Recombinant Geobacter sulfurreducens 50S ribosomal protein L31 (rpmE)

What is the structure and function of the 50S ribosomal protein L31 in Geobacter sulfurreducens?

The 50S ribosomal protein L31 (rpmE) in Geobacter sulfurreducens is a small (approximately 65 amino acids), basic protein that is a component of the large ribosomal subunit. The protein is characterized by:

• A highly disordered structure with an unstructured amino-terminal region (residues 2-8) that is enriched in lysine residues

• Several cysteine residues that can form various intramolecular disulfide bonds

• Sequence: MREGIHPKYN EVTVKCLCGN TFQTRSTKPE ISTEICSACH PFFTGKQKLV DTAGRVERFK KRYGM

Functionally, L31 contributes to:

• Formation of the protein-protein intersubunit bridge B1b, a key connecting link between ribosomal subunits

• Maintaining ribosome stability during the translation process

• Supporting ribosome dynamics through interactions with other ribosomal proteins

Unlike in E. coli, where L31 interacts with the 30S subunit via its carboxy-terminal part and with the 50S subunit via its amino-terminal domain, the specific interactions in G. sulfurreducens have not been fully characterized but are likely similar based on homology .

How is the rpmE gene regulated in bacterial systems, and does G. sulfurreducens exhibit similar regulatory mechanisms?

The rpmE gene in bacterial systems, particularly in E. coli, exhibits autogenous regulation where the protein product (L31) regulates its own expression. Based on research evidence:

• Transcription of rpmE occurs from two promoter regions in E. coli

• Translation of both mRNA transcripts is feedback regulated by L31 itself

• The autogenous operator is located within the shorter transcript

• A highly conserved stem-loop structure in the rpmE 5′UTR serves as a translational operator targeted by L31

• The unstructured amino-terminal part of L31 enriched in lysine is necessary for its repressor activity

While the specific regulatory mechanisms in G. sulfurreducens have not been extensively characterized in the provided search results, evidence from related bacteria suggests similar autogenous regulation likely occurs. A key difference may relate to the unique metabolism of G. sulfurreducens, which might influence the expression patterns of ribosomal proteins during different growth conditions (e.g., when using electrodes versus soluble electron acceptors) .

What are the optimal purification methods for recombinant G. sulfurreducens L31 protein?

Purification of recombinant G. sulfurreducens L31 protein presents several challenges due to its small size and biochemical properties. Based on published protocols and commercial information, the following methodologies are recommended:

Expression System Selection:

• Mammalian cell expression systems have been successfully used for G. sulfurreducens L31

• E. coli expression systems with GST or His tags are viable alternatives

Purification Protocol:

• Cell lysis using buffer containing protease inhibitors

• Initial purification using affinity chromatography (GST-tag or His-tag based)

• Tag removal using specific proteases if necessary

• Further purification by size exclusion chromatography

• Final concentration determination by SDS-PAGE or spectrophotometric methods

Critical Considerations:

• Maintain reducing conditions during purification to prevent disulfide bond formation

• Avoid repeated freeze-thaw cycles; store working aliquots at 4°C for up to one week

• For long-term storage, add 5-50% glycerol and store at -20°C/-80°C

• Lyophilized form has longer shelf life (12 months) compared to liquid form (6 months)

Protein purity above 85% (as measured by SDS-PAGE) should be achievable using these methods .

How does L31 from G. sulfurreducens compare structurally and functionally to its homologs in other bacterial species?

L31 proteins from different bacterial species share similar core functions but exhibit notable differences in sequence, structure, and specific interactions:

Sequence Comparison:

Functional Differences:

• E. coli L31 has been extensively characterized for its role in ribosomal subunit association and translation initiation

• G. sulfurreducens L31 likely functions similarly but may have adaptations related to the organism's unique electron transfer mechanisms

• The loosely associated nature of L31 with the ribosome is common across species, but the strength of this association may vary

Research Implications:
The differences in sequence and potentially in function suggest that L31 from G. sulfurreducens may have evolved specific adaptations related to the organism's unique metabolism involving extracellular electron transfer. These differences could influence protein-protein interactions within the ribosome or with other cellular components .

What role might L31 play in the unique extracellular electron transfer capabilities of G. sulfurreducens?

While L31 is primarily characterized as a ribosomal protein, its potential influence on the unique metabolism of G. sulfurreducens warrants consideration:

Direct Connections to Electron Transfer:

• No direct evidence links L31 specifically to extracellular electron transfer (EET) pathways

• As a ribosomal protein, L31 would influence the translation of proteins involved in EET pathways

Indirect Effects through Translational Regulation:

• G. sulfurreducens has multiple redundant pathways in its central metabolism that are precisely regulated

• Translation efficiency affected by L31 could influence the balance between these pathways

• Key cytochromes involved in EET (like PgcA) require proper translation

Transcriptomic Evidence:

• Studies of G. sulfurreducens under different electron acceptor conditions (fumarate vs. Fe(III) citrate) show differential expression of numerous genes

• The expression of ribosomal proteins, potentially including L31, might be coordinated with the expression of EET components

What are the key considerations for designing experiments to study L31 interactions with RNA or other proteins?

When designing experiments to study L31 interactions with RNA or other proteins in G. sulfurreducens, researchers should consider:

Methodological Approaches:

• NMR Chemical Shift Perturbation: Successfully used to identify protein-protein interactions between periplasmic cytochromes in G. sulfurreducens

• Site-Directed Mutagenesis: Essential for identifying key residues involved in interactions (e.g., the importance of the N-terminal region for autogenous regulation)

• RNA Footprinting: To map the precise RNA binding sites

• Biophysical Techniques: Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) for binding kinetics

Specific Experimental Design Considerations:

• Buffer Conditions: Maintaining physiologically relevant pH and salt concentrations is crucial for studying native interactions

• Redox State Management: L31 contains multiple cysteines that can form disulfide bonds, affecting its conformation and interactions

• Expression Tags: Selection of appropriate tags (GST, His) that minimize interference with native interactions

• Temperature Controls: G. sulfurreducens optimal growth temperature is 30°C, which should be considered for interaction studies

Potential Interaction Partners to Investigate:

• 23S rRNA binding has been documented for E. coli L31

• Other ribosomal proteins, particularly those involved in intersubunit bridge formation

• mRNA transcripts containing the putative L31 binding site from the rpmE 5′UTR

Research has shown that the unstructured amino-terminal region (residues 2-8) of L31 is necessary for autogenous regulation, suggesting this region is critical for RNA interactions .

How do different electron acceptor conditions affect the expression of ribosomal proteins, including L31, in G. sulfurreducens?

G. sulfurreducens can utilize various electron acceptors, including fumarate, Fe(III) citrate, and electrodes. The choice of electron acceptor has significant impacts on gene expression patterns:

Expression Patterns Under Different Electron Acceptors:

• Transcriptomic analysis reveals substantial differences in gene expression when G. sulfurreducens grows with fumarate versus Fe(III) citrate as electron acceptors

• During Fe(III) reduction, G. sulfurreducens induces specific genes as the redox potential decreases, suggesting a sensing mechanism that could also affect ribosomal protein expression

Potential Impact on L31 Expression:

• While specific data on L31 expression changes are not directly reported in the search results, ribosomal proteins generally show coordinated expression patterns

• The induction of genes such as cbcBA with the aid of the regulator BccR during Fe(III) reduction suggests complex regulatory networks that could influence ribosomal protein expression

Methodological Approach to Study These Effects:

• Grow G. sulfurreducens cultures under standardized conditions with different electron acceptors:

  • Fumarate (40 mM)

  • Fe(III) citrate (56 mM)

  • Poised electrodes at various potentials (-0.03 V to +0.24 V vs. SHE)

• Extract RNA at defined growth phases

• Perform RT-qPCR targeting rpmE and related genes

• Conduct proteomics to correlate transcript levels with protein abundance

• Compare results across conditions to identify regulatory patterns

Understanding these expression patterns could provide insights into how G. sulfurreducens adapts its translational machinery to different electron acceptor environments.

What are the implications of L31's loose association with the ribosome in G. sulfurreducens?

The loose association of L31 with the ribosome has significant implications for both experimental approaches and biological function:

Experimental Challenges:

• Difficulty in isolating intact ribosomes with L31 still attached

• Variable detection of L31 in ribosome preparations due to its tendency to dissociate

• Need for specialized techniques to preserve the native state of L31-ribosome complexes

Biological Significance:

• Potential for L31 to serve dual roles - both as a ribosomal component and as a free protein with regulatory functions

• Dynamic association/dissociation that may be responsive to cellular conditions

• Possible role in ribosome assembly/disassembly processes

Research Findings from E. coli Relevant to G. sulfurreducens:

• "The failure to identify L31 in many ribosome preparations is probably due to the protein's loose association with the ribosome and its ability to form various intramolecular disulfide bonds, leading to L31 forms with distinct mobilities in gels"

• This property creates challenges for studying the protein but also suggests regulatory flexibility

Methodological Recommendations:

• Use gentle ribosome isolation procedures that minimize L31 dissociation

• Consider crosslinking approaches to stabilize L31-ribosome interactions

• Compare L31 distribution between ribosome-bound and free pools under different growth conditions

• Investigate potential regulatory roles of free L31 outside the ribosome

The loose association might be especially relevant in G. sulfurreducens, which has unique metabolic adaptations that could require specialized ribosome dynamics or regulation.

How can researchers effectively study the autogenous regulation of the rpmE gene in G. sulfurreducens?

To effectively study the autogenous regulation of the rpmE gene in G. sulfurreducens, researchers can adapt approaches used successfully with E. coli and other bacteria:

Recommended Experimental Approaches:

• Reporter Gene Fusions:

  • Create chromosomally integrated fusions of rpmE with the lacZ reporter gene

  • Measure β-galactosidase activity under various conditions and in different genetic backgrounds

  • Compare expression levels in wild-type versus rpmE deletion mutants

• Identification of Regulatory Elements:

  • Analyze the 5′UTR of rpmE for conserved stem-loop structures similar to those found in E. coli

  • Perform site-directed mutagenesis of predicted regulatory elements

  • Use RNA structure prediction algorithms to identify potential L31 binding sites

• In vitro Binding Studies:

  • Express and purify recombinant L31 protein

  • Synthesize RNA transcripts containing the predicted regulatory regions

  • Conduct gel shift assays to quantify binding affinity and specificity

  • Perform RNA footprinting to identify exact binding sites

Key Findings from Related Research:
In E. coli, researchers have shown that:

• The rpmE gene is transcribed from two promoter regions

• A highly conserved stem-loop structure in the 5′UTR serves as the translational operator

• The unstructured amino-terminal region of L31 is necessary for its repressor activity

Similar mechanisms likely exist in G. sulfurreducens, though they may have evolved specific features related to the organism's unique metabolism and environmental adaptations.

What techniques can help resolve contradictions in sequence data for recombinant L31 protein?

Contradictions in sequence data for recombinant proteins can arise from various sources, including post-translational modifications, technical artifacts, or actual genetic variations. To resolve such contradictions for L31:

Common Sources of Sequence Discrepancies:

• Post-translational processing (as observed with E. coli L31)

• RNA editing events

• Strain-specific variations

• Technical errors in sequencing or annotation

Methodological Approaches to Resolve Contradictions:

• Multi-Method Protein Sequencing:

  • N-terminal sequencing by Edman degradation

  • Mass spectrometry (LC-MS/MS) for full protein characterization

  • Analysis of tryptic peptides, particularly from the C-terminal region

  In E. coli, this approach revealed that "...RFNKRFNIPGSK represents the true C-terminus of L31" despite earlier contradictory reports .

• Comparative Genomics:

  • Sequence analysis across multiple G. sulfurreducens strains

  • Comparison with closely related Geobacter species

  • Phylogenetic analysis to identify conserved regions

• Experimental Validation:

  • Construct expression vectors with different predicted sequences

  • Assess functional complementation of deletion mutants

  • Compare migration patterns on high-resolution gels

• Structural Analysis:

  • X-ray crystallography or NMR to determine actual protein structure

  • Compare experimental structure with predicted models from different sequences

When investigating sequence discrepancies, it's important to consider that L31 can form various intramolecular disulfide bonds, potentially leading to mobility differences in gels that might be misinterpreted as sequence variations .

How does the metal content of G. sulfurreducens affect the expression and function of ribosomal proteins like L31?

G. sulfurreducens has a unique metal composition profile that may influence ribosomal protein expression and function:

Metal Content Observations:

• G. sulfurreducens contains significantly higher iron content compared to E. coli (approximately an order of magnitude higher)

• There are notable differences in trace metal content between G. sulfurreducens grown with different electron acceptors

• The organism has "a large amount of lipid and iron compared to other bacteria"

Potential Impacts on L31 and Ribosomal Function:

• Direct Effects on Protein Folding:

  • L31 contains multiple cysteine residues that form disulfide bonds

  • Metal ions, particularly zinc, can influence disulfide bond formation and protein stability

  • The redox environment affected by metal content could impact L31 folding and ribosome association

• Regulatory Effects:

  • Metal-responsive transcriptional regulators might influence rpmE expression

  • Iron limitation has been shown to inhibit extracellular electron transfer in G. sulfurreducens

  • Changes in metal availability could trigger stress responses affecting translation

• Experimental Approaches to Study These Relationships:

  • Compare L31 expression and ribosome association under varying metal concentrations

  • Use metallomics and transcriptomics to correlate metal content with gene expression patterns

  • Investigate metal-binding properties of L31 through spectroscopic methods

The unique metabolism of G. sulfurreducens, particularly its high cytochrome content for electron transfer, creates a cellular environment with distinct metal requirements that likely influence ribosomal function in ways that differ from model organisms like E. coli.

What are the best approaches for engineering mutations in the rpmE gene of G. sulfurreducens?

Engineering mutations in G. sulfurreducens requires specialized approaches due to the organism's unique metabolism and genetic characteristics:

Established Genetic Modification Methods:

• Markerless Deletion System:

  • Successfully used for creating the ΔpgcA mutant in G. sulfurreducens

  • Process involves:
    a) Cloning ~1 kb up and downstream of target gene into pk18mobsacB vector
    b) Mating into G. sulfurreducens via E. coli strain S17-1
    c) First selection on kanamycin plates
    d) Second selection on sucrose for recombination events
    e) PCR screening for gene deletion

• Complementation Strategies:

  • pRK2-Geo2 backbone with a constitutive promoter from G. sulfurreducens acpP gene (GSU1604)

  • Allows for expression of the target gene from a plasmid in deletion mutants

• Site-Directed Mutagenesis Approaches:

  • Mutations can be introduced into specific regions of the rpmE gene

  • For L31, targeting the N-terminal lysine-rich region or the cysteine residues would be particularly informative

Growth Considerations for Mutant Selection:

• Use NBAFYE plates (NBAF medium amended with yeast extract)

• Supplement with appropriate antibiotics (200 μg/ml kanamycin, 20 μg/ml gentamycin, or 10 μg/ml chloramphenicol)

• Add 0.1% peptone and 15 mM pyruvate to alleviate possible metabolic limitations generated by gene inactivations

• Incubate in an anaerobic chamber under 7% H₂, 10% CO₂, and 83% N₂ atmosphere at 30°C

Phenotypic Characterization:
Compare growth of mutants under different electron donor/acceptor conditions, including:

• Acetate (15 mM) with Fe(III) citrate (56 mM)

• Acetate (15 mM) with fumarate (40 mM)

• Electrode-based growth systems