Recombinant Sulfurovum sp. Elongation factor G (fusA), partial

What is the primary role of Elongation Factor G (fusA) in bacterial protein synthesis?

Elongation Factor G (fusA) plays critical roles in two essential steps of bacterial translation. During elongation, fusA catalyzes the translocation of peptidyl-tRNA from the A-site to the P-site after peptide bond formation. During ribosome recycling, fusA works with ribosome recycling factor (RRF) through multiple rounds of GTP hydrolysis to split the 70S ribosome into subunits . In Sulfurovum, which dominates deep-sea cold seep microbial communities, fusA likely maintains these conserved functions while potentially exhibiting adaptations to cold, sulfur-rich environments .

Methodological approach: To characterize fusA function, researchers should:

• Express recombinant protein with appropriate tags (His, GST)

• Purify using affinity chromatography followed by size exclusion

• Perform in vitro translation assays using purified ribosomes

• Measure GTP hydrolysis rates under various conditions using malachite green assays

How can researchers validate the structural integrity of purified recombinant Sulfurovum sp. fusA?

Structural validation is essential before functional characterization, particularly for proteins from extremophiles like Sulfurovum that may require specific conditions for proper folding.

Methodological approach:

• Perform circular dichroism (CD) spectroscopy to assess secondary structure content

• Use thermal shift assays to determine protein stability under various buffer conditions

• Conduct limited proteolysis to confirm compact, folded domains resistant to digestion

• Compare with known EF-G structures using homology modeling based on the fusA sequence from Sulfurovum

• Verify nucleotide binding capacity using fluorescent GTP analogs or isothermal titration calorimetry

What expression systems are most suitable for producing functional Sulfurovum sp. fusA?

Expressing functional recombinant fusA from Sulfurovum presents challenges due to its extremophilic origin and potential codon bias.

Methodological approach:

• Test multiple expression vectors with different promoters (T7, tac, arabinose-inducible)

• Optimize expression conditions:

  • Temperature (12-30°C, with emphasis on lower temperatures)

  • Induction time (4-24 hours)

  • Inducer concentration

• Consider specialized E. coli strains:

  • ArcticExpress for cold-adapted expression

  • Rosetta for rare codon optimization

  • C41/C43 for toxic or membrane-associated proteins

• Evaluate solubility enhancement strategies:

  • Fusion partners (MBP, SUMO, Trx)

  • Co-expression with chaperones (GroEL/ES)

How do conformational dynamics influence Sulfurovum sp. fusA function on the ribosome?

The conformational changes of EF-G are crucial for its function. The F88 residue in S. aureus (corresponding to F90 in T. thermophilus) is important for transmitting conformational changes between GTP and GDP forms , suggesting similar dynamics may be critical in Sulfurovum sp. fusA.

Methodological approach:

• Generate single cysteine variants at key positions for fluorescent labeling

• Perform FRET analysis to track conformational changes during GTP hydrolysis

• Use cryo-EM to capture different conformational states of fusA on the ribosome

• Compare dynamics at different temperatures relevant to deep-sea environments (4-15°C)

• Create point mutations in switch regions and measure effects on:

  • GTP hydrolysis rate

  • Ribosome binding affinity

  • Translocation efficiency

How does Sulfurovum sp. fusA resist inhibition by fusidic acid?

Fusidic acid works by blocking EF-G on the ribosome with GDP, preventing its release from post-translocation complexes . Understanding potential natural resistance in Sulfurovum sp. fusA could provide insights into antibiotic resistance mechanisms.

Methodological approach:

• Compare sequence alignments focusing on known resistance sites (e.g., F88 in S. aureus)

• Test susceptibility to fusidic acid in vitro using:

  • GTPase activity assays

  • Ribosome binding assays

  • Translocation assays

• Create chimeric proteins swapping domains between Sulfurovum sp. fusA and susceptible EF-Gs

• Perform computational docking and molecular dynamics simulations to identify potential structural differences affecting drug binding

What kinetic parameters characterize GTP hydrolysis by Sulfurovum sp. fusA compared to mesophilic homologs?

As a protein from a deep-sea bacterium, Sulfurovum sp. fusA likely exhibits adaptations for function at low temperatures and high pressure.

Methodological approach:

• Measure steady-state kinetic parameters (Km, kcat, kcat/Km) across temperature range (4-37°C)

• Determine activation energies using Arrhenius plots

• Assess effects of pressure using specialized high-pressure equipment

• Compare with EF-G from model organisms and other extremophiles using a comparative table:

How does the amino acid composition of Sulfurovum sp. fusA contribute to cold adaptation?

Cold-adapted proteins typically exhibit specific amino acid substitutions that maintain flexibility at low temperatures.

Methodological approach:

• Perform comparative sequence analysis between Sulfurovum sp. fusA and mesophilic homologs focusing on:

  • Reduced proline content in loops

  • Reduced arginine/increased lysine ratio

  • Increased glycine content

  • Reduced hydrophobic core packing

• Create a position-specific scoring matrix to identify cold-adaptation signatures

• Generate thermal stability curves using differential scanning fluorimetry

• Measure activity at different temperatures (4-37°C) and correlate with structural features

How does fusA expression in Sulfurovum sp. respond to environmental stressors?

Based on studies of fusA in other bacteria, expression levels may change in response to environmental conditions. In P. plecoglossicida, fusA expression was significantly affected by temperature, pH, and heavy metals .

Methodological approach:

• Culture Sulfurovum under varying conditions:

  • Temperature range (4-30°C)

  • pH (5-9)

  • Oxidative stress (H₂O₂)

  • Heavy metals (Cu²⁺, others found in deep-sea environments)

  • Iron availability (using chelating agents)

• Quantify fusA expression using qRT-PCR

• Perform RNA-seq to identify co-regulated genes

• Map regulatory elements in the fusA promoter region

What role might fusA play in the ecological success of Sulfurovum in deep-sea cold seeps?

Sulfurovum dominates microbial communities in deep-sea cold seeps, including seawater close to seepage, surface sediments, and association with animal hosts .

Methodological approach:

• Compare fusA sequences from different Sulfurovum strains isolated from various microhabitats

• Measure fusA expression levels in situ using environmental transcriptomics

• Assess co-occurrence patterns between fusA expression and biogeochemical processes

• Investigate potential moonlighting functions beyond translation (as seen in P. plecoglossicida, where fusA affects biofilm formation )

What approaches are most effective for determining the structure of Sulfurovum sp. fusA?

Understanding the structure of Sulfurovum sp. fusA is essential for insights into its function and adaptations.

Methodological approach:

• X-ray crystallography:

  • Perform systematic crystallization screening with various nucleotides (GDP, GTP, GMP-PNP)

  • Consider co-crystallization with ribosome components

  • Use crystallization conditions optimized for cold-adapted proteins (lower temperatures, specific precipitants)

• Cryo-electron microscopy:

  • Prepare fusA-ribosome complexes trapped in different states

  • Use vitrification conditions optimized for cold-active complexes

• Small-angle X-ray scattering (SAXS):

  • Characterize solution structure and conformational flexibility

  • Compare different nucleotide-bound states

• Integrative structural biology approach:

  • Combine data from multiple methods with computational modeling

  • Validate with hydrogen-deuterium exchange mass spectrometry

How can researchers identify domain-domain interactions critical for Sulfurovum sp. fusA function?

EF-G typically consists of five domains, with interdomain interactions being crucial for function. In S. aureus, FA-resistant mutations are found scattered at domain interfaces .

Methodological approach:

• Create domain truncation constructs to identify minimal functional units

• Perform cross-linking mass spectrometry to map domain interfaces

• Use site-directed mutagenesis targeting predicted interface residues

• Apply molecular dynamics simulations to analyze domain movements during the GTPase cycle

• Develop FRET-based assays with domain-specific labels to track conformational changes in real-time

What structural features might explain Sulfurovum sp. fusA adaptation to deep-sea environments?

Sulfurovum thrives in the deep sea where it plays important roles in carbon, sulfur, and nitrogen cycles . Structural adaptations in fusA likely contribute to its functionality in this environment.

Methodological approach:

• Identify unique insertions/deletions through multiple sequence alignment with mesophilic EF-Gs

• Analyze surface charge distribution and compare with homologs from different temperature niches

• Examine hydrogen bonding networks and salt bridge patterns that might confer cold stability

• Look for structural elements that could provide resistance to pressure effects

• Model the hydration shell and analyze water-protein interactions that might differ from mesophilic homologs

How can recombinant Sulfurovum sp. fusA be utilized in cell-free protein synthesis systems?

Cell-free protein synthesis systems require efficient translation factors. Cold-adapted fusA could enable protein production at lower temperatures.

Methodological approach:

• Develop a cell-free translation system incorporating purified Sulfurovum sp. fusA

• Compare translation efficiency at different temperatures (4-37°C)

• Test expression of difficult-to-express proteins that may benefit from low-temperature synthesis

• Optimize buffer conditions for maximum activity of Sulfurovum sp. fusA

• Create hybrid systems combining components from different temperature-adapted organisms

What insights can comparative analysis of fusA across Sulfurovum species provide about their evolutionary adaptation?

Comparative genomics of fusA from different Sulfurovum species could reveal signatures of adaptation to specific deep-sea niches.

Methodological approach:

• Sequence fusA from multiple Sulfurovum isolates from various deep-sea environments

• Perform phylogenetic analysis to identify clade-specific adaptations

• Calculate dN/dS ratios to identify sites under positive selection

• Correlate sequence variations with environmental parameters

• Reconstruct ancestral sequences to understand the evolutionary trajectory of fusA in this genus

How can knowledge of Sulfurovum sp. fusA structure and function inform studies of antibiotic resistance?

Understanding the molecular basis of natural antibiotic resistance in environmental bacteria can provide insights into clinical resistance mechanisms.

Methodological approach:

• Compare the fusidic acid binding site between Sulfurovum sp. fusA and clinical isolates

• Test novel fusidic acid derivatives against wild-type and mutant fusA proteins

• Identify structural features that could be targeted by new translation inhibitors

• Investigate horizontal gene transfer potential of resistance-conferring fusA variants

• Develop predictive models for resistance emergence based on structural constraints