gene within this context could reveal its potential role in adaptation to specific redox conditions.

What is the phylogenetic distribution of crcB across Shewanella species and related genera?

The phylogenetic distribution of crcB genes across Shewanella species reflects both vertical inheritance and potential horizontal gene transfer events. To comprehensively analyze this distribution:

• Phylogenetic analysis methodology:

  • Construction of phylogenetic trees using maximum likelihood or Bayesian methods

  • Comparison of crcB gene trees with species trees to identify discordance

  • Assessment of selective pressures using dN/dS ratios

• Genomic context examination:

  • Analysis of genes flanking crcB in different species

  • Identification of conserved synteny or disruption patterns

  • Detection of mobile genetic elements that might facilitate transfer

• Taxonomic range consideration:

  • Core presence in Shewanella species (S. baltica, S. putrefaciens, S. amazonensis)

  • Distribution in related Gammaproteobacteria

  • Comparison with more distant bacterial lineages

Studies have shown that S. baltica dominates H2S-producing bacterial populations in iced marine fish, particularly after cold storage . Meanwhile, transcriptional variation exists even between closely related S. baltica ecotypes . This suggests that while crcB may be conserved across the genus as part of the core genome essential for ion homeostasis, functional variations might exist that contribute to niche specialization and environmental adaptation.

How can structural biology techniques be applied to study CrcB conformation?

Structural characterization of CrcB presents challenges typical of membrane proteins but is essential for understanding its mechanism. The following methodological approaches are recommended:

• Cryo-electron microscopy (cryo-EM):

  • Sample preparation in nanodiscs or amphipols to maintain native-like environment

  • Single-particle analysis for 3D reconstruction

  • Conditions: 1-3 mg/ml protein, vitrification on holey carbon grids

  • Data collection at 300 kV with direct electron detectors

• X-ray crystallography optimization:

  • Lipidic cubic phase (LCP) or bicelle crystallization methods

  • Screening of detergents, lipids, and stabilizing compounds

  • Use of antibody fragments or nanobodies as crystallization chaperones

  • Synchrotron radiation for data collection

• NMR spectroscopy approaches:

  • Solution NMR for smaller domains or flexible regions

  • Solid-state NMR for full-length protein in lipid bilayers

  • Selective isotopic labeling (15N, 13C) strategies

  • Magic angle spinning techniques for enhanced resolution

• Computational modeling:

  • Homology modeling based on related structures

  • Molecular dynamics simulations in explicit membrane environments

  • Integration of experimental constraints from limited resolution data

  • Refinement of models using machine learning approaches

For the commercially available His-tagged recombinant CrcB proteins , initial structural studies would benefit from optimization of reconstitution conditions to ensure proper folding in membrane mimetics before applying these structural biology techniques.

What methods can resolve contradictory data regarding CrcB function across different experimental systems?

Resolving contradictory data about CrcB function requires systematic troubleshooting and standardized approaches:

• Experimental standardization protocol:

  • Define consistent buffer compositions, pH, temperature, and ion concentrations

  • Establish standardized protein preparation methods

  • Use identical expression constructs and purification protocols

  • Implement blinded analysis to eliminate experimenter bias

• Multi-laboratory validation approach:

  • Cross-laboratory testing of identical samples and protocols

  • Round-robin testing of critical experiments

  • Statistical power analysis to determine adequate sample sizes

  • Meta-analysis of compiled data sets

• Systematic variation testing:

  • Controlled variation of experimental parameters (temperature, pH, ionic strength)

  • Testing across multiple S. baltica strains and related species

  • Evaluation of different membrane/lipid environments

  • Assessment of equipment-specific variations

• Integrated data analysis framework:

  • Bayesian analysis to incorporate prior knowledge

  • Machine learning approaches to identify hidden variables

  • Development of quantitative models that account for experimental variations

  • Standardized reporting of all experimental conditions and controls

Studies have shown that even within S. baltica species, substantial transcriptional variation exists between strains under identical laboratory conditions . This natural variation could explain some contradictory results and highlights the importance of using well-characterized strains and reporting detailed experimental conditions when studying CrcB function.

How does CrcB function relate to Shewanella baltica's role in marine ecosystems?

Understanding CrcB's role in Shewanella baltica's marine ecosystem function requires ecological context and specialized methodological approaches:

• Environmental sampling and expression analysis:

  • Collection of samples across Baltic Sea redox gradients

  • RNA extraction and qPCR targeting crcB transcripts

  • Correlation of expression with environmental parameters

  • Metatranscriptomic analysis of natural communities

• Microcosm experiments:

  • Simulation of marine conditions in laboratory settings

  • Tracking of wild-type vs. crcB mutant population dynamics

  • Response to environmental stressors (temperature, salinity, pollutants)

  • Interaction with other marine microorganisms

• Fluoride resistance phenotyping:

  • Comparison of S. baltica strains from different marine environments

  • Determination of minimum inhibitory concentrations for fluoride

  • Correlation of resistance with crcB sequence variants

  • Testing adaptation capacity through experimental evolution

S. baltica has been identified as the dominant culturable nitrate-reducing bacterium in the Baltic Sea, particularly in the oxic-anoxic transition zone . This ecological niche is characterized by specific redox conditions where S. baltica likely plays important roles in nitrogen cycling. The CrcB protein, as an ion transporter, may contribute to S. baltica's ability to maintain ion homeostasis under the fluctuating conditions of this interface zone.

Recent studies with coastal Shewanella have examined their response to different food substrates, including planktonic and terrestrial carbon sources , which is relevant given projections of increased land runoff due to climate change. Understanding how CrcB contributes to adaptation to changing marine conditions could provide insights into microbial community dynamics in future Baltic Sea scenarios.

What implications does CrcB research have for seafood safety and preservation?

CrcB research has several potential implications for seafood safety and preservation strategies:

• Spoilage mechanism elucidation:

  • Investigation of CrcB's potential role in cold adaptation of S. baltica

  • Correlation between CrcB function and production of spoilage compounds

  • Assessment of CrcB involvement in biofilm formation on seafood surfaces

• Preservation technology development:

  • Screening for compounds that inhibit CrcB function

  • Testing of fluoride-based preservatives as potential CrcB inhibitors

  • Development of targeted antimicrobial approaches against S. baltica

• Quality indicator development:

  • Evaluation of crcB expression as a potential biomarker for spoilage prediction

  • Development of molecular diagnostic tools targeting crcB

  • Correlation of crcB variants with spoilage potential in different fish species

• Risk assessment protocols:

  • Monitoring of S. baltica strains in seafood processing environments

  • Tracking of CrcB variants associated with enhanced cold adaptation

  • Development of predictive models for spoilage based on S. baltica detection

Studies have established that S. baltica is the most important H2S-producing organism in iced Danish marine fish , and the RpoS sigma factor positively affects its spoilage activity . The spoilage reactions primarily involve trimethylamine-N-oxide reduction and H2S production. Understanding CrcB's potential contribution to these processes, particularly in relation to cold adaptation, could lead to improved preservation methods targeting specific molecular mechanisms of spoilage.