Recombinant Geobacter sulfurreducens Elongation factor P 2 (efp2)

What is Elongation Factor P 2 (Efp-2) in G. sulfurreducens?

Efp-2 (encoded by gene GSU1752) is one of two Elongation Factor P homologs in G. sulfurreducens. It functions as a specialized translation factor that prevents ribosomal stalling during the synthesis of proteins containing consecutive proline residues. Tn-Seq analysis has demonstrated that Efp-2 becomes particularly important during growth with electrodes as terminal electron acceptors .

How does Efp-2 differ from other bacterial elongation factors?

Unlike the standard elongation factors (EF-Tu, EF-G) that function in all protein synthesis, Efp-2 in G. sulfurreducens specifically alleviates ribosome stalling when translating consecutive proline codons. This specialization is significant in G. sulfurreducens due to its unique metabolism requiring proline-rich proteins involved in extracellular electron transfer (EET) processes .

What is the genomic context of efp2 in G. sulfurreducens?

The efp2 gene (GSU1752) exists in a gene cluster with GSU1753 (encoding Ef-P lysine-lysyltransferase) and GSU1754 (encoding Ef-P lysyl-lysine 2,3-aminomutase) . This clustering suggests coordinated expression and function, as these enzymes work together to post-translationally modify Efp-2, which is essential for its proper function in translation.

What post-translational modifications are required for Efp-2 activity?

Efp-2 requires post-translational modifications by two enzymes: Ef-P lysine-lysyltransferase (GSU1753) and Ef-P lysyl-lysine 2,3-aminomutase (GSU1754) . These modifications create a unique extended amino acid side chain necessary for Efp-2 to interact effectively with the ribosome and resolve polyproline-induced stalling during translation.

How does the structure of Efp-2 relate to its function in translation?

Efp-2 adopts a structure mimicking the L-shaped tRNA molecule, allowing it to enter the ribosome and position its modified lysine residue near the peptidyl transferase center. This positioning facilitates the synthesis of peptide bonds between consecutive proline residues. Structural studies of recombinant Efp-2 would typically employ X-ray crystallography or cryo-EM techniques to elucidate these structural features.

What proteins dependent on Efp-2 are particularly important in G. sulfurreducens?

Efp-2 is likely crucial for the translation of proline-rich proteins involved in extracellular electron transfer, including:

• PilA, the structural protein for electrically conductive pili

• Specific c-type cytochromes such as OmcM, OmcH, and PpcA, which are upregulated during metal reduction

• Proteins involved in biofilm formation on electrodes

What expression systems are effective for producing recombinant Efp-2?

For recombinant expression of G. sulfurreducens Efp-2:

• Expression vector selection: pET21d vector is recommended as it contains unique restriction sites (NheI and XhoI) and a His-tag sequence that simplifies purification .

• Host strain selection: BL21(DE3) E. coli cells are suitable hosts due to their reduced protease activity and compatibility with T7-based expression systems .

• Induction protocol: Optimal induction conditions include 1mM IPTG for 3 hours at 37°C .

Expected yield: ~10-15 mg of purified protein per liter of culture when using this system.

What purification strategy is most efficient for recombinant Efp-2?

A multi-step purification protocol is recommended:

• Initial capture: Ni-NTA affinity chromatography using the C-terminal His-tag

  • Equilibration buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole

  • Elution buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 250 mM imidazole

• Polishing step: Size exclusion chromatography

  • Running buffer: 50 mM Tris-HCl pH 8.0, 150 mM NaCl

• Quality control: SDS-PAGE analysis should show a single band at approximately 21 kDa .

How can the activity of recombinant Efp-2 be assessed in vitro?

Functional assessment of recombinant Efp-2 can be performed using:

• In vitro translation assays: Using a cell-free translation system with reporters containing polyproline sequences

  • Control: Standard reporter without polyproline stretches

  • Test: Reporter with (PPP)n motifs

  • Readout: Translation efficiency measured by luminescence or fluorescence

• Ribosome binding assays: Using purified ribosomes and fluorescently labeled Efp-2 to measure binding kinetics

  • Kd values typically range from 0.1-1 μM for functional Efp-2

How does Efp-2 contribute to extracellular electron transfer in G. sulfurreducens?

Efp-2 facilitates the translation of proteins critical for extracellular electron transfer. Transcriptome analysis during Pd(II) reduction revealed upregulation of several proteins dependent on efficient translation of polyproline motifs, including:

The efficient translation of these proteins is dependent on functional Efp-2, making it an indirect but crucial component of the electron transfer network .

What is the relationship between Efp-2 and microaerobic growth in G. sulfurreducens?

G. sulfurreducens can grow under microaerobic conditions with oxygen as terminal electron acceptor . Efp-2 likely plays a role in adapting to these conditions by facilitating the translation of proteins involved in:

• Oxygen reduction pathways involving cytochrome c oxidase and cytochrome d ubiquinol oxidase

• Stress response proteins that contain polyproline motifs and are upregulated under oxidative stress conditions

• Proteins involved in biofilm formation, which increases under oxygen exposure

How does Efp-2 activity compare between different electron acceptor conditions?

The importance of Efp-2 varies depending on the electron acceptor used by G. sulfurreducens:

These differences reflect the varying protein requirements for different respiratory pathways in G. sulfurreducens.

What methods are effective for creating efp2 mutants in G. sulfurreducens?

Creating efp2 mutants requires specialized approaches due to G. sulfurreducens' unique physiology:

• Homologous recombination strategy:

  • Design primers with 50 bp homology arms flanking efp2

  • Amplify antibiotic resistance cassette (typically kanamycin)

  • Transform G. sulfurreducens using electroporation (1.5 kV, 400 Ω, 25 μF)

  • Select on fumarate medium with appropriate antibiotic

  • Verify by PCR and sequencing

• CRISPR-Cas9 approach:

  • Design sgRNA targeting efp2

  • Provide repair template with desired mutations

  • Transform using shuttle vectors adapted for G. sulfurreducens

• Complementation testing:

  • Clone wild-type efp2 into pRG5.1 vector

  • Transform into efp2 mutant strain

  • Assess restoration of phenotype

How can efp2 expression be regulated for functional studies?

To regulate efp2 expression in G. sulfurreducens:

• Inducible promoter systems:

  • The RpoN-dependent promoter system can be adapted for controlled expression

  • Integration host factor (IHF) binding sites can be incorporated for additional regulation

• Molecular beacons for expression monitoring:

  • Design RNA sensors that fluoresce when binding efp2 mRNA

  • Use RT-qPCR to quantify expression levels under different conditions

• Reporter fusion constructs:

  • Create translational fusions with fluorescent proteins

  • Monitor localization and expression patterns in vivo

What phenotypes are associated with efp2 mutations in G. sulfurreducens?

Expected phenotypes from efp2 mutations include:

How might Efp-2 engineering improve G. sulfurreducens performance in microbial fuel cells?

Engineering optimized Efp-2 variants could enhance G. sulfurreducens performance through:

• Improved translation efficiency of key EET proteins:

  • Enhanced production of PilA for better conductive nanowires

  • Increased synthesis of outer membrane cytochromes

• Oxygen tolerance enhancement:

  • Better translation of proteins involved in oxygen defense mechanisms

  • Engineering an Efp-2 variant that maintains functionality under oxidative stress

• Metal reduction capacity improvement:

  • Optimizing the translation of proteins specific to metal reduction pathways

  • Potential 20-40% increase in Pd(II) reduction capacity based on comparable pili mutant studies

What is the evolutionary significance of having two Efp homologs in G. sulfurreducens?

The presence of two Efp homologs (including Efp-2) in G. sulfurreducens suggests evolutionary adaptation:

• Functional specialization:

  • Efp-2 appears specialized for electrode/metal respiration conditions

  • The second homolog may be optimized for other metabolic modes

• Redundancy for environmental resilience:

  • Provides backup systems for different growth conditions

  • Allows for survival in fluctuating environments like soil-water interfaces

• Comparative analysis with other Geobacter species:

  • Related species like G. metallireducens and G. chapellei show varying Efp distributions

  • This divergence likely reflects adaptation to specific ecological niches

What are the key research priorities for advancing our understanding of Efp-2 function in G. sulfurreducens?

Future research should focus on:

• Structural elucidation of G. sulfurreducens Efp-2:

  • X-ray crystallography or cryo-EM studies

  • Comparative analysis with Efp proteins from other species

• Comprehensive proline-rich proteome analysis:

  • Identification of all proteins dependent on Efp-2 for translation

  • Correlation with electroactive phenotypes

• Integration with global regulatory networks:

  • Interaction with RpoS and RpoN regulons

  • Role in IHF-mediated gene regulation

• Development of Efp-2 variants for biotechnological applications:

  • Enhanced metal reduction for bioremediation

  • Improved electron transfer for bioelectrochemical systems

  • Increased tolerance to environmental stressors