Recombinant Geobacter sulfurreducens 50S ribosomal protein L23 (rplW)

What is Geobacter sulfurreducens and why is its 50S ribosomal protein L23 of interest?

Geobacter sulfurreducens is an anaerobic, gram-negative bacterium known for its unique ability to transfer electrons to insoluble materials such as iron oxides and electrodes. This capability makes it essential in biogeochemical iron cycling and microbial electrochemical systems . The bacterium has an extraordinary metabolism heavily dependent on cytochromes, resulting in cells with distinctive composition, including high iron content (2 ± 0.2 μg/g dry weight) and lipid content (32 ± 0.5% dry weight) .

The 50S ribosomal protein L23 (rplW) is a component of the large ribosomal subunit involved in protein synthesis. While not directly linked to electron transfer, understanding ribosomal proteins in G. sulfurreducens could provide insights into how this organism regulates protein expression under various growth conditions, particularly when shifting between soluble and insoluble electron acceptors. The protein may also serve as a target for genetic manipulation studies to understand ribosomal function in this metabolically unique organism.

How does the structure of G. sulfurreducens 50S ribosomal protein L23 compare with other bacterial species?

While specific structural data for G. sulfurreducens L23 is not widely documented in current literature, we can make educated comparisons based on the closely related Geobacter uraniireducens L23 protein. The G. uraniireducens L23 protein consists of 94 amino acids with the sequence: MNIYSVIKKP LITEKTTIEK DERNIISFVV SSDANKIEIK DAVKTLFNVD VASVKTVNVA GKVKRVGKNI GKRSNWKKAY VTLKEGSNVD FFEA .

This sequence likely shares high homology with G. sulfurreducens L23 due to their phylogenetic proximity. The conserved regions would be expected to maintain the core structural elements essential for ribosomal function, including RNA binding sites and interactions with other ribosomal proteins. Structural conservation among L23 proteins is typically high across bacterial species, with variability often concentrated in surface-exposed regions.

What expression systems are suitable for producing recombinant G. sulfurreducens ribosomal proteins?

Based on research with other Geobacter proteins, E. coli expression systems are commonly used for producing recombinant proteins from G. sulfurreducens. The related G. uraniireducens L23 protein has been successfully expressed in E. coli systems , suggesting similar approaches would work for G. sulfurreducens L23.

For expression in E. coli, the following methodological considerations are important:

• Vector selection: The IncQ plasmid pCD342 has been demonstrated as a suitable expression vector for G. sulfurreducens proteins . This class of broad-host-range vectors can effectively replicate in Geobacter species.

• Codon optimization: Consider codon optimization for expression in E. coli, as G. sulfurreducens may have different codon usage patterns.

• Purification strategy: Incorporate an appropriate affinity tag (His-tag is commonly used) that can be determined during the manufacturing process to facilitate protein purification .

• Expression conditions: Optimize temperature, IPTG concentration, and induction time for maximum protein yield while minimizing inclusion body formation.

What are the recommended storage conditions for recombinant G. sulfurreducens L23 protein?

Based on protocols for similar ribosomal proteins, the following storage recommendations apply:

• For lyophilized protein: Store at -20°C/-80°C for up to 12 months .

• For liquid formulations: Store at -20°C/-80°C for up to 6 months .

• For working aliquots: Store at 4°C for up to one week .

• Reconstitution procedure:

  • Briefly centrifuge the vial before opening

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add 5-50% glycerol (final concentration) for long-term storage

  • Avoid repeated freeze-thaw cycles

How might the expression of rplW in G. sulfurreducens vary under different electron acceptor conditions?

When studying rplW expression specifically:

What methodologies are most effective for studying interactions between L23 and other components of the G. sulfurreducens translation machinery?

To investigate the role of L23 in the context of G. sulfurreducens translation:

• Co-immunoprecipitation (Co-IP) studies:

  • Express tagged versions of L23 in G. sulfurreducens using the genetic system developed for this organism

  • Use antibodies against the tag to pull down L23 and associated components

  • Identify interaction partners using mass spectrometry

• Cryo-electron microscopy:

  • Isolate intact ribosomes from G. sulfurreducens under different growth conditions

  • Determine structural arrangements including L23 positioning

  • Compare with ribosome structures from other organisms

• In vitro reconstitution:

  • Express and purify recombinant G. sulfurreducens ribosomal proteins

  • Study assembly kinetics and interactions

  • Evaluate effects of mutations in L23 on ribosome assembly

How does the presence of conjugative plasmids affect the expression of ribosomal proteins in G. sulfurreducens?

Recent research has shown that conjugative plasmids inhibit extracellular electron transfer in G. sulfurreducens . This inhibition occurs at the transcriptional level, affecting genes involved in nanowire formation and extracellular electron transfer.

To investigate potential effects on ribosomal protein expression:

• Comparative transcriptomics:

  • Compare expression levels of rplW and other ribosomal protein genes between wild-type G. sulfurreducens and strains carrying conjugative plasmids (pKJK5, RP4, or pB10)

  • Analyze under both fumarate and Fe(III)-reducing conditions

  • Correlate with growth rates and protein synthesis capacities

• Proteomics approach:

  • Quantify relative abundance of L23 and other ribosomal proteins in plasmid-bearing versus plasmid-free cells

  • Assess post-translational modifications that might affect ribosomal assembly or function

• Functional assays:

  • Compare in vitro translation efficacy using ribosomes isolated from plasmid-bearing versus plasmid-free cells

  • Measure amino acid incorporation rates as indicators of translation efficiency

What are the challenges in differentiating the functions of ribosomal protein L23 from other ribosomal proteins in G. sulfurreducens?

Investigating the specific functions of L23 in G. sulfurreducens presents several methodological challenges:

• Genetic manipulation approaches:

  • Since ribosomal proteins are essential, complete knockout is likely lethal

  • Site-directed mutagenesis targeting conserved versus variable regions

  • Development of conditional expression systems to control L23 levels

  • Use of the genetic system developed for G. sulfurreducens to create these mutations

• Structural biology considerations:

  • Difficulty in isolating homogeneous ribosome populations

  • Potential for structural heterogeneity affecting crystallization

  • Need for high-resolution techniques to distinguish subtle structural differences

• Experimental design for functional studies:

  • Establishing in vitro translation systems specific for G. sulfurreducens

  • Designing reporter systems to monitor translation accuracy and efficiency

  • Controlling for indirect effects when manipulating ribosomal components

How might the high iron and lipid content of G. sulfurreducens affect studies of its ribosomal proteins?

G. sulfurreducens has unusually high iron content (2 ± 0.2 μg/g dry weight) and lipid content (32 ± 0.5% dry weight) , which presents unique challenges for ribosomal protein studies:

• Protein purification considerations:

  • High lipid content may require modified lysis and extraction protocols

  • Lipid contamination could affect protein folding and activity assays

  • Need for additional purification steps to remove lipid interactions

• Iron interference issues:

  • Potential for iron-protein interactions affecting structural studies

  • Iron contamination in protein preparations affecting spectroscopic analyses

  • Metal chelators may be required during purification, with careful control to avoid affecting metalloproteins

• Methodological adaptations:

  • Modified Bradford or BCA assays accounting for lipid interference

  • Specialized membrane disruption techniques for efficient ribosome isolation

  • Additional controls in functional assays to account for iron and lipid effects

What are the optimal conditions for expressing G. sulfurreducens L23 in heterologous systems?

Based on protocols for similar ribosomal proteins and the unique characteristics of G. sulfurreducens proteins:

• Expression system selection:

  • E. coli BL21(DE3) for high-level expression

  • Consider the pCD342 vector (IncQ plasmid) proven effective for G. sulfurreducens

  • Cold-adapted expression strains for potentially better folding

• Induction parameters:

  • Lower induction temperatures (16-20°C) to enhance proper folding

  • Reduced IPTG concentrations (0.1-0.5 mM) for slower expression

  • Extended induction times (overnight) at lower temperatures

• Media considerations:

  • Rich media (LB or TB) for maximum biomass

  • Defined media for specific isotopic labeling

  • Supplementation with iron may be beneficial given G. sulfurreducens' high iron content

What purification strategies are most effective for isolating recombinant G. sulfurreducens L23?

To achieve high purity (>85% by SDS-PAGE) as reported for similar proteins :

• Initial capture step:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged protein

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5-20 mM imidazole

• Secondary purification:

  • Ion exchange chromatography to remove remaining contaminants

  • Size exclusion chromatography for final polishing and buffer exchange

• Quality control procedures:

  • SDS-PAGE to confirm >85% purity

  • Mass spectrometry to verify protein identity and integrity

  • Circular dichroism to assess proper folding

How can researchers optimize functional assays for recombinant G. sulfurreducens L23?

When designing functional assays:

• RNA binding studies:

  • Filter binding assays with labeled rRNA fragments

  • Electrophoretic mobility shift assays (EMSA)

  • Surface plasmon resonance for binding kinetics

• Protein-protein interaction assays:

  • Pull-down assays with other recombinant ribosomal proteins

  • Yeast two-hybrid screening for interaction partners

  • Fluorescence resonance energy transfer (FRET) for real-time interaction studies

• Ribosome reconstitution:

  • In vitro assembly of partial ribosomal complexes

  • Assessment of L23 incorporation into native ribosomal subunits

  • Functional translation assays with reconstituted ribosomes

What are the most promising applications of research on G. sulfurreducens L23?

Research on G. sulfurreducens L23 has several potential applications:

• Understanding ribosomal adaptation in metabolically unique organisms:

  • Insights into how protein synthesis machinery adapts to electron transport needs

  • Potential correlations between ribosomal protein structure and extracellular electron transfer capabilities

• Biotechnological applications:

  • Development of optimized expression systems for G. sulfurreducens proteins

  • Engineering ribosomes for enhanced production of electron transfer components

  • Using ribosomal proteins as targets for enhancing G. sulfurreducens capabilities in microbial fuel cells

• Evolutionary studies:

  • Comparative analysis of ribosomal proteins across Geobacteraceae

  • Insights into specialization of protein synthesis machinery in environmentally important bacteria