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

What is Elongation Factor G (fusA) and what is its role in Synechococcus sp.?

Elongation Factor G (EF-G), encoded by the fusA gene, is a ribosomal translocation enzyme crucial for bacterial protein synthesis. In Synechococcus sp., as in other bacteria, EF-G functions in the translocation step of protein synthesis, moving the tRNA and mRNA through the ribosome after peptide bond formation.

The fusA gene (typically around 2,000-2,100 bp) encodes a protein that catalyzes GTP hydrolysis to drive the translocation process. EF-G is also the target of fusidic acid, an antibiotic derived from Fusidium coccineum that inhibits protein synthesis . The gene has been described in numerous bacterial species including various Bacillus and Clostridium species .

Methodologically, researchers can identify and study fusA in Synechococcus sp. through:

• Genomic analysis using bioinformatics tools

• Comparative sequence analysis with homologs from other species

• Structural modeling to identify conserved functional domains

• Expression analysis under different growth conditions

How can I clone and express the fusA gene from Synechococcus sp.?

Cloning and expressing fusA from Synechococcus sp. requires a methodical approach:

• DNA Extraction:

  • Cultivate Synechococcus sp. under optimal conditions

  • Extract genomic DNA using methods optimized for cyanobacteria (accounting for their unique cell wall characteristics)

• PCR Amplification:

  • Design primers based on available sequence data or consensus regions

  • Include appropriate restriction sites for subsequent cloning

  • Optimize PCR conditions (higher GC content may require specialized polymerases)

• Vector Selection:

  • Choose expression systems compatible with the CyanoGate MoClo system for Synechococcus sp.

  • Consider neutral sites suitable for stable genomic integration

  • Evaluate inducible promoter systems such as the 2,4-diacetylphloroglucinol (DAPG)-inducible PhlF repressor system

• Transformation and Expression:

  • Transform into E. coli initially for construct verification

  • Transfer verified constructs into cyanobacterial hosts

  • Optimize expression conditions (light intensity, temperature, induction parameters)

• Verification:

  • Confirm expression through Western blotting

  • Validate functionality through activity assays

What are the common challenges in purifying recombinant Synechococcus sp. EF-G?

Purification of recombinant Synechococcus sp. EF-G presents several methodological challenges:

• Protein Solubility Issues:

  • EF-G (approximately 75-80 kDa) often forms inclusion bodies when overexpressed

  • Optimization strategies include reducing expression temperature (16-20°C), using weaker promoters, or employing solubility tags

• Maintaining Native Conformation:

  • EF-G requires proper folding for GTPase activity

  • Purification buffers must include Mg²⁺ (essential for structural integrity)

  • Avoid harsh elution conditions that may denature the protein

• Co-purification of Contaminants:

  • Host EF-G may co-purify due to sequence similarity

  • Multi-step purification protocols are typically required

  • Combination of affinity, ion-exchange, and size-exclusion chromatography yields best results

• Stability Concerns:

  • EF-G is prone to aggregation and degradation

  • Add protease inhibitors during purification

  • Store with glycerol (20-30%) at -80°C in small aliquots

• Activity Preservation:

  • Monitor GTPase activity throughout purification

  • Test functionality in translation assays

How can I assess the functionality of purified recombinant Synechococcus sp. EF-G?

Functionality assessment of purified Synechococcus sp. EF-G should include multiple complementary approaches:

• GTPase Activity Assay:

  • Measure GTP hydrolysis rates using malachite green assay (phosphate detection)

  • Compare intrinsic vs. ribosome-stimulated GTPase activity

  • Generate kinetic parameters (Km, Vmax) under different conditions

• Ribosome Binding Studies:

  • Assess binding to Synechococcus sp. ribosomes using filter binding assays

  • Determine binding constants through surface plasmon resonance (SPR)

  • Characterize GTP-dependent vs. GDP-dependent binding profiles

• Translocation Assays:

  • Use reconstituted in vitro translation systems

  • Measure toeprinting assays to detect ribosome movement

  • Quantify translocation rates under different conditions

• Antibiotic Sensitivity Testing:

  • Determine fusidic acid sensitivity profile

  • Compare with EF-G from other bacterial species

  • Establish IC₅₀ values for various translation inhibitors

• Conformational Analysis:

  • Apply multi-channel single-molecule FRET (smFRET) microscopy to examine conformational changes during function

  • Monitor structural integrity using circular dichroism spectroscopy

  • Use limited proteolysis to verify proper folding

How do mutations in the fusA gene affect antibiotic resistance, and what experimental approaches can characterize these effects?

Mutations in fusA are associated with resistance to fusidic acid in multiple bacterial species, including Clostridium difficile . These mutations typically involve nonsynonymous substitutions or, in some cases, codon deletions . To characterize these effects in Synechococcus sp., consider these methodological approaches:

• Mutation Identification and Characterization:

  • Sequence fusA from wild-type and laboratory-evolved resistant strains

  • Perform comparative analysis with known resistance mutations in other species

  • Map mutations onto structural models of EF-G

• Systematic Mutagenesis Studies:

  • Create a library of site-directed mutants based on:

    • Mutations identified in resistant strains

    • Conserved mutations found in other species

    • Novel mutations in predicted functional sites

  • Express and purify mutant proteins for biochemical characterization

• Resistance Phenotype Analysis:

  Mutation Type	Typical MIC Change	Growth Rate Effect	Translation Efficiency
  Domain I (GTPase)	4-16× increase	Moderate reduction	Slightly compromised
  Domain III/V interface	8-64× increase	Minimal reduction	Near wild-type
  Domain II	2-8× increase	Variable	Variable
  Multiple mutations	>64× increase	Severe reduction	Significantly compromised

• Structural and Functional Analysis:

  • Compare GTPase activity of wild-type vs. mutant proteins

  • Characterize ribosome binding properties

  • Evaluate effects on translocation efficiency

  • Assess thermal stability and conformational dynamics

• Cross-Resistance Profiles:

  • Test sensitivity to other translation inhibitors

  • Identify potential compensatory mutations

What approaches can be used to study the interaction between Synechococcus sp. EF-G and the ribosome during translocation?

Understanding the EF-G-ribosome interaction requires sophisticated methodological approaches:

• Cryo-Electron Microscopy (Cryo-EM):

  • Prepare complexes of Synechococcus sp. ribosomes with EF-G in different functional states

  • Capture high-resolution structures of translocation intermediates

  • Compare with available structures from model organisms

• Single-Molecule Fluorescence Techniques:

  • Implement multi-channel smFRET microscopy as described for E. coli EF-G

  • Label specific domains of EF-G and ribosomal components with fluorescent dyes

  • Monitor real-time conformational changes during translocation

  • Quantify kinetic parameters under different environmental conditions

• Chemical Cross-linking Coupled with Mass Spectrometry (XL-MS):

  • Use bifunctional cross-linkers to capture transient interactions

  • Identify interaction sites by mass spectrometry analysis

  • Create detailed interaction maps specific to Synechococcus sp.

• Molecular Dynamics Simulations:

  • Develop computational models based on structural data

  • Simulate the translocation process under conditions relevant to Synechococcus sp. habitats

  • Identify species-specific interaction networks

• Hybrid Approaches:

  • Combine structural, biochemical, and computational methods for comprehensive understanding

  • Integrate data into mechanistic models of translocation

How can recombinant Synechococcus sp. EF-G be incorporated into synthetic biology applications?

Recombinant Synechococcus sp. EF-G offers several opportunities for synthetic biology applications:

• Enhanced Protein Production Systems:

  • Engineer Synechococcus sp. EF-G variants with increased translocation efficiency

  • Develop specialized cell-free protein synthesis systems using components from Synechococcus sp.

  • Optimize translation under conditions relevant to biotechnology applications

• Development of Selection Markers:

  • Create fusidic acid resistance markers based on specific fusA mutations

  • Design selection systems compatible with the genetic tools available for Synechococcus sp.

  • Integrate with existing transformation protocols

• Chassis Engineering for Biotechnology:

  • Modify fusA in Synechococcus sp. PCC 11901, which has capacity for sustained biomass accumulation to high cell densities

  • Combine with the CyanoGate MoClo system and other genetic tools described for cyanobacteria

  • Optimize for heterologous protein expression

• Applications in Bioenergy and Biomaterials:

  Application	EF-G Engineering Approach	Expected Outcome	Evaluation Methods
  Biofuel production	Optimize translation efficiency under photosynthetic conditions	Increased carbon fixation and product yield	Metabolic flux analysis
  Protein-based materials	Engineer EF-G for non-canonical amino acid incorporation	Novel biomaterials with unique properties	Material characterization assays
  Biosensors	Create EF-G-based reporters sensitive to translation inhibitors	Detection systems for environmental contaminants	Sensitivity and specificity testing

What methodologies can be used to study the impact of environmental factors on fusA expression and EF-G function in Synechococcus sp.?

Investigating environmental impacts on fusA expression and EF-G function requires multifaceted approaches:

• Transcriptomic Analysis:

  • Perform RNA-Seq on Synechococcus sp. cultures under varying:

    • Light intensities and qualities

    • Temperature ranges

    • Nutrient limitations (particularly N, P, Fe)

    • CO₂ concentrations

  • Quantify fusA transcript levels relative to reference genes

  • Identify co-regulated genes that may form functional networks

• Proteomics Approach:

  • Use quantitative proteomics to measure EF-G protein levels

  • Compare protein abundance with transcript levels to identify post-transcriptional regulation

  • Characterize post-translational modifications under different conditions

• Functional Assays Under Environmental Stress:

  • Measure translation rates in vivo using reporter systems

  • Assess polysome profiles under different environmental conditions

  • Purify EF-G from cells grown under different conditions for activity assays

• Ecological Context Analysis:

  • Compare responses across different Synechococcus strains from diverse aquatic environments

  • Relate laboratory findings to environmental parameters in natural habitats

  • Develop predictive models for translation efficiency under changing environmental conditions

• Combined Approach Data Table:

  Environmental Factor	Expected Effect on fusA Expression	Expected Effect on EF-G Function	Methodological Approach
  High light intensity	Potential upregulation	Possible increased activity	RNA-Seq + activity assays
  Nutrient limitation	Strain-dependent response	Reduced activity	Proteomics + ribosome profiling
  Temperature stress	Transient induction	Conformational changes	qRT-PCR + thermal stability assays
  Salinity variation	Marine strain-specific regulation	Altered ribosome binding	Comparative transcriptomics

How can CRISPR-Cas9 technology be utilized to study or modify fusA in Synechococcus sp.?

CRISPR-Cas9 offers powerful approaches for fusA modification in Synechococcus sp.:

• CRISPR System Optimization:

  • Adapt CRISPR-Cas9 systems for efficient editing in Synechococcus sp.

  • Utilize expression systems compatible with cyanobacterial hosts

  • Integrate with the genetic tools described for Synechococcus sp. PCC 11901

• Targeted fusA Modifications:

  • Generate precise point mutations corresponding to known functional residues

  • Create domain swaps between fusA genes from different bacterial species

  • Introduce fluorescent or affinity tags for localization and interaction studies

• Functional Analysis Approaches:

  • Create a library of fusA variants with systematic mutations

  • Perform high-throughput phenotyping under various growth conditions

  • Characterize translation efficiency and accuracy in each variant

• Gene Regulation Studies:

  • Implement CRISPR interference (CRISPRi) to modulate fusA expression

  • Create inducible knockdown systems

  • Correlate expression levels with physiological parameters

• Genome-Wide Interaction Studies:

  • Perform CRISPR-based screens to identify genetic interactions with fusA

  • Map suppressors of fusA mutant phenotypes

  • Identify synthetic lethal interactions

How does the structure of Synechococcus sp. EF-G compare to that of other bacterial species?

Understanding the structural features of Synechococcus sp. EF-G requires comparative analysis:

• Sequence-Structure Relationships:

  • Perform multiple sequence alignment of fusA genes across diverse bacterial phyla

  • Identify conserved domains and Synechococcus-specific variations

  • Use homology modeling to predict structural differences

• Structural Analysis Approaches:

  • Apply X-ray crystallography or cryo-EM to determine high-resolution structures

  • Compare with available structures from model organisms

  • Focus on specific features of domains I-V

• Domain-Specific Structural Features:

  Domain	Primary Function	Expected Synechococcus sp. Features	Structural Analysis Methods
  Domain I (G domain)	GTP binding/hydrolysis	Highly conserved	GTPase activity assays
  Domain II	EF-G-specific	Moderate conservation	Ribosome interaction studies
  Domain III	Resembles EF-Tu domain II	Cyanobacteria-specific features	Comparative modeling
  Domain IV	Mimics tRNA	Potential adaptation to marine environment	smFRET analysis
  Domain V	Ribosome interaction	Variable conservation	Cross-linking studies

• Conformational Dynamics:

  • Characterize conformational changes during the GTPase cycle

  • Compare compact and extended conformations as mentioned for E. coli EF-G

  • Identify unique dynamic properties related to Synechococcus sp. habitat

• Structure-Function Correlations:

  • Map functional properties to structural features

  • Identify residues responsible for species-specific activities

  • Relate structural adaptations to environmental conditions

What evolutionary insights can be gained from studying Synechococcus sp. fusA in relation to other cyanobacteria?

Evolutionary analysis of Synechococcus sp. fusA provides important insights:

• Phylogenetic Analysis:

  • Construct robust phylogenetic trees based on fusA sequences

  • Compare with organismal phylogeny and other essential genes

  • Identify potential horizontal gene transfer events

• Selection Pressure Analysis:

  • Calculate Ka/Ks ratios to identify sites under selection

  • Compare evolutionary rates across different domains

  • Correlate evolutionary patterns with ecological niches

• Comparative Genomics:

  • Analyze fusA gene context across cyanobacterial genomes

  • Identify conserved operonic structures

  • Compare gene organization in marine vs. freshwater strains

• Molecular Clock Analysis:

  • Estimate divergence times for key evolutionary events

  • Correlate with geological and environmental history

  • Relate to the evolution of photosynthetic apparatus

• Adaptive Evolution Assessment:

  • Identify signatures of adaptation in different Synechococcus lineages

  • Correlate with habitat transitions (marine/freshwater)

  • Evaluate co-evolution with other translation components