Recombinant Carboxydothermus hydrogenoformans Protein CrcB homolog 1 (crcB1)

What is Carboxydothermus hydrogenoformans and why is it significant for research?

Carboxydothermus hydrogenoformans is a thermophilic bacterium isolated from a hot spring in Kunashir Island, Russia. It is significant for research for three major reasons: it grows at very high temperatures (optimally at 78°C), it utilizes carbon monoxide as almost its sole carbon source, and it converts water to hydrogen gas as part of its metabolism . The organism belongs to the Firmicutes phylum and has become a model for studying hydrogenogens, which are bacteria and archaea that grow anaerobically using CO as their carbon source and water as an electron acceptor . Its genome sequence has revealed remarkable features, including five different carbon monoxide dehydrogenase complexes, which likely enable it to utilize CO for diverse cellular processes and compete effectively when CO is limiting .

What is the molecular structure of CrcB homolog 1 protein?

The CrcB homolog 1 protein (crcB1) from C. hydrogenoformans consists of 123 amino acids with the sequence: "MVDLLLIGLGGSIGAILRYTLTKKIGERYQGDWPLATFLINIIGSFGLGLLYGFKLNQVIWLLLGTGFFGGFTTFSTYIYEAIFLMEEGLFWKNVNYLLTSIFTGVVFFAAGMWLANFFKGGV" . This protein is encoded by the gene crcB1 with the ordered locus name CHY_2103 in the C. hydrogenoformans genome . Structural analysis suggests that this protein likely contains multiple transmembrane domains, which is consistent with CrcB proteins in other organisms that are typically membrane-associated. Detailed three-dimensional structural information would require X-ray crystallography or NMR studies, which have not been reported in the provided search results.

What expression systems are recommended for recombinant production of C. hydrogenoformans proteins?

For recombinant production of C. hydrogenoformans proteins, E. coli expression systems have been successfully employed. As demonstrated with Carbon Monoxide Dehydrogenase II (CODH-II), a protocol for high-activity recombinant expression in E. coli has been established . For CrcB1 specifically, a similar approach may be viable with appropriate modifications. When expressing thermophilic proteins in mesophilic hosts like E. coli, researchers should consider:

• Use of expression vectors with strong, inducible promoters (e.g., T7)

• Optimization of codon usage for the host organism

• Incorporation of appropriate tags for purification (tag type can be determined during the production process)

• Implementation of strategies to prevent inclusion body formation, such as co-expression with chaperones

• Cultivation at lower temperatures (15-25°C) after induction to enhance proper folding

The expression strategy should be tailored based on the specific properties of CrcB1, including its hydrophobicity and potential membrane association.

What are the optimal storage conditions for recombinant CrcB1?

Based on information provided for commercially available recombinant CrcB1, the optimal storage conditions include:

• Primary storage: -20°C for regular use or -80°C for extended storage

• Storage buffer: Tris-based buffer with 50% glycerol, optimized for protein stability

• Working aliquots: Can be stored at 4°C for up to one week

• Avoiding repeated freeze-thaw cycles, as this can lead to protein denaturation

How might CrcB1 function in relation to the carbon monoxide metabolism of C. hydrogenoformans?

While the search results don't directly address CrcB1's role in carbon monoxide metabolism, we can analyze its potential function through comparative genomics and functional context. CrcB homologs in other organisms are often associated with ion transport, particularly fluoride ion channels that protect against environmental toxicity.

In C. hydrogenoformans, which has a specialized carbon monoxide metabolism, CrcB1 might play several potential roles:

• Maintaining ion homeostasis during CO metabolism, which produces H₂ and CO₂ that could affect intracellular pH

• Contributing to membrane integrity under the extreme thermophilic conditions where the organism thrives

• Potentially participating in a specialized transport system associated with the five CODH complexes encoded in the genome

The genome of C. hydrogenoformans reveals remarkable specialization for CO utilization, including five different CODH complexes that likely enable diverse metabolic capabilities . Research to elucidate CrcB1's specific role would require techniques like gene knockout studies, protein-protein interaction analysis, and metabolic flux analysis under varying CO concentrations.

What kinetic parameters should be considered when studying CrcB1 in relation to C. hydrogenoformans metabolism?

Understanding kinetic parameters is crucial when studying any protein involved in C. hydrogenoformans metabolism. Based on studies of CO metabolism in this organism, several kinetic considerations would be relevant for CrcB1 research:

• Temperature dependence: C. hydrogenoformans grows optimally at 70°C, and its enzymatic activities show distinct temperature profiles. Research shows that its CO conversion activities are highest at elevated temperatures .

• Substrate concentration effects: Studies on C. hydrogenoformans have shown that CO acts as both a substrate and an inhibitor at elevated concentrations . The kinetic model that best describes this behavior is the Han and Levenspiel model for substrate inhibition kinetics .

• Gas-liquid mass transfer limitations: At high biomass concentrations, the gas-liquid mass transfer of CO becomes limiting. For optimal substrate/biomass ratio, exceeding 5 mol CO g⁻¹ biomass VSS is recommended to avoid gas-liquid substrate transfer limitation .

• Inhibition parameters: Research has shown that the maximum specific CO consumption rate (kₘₐₓ) could reach 8.2 mol CO g⁻¹ VSS day⁻¹ if CO were not an inhibitor .

A table summarizing reported kinetic parameters for C. hydrogenoformans:

How does the genomic context of the crcB1 gene inform our understanding of its function?

• The genome contains genes with diverse evolutionary origins, as evidenced by the cooF and cooS genes (encoding carbon monoxide dehydrogenase components) which show different codon usage patterns suggesting lateral gene transfer from different donor species .

• The cooF gene particularly shows an archaeal-like codon usage pattern dominated by AGA and AGG for arginine codons .

• The genome encodes five different CODH complexes, suggesting specialized functions for CO utilization .

To fully understand crcB1's genomic context, researchers should:

• Perform synteny analysis to identify conserved gene neighborhoods across related species

• Analyze promoter regions for regulatory elements that might indicate co-regulation with CO metabolism genes

• Examine transcriptomic data to identify co-expressed genes under various growth conditions

• Use phylogenetic profiling to identify species with similar gene complements

These approaches could reveal functional associations not immediately apparent from sequence data alone.

What protein purification strategies are effective for isolating recombinant CrcB1?

Purifying recombinant CrcB1 requires specialized approaches due to its likely membrane-associated nature and thermophilic origin. Based on successful purification protocols for other C. hydrogenoformans proteins, the following methodology is recommended:

• Cell lysis optimization: For thermophilic membrane proteins, use a combination of mechanical disruption (sonication or French press) and detergent solubilization. Consider testing multiple detergents including n-dodecyl-β-D-maltoside (DDM), CHAPS, or Triton X-100 at varying concentrations.

• Affinity chromatography: Utilize the affinity tag incorporated during expression (the tag type will be determined during the production process) . Common options include His-tag purification using Ni-NTA resin or GST-tag purification.

• Thermostability advantage: Exploit the thermostability of C. hydrogenoformans proteins by incorporating a heat treatment step (60-70°C for 10-20 minutes) to denature less stable E. coli proteins while preserving CrcB1.

• Size exclusion chromatography: As a polishing step, use size exclusion chromatography to remove aggregates and obtain homogeneous protein.

• Buffer optimization: The final buffer composition should maintain protein stability. A Tris-based buffer with 50% glycerol has been reported as suitable for storage .

A notable protocol for purifying C. hydrogenoformans proteins is described for CODH-II, which could serve as a starting template with appropriate modifications for membrane protein purification .

What experimental approaches can determine the physiological role of CrcB1 in C. hydrogenoformans?

Determining the physiological role of CrcB1 requires a multi-faceted experimental approach:

• Gene knockout or CRISPR interference: Generate crcB1 deletion mutants or use CRISPRi to downregulate expression, then assess phenotypic changes in:

  • Growth rates under varying CO concentrations

  • CO consumption kinetics

  • H₂ production efficiency

  • Cell survival under different ionic conditions

• Heterologous expression and complementation: Express C. hydrogenoformans crcB1 in model organisms lacking CrcB homologs and assess phenotypic rescue.

• Protein localization: Use fluorescently tagged CrcB1 or immunolocalization to determine subcellular localization, particularly in relation to CODH complexes.

• Protein-protein interactions: Implement pull-down assays, bacterial two-hybrid systems, or crosslinking studies to identify interaction partners, particularly focusing on components of the CODH complexes.

• Transport studies: If CrcB1 functions as an ion transporter, use fluorescent ion indicators or radioisotope flux assays to measure transport activity in reconstituted liposomes or whole cells.

• Transcriptomic and proteomic analysis: Compare expression patterns of crcB1 and potential interacting partners under various growth conditions, particularly varying CO concentrations.

• Structural studies: Determine the three-dimensional structure using X-ray crystallography or cryo-electron microscopy to gain insights into functional mechanisms.

These approaches would need to be conducted at elevated temperatures (60-70°C) to mimic the natural growth conditions of C. hydrogenoformans .

How can researchers effectively analyze the thermal stability of CrcB1?

Analyzing the thermal stability of CrcB1 from a thermophilic organism like C. hydrogenoformans requires specialized techniques:

• Differential Scanning Calorimetry (DSC): Measure heat capacity changes during protein unfolding at increasing temperatures. For thermophilic proteins like CrcB1, the temperature range should extend to 100°C or higher. This provides the melting temperature (Tm) and thermodynamic parameters of unfolding.

• Circular Dichroism (CD) Spectroscopy: Monitor changes in secondary structure as a function of temperature. For thermophilic proteins, measurements should be taken at 5°C intervals from 25°C to at least 95°C.

• Thermal Shift Assays: Use fluorescent dyes like SYPRO Orange that bind to hydrophobic regions exposed during protein unfolding. This technique can also screen buffer conditions that enhance thermal stability.

• Activity Assays at Various Temperatures: Measure protein function across a temperature gradient (30-95°C) to determine the temperature optimum and the temperature at which activity is lost.

• Limited Proteolysis: Incubate the protein at various temperatures followed by brief exposure to proteases. Increased susceptibility to proteolysis indicates conformational changes or unfolding.

• Dynamic Light Scattering (DLS): Monitor protein aggregation as temperature increases, which can indicate the onset of thermal denaturation.

Given that C. hydrogenoformans grows optimally at 78°C , CrcB1 is expected to maintain stability and function at elevated temperatures. Comparative analysis with CrcB homologs from mesophilic organisms could provide insights into the structural determinants of thermostability.

How does CrcB1 compare with other proteins in the C. hydrogenoformans proteome?

Comparing CrcB1 with other proteins in the C. hydrogenoformans proteome requires comprehensive analysis:

• Codon usage analysis: Similar to the analysis performed for cooF and cooS genes , examine whether crcB1 shows unusual codon usage patterns that might suggest lateral gene transfer. This could indicate evolutionary adaptation or acquisition of specialized functions.

• Comparative expression analysis: Determine if crcB1 is co-expressed with genes involved in CO metabolism, particularly the five CODH complexes encoded in the genome . This would suggest functional association with carbon monoxide utilization pathways.

• Protein domain architecture: Analyze whether unique structural features distinguish CrcB1 from other membrane proteins in C. hydrogenoformans, potentially relating to function under extreme thermophilic conditions.

• Evolutionary rate analysis: Compare the evolutionary rate of crcB1 with other genes to determine if it's under stronger selective pressure, which might indicate essential function.

• Post-translational modification prediction: Identify potential sites for post-translational modifications that might regulate CrcB1 function in response to environmental conditions.

The extraordinary metabolic capabilities of C. hydrogenoformans, particularly its five different CODH systems , suggest specialized adaptation to a CO-dependent lifestyle. Understanding where CrcB1 fits within this specialized system requires integrative analysis across multiple data types.

What bioinformatic tools are most effective for predicting CrcB1 function?

Predicting CrcB1 function requires a strategic combination of bioinformatic approaches:

• Transmembrane topology prediction: Tools like TMHMM, MEMSAT, and Phobius can predict membrane-spanning regions, which are likely important for CrcB1 function.

• Homology modeling: Using structural templates from the Protein Data Bank, construct 3D models to predict functional sites and potential ligand-binding regions.

• Molecular dynamics simulations: Simulate CrcB1 behavior within a membrane environment at elevated temperatures (70-80°C) to understand conformational dynamics under physiological conditions.

• Phylogenetic profiling: Identify co-evolving genes across multiple genomes to predict functional associations and potential interaction partners.

• Protein-protein interaction prediction: Tools like STRING, PSICQUIC, and InterPreTS can predict interaction partners based on sequence co-evolution, domain-domain interactions, and experimental data from related proteins.

• Functional domain analysis: Tools like InterProScan, Pfam, and PROSITE can identify conserved domains that might suggest function.

• Genomic context analysis: Examine neighboring genes, as functionally related genes are often clustered in bacterial genomes.

For thermophilic proteins like those from C. hydrogenoformans, specialized tools that account for adaptations to high temperatures should be prioritized when available.

What are the key unanswered questions about CrcB1 that merit further investigation?

Several critical questions about CrcB1 remain unanswered and represent promising avenues for future research:

• Physiological function: What is the precise role of CrcB1 in C. hydrogenoformans physiology? Does it participate directly in CO metabolism or play a supportive role in maintaining cellular homeostasis under thermophilic, CO-rich conditions?

• Structural adaptations: What structural features enable CrcB1 to function at the extreme temperatures (optimally 78°C) where C. hydrogenoformans thrives ? How do these compare with CrcB homologs from mesophilic organisms?

• Interaction with CODH complexes: Does CrcB1 interact directly or indirectly with any of the five CODH complexes identified in the C. hydrogenoformans genome ? If so, what is the nature and functional significance of these interactions?

• Evolutionary history: Was crcB1 acquired through lateral gene transfer like the cooF gene , or is it an ancestral gene that has co-evolved with the CO utilization machinery?

• Biotechnological applications: Can CrcB1 be engineered for applications in biotechnology, particularly in processes requiring thermostable membrane proteins?

Addressing these questions would not only advance our understanding of C. hydrogenoformans biology but also contribute to broader knowledge of extremophile adaptation and carbon monoxide metabolism.

How might CrcB1 be leveraged in biotechnological applications?

The thermostable nature of proteins from C. hydrogenoformans makes them candidates for various biotechnological applications:

• Bioremediation: C. hydrogenoformans proteins, potentially including CrcB1, might be engineered for high-temperature bioremediation processes, particularly those involving carbon monoxide or other toxic gases.

• Hydrogen production: The remarkable ability of C. hydrogenoformans to produce hydrogen from CO and water suggests potential applications in biohydrogen production. If CrcB1 plays a role in this process, it could be engineered to enhance efficiency.

• Carbon capture: C. hydrogenoformans utilizes the Wood-Ljungdahl pathway, which fixes CO₂ . Understanding the role of all proteins in this organism's carbon metabolism, including potential supporting roles of CrcB1, could inform biocatalytic approaches to carbon capture.

• Thermostable biosensors: If CrcB1 functions as an ion transporter or sensor, it could be engineered as a component of thermostable biosensors for industrial processes operating at elevated temperatures.

• Synthetic biology platforms: The ability of C. hydrogenoformans to grow using only CO as a carbon source represents a minimalist metabolism that could inform design of synthetic organisms for specialized industrial applications.