Recombinant Geobacter sulfurreducens 30S ribosomal protein S13 (rpsM)

What is the functional significance of 30S ribosomal protein S13 (rpsM) in Geobacter sulfurreducens?

The 30S ribosomal protein S13 is a critical component of the small ribosomal subunit in G. sulfurreducens. This protein plays a vital role in maintaining ribosomal structure and facilitating translation by helping coordinate interactions between tRNA and mRNA at the decoding center. In G. sulfurreducens, rpsM may have additional significance given the organism's unique metabolic capabilities, including dissimilatory metal reduction. Similar to other bacteria, rpsM in G. sulfurreducens likely participates in the assembly of the 30S subunit and contributes to translational fidelity. Research suggests that ribosomal proteins in G. sulfurreducens may show differential expression under various environmental conditions, particularly during stress response, similar to how RpoS regulates gene expression under suboptimal conditions in subsurface environments .

What is known about rpsM conservation across different Geobacter species?

Ribosomal proteins are generally highly conserved across bacterial species due to their fundamental role in protein synthesis. While specific comparative studies of rpsM across different Geobacter species are not extensively documented, the protein likely shows high sequence conservation reflecting its essential function. G. sulfurreducens serves as a model organism for the Geobacter genus because it is closely related to Geobacter species that predominate in subsurface environments, can be cultured in laboratory conditions, and has an available genome sequence and genetic system . This makes comparative analysis of ribosomal proteins like rpsM more feasible. Any variations in rpsM sequence between different Geobacter species would be particularly interesting as they might reflect adaptations to specific environmental conditions or metabolic capabilities.

How does recombinant rpsM expression differ from native expression in G. sulfurreducens?

Recombinant expression of G. sulfurreducens rpsM typically involves heterologous systems such as E. coli, which presents several differences compared to native expression. In heterologous systems, rpsM may lack post-translational modifications that occur naturally in G. sulfurreducens. Additionally, codon usage differences between G. sulfurreducens and expression hosts can affect translation efficiency and protein folding. The reducing environment inside G. sulfurreducens cells may also differ from expression hosts, potentially affecting disulfide bond formation in the recombinant protein.

For optimal expression, researchers should consider codon optimization and the use of specific E. coli strains designed for expression of proteins from organisms with different GC content. Expression conditions should be carefully optimized, as demonstrated in studies with other bacterial proteins where temperature, induction parameters, and media composition significantly affected recombinant protein yield and solubility .

What role might rpsM play in stress response mechanisms in Geobacter sulfurreducens?

While direct evidence for rpsM involvement in stress response is limited, several lines of research suggest potential roles. G. sulfurreducens possesses sophisticated stress response mechanisms, as evidenced by studies on RpoS sigma factor, which contributes to survival in stationary phase and upon oxygen exposure . Ribosomal proteins like rpsM may participate in these responses through:

• Selective translation of stress-response transcripts

• Interactions with regulatory RNAs

• Extraribosomal functions during stress conditions

The RpoS sigma factor in G. sulfurreducens regulates genes specifically required for the reduction of insoluble Fe(III) oxide, the primary form of Fe(III) in most sedimentary environments . Changes in the pattern of c-type cytochromes have been observed in rpoS mutants, suggesting altered protein expression profiles during stress . As a component of the translation machinery, rpsM could be involved in modulating protein synthesis under these conditions, potentially participating in the regulated expression of stress-response proteins.

How can structural studies of rpsM contribute to understanding G. sulfurreducens adaptation to extreme environments?

Structural studies of rpsM can provide valuable insights into G. sulfurreducens adaptation mechanisms through several approaches:

• Comparative structural analysis with mesophilic homologs may reveal adaptations in protein stability

• Identification of unique structural features that facilitate function under stress conditions

• Analysis of interaction surfaces with rRNA and other ribosomal proteins

G. sulfurreducens demonstrates remarkable adaptability, including tolerance to long-term oxygen exposure despite being originally designated as a strict anaerobe . This adaptability may be reflected in the structure of key proteins including ribosomal components. For example, analysis of the rpsM structure might reveal modifications that enhance ribosome stability under oxidative stress conditions.

What interactions exist between rpsM and stress-response regulatory elements?

Potential interactions between rpsM and stress-response regulatory elements in G. sulfurreducens remain an important area for investigation. Research on other bacteria suggests that ribosomal proteins can interact with regulatory RNAs and proteins outside their primary ribosomal function. In G. sulfurreducens, stress responses are partly regulated by RpoS, which contributes to survival in stationary phase and upon oxygen exposure .

Investigations into potential interactions might include:

• Co-immunoprecipitation studies to identify protein-protein interactions between rpsM and stress-response regulators

• RNA-protein interaction studies to detect binding of rpsM to regulatory RNAs

• Transcriptomics and proteomics analyses of rpsM mutants under stress conditions

G. sulfurreducens contains additional regulatory systems beyond RpoS, including a sigma factor belonging to the family having extracytoplasmic functions (RpoE), which is likely involved in resistance to oxidative stress based on its role in other gram-negative bacteria . Understanding how rpsM interacts with these regulatory networks could provide insights into the integrated stress response mechanisms in G. sulfurreducens.

What are the optimal conditions for recombinant expression and purification of G. sulfurreducens rpsM?

Optimizing recombinant expression and purification of G. sulfurreducens rpsM requires careful consideration of several factors:

Expression System Selection:

• E. coli BL21(DE3) or Rosetta strains are recommended to address potential codon usage differences

• pET expression vectors with T7 promoter systems offer controllable induction

Expression Conditions:

• Lower temperatures (16-25°C) often improve protein solubility

• Induction at mid-log phase (OD600 of 0.6-0.8) using 0.1-0.5 mM IPTG

• Supplemented media (e.g., with trace metals) may improve yield

Purification Strategy:

• Cell lysis using buffer containing 50 mM HEPES instead of Tris to prevent interference with lysine residue acylation reactions

• Affinity chromatography using His-tag or alternative tags

• Size exclusion chromatography for final purification

• Consider ion-exchange chromatography if nucleic acid contamination occurs

The purification procedure should be adapted based on the unique characteristics of rpsM, including its potential to bind RNA. RNA-binding proteins often co-purify with cellular RNA, which may require additional purification steps such as high-salt washes or RNase treatment.

How can researchers effectively analyze interactions between rpsM and other cellular components?

To analyze interactions between G. sulfurreducens rpsM and other cellular components, researchers can employ several complementary approaches:

For Protein-Protein Interactions:

• Co-immunoprecipitation using antibodies against rpsM

• Bacterial two-hybrid systems

• Cross-linking mass spectrometry (XL-MS)

• Surface plasmon resonance (SPR) for in vitro interaction kinetics

For Protein-RNA Interactions:

• RNA immunoprecipitation (RIP)

• Electrophoretic mobility shift assays (EMSA)

• UV cross-linking studies

• CLIP-seq (cross-linking immunoprecipitation-sequencing)

For In Vivo Studies:

• Fluorescence microscopy using GFP-tagged rpsM

• Chromatin immunoprecipitation (ChIP) if extraribosomal DNA interactions are suspected

• Ribosome profiling to assess translation impacts

When conducting immunoprecipitation studies, researchers should develop specific antibodies against rpsM or use epitope tagging approaches. Western immunoblot analysis can be performed using standardized protocols where lanes contain 10 μg/ml of protein as determined by protein assays, similar to approaches used in other G. sulfurreducens studies .

What techniques are recommended for studying rpsM expression under different stress conditions?

To study rpsM expression under various stress conditions in G. sulfurreducens, researchers should consider the following techniques:

Transcriptomic Analysis:

• qRT-PCR using specific primers designed for rpsM

• RNA-Seq for genome-wide expression analysis

• Northern blotting for transcript size and stability analysis

For qRT-PCR analysis, researchers should follow validated protocols including:

• RNA isolation using TRIzol reagent followed by DNase treatment to remove contaminating genomic DNA

• cDNA conversion using high-capacity cDNA archive kits

• qRT-PCR using SYBR green with optimized thermocycling parameters (95°C for 15 min, followed by 40 cycles of 94°C for 15s, 60°C for 30s, and 72°C for 30s)

• Data transformation using the 2^(-ΔΔCT) method

Proteomic Analysis:

• Western blotting for specific protein detection

• Mass spectrometry-based proteomics

• Pulse-chase experiments to measure protein turnover rates

Stress Conditions to Test:

• Oxygen exposure (G. sulfurreducens can tolerate long-term oxygen exposure despite being originally designated as a strict anaerobe)

• Stationary phase (relevant to subsurface growth conditions)

• Metal limitation/excess

• Alternative electron acceptors (soluble vs. insoluble Fe(III))

• Temperature and pH stresses

When analyzing membrane-associated proteins, use isolation protocols similar to those employed in other G. sulfurreducens studies, with proteins resuspended in modified buffer Z containing 50 mM HEPES to prevent interference with the lysine residue acylation reaction .

How can rpsM mutants be used to study translational regulation in G. sulfurreducens?

rpsM mutants offer valuable tools for investigating translational regulation mechanisms in G. sulfurreducens:

Construction Approaches:

• Site-directed mutagenesis targeting functional domains

• Deletion mutants with complementation systems

• Tag insertion for tracking and purification

Applications:

• Investigating the impact on global protein synthesis rates

• Identifying differentially translated mRNAs under various conditions

• Examining effects on ribosome assembly and stability

• Studying translation fidelity and error rates

Researchers can construct G. sulfurreducens mutants using established genetic techniques. For example, the approach used for creating rpoS mutants in G. sulfurreducens provides a valuable template, where PCR-amplified fragments containing the gene of interest interrupted by an antibiotic resistance cassette are used for transformation . Complementation can be achieved by cointegration of plasmids carrying the entire gene .

Phenotypic analysis of rpsM mutants should examine:

• Growth rates under various conditions

• Stress tolerance profiles

• Metal reduction capabilities

• Proteome alterations using comparative proteomics

What potential does rpsM have as a target for understanding ribosome specialization in environmentally adaptive bacteria?

The rpsM protein presents significant potential for understanding ribosome specialization in environmentally adaptive bacteria like G. sulfurreducens:

Research Directions:

• Comparative structural analysis across bacteria from different environments

• Investigation of condition-specific ribosome heterogeneity

• Examination of specialized translation during stress responses

• Study of potential extraribosomal functions

G. sulfurreducens demonstrates remarkable environmental adaptability, including tolerance to oxygen exposure and the ability to reduce various metals and grow via reductive dehalogenation . This adaptability suggests potential specialization in translational machinery to support metabolic flexibility.

This research could reveal how ribosomal proteins like rpsM contribute to the remarkable metabolic versatility of G. sulfurreducens, which allows it to thrive in diverse subsurface environments and participate in bioremediation of various contaminants .

How can studies of rpsM contribute to understanding G. sulfurreducens' role in bioremediation applications?

Studies of rpsM can provide valuable insights into G. sulfurreducens' bioremediation capabilities through several research avenues:

Translational Control of Bioremediation Pathways:

• Investigating how rpsM contributes to the expression of key enzymes involved in metal reduction

• Examining translational efficiency of bioremediation-related genes under relevant environmental conditions

• Analyzing how stress responses affect the synthesis of proteins involved in contaminant metabolism

G. sulfurreducens has significant bioremediation potential due to its ability to oxidize aromatic contaminants with the reduction of Fe(III), reductively precipitate uranium, and grow via reductive dehalogenation . These capabilities suggest that they can contribute to the bioremediation of various contaminants in subsurface environments, including aromatic hydrocarbons and uranium .

Research focusing on rpsM could explore:

• How translational regulation affects expression of c-type cytochromes involved in electron transfer to Fe(III)

• Whether specialized ribosomes containing modified rpsM exist during growth on specific contaminants

• How translation efficiency under oxygen stress affects bioremediation capacity

Such studies would contribute to optimizing bioremediation strategies by understanding the fundamental translational mechanisms that support G. sulfurreducens' metabolic versatility in contaminated environments.