Recombinant Geobacter sulfurreducens 50S ribosomal protein L5 (rplE)

What is the function of 50S ribosomal protein L5 in Geobacter sulfurreducens?

Based on comparative studies with other bacteria, L5 in G. sulfurreducens likely serves as a critical component for large ribosomal subunit assembly. In E. coli, L5 plays a key role in the formation of the central protuberance (CP) of the 50S subunit . When L5 synthesis is arrested in E. coli, cells accumulate defective 45S particles lacking CP components (5S rRNA and proteins L5, L16, L18, L25, L27, L31, L33, and L35) that cannot associate with the small ribosomal subunit .

In G. sulfurreducens, with its extensive cytochrome network and unique metabolism, proper ribosome assembly through L5 would be essential for synthesizing the proteins involved in its distinctive extracellular electron transfer pathways. This is particularly important considering G. sulfurreducens' ability to reduce extracellular metals like Fe(III) and Mn(IV) requires properly expressed cytochromes .

How does the structure of G. sulfurreducens L5 compare to homologs in other bacteria?

While detailed structural comparisons specific to G. sulfurreducens L5 are not directly provided in the available literature, ribosomal proteins typically maintain conserved functional domains across bacterial species. G. sulfurreducens' L5 would be expected to maintain the core 5S rRNA binding domain and interfaces for interaction with other ribosomal proteins.

The structural investigation methodology would involve:

• Expressing recombinant G. sulfurreducens L5 using RK2-based vectors, which show superior stability in Geobacter (maintained for over 15 generations without antibiotic selection)

• Purifying the protein using affinity chromatography

• Employing structural determination techniques such as X-ray crystallography or cryo-electron microscopy

• Comparing the resulting structure with known bacterial L5 structures to identify G. sulfurreducens-specific features

Any unique structural features might reflect adaptations to G. sulfurreducens' metal-rich environment and specialized metabolism.

What role might L5 play in G. sulfurreducens' metal reduction pathways?

While L5 does not directly participate in electron transfer to metals, its role in ribosome assembly creates an indirect but critical connection to G. sulfurreducens' metal reduction capabilities. The methodology to understand this connection involves:

• Creating conditional L5 depletion strains using inducible expression systems such as the VanR-dependent vanillate-inducible system developed for G. sulfurreducens

• Measuring Fe(III) reduction capability in wild-type versus L5-depleted conditions

• Monitoring expression levels of key cytochromes involved in extracellular electron transfer

L5 depletion would likely impair the synthesis of critical cytochromes such as PgcA, which has been shown to be essential for Fe(III) and Mn(IV) oxide reduction . Similar effects have been observed in RelGsu mutants, where Fe(III) reduction capacity was substantially diminished, accompanied by down-regulation of electron transport genes .

How does G. sulfurreducens ribosome assembly differ from other bacteria under metal-reducing conditions?

G. sulfurreducens shows unique adaptations to its metal-reducing lifestyle. Its ribosomes likely assemble under distinctive conditions compared to other bacteria:

• High intracellular iron content: G. sulfurreducens has significantly higher metal concentrations, particularly iron, compared to organisms like E. coli . This metal-rich intracellular environment might necessitate specialized assembly factors or protective mechanisms for ribosome biogenesis.

• Stress response integration: The RelGsu-mediated stringent response in G. sulfurreducens affects Fe(III) reduction . L5 expression may be regulated as part of this response, with different patterns of ribosomal protein synthesis during metal reduction versus growth with other electron acceptors.

• Redox sensitivity: Given G. sulfurreducens' sensitivity to oxidative stress , ribosome assembly might incorporate mechanisms to maintain functionality under varying redox conditions, potentially involving L5 adaptations.

Research methodologies should combine ribosome profiling under different electron acceptor conditions with comparative analyses of L5-associated assembly factors between G. sulfurreducens and non-metal-reducing bacteria.

How do regulatory networks coordinate L5 expression with cytochrome production in G. sulfurreducens?

The coordination between ribosome assembly and cytochrome production represents a fascinating research area. Experimental approaches should include:

• Transcriptome analysis comparing expression patterns of L5 and cytochrome genes under different growth conditions (Fe(III) reduction versus fumarate respiration)

• Investigation of potential regulators that might co-regulate ribosomal and cytochrome genes

• Analysis of the RelGsu regulon, as RelGsu has been shown to affect both growth regulation and Fe(III) reduction

Research in G. sulfurreducens has shown that during stationary phase, protein synthesis genes were up-regulated in RelGsu mutants while genes involved in stress responses and electron transport were down-regulated . This suggests regulatory links between translational machinery (including L5) and electron transfer components that could be further explored through targeted genetic studies.

What structural adaptations in L5 might facilitate ribosome function under high metal concentrations?

G. sulfurreducens functions in environments with high metal concentrations and maintains higher intracellular iron levels than typical bacteria . This unique environment might necessitate specific adaptations in L5:

• Modified metal-binding sites that prevent non-specific interactions with free metals

• Altered surface properties affecting interactions with 5S rRNA and neighboring proteins

• Specific post-translational modifications that enhance stability

Research methodology would involve site-directed mutagenesis of potential metal-interaction sites in L5, followed by functional assays measuring ribosome assembly efficiency and translation fidelity under varying metal concentrations. Complementation studies with L5 variants in L5-depleted strains would reveal which features are essential for G. sulfurreducens' unique physiology.

What are optimal strategies for expressing and purifying recombinant G. sulfurreducens L5?

Based on the available literature, the following methodological approach is recommended:

Expression system selection:

• Use RK2-based vectors rather than pBBR1 plasmids, as the former show superior stability in G. sulfurreducens (maintained for over 15 generations without selection)

• Consider both constitutive expression using native Geobacter promoters and controlled expression using the vanillate-inducible system

Host selection:

• Heterologous expression in E. coli for high yield but potential folding issues

• Homologous expression in G. sulfurreducens for proper folding but lower yield

• Expression in Shewanella oneidensis as an alternative (similar to successful PgcA expression)

Purification strategy:

• Affinity tags should be carefully selected to minimize interference with L5 function

• Include metal chelators in buffers to prevent non-specific metal binding

• Monitor for co-purification of 5S rRNA, as observed with other ribosomal proteins

These approaches should be optimized based on the intended application, with structural studies requiring higher purity than functional assays.

How can L5 function be studied through genetic manipulation of G. sulfurreducens?

Given that L5 is likely essential (based on E. coli studies) , complete gene deletion might not be viable. Alternative genetic approaches include:

• Conditional expression systems:

  • Implement the vanillate-inducible system demonstrated effective for G. sulfurreducens

  • Create strains where L5 expression can be gradually reduced to study threshold effects

• Domain mapping through partial deletions:

  • Target non-essential regions identified through comparative analysis

  • Create chimeric L5 proteins by domain swapping with L5 from other species

• Point mutations:

  • Target conserved residues involved in 5S rRNA binding or protein-protein interactions

  • Engineer metal-binding sites to test hypotheses about metal tolerance

• Reporter fusions:

  • Create L5-fluorescent protein fusions to track localization

  • Develop split reporter systems to monitor L5 interactions with assembly partners

Genetic manipulations can be performed using the SacB/sucrose counterselection method proven effective in G. sulfurreducens , though care must be taken to maintain expression at levels compatible with cell viability.

What techniques are most effective for analyzing the impact of L5 on G. sulfurreducens metal reduction?

A systematic approach combining multiple techniques would be most effective:

Growth and metal reduction assays:

• Monitor Fe(III) reduction using methods established in previous studies

• Compare reduction of soluble (Fe(III) citrate) versus insoluble Fe(III) oxides

• Extend to other metals including Mn(IV) oxides

Protein expression analysis:

• Quantify levels of key cytochromes (such as PgcA) known to be involved in extracellular electron transfer

• Monitor expression of copper-containing proteins like OmpB and OmpC required for Fe(III) oxide reduction

Ribosome profiling:

• Analyze translation efficiency of cytochrome mRNAs under varying L5 levels

• Compare translational profiles between wild-type and L5-depleted conditions

Electrochemical techniques:

• Use poised electrode systems (+0.24V vs. SHE) similar to those described in previous G. sulfurreducens studies

• Perform cyclic voltammetry over a wide potential range (-0.4V to +0.3V)

This multi-faceted approach would distinguish direct effects on metal reduction from secondary effects resulting from altered protein synthesis.

How should researchers interpret changes in metal reduction upon L5 manipulation?

When interpreting experimental data on the relationship between L5 and metal reduction, researchers should consider:

• Direct versus indirect effects:

  • Primary effects on ribosome assembly and general protein synthesis

  • Secondary effects on specific cytochrome expression

  • Tertiary effects on cellular energetics and redox state

• Comparison with known regulatory mutants:

  • RelGsu mutants show decreased Fe(III) reduction and altered gene expression patterns

  • Compare phenotypic similarities and differences with L5-depleted strains

• Electron acceptor specificity:

  • Differentiate between effects on Fe(III) oxide, Fe(III) citrate, and electrode reduction

  • PgcA deletion specifically affects Fe(III) and Mn(IV) oxide reduction but not electrode reduction

  • Similar electron acceptor specificity in L5-compromised strains would suggest targeted effects rather than general translation deficiency

• Quantitative analysis framework:

This interpretative framework helps distinguish between general growth defects and specific impacts on metal reduction pathways.

What controls are essential when studying L5 function in G. sulfurreducens?

Robust experimental design requires comprehensive controls:

• Genetic controls:

  • Empty vector controls for plasmid-based studies

  • Complementation with wild-type L5 to confirm phenotype specificity

  • Non-ribosomal protein mutations as controls for general translation effects

• Growth condition controls:

  • Compare multiple electron acceptors (Fe(III) oxide, Fe(III) citrate, fumarate, electrodes)

  • Test under different nutritional states (acetate limitation, nitrogen limitation)

  • Evaluate under varying oxygen exposures, as G. sulfurreducens shows distinct responses to oxidative stress

• Time course considerations:

  • Monitor effects immediately after L5 depletion versus after several generations

  • Account for adaptation mechanisms that might compensate for L5 deficiency

• Reporter controls:

  • Include translation efficiency controls using reporter genes

  • Monitor general versus specific effects on protein synthesis

These controls help differentiate L5-specific effects from general stress responses or artifacts of experimental manipulation.

How can contradictory results between in vitro and in vivo L5 studies be reconciled?

When facing discrepancies between in vitro reconstitution experiments and in vivo genetic studies, researchers should:

• Identify potential factors present in vivo but absent in vitro:

  • High metal concentrations characteristic of G. sulfurreducens

  • Interactions with assembly factors or chaperones

  • Post-translational modifications

• Develop intermediate systems to bridge the gap:

  • Cell-free translation systems using G. sulfurreducens extracts

  • Reconstitution experiments with increasing complexity

  • Hybrid approaches combining purified components with cellular extracts

• Consider G. sulfurreducens-specific factors:

  • Unique lipid composition (high C:O and H:O ratios)

  • Metal sequestration mechanisms

  • Interaction with the stringent response system

• Methodical approach to reconciliation:

By systematically moving between these levels of analysis, apparently contradictory results can often be resolved into a more comprehensive understanding of L5 function in the complex cellular environment of G. sulfurreducens.