Recombinant Desulfobacterium autotrophicum Protein CrcB homolog (crcB)

What is the genomic context of the crcB homolog in Desulfobacterium autotrophicum HRM2?

The crcB homolog exists within the 5.6 megabasepair genome of Desulfobacterium autotrophicum HRM2, which is notably about 2 Mbp larger than genomes of other sulfate-reducing bacteria (SRB) . This genome contains a high number of plasticity elements (>100 transposon-related genes) and repetitive elements (132 paralogous genes Mbp⁻¹), which suggests a distinct evolutionary path compared to Desulfovibrio species . The genomic organization around crcB may provide insights into its functional relationships with other genes involved in ion transport or energy metabolism pathways characteristic of this metabolically versatile organism.

What is the predicted structural and functional role of CrcB homolog in Desulfobacterium autotrophicum?

The CrcB homolog in D. autotrophicum is predicted to function as a fluoride channel/transporter based on homology with characterized CrcB proteins. Given D. autotrophicum's ability to grow in various environmental conditions including chemolithoautotrophically with H₂, CO₂, and sulfate , the CrcB homolog likely plays a role in maintaining ion homeostasis under changing environmental challenges. Structural predictions suggest a membrane-spanning protein with multiple transmembrane domains, potentially functioning in conjunction with the organism's extensive regulatory network (represented by more than 250 sensory/regulatory proteins) .

How does the expression of crcB homolog compare between autotrophic and heterotrophic growth conditions in the native organism?

Under native conditions, the expression of crcB homolog likely varies with growth conditions. During chemolithoautotrophic growth with H₂, CO₂, and sulfate, expression patterns would differ from those during heterotrophic growth on organic compounds. The regulatory mechanisms may involve some of the extensive sensory/regulatory protein families present in D. autotrophicum HRM2, which enable efficient adaptation to changing environmental conditions . Quantitative expression studies comparing growth on different carbon sources would help elucidate these regulatory patterns.

What are the optimal expression systems for producing recombinant D. autotrophicum CrcB homolog?

Expression System Comparison:

How can translation initiation site accessibility be optimized for successful expression of crcB?

To optimize expression of D. autotrophicum CrcB homolog, researchers should focus on the accessibility of translation initiation sites, which has been shown to be a critical determinant of expression success . Accessibility can be modeled using mRNA base-unpairing across the Boltzmann's ensemble, and optimized through:

• Synonymous codon substitutions within the first nine codons of the mRNA, which can be designed using tools like TIsigner

• Modification of the 5' untranslated region to reduce secondary structure formation

• Adjustment of the spacing between the Shine-Dalgarno sequence and start codon

These approaches can increase expression success without altering the amino acid sequence of the target protein. Analysis of 11,430 expression experiments has demonstrated that higher accessibility correlates strongly with successful protein expression, achieving AUC scores significantly higher than other features such as codon adaptation index or GC content .

What fusion partners have proven most effective for improving solubility and purification of D. autotrophicum membrane proteins?

While specific data for D. autotrophicum CrcB homolog is limited, general principles for membrane protein expression suggest several effective fusion partners:

Selection should be guided by experimental goals and downstream applications, with N-terminal tags generally preferred for membrane proteins to avoid disrupting membrane insertion sequences.

How can researchers address growth inhibition issues when expressing recombinant crcB in E. coli?

Expression of membrane proteins like CrcB homologs can impose metabolic burdens on host cells. Research shows that higher accessibility of translation initiation sites leads to higher protein production but slower cell growth, supporting the concept of protein cost where cell growth is constrained during overexpression . To address growth inhibition:

• Use tunable expression systems with tight regulation (e.g., pBAD)

• Lower induction temperature to 18-20°C to slow protein production

• Supplement media with additional osmolytes or specific ions based on CrcB's function

• Consider C41/C43 E. coli strains specifically developed for toxic membrane proteins

• Implement fed-batch cultivation strategies to maintain optimal growth conditions

The growth rate should be monitored throughout expression, with harvest timing optimized to balance protein yield and cell viability.

What detergent extraction and purification protocols are most effective for maintaining functional crcB protein?

Recommended Detergent Screening Protocol:

• Initial extraction screening: Test panel of detergents including DDM (n-Dodecyl β-D-maltoside), LMNG, LDAO, and Fos-choline-12

• Solubilization efficiency assessment: Western blot analysis of supernatant vs. pellet fractions

• Stability evaluation: SEC (Size Exclusion Chromatography) profiles after 24, 48, and 72 hours at 4°C

• Functional validation: Fluoride binding assays with purified protein in different detergent environments

For D. autotrophicum CrcB homolog, mild detergents like DDM are typically recommended for initial extraction, with concentration optimization required for each preparation. Purification should proceed via IMAC (Immobilized Metal Affinity Chromatography) with His-tag, followed by SEC for oligomeric state assessment and homogeneity verification.

What are the key considerations for assessing proper folding and functionality of recombinantly expressed CrcB homolog?

Proper folding and functionality assessment requires multiple approaches:

• Secondary structure analysis: Circular dichroism (CD) spectroscopy to confirm predicted alpha-helical content characteristic of membrane channels

• Thermostability assessment: Differential scanning fluorimetry with varying fluoride concentrations

• Ion transport assays: Liposome reconstitution with fluoride-sensitive probes to verify channel activity

• Binding studies: Isothermal titration calorimetry or fluorescence-based assays to determine binding affinities for fluoride ions

Researchers should also consider that D. autotrophicum's adaptation to marine environments may influence the optimal conditions for CrcB function, potentially requiring buffers that mimic the native ionic environment.

How can researchers effectively study the role of crcB in fluoride resistance mechanisms of D. autotrophicum?

To investigate crcB's role in fluoride resistance:

• Generate gene deletion mutants in model organisms expressing the D. autotrophicum crcB homolog

• Perform complementation studies with wild-type and mutant variants

• Conduct growth inhibition assays with increasing fluoride concentrations

• Measure intracellular fluoride concentrations using fluoride-sensitive probes

• Combine with transcriptomic analysis to identify co-regulated genes in response to fluoride exposure

These approaches would help establish whether the D. autotrophicum CrcB homolog functions similarly to characterized fluoride channels, and how it might contribute to the organism's adaptation to its native environment.

What structural techniques are most promising for resolving the 3D structure of D. autotrophicum CrcB homolog?

How does sequence variation in crcB homologs across sulfate-reducing bacteria correlate with their environmental niches?

Comparative genomics approaches can reveal how CrcB homologs have evolved across sulfate-reducing bacteria from different environments. D. autotrophicum HRM2's genome shows evidence of different evolutionary history compared to Desulfovibrio species, with a high number of genome plasticity elements and repetitive elements . A systematic analysis of CrcB sequence conservation, particularly in transmembrane domains and putative ion selectivity regions, could reveal adaptation signatures related to:

• Halotolerance in marine vs. freshwater SRB species

• pH adaptation in different sediment environments

• Co-evolution with other ion transport systems

• Functional diversification beyond fluoride transport

How can researchers integrate transcriptomic and proteomic analyses to understand crcB regulation in D. autotrophicum?

An integrative approach to understanding CrcB regulation should:

• Perform RNA-Seq analysis comparing expression under various growth conditions (autotrophic vs. heterotrophic, different ion concentrations)

• Combine with quantitative proteomics to correlate transcript and protein abundance

• Identify transcription factors potentially binding to the crcB promoter region

• Map crcB expression patterns to the comprehensive regulatory network involving the >250 sensory/regulatory proteins found in D. autotrophicum

• Correlate expression patterns with metabolic shifts in carbon and energy metabolism

This multi-omics approach would contextualize CrcB function within the broader adaptive strategies of D. autotrophicum to its changing environment.

What are the implications of D. autotrophicum's unique genome plasticity for studying the evolution of the crcB gene family?

D. autotrophicum HRM2's genome contains >100 transposon-related genes, regions of GC discontinuity, and a high number of repetitive elements (132 paralogous genes Mbp⁻¹) , suggesting genomic plasticity that may influence crcB evolution. Research approaches should:

• Analyze the genomic neighborhood of crcB for evidence of horizontal gene transfer

• Compare synteny of crcB and surrounding genes across related species

• Investigate the distribution of crcB homologs in relation to the organism's metabolic capabilities

• Perform phylogenetic analyses to determine if crcB evolution tracks with species evolution or shows evidence of independent acquisition

This evolutionary context would help researchers understand how the crcB homolog contributes to D. autotrophicum's ecological success in marine sediment environments.

How does the functionality of crcB relate to the unique energy conservation mechanisms in D. autotrophicum?

The functionality of CrcB should be investigated in relation to D. autotrophicum's energy conservation mechanisms, particularly considering:

• Potential interactions with the heterodisulfide reductase system, which has proposed roles in energy conservation during dissimilatory sulfate reduction

• Relationships to transmembrane complexes (TpII-c3, Hme, Rnf) involved in electron transfer

• Ion homeostasis requirements during chemolithoautotrophic growth versus heterotrophic metabolism

• Potential role in maintaining membrane potential under different growth conditions

Understanding these relationships would provide insights into how ion transport systems like CrcB integrate with the core bioenergetic machinery that enables D. autotrophicum's metabolic versatility.