Recombinant Nitratiruptor sp. Elongation factor Ts (tsf)

What is Nitratiruptor and why is it of scientific interest?

Nitratiruptor is a genus of thermophilic bacteria found in deep-sea hydrothermal vent environments. The most studied species include Nitratiruptor labii HRV44 T and Nitratiruptor tergarcus. These bacteria are of particular interest because they possess the ability to reduce nitrous oxide (N2O) to dinitrogen gas (N2) at high temperatures, contributing to nitrogen cycling in extreme environments . Nitratiruptor labii HRV44 T, isolated from a deep-sea hydrothermal vent in the Mid-Okinawa Trough, is particularly notable for its outstanding N2O reduction activity . This genus belongs to the class Campylobacteria and represents one of the first thermophilic organisms capable of growth by hydrogen oxidation coupled to N2O reduction .

What are the growth characteristics of Nitratiruptor species?

Nitratiruptor species exhibit the following growth characteristics:

These bacteria are chemolithoautotrophs, utilizing hydrogen as an electron donor and carbon dioxide as a carbon source . They show remarkable growth when utilizing N2O as an electron acceptor, with N. labii HRV44 T demonstrating the highest N2O consumption rate (approximately 9.9 ± 1.4 μmol h-1 per mL of culture) among related strains .

What is the biological role of Elongation Factor Ts in bacterial protein synthesis?

Elongation Factor Ts (EF-Ts) serves as a guanosine nucleotide exchange factor (GEF) for Elongation Factor Tu (EF-Tu). During protein synthesis, aminoacyl-tRNA enters the ribosome in a ternary complex with EF-Tu and GTP . After GTP hydrolysis, EF-Tu-GDP dissociates from the ribosome. EF-Ts then facilitates the exchange of GDP for GTP on EF-Tu, enabling the formation of new ternary complexes for subsequent rounds of elongation .

Recent studies have revealed that EF-Ts plays a more complex role than previously thought. Beyond its nucleotide exchange function, EF-Ts directly facilitates both the formation and disassembly of the EF-Tu·GTP·aminoacyl-tRNA ternary complex . This additional regulatory role contributes to the rapid and faithful protein synthesis required in living cells.

How does thermophilic bacterial EF-Ts likely differ from mesophilic homologs?

While specific structural data for Nitratiruptor sp. EF-Ts is not explicitly detailed in the search results, thermophilic proteins typically exhibit several adaptations that contribute to their thermal stability:

• Increased number of salt bridges and hydrogen bonds

• Higher proportion of charged amino acids (Arg, Glu, Lys)

• Reduced number of thermolabile residues (Asn, Gln, Cys)

• More compact hydrophobic core

• Reduced surface loop regions

Given Nitratiruptor's optimal growth temperature of 53-55°C , its EF-Ts would likely incorporate these thermostability features while maintaining the conserved functional domains required for interaction with EF-Tu and nucleotide exchange.

What expression systems are most suitable for producing recombinant thermophilic proteins like Nitratiruptor EF-Ts?

When expressing recombinant thermophilic proteins, several expression systems can be considered, each with advantages and limitations:

For Nitratiruptor EF-Ts, an initial approach using E. coli BL21(DE3) with a reduced induction temperature (20-25°C) would balance yield and proper folding. Given that Nitratiruptor grows optimally at moderate thermophilic temperatures (53-55°C) , its proteins may express reasonably well in mesophilic hosts with appropriate optimization.

What purification strategies would be effective for recombinant Nitratiruptor EF-Ts?

A multi-step purification strategy would likely be effective for recombinant Nitratiruptor EF-Ts:

• Affinity Chromatography: Using a His-tag or other affinity tag for initial capture

• Heat Treatment: Exploiting the thermostability of Nitratiruptor EF-Ts (potentially at 50-55°C) to denature contaminating E. coli proteins

• Ion Exchange Chromatography: Further purification based on charge properties

• Size Exclusion Chromatography: Final polishing step and buffer exchange

The heat treatment step is particularly advantageous when purifying thermophilic proteins from mesophilic expression hosts, as it can significantly reduce contaminating proteins while leaving the target protein intact.

How can researchers assess the activity of recombinant Nitratiruptor EF-Ts?

Several assays can be employed to assess the functionality of recombinant Nitratiruptor EF-Ts:

• GDP/GTP Exchange Assay: Measuring the rate of nucleotide exchange on EF-Tu in the presence of EF-Ts using fluorescently labeled nucleotides or radioactive tracers

• Ternary Complex Formation Assay: Monitoring the formation and decay rates of EF-Tu·GTP·aminoacyl-tRNA ternary complexes, which are accelerated in the presence of EF-Ts

• Thermal Stability Assessment: Determining the temperature at which the protein retains activity, which should align with Nitratiruptor's growth temperature range (45-60°C)

• Protein Translation Assay: Evaluating the ability of Nitratiruptor EF-Ts to support in vitro translation systems at elevated temperatures

Based on studies with E. coli EF-Ts, fluorescence-based assays have been successfully used to quantitatively explore the kinetic features of ternary complex formation and decay , which could be adapted for Nitratiruptor EF-Ts.

How might the transcriptional regulation of tsf differ in Nitratiruptor compared to model organisms?

The transcriptional regulation of translation-related genes in Nitratiruptor likely reflects adaptations to its unique ecological niche. Based on transcriptomic studies of Nitratiruptor labii HRV44 T, many genes involved in basic cellular processes are constitutively expressed under anaerobic conditions . This suggests that translation machinery genes, potentially including tsf, may be continuously expressed to maintain protein synthesis capacity in these extreme environments.

Studies on N. labii HRV44 T have identified Crp/Fnr transcriptional regulators that control the expression of genes involved in electron transport and energy metabolism . These regulators might also influence the expression of translation-related genes like tsf, especially during shifts in environmental conditions such as oxygen availability or temperature fluctuations.

What role might Nitratiruptor EF-Ts play in thermal adaptation of the translation machinery?

In thermophilic bacteria like Nitratiruptor, protein synthesis must function efficiently at elevated temperatures (45-60°C for N. labii) . EF-Ts likely plays a crucial role in maintaining translation efficiency under these conditions through several potential mechanisms:

The interaction between EF-Ts and EF-Tu might exhibit distinct kinetic properties optimized for the thermophilic lifestyle of Nitratiruptor, potentially with higher affinity or altered nucleotide exchange rates compared to mesophilic counterparts.

How might the interaction between Nitratiruptor EF-Ts and EF-Tu relate to N2O reduction pathways?

Nitratiruptor species are notable for their ability to grow by hydrogen oxidation coupled to N2O reduction . While direct evidence linking translation factors to N2O metabolism is not presented in the search results, protein synthesis machinery undoubtedly plays a critical role in expressing the enzymes involved in this pathway.

Transcriptomic studies have shown that N. labii HRV44 T constitutively expresses most transcripts related to denitrification, even in the absence of nitrogen oxides as electron acceptors . When N2O is added to cultures, gene expressions involved in electron transport to NosZ (N2O reductase) are upregulated within 3 hours, rather than upregulation of nos genes themselves .

This suggests that Nitratiruptor maintains a readiness to rapidly respond to N2O availability, which would require efficient translation machinery (including EF-Ts and EF-Tu) to quickly synthesize the necessary electron transport proteins when needed.

What are the optimal conditions for assaying Nitratiruptor EF-Ts activity in vitro?

Based on the growth characteristics of Nitratiruptor species, the following conditions would likely be optimal for assaying EF-Ts activity in vitro:

These conditions would mimic the natural environment in which Nitratiruptor EF-Ts functions, likely yielding the most physiologically relevant activity measurements. Furthermore, assays measuring interaction with EF-Tu should consider using either homologous EF-Tu from Nitratiruptor or thermostable EF-Tu from related thermophiles to avoid temperature-related denaturation of interaction partners.

What structural analysis techniques are most informative for characterizing thermophilic translation factors?

Several complementary structural analysis techniques can provide valuable insights into the structure and function of thermophilic translation factors like Nitratiruptor EF-Ts:

Each of these techniques provides complementary information, and a multi-technique approach would yield the most comprehensive understanding of Nitratiruptor EF-Ts structure and function.

How might recombinant Nitratiruptor EF-Ts contribute to thermostable in vitro translation systems?

Recombinant Nitratiruptor EF-Ts could significantly enhance the development of thermostable in vitro translation systems for biotechnological applications. Current cell-free protein synthesis systems often operate at moderate temperatures (30-37°C) and can suffer from reduced efficiency during prolonged incubations. A thermostable system incorporating Nitratiruptor EF-Ts could offer several advantages:

• Increased reaction rates at elevated temperatures (45-55°C)

• Reduced risk of microbial contamination during extended runs

• Potential compatibility with thermostable mRNA templates

• Enhanced synthesis of proteins that fold optimally at higher temperatures

The unique properties of EF-Ts from a thermophilic deep-sea bacterium like Nitratiruptor could provide stability without sacrificing the critical nucleotide exchange activity and ternary complex regulation functions described for E. coli EF-Ts .

What insights could comparative analysis of Nitratiruptor EF-Ts provide about protein evolution in extreme environments?

Comparative analysis of Nitratiruptor EF-Ts with homologs from mesophilic and other extremophilic bacteria could reveal key adaptations driving protein evolution in extreme environments. Such analysis might uncover:

• Specific amino acid substitutions that confer thermostability without compromising function

• Conservation patterns in nucleotide binding and EF-Tu interaction domains

• Evolutionary relationships between translation factors from organisms inhabiting different extreme environments

Nitratiruptor species occupy a fascinating ecological niche at deep-sea hydrothermal vents, where they must contend with not only high temperatures but also unique chemical conditions and pressure effects . Their translation machinery, including EF-Ts, likely represents a specialized adaptation to this multifaceted extreme environment.

How can molecular dynamics simulations enhance our understanding of Nitratiruptor EF-Ts function?

Molecular dynamics (MD) simulations offer powerful tools for investigating the structural and functional properties of proteins like Nitratiruptor EF-Ts, particularly when experimental approaches may be challenging. MD simulations could:

• Model the behavior of Nitratiruptor EF-Ts at different temperatures to identify thermostability determinants

• Simulate the interaction between EF-Ts and EF-Tu during nucleotide exchange

• Predict the effects of pH and salt concentration on protein dynamics

• Investigate the molecular basis for the dual role of EF-Ts in both facilitating ternary complex formation and dissociation

These computational approaches, combined with experimental validation, could provide unprecedented insights into the molecular mechanisms underlying Nitratiruptor EF-Ts function in its extreme natural environment.