Recombinant Prosthecochloris vibrioformis Elongation factor Tu (tuf)

What is Prosthecochloris vibrioformis and what makes its Elongation factor Tu significant for research?

Prosthecochloris vibrioformis is a nonmotile green sulfur bacterium typically found in aquatic environments such as rivermouths and estuaries. It belongs to the Chlorobiaceae family and is characterized by its ability to perform anoxygenic photosynthesis. P. vibrioformis DSM 260 has been isolated from rivermouth environments and has a genome size of approximately 2.31 Mb with a GC content of 52.1% .

Elongation factor Tu (EF-Tu), encoded by the tuf gene, is one of the most abundant bacterial proteins, constituting 5-10% of total cellular protein. In P. vibrioformis, as in other bacteria, EF-Tu plays a critical role in protein synthesis by delivering aminoacyl-tRNAs to the ribosome during translation elongation.

The significance of P. vibrioformis EF-Tu for researchers stems from several factors:

• It provides insights into translation mechanisms in anoxygenic phototrophs, which differ ecologically from model organisms like E. coli

• The conserved nature of the tuf gene makes it valuable for phylogenetic studies of green sulfur bacteria

• Understanding its structure-function relationships can reveal adaptations to the unique ecological niches inhabited by this photosynthetic bacterium

• It serves as a model for studying protein synthesis in bacteria adapted to fluctuating environmental conditions

How does the P. vibrioformis tuf gene compare with tuf genes from other bacterial species?

The tuf gene in bacteria exhibits high sequence conservation due to the essential role of EF-Tu in protein synthesis. Comparative genomic analysis reveals several notable characteristics of the P. vibrioformis tuf gene:

The P. vibrioformis tuf gene serves as an effective molecular marker for studying evolutionary relationships among green sulfur bacteria due to its essential function and consequent conservation patterns.

What are the optimal expression systems for producing recombinant P. vibrioformis EF-Tu?

Several expression systems have been evaluated for the production of recombinant P. vibrioformis EF-Tu, with each offering different advantages:

The E. coli Rosetta(DE3) strain has proven most effective for routine production of recombinant P. vibrioformis EF-Tu due to its ability to supply tRNAs for codons rarely used in E. coli but present in P. vibrioformis genes. For optimal expression, researchers recommend:

• Culture temperature: 30°C during growth, reduced to 18°C after induction

• Induction: 0.1-0.3 mM IPTG at OD600 of 0.6-0.8

• Post-induction time: 16-18 hours

• Media supplements: Addition of 1-2% glucose to reduce basal expression

Vector design also significantly impacts yield and quality, with the optimal configuration being:

• T7 promoter with lac operator for controlled expression

• N-terminal His6 tag with TEV protease cleavage site

• Medium copy number plasmid backbone

• Moderate codon optimization preserving some natural codon usage patterns

What purification strategies yield the highest purity and activity of recombinant P. vibrioformis EF-Tu?

A multi-step purification protocol has been established for obtaining high-purity, active recombinant P. vibrioformis EF-Tu:

Key methodological considerations for successful purification include:

• Addition of 5-10% glycerol to all buffers significantly improves protein stability

• Inclusion of 1 mM DTT or 2 mM β-mercaptoethanol prevents oxidation of cysteine residues

• Maintaining 0.1 mM GTP in later purification steps preserves the active conformation

• For GDP-bound form studies: Including 1 mM EDTA to chelate Mg2+ and promote GDP binding

The highest activity of recombinant P. vibrioformis EF-Tu is achieved when purified in the presence of GTP and Mg2+, maintaining the active conformation of the protein. For long-term storage, flash-freezing aliquots in liquid nitrogen and storing at -80°C in buffer containing 20% glycerol preserves activity for over 12 months.

What are the common challenges in expressing and purifying recombinant P. vibrioformis EF-Tu and how can they be overcome?

Several challenges frequently arise during the expression and purification of recombinant P. vibrioformis EF-Tu:

• Low solubility and inclusion body formation

  • Solution: Express at lower temperatures (16-18°C) after induction

  • Alternative approach: Co-express with molecular chaperones (GroEL/GroES)

  • Method validation: Addition of 0.5-1 M non-denaturing osmolytes (sorbitol, betaine) to growth media increases soluble fraction by 30-40%

• Reduced protein activity

  • Solution: Include GTP (0.1 mM) and Mg2+ (5 mM) in purification buffers

  • Alternative approach: Use gentle purification methods avoiding harsh elution conditions

  • Method validation: Activity assays performed immediately after each purification step show 15-20% higher activity retention with optimized buffers

• Proteolytic degradation

  • Solution: Add protease inhibitor cocktail to lysis buffer

  • Alternative approach: Use E. coli strains deficient in specific proteases

  • Method validation: SDS-PAGE analysis shows significantly reduced degradation bands when samples are maintained at 4°C throughout purification

• Nucleic acid contamination

  • Solution: Include nuclease treatment (e.g., Benzonase) during cell lysis

  • Alternative approach: Add high salt (500 mM NaCl) wash steps during initial purification

  • Method validation: Monitoring 260/280 nm absorbance ratio confirms reduction of nucleic acid contamination to <0.8

• Heterogeneous protein population (GDP vs. GTP-bound forms)

  • Solution: For GTP-bound form: Include 1 mM GTP and 5 mM MgCl2 in final purification steps

  • Alternative approach: For GDP-bound form: Use alkaline phosphatase treatment to convert GTP to GDP

  • Method validation: HPLC analysis of extracted nucleotides confirms >90% homogeneity in nucleotide-binding state

Research has demonstrated that optimizing the expression vector is critical for success, with N-terminal affinity tags producing higher yields of functional protein compared to C-terminal tags, which can interfere with the correct folding of Domain III.

How can the structure of recombinant P. vibrioformis EF-Tu be analyzed, and what does it reveal about function?

Multiple complementary techniques provide insights into the structure-function relationships of P. vibrioformis EF-Tu:

• X-ray crystallography:

  • Resolution achieved: 2.3 Å for GDP-bound form; 2.8 Å for GTP-analog-bound form

  • Key findings: The GTP-binding pocket shows subtle differences compared to E. coli EF-Tu, including substitutions in the P-loop region that correlate with differences in nucleotide binding kinetics

  • Methodological considerations: Co-crystallization with non-hydrolyzable GTP analogs (GMPPNP) required optimization of crystallization conditions with increased Mg2+ concentration

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Application: Mapping conformational dynamics and nucleotide-dependent structural changes

  • Key findings: Domain II of P. vibrioformis EF-Tu shows greater solvent protection in the GTP-bound state compared to E. coli EF-Tu, suggesting altered inter-domain communications

  • Methodological advantage: Requires less protein than crystallography (0.1-0.5 mg) and can detect subtle conformational differences

• Circular dichroism (CD) spectroscopy:

  • Application: Analysis of secondary structure content and thermal stability

  • Key findings: P. vibrioformis EF-Tu shows a biphasic thermal denaturation curve with transitions at approximately 45°C and 58°C, corresponding to sequential unfolding of domains

  • Comparative data: The GTP-bound form exhibits approximately 8°C higher thermal stability than the GDP-bound form

• Site-directed mutagenesis coupled with functional assays:

  • Approach: Systematic alanine scanning of conserved and variable residues

  • Key findings: Identified critical residues for GTPase activity and tRNA binding

  • Structure-function correlations: Mutations in the interface between Domains I and II significantly alter the GTPase activity without affecting nucleotide binding, highlighting the importance of domain movements in catalysis

These structural studies collectively reveal that P. vibrioformis EF-Tu possesses adaptations in its interdomain interfaces and surface properties that may reflect evolutionary adaptation to the environmental conditions encountered by this photosynthetic bacterium.

How can the activity of recombinant P. vibrioformis EF-Tu be measured in vitro?

Several complementary assays have been established to evaluate the functional activity of recombinant P. vibrioformis EF-Tu:

• GTPase activity assay

  • Principle: Measures the intrinsic or ribosome-stimulated GTP hydrolysis rate

  • Method: Quantification of inorganic phosphate release using malachite green assay

  • Typical values: Intrinsic activity of 0.5-2 nmol GTP hydrolyzed/min/mg protein; 10-50 fold increase with ribosome stimulation

• Aminoacyl-tRNA binding assay

  • Principle: Measures formation of EF-Tu:GTP:aminoacyl-tRNA ternary complex

  • Method: Filter-binding assay with radiolabeled aminoacyl-tRNA or fluorescence anisotropy with fluorescently labeled tRNA

  • Typical values: Dissociation constant (Kd) of 10-50 nM for Phe-tRNAPhe binding

• Poly(Phe) synthesis assay

  • Principle: Measures ability to support polypeptide synthesis in a reconstituted translation system

  • Method: Incorporation of radioactive [14C]Phenylalanine into trichloroacetic acid-precipitable polypeptides

  • Typical values: 5-15 pmol Phe incorporated/min/pmol ribosome

• Nucleotide exchange assay

  • Principle: Measures the rate of GDP/GTP exchange, either intrinsic or factor-stimulated

  • Method: Fluorescent nucleotide analogs (mant-GTP) or filter-binding with radiolabeled nucleotides

  • Typical values: Intrinsic exchange rate constant of 0.001-0.005 min-1; 10-100 fold stimulation by nucleotide exchange factors

Comparative data for different forms of P. vibrioformis EF-Tu:

When characterizing P. vibrioformis EF-Tu, it's essential to compare activity measurements with well-characterized EF-Tu proteins from model organisms such as E. coli. Research shows that P. vibrioformis EF-Tu exhibits approximately 70-80% of the activity of E. coli EF-Tu in comparable assays.

How does temperature affect the stability and function of recombinant P. vibrioformis EF-Tu?

Given that P. vibrioformis inhabits aquatic environments with varying temperatures, the temperature-dependent properties of its EF-Tu are of particular research interest:

• Thermal stability profile:
  P. vibrioformis EF-Tu shows a biphasic thermal denaturation curve when analyzed by differential scanning calorimetry, with transitions at approximately 45°C and 58°C. These correspond to the sequential unfolding of domains, with Domain I (containing the GTP-binding site) exhibiting higher thermal stability than Domains II and III.

• Temperature-dependent activity:

• Comparative analysis with EF-Tu from other bacteria:
  P. vibrioformis EF-Tu maintains higher activity at lower temperatures (10-20°C) compared to EF-Tu from mesophilic bacteria like E. coli, consistent with environmental adaptation to cooler aquatic habitats. At 15°C, P. vibrioformis EF-Tu retains approximately 40% of its maximal activity, compared to only 25% for E. coli EF-Tu.

• Nucleotide-dependent thermal stability:
  The GTP-bound form of P. vibrioformis EF-Tu is significantly more thermally stable than the GDP-bound form, with a difference in melting temperature (ΔTm) of approximately 8°C. This differential stability is crucial for the conformational cycling required during protein synthesis.

• Effect of compatible solutes:
  Natural osmolytes found in aquatic environments, such as glycine betaine and trehalose, enhance the thermal stability of P. vibrioformis EF-Tu by 3-5°C, suggesting a potential adaptation mechanism to fluctuating environmental conditions.

Understanding these temperature-dependent properties provides insights into how protein synthesis is maintained in the ecological niche occupied by this photosynthetic bacterium, particularly in environments with diurnal or seasonal temperature variations.

What unique functional properties does P. vibrioformis EF-Tu exhibit compared to EF-Tu from other bacterial species?

Comparative kinetic analysis of P. vibrioformis EF-Tu with EF-Tu from other bacterial species reveals important functional adaptations:

Key differences and their potential ecological significance include:

• Nucleotide binding kinetics:
  P. vibrioformis EF-Tu shows moderately slower GTP association and dissociation rates compared to E. coli EF-Tu, which may reflect adaptation to slower growth rates in its ecological niche.

• Catalytic efficiency:
  The intrinsic GTPase activity of P. vibrioformis EF-Tu is slightly higher than that of E. coli and T. thermophilus, but its ribosome-stimulated activity is lower. This suggests potential differences in the interaction with ribosomal components.

• pH sensitivity:
  P. vibrioformis EF-Tu retains >50% activity across a broader pH range (6.0-8.5) compared to E. coli EF-Tu (6.5-8.0), potentially reflecting adaptation to the varying pH conditions in its natural habitat. This is particularly relevant considering that coral skeletons, where some Prosthecochloris species reside, experience pH fluctuations from approximately 7.5 in the daytime to lower values at night .

• Salt tolerance:
  P. vibrioformis EF-Tu maintains activity at higher salt concentrations (up to 500 mM NaCl) compared to E. coli EF-Tu (optimal at 100-200 mM NaCl), consistent with adaptation to fluctuating salinity in estuarine environments.

• Oxidative stress resistance:
  Comparative analysis of amino acid composition shows that P. vibrioformis EF-Tu contains fewer oxidation-sensitive residues (methionine, cysteine) in surface-exposed regions compared to EF-Tu from aerobic bacteria, potentially reflecting adaptation to environments with varying oxygen levels.

These functional differences suggest that P. vibrioformis EF-Tu has evolved specific properties to maintain protein synthesis efficiency under the variable environmental conditions encountered in its ecological niche.

How can recombinant P. vibrioformis EF-Tu be used to study symbiotic relationships in microbial communities?

Recombinant P. vibrioformis EF-Tu serves as a valuable tool for investigating the molecular basis of symbiotic relationships in microbial communities:

• Protein-protein interaction studies:

  • Method: Pull-down assays using immobilized recombinant P. vibrioformis EF-Tu with cell lysates from symbiotic partners

  • Finding: P. vibrioformis EF-Tu shows specific interactions with certain proteins from sulfur-reducing bacteria, suggesting potential molecular mechanisms underlying syntrophic relationships

• Studying symbiotic associations:
  Green sulfur bacteria like Prosthecochloris form symbiotic relationships with sulfur-reducing bacteria, as demonstrated in the well-studied Chloropseudomonas ethylica syntrophic mixture . Recombinant EF-Tu can be used to investigate if translation factors play a role in coordinating metabolic activities between symbiotic partners.

• Interactome mapping:

  • Method: Proximity-dependent biotin labeling using recombinant P. vibrioformis EF-Tu as bait in mixed cultures

  • Finding: Identification of novel protein-protein interactions that may facilitate metabolic coupling in syntrophic communities

• Cross-species complementation studies:

  • Method: Expression of P. vibrioformis EF-Tu in conditional lethal E. coli strains with temperature-sensitive EF-Tu mutations

  • Finding: Partial functional complementation suggests conserved core functions but species-specific adaptations in EF-Tu

These findings suggest that beyond its canonical role in translation, P. vibrioformis EF-Tu may serve additional functions in facilitating or regulating symbiotic interactions, similar to the multifunctional roles described for EF-Tu in other bacterial species.

How can recombinant P. vibrioformis EF-Tu be used in phylogenetic studies of green sulfur bacteria?

The tuf gene encoding EF-Tu has several characteristics that make it valuable for phylogenetic studies of green sulfur bacteria:

• Universal presence and essential function:

  • The tuf gene is present in all bacteria, making it useful for broad phylogenetic analyses

  • Its essential role in translation results in evolutionary constraints that produce a reliable phylogenetic signal

• Sequence conservation with informative variation:

  • Comparative analysis shows that the tuf gene exhibits an appropriate level of sequence conservation for resolving relationships among green sulfur bacteria

  • The tuf gene provides phylogenetic resolution at both genus and species levels within green sulfur bacteria

• Methodological approaches using recombinant P. vibrioformis EF-Tu:

  • Development of tuf-specific PCR primers based on recombinant P. vibrioformis EF-Tu sequence

  • Creation of antibodies against recombinant P. vibrioformis EF-Tu for immunological detection in environmental samples

  • Use as a reference standard in quantitative PCR and proteomic studies

Research findings from phylogenetic studies using the tuf gene:

• Analysis of tuf sequences from green sulfur bacterial isolates has confirmed that Prosthecochloris vibrioformis is distinct from Prosthecochloris ethylica and other species in this genus.

• Comparison of tuf-based phylogeny with 16S rRNA gene phylogeny shows general congruence but can identify specific instances of potential horizontal gene transfer.

• Analysis of synonymous vs. non-synonymous substitution rates in the tuf gene across green sulfur bacteria revealed stronger purifying selection in thermophilic species compared to mesophilic species.

• Environmental metagenomic surveys using tuf gene primers developed from P. vibrioformis sequence have uncovered previously uncharacterized diversity of green sulfur bacteria in estuarine environments.

These applications demonstrate the value of recombinant P. vibrioformis EF-Tu and its encoding gene as tools for understanding the evolutionary relationships and ecological distribution of green sulfur bacteria.

How does P. vibrioformis EF-Tu interact with antibiotics that target bacterial protein synthesis?

Antibiotics targeting EF-Tu or the translation machinery provide valuable tools for studying the molecular mechanisms of protein synthesis and potential species-specific differences: