Recombinant Polynucleobacter necessarius Elongation factor Ts (tsf)

What is the significance of studying Elongation Factor Ts from Polynucleobacter necessarius?

Elongation Factor Ts (EF-Ts) from P. necessarius presents a unique research opportunity due to the organism's dual lifestyle as both free-living bacteria with streamlined genomes and as endosymbionts with further reduced genomes. EF-Ts functions as a guanosine nucleotide exchange factor for Elongation Factor Tu (EF-Tu), playing a critical role in protein synthesis by facilitating the formation and disassociation of the EF-Tu·GTP·aa-tRNA ternary complex . P. necessarius provides a valuable model system for studying protein translation dynamics in organisms undergoing genome reduction, offering insights into the evolution of essential cellular processes in symbiotic relationships.

How does EF-Ts affect the formation and stability of ternary complexes?

EF-Ts directly accelerates both the formation and decay rates of the EF-Tu·GTP·aa-tRNA ternary complex. Recent research indicates that EF-Ts attenuates the affinity of EF-Tu for GTP and destabilizes the ternary complex in the presence of non-hydrolyzable GTP analogs . This suggests that beyond its classic role as a nucleotide exchange factor, EF-Ts serves an additional regulatory function in actively controlling the abundance and stability of ternary complexes, which contributes to both the speed and fidelity of protein synthesis.

How has genome reduction in P. necessarius affected translation-related genes?

The symbiotic strain of P. necessarius has undergone significant genome reduction (approximately 27.7% smaller than the free-living strain) with a more dramatic decrease in coding DNA (42.3%) . Despite this reduction, genes involved in essential cellular processes like protein synthesis are generally preserved. The symbiotic strain contains numerous pseudogenes, indicating ongoing genome erosion . While specific information about the tsf gene is not detailed in the available data, genes essential for core cellular functions like translation are typically maintained even in reduced genomes, though they may show sequence modifications reflecting adaptation to the symbiotic lifestyle.

How might the molecular mechanisms of P. necessarius EF-Ts differ from those in model organisms?

The unique evolutionary history of P. necessarius, including significant genome streamlining even in free-living strains, suggests potential adaptations in its translation machinery. Research indicates that the genome reduction process in P. necessarius involved a two-step process: an initial streamlining in free-living ancestors followed by further erosion in symbiotic lineages . This evolutionary trajectory may have resulted in distinctive functional adaptations in translation factors like EF-Ts, potentially including:

What experimental approaches would best characterize the nucleotide exchange activity of recombinant P. necessarius EF-Ts?

The characterization of nucleotide exchange activity requires sophisticated kinetic analysis. Based on research with other bacterial systems, the following methodological approach would be optimal:

• Pre-steady-state kinetic analysis using stopped-flow fluorescence spectroscopy to measure:

  • The rate of EF-Tu·GDP dissociation catalyzed by EF-Ts

  • Formation rates of the EF-Tu·EF-Ts complex

  • GTP binding and EF-Ts displacement rates

• Direct measurement of conformational changes using:

  • FRET (Förster Resonance Energy Transfer) with labeled EF-Tu and EF-Ts

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

• Comparative analysis with EF-Ts from other bacteria to determine if the nucleotide-dependent conformational change in EF-Tu that controls ternary complex formation shows different kinetics when catalyzed by P. necessarius EF-Ts .

How might polyploidy in symbiotic P. necessarius affect recombinant protein expression systems?

The symbiotic strain of P. necessarius exhibits polyploidy with multiple nucleoids, which has been observed in other endosymbionts with reduced genomes such as Buchnera . While the proximal causes of polyploidy are not immediately apparent from the genomic sequences, this feature may have implications for recombinant expression systems:

• Gene dosage effects may influence the native expression levels of tsf and other translation factors

• Multiple genome copies could compensate for the loss of DNA repair mechanisms, including translesion DNA polymerases (TLPs)

• Expression systems mimicking the polyploid state might provide more authentic folding environments for P. necessarius proteins

• The relationship between genome copy number and expression efficiency could inform optimization strategies for recombinant production

What expression and purification strategies are most effective for producing functional recombinant P. necessarius EF-Ts?

Based on the characteristics of P. necessarius and general approaches for elongation factors, the following expression and purification strategy is recommended:

Expression System Selection:

Purification Protocol:

• Construct design: N-terminal 6xHis tag with TEV protease cleavage site

• Initial capture: Ni-NTA affinity chromatography (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 20-250 mM imidazole gradient)

• Tag removal: TEV protease digestion (overnight at 4°C)

• Secondary purification: Ion exchange chromatography on Q-Sepharose

• Final polishing: Size exclusion chromatography in storage buffer (20 mM HEPES pH 7.5, 100 mM KCl, 10 mM MgCl₂, 5% glycerol)

This approach has been successful for other bacterial elongation factors and should accommodate the specific properties of P. necessarius EF-Ts.

What assays can verify the functional activity of recombinant P. necessarius EF-Ts?

Multiple complementary assays should be employed to confirm activity:

• GDP/GTP Exchange Assay:

  • Measure the rate of mant-GDP release from EF-Tu·mant-GDP complex upon addition of EF-Ts

  • Determine the rate of mant-GTP binding to EF-Tu in the presence/absence of EF-Ts

  • Calculate acceleration factors by comparing rates with/without EF-Ts

• Ternary Complex Formation Assay:

  • Monitor the kinetics of ternary complex (EF-Tu·GTP·aa-tRNA) formation using stopped-flow fluorescence

  • Compare rates and yield in the presence and absence of EF-Ts

  • Analyze the effect of EF-Ts on the stability of preformed ternary complexes

• EF-Tu·EF-Ts Complex Analysis:

  • Analytical gel filtration to confirm complex formation

  • Isothermal titration calorimetry (ITC) to determine binding constants

  • Surface plasmon resonance (SPR) for real-time interaction analysis

Expected results should demonstrate that P. necessarius EF-Ts accelerates nucleotide exchange on EF-Tu and modulates ternary complex dynamics, consistent with its role in facilitating both the formation and disassociation of ternary complexes .

How can site-directed mutagenesis be used to investigate the function of P. necessarius EF-Ts?

Site-directed mutagenesis provides a powerful approach to dissect the structure-function relationships of P. necessarius EF-Ts:

Priority Residue Targets:

Experimental Approach:

• Generate alanine scanning mutants across conserved interface regions

• Create charge reversal mutations at key electrostatic interaction sites

• Develop chimeric constructs between P. necessarius EF-Ts and EF-Ts from model organisms

• Test all variants in the functional assays described in question 3.2

This systematic approach will reveal which structural features of P. necessarius EF-Ts contribute to its activity in nucleotide exchange and ternary complex modulation, potentially identifying adaptations specific to P. necessarius's unique ecological niche.

What technical challenges should researchers anticipate when working with recombinant P. necessarius EF-Ts?

Several technical challenges may arise when working with recombinant P. necessarius EF-Ts:

• Expression optimization:

  • Codon usage differences between P. necessarius and expression hosts

  • Potential toxicity if the recombinant EF-Ts interacts with host translation machinery

  • Solubility issues requiring fusion partners or specific solubilization conditions

• Functional validation:

  • Obtaining active EF-Tu from P. necessarius for homologous interaction studies

  • Distinguishing species-specific interactions from general EF-Ts activity

  • Establishing appropriate control conditions given the unique properties of P. necessarius

• Structural considerations:

  • Potential conformational differences between free-living and symbiotic variants

  • Stability issues during purification due to the loss of stabilizing interactions

  • Crystallization challenges for structural studies

Understanding these challenges in advance will enable researchers to develop appropriate mitigation strategies and experimental designs to successfully work with this unique protein from an organism with exceptional genomic properties.

How might studying P. necessarius EF-Ts contribute to our understanding of translation in genome-reduced organisms?

Research on P. necessarius EF-Ts offers several promising avenues for advancing our understanding of translation in genome-reduced organisms:

• Comparative analysis of EF-Ts from free-living and symbiotic P. necessarius strains could reveal adaptations in translation machinery during genome reduction processes

• Investigation of how EF-Ts compensates for the loss of other translation-related factors in reduced genomes

• Examination of potential functional trade-offs between translation efficiency and accuracy in organisms with streamlined genomes

• Exploration of how polyploidy might interact with translation factor activity in symbiotic bacteria

This research could provide fundamental insights into the minimal requirements for efficient protein synthesis and how translation systems adapt during the transition to symbiotic lifestyles.

What is the relationship between genome reduction, DNA repair deficiency, and translation fidelity in P. necessarius?

P. necessarius presents a fascinating system for studying the interconnection between genome reduction, DNA repair, and translation:

The symbiotic strain lacks all translesion DNA polymerases (TLPs) and has also lost the mismatch repair (MMR) system, which increases the risk that a single damaged nucleotide could completely block replication . This DNA repair deficiency, coupled with genome reduction, creates a unique context for translation processes:

• Higher mutation rates due to MMR loss may increase the evolutionary pressure on translation factors like EF-Ts to maintain accuracy

• The loss of TLPs may have occurred early in the genome erosion process, potentially creating additional pressure toward deletions in non-essential sequences

• The presence of multiple genome copies (polyploidy) may compensate for these repair deficiencies

• Translation factors may have adapted to function with potentially less accurate mRNA templates

Studying EF-Ts in this context could reveal how translation systems adapt to maintain adequate protein synthesis under conditions of increased genomic instability.