Recombinant Thermus thermophilus Protein CrcB homolog (crcB)

What is the function of CrcB homolog in Thermus thermophilus?

CrcB homologs in bacteria typically function as fluoride channels that provide resistance to fluoride toxicity by exporting fluoride ions from the cell. In Thermus thermophilus, the CrcB homolog likely serves a similar protective function, particularly important given that fluoride can inhibit enzymes involved in phosphoryl transfer reactions and energy metabolism.

The protein may have adapted to function optimally at the high temperatures in which T. thermophilus thrives (65-80°C). Like other T. thermophilus proteins, the stability of CrcB at elevated temperatures makes it valuable for structural and functional studies, similar to other characterized proteins from this organism such as RecA and RlmO .

How does the structure of Thermus thermophilus CrcB homolog compare to mesophilic homologs?

While the specific crystal structure of T. thermophilus CrcB homolog has not been fully characterized in the provided literature, we can draw parallels from other T. thermophilus proteins. T. thermophilus proteins typically exhibit increased rigidity, more extensive ion pair networks, and reduced flexibility in loop regions compared to mesophilic homologs, contributing to their thermostability.

The structural analysis approach would be similar to that used for the hypothetical protein TTHB192 from T. thermophilus HB8, which was determined at 1.9 Å resolution . The CrcB homolog likely maintains the core structural elements found in mesophilic homologs while incorporating adaptations that enhance stability at high temperatures, potentially including a higher proportion of charged residues forming salt bridges and more extensive hydrophobic core packing.

What are the optimal growth conditions for Thermus thermophilus when expressing native CrcB?

Thermus thermophilus is a hyperthermophilic bacterium that grows optimally at temperatures between 65°C and 80°C. For native expression of CrcB and other proteins, the organism is typically cultured in rich medium such as Thermus Medium or modified versions of Luria-Bertani broth adapted for thermophiles.

Growth conditions for optimal native expression of T. thermophilus proteins generally include:

Similar to studies with T. thermophilus methyltransferases, temperature sensitivity experiments may be conducted at 60-80°C to assess the function of CrcB under various conditions .

How can CRISPR-Cas9 be utilized to study the functional role of CrcB homolog in Thermus thermophilus?

CRISPR-Cas9 gene editing in T. thermophilus requires modifications to standard protocols due to the organism's high growth temperature and natural competence. To study CrcB homolog function:

• Design a thermostable Cas9 variant or use native T. thermophilus CRISPR systems, which are adapted to function at high temperatures.

• Create specific guide RNAs targeting the CrcB homolog gene with appropriate promoters functional in T. thermophilus.

• Develop a knockout construct containing selectable markers (typically thermostable antibiotic resistance genes).

• Transform T. thermophilus using natural competence protocols by adding DNA to cells in exponential growth phase.

• Perform phenotypic assays under varying fluoride concentrations to assess the role of CrcB in fluoride resistance.

• Complement the knockout with wild-type or mutant versions of CrcB to confirm phenotypic observations.

This approach parallels methods used to create and characterize the ΔTTHA1493 and ΔTTHA1280 strains in T. thermophilus for studying methyltransferase functions . Temperature sensitivity assays (at 60°C, 70°C, and 80°C) would be particularly relevant, as they could reveal whether CrcB contributes to thermal adaptation, similar to investigations of other T. thermophilus proteins .

What structural adaptations enable the thermostability of Thermus thermophilus CrcB homolog compared to mesophilic versions?

The thermostability of T. thermophilus CrcB homolog likely results from several structural adaptations:

Circular dichroism measurements, similar to those performed for ttRecX which showed stability up to 80°C at neutral pH with an α-helical content of 54%, would be valuable for characterizing the thermal stability profile of CrcB . X-ray crystallography at high resolution (1.7-1.9 Å) would provide detailed structural information, as was achieved for RlmO and TTHB192 .

How does the genomic context of the CrcB homolog in Thermus thermophilus provide insights into its evolutionary history and functional relationships?

The genomic context analysis of CrcB homolog in T. thermophilus can reveal important evolutionary and functional insights:

• Operon Structure: Determine whether CrcB is part of a larger operon, which might indicate functional relationships with co-transcribed genes.

• Proximity to Replication Terminus: Similar to TtAgo, which was found to be associated with the chromosomal region where replication terminates, the genomic position of CrcB relative to replication landmarks may provide functional clues .

• Comparative Genomics: Analysis of synteny with related thermophiles and mesophiles can reveal conservation patterns that indicate evolutionary importance.

• GC Skew Analysis: Using methods similar to those employed for identifying origin and terminus of replication in T. thermophilus, GC skew analysis ([G-C]/[G+C]) can provide insights into the evolutionary pressures on the genomic region containing CrcB .

• Horizontal Gene Transfer Evidence: Assessment of codon usage bias and GC content compared to the rest of the genome may indicate if CrcB was acquired through horizontal gene transfer.

This genomic context analysis parallels approaches used for other T. thermophilus proteins, such as TtAgo, where cumulative GC skew analysis placed the origin of replication at 1,541,431 bp and identified the chromosomal terminus site at 626,088 bp .

What are the optimal conditions for heterologous expression and purification of recombinant Thermus thermophilus CrcB homolog?

Optimal expression and purification of recombinant T. thermophilus CrcB homolog typically involves:

Expression System:

Purification Protocol:

• Heat Treatment: Exploit the thermostability by heating the cell lysate to 65-70°C for 15-20 minutes to precipitate most E. coli proteins while keeping CrcB soluble, similar to the approach used for ttRecX purification .

• Column Chromatography Sequence:

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

  • Size exclusion chromatography to ensure monodispersity

  • Optional ion exchange chromatography for further purification

• Buffer Optimization: Use high-salt buffers (300-500 mM NaCl) with mild detergents if CrcB proves to be membrane-associated, maintaining pH 7.5-8.0 throughout purification.

• Quality Assessment: Verify purity by SDS-PAGE, structural integrity by circular dichroism, and functional activity through fluoride ion transport assays.

This approach is comparable to that used for ttRecX purification, which employed heat treatment and multiple column chromatography steps, with size-exclusion chromatography confirming its monomeric state in solution .

What biophysical techniques are most effective for characterizing the thermal stability and structural dynamics of Thermus thermophilus CrcB homolog?

Several biophysical techniques are particularly effective for characterizing the thermostable CrcB homolog:

These techniques should be performed at a range of temperatures (25-85°C) to fully characterize the thermostability profile and structural transitions of the CrcB homolog.

How can site-directed mutagenesis be applied to identify key residues for fluoride selectivity and transport in Thermus thermophilus CrcB homolog?

A systematic site-directed mutagenesis approach for the T. thermophilus CrcB homolog would include:

• Sequence Alignment and Structure Prediction:

  • Align T. thermophilus CrcB with characterized CrcB proteins from other organisms

  • Identify conserved residues likely involved in fluoride binding and transport

  • Use homology modeling based on known structures (if available) to predict the 3D arrangement of these residues

• Mutant Design Strategy:

• Expression and Purification of Mutants: Using the optimized protocol for wild-type protein, with special attention to potential changes in stability or expression levels.

• Functional Characterization:

  • Fluoride transport assays using liposomes or whole-cell systems

  • Ion selectivity measurements comparing fluoride transport to other halides

  • Thermal stability assessments of each mutant compared to wild-type

• Structural Validation: X-ray crystallography of key mutants to correlate functional changes with structural alterations.

This approach parallels the characterization of RlmO in T. thermophilus, where key residues in the active site were identified and compared to related methyltransferases to understand the enzymatic mechanism .