Recombinant Methanococcus maripaludis 30S ribosomal protein S9P (rps9p)

What is Methanococcus maripaludis and why is it important as a model organism?

Methanococcus maripaludis is a rapidly growing, fully sequenced, genetically tractable model organism among hydrogenotrophic methanogens. It belongs to the domain Archaea and has the ability to convert CO₂ and H₂ into methane (CH₄). The organism has become a workhorse similar to Escherichia coli and S. cerevisiae for fundamental and experimental biotechnology studies due to its unique metabolic capabilities, fully sequenced genome, and availability of genetic tools . It is non-pathogenic, gram-negative, weakly motile, non-spore-forming, and a strictly anaerobic mesophile with a pleomorphic coccoid-rod shape averaging 1.2 by 1.6 μm in size .

What is the 30S ribosomal protein S9P and what is its role in M. maripaludis?

The 30S ribosomal protein S9P (rps9p), encoded by the MMP1325 gene in M. maripaludis S2, is a component of the small ribosomal subunit that participates in the translation process. It is part of a larger ribosomal gene neighborhood that includes other ribosomal proteins and RNA polymerase subunits . The gene is located in close proximity to other ribosomal components and translation machinery genes, suggesting coordinated expression and function:

What is the genomic context of the rps9p gene in M. maripaludis?

The rps9p gene (MMP1325) in M. maripaludis S2 is located within a ribosomal protein and RNA polymerase gene cluster. According to module neighborhood information, MMP1325 has genetic associations with 13 gene neighbors in modules 103 and 118 . This genomic organization suggests coordinated regulation of translation and transcription machinery, as these genes encode components of both the ribosome and RNA polymerase.

What are the optimal expression systems for producing recombinant M. maripaludis rps9p?

Based on methodologies used for other M. maripaludis proteins, recombinant rps9p can be effectively expressed using several systems:

• Yeast expression system: This approach has been successfully used for other M. maripaludis ribosomal proteins such as 30S ribosomal protein S2P .

• Baculovirus expression: This system has been reported for expression of M. maripaludis ribosomal proteins and offers advantages for archaeal proteins that may require specific folding conditions .

• E. coli-based expression: While not specifically mentioned for rps9p, this is a common approach for archaeal proteins when combined with appropriate optimization of codon usage and growth conditions.

What purification strategies are most effective for recombinant M. maripaludis rps9p?

For purification of recombinant archaeal ribosomal proteins like rps9p, the following strategies have been shown to be effective:

• Affinity chromatography: Using tags like His-tags for initial capture, though for functional studies, it's recommended to express the protein without additional tags to minimize interference with the native structure and function .

• Size exclusion chromatography: For further purification after initial capture.

• Ion exchange chromatography: Particularly useful for separating ribosomal proteins from nucleic acids that may co-purify.

For optimal results, a purity target of >85% (as assessed by SDS-PAGE) should be considered baseline for functional studies, consistent with standards for other M. maripaludis ribosomal proteins .

How should recombinant M. maripaludis rps9p be stored for maximum stability?

Based on storage recommendations for other archaeal ribosomal proteins:

• Short-term storage: Aliquots can be stored at 4°C for up to one week .

• Long-term storage:

  • Liquid form: 6 months at -20°C/-80°C

  • Lyophilized form: 12 months at -20°C/-80°C

• Reconstitution protocol:

  • 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 can researchers assess the functional activity of recombinant M. maripaludis rps9p?

Functional characterization of recombinant rps9p can be performed using:

• In vitro translation assays: Comparing translation efficiency with and without the recombinant protein.

• Ribosome assembly assays: Monitoring incorporation into partial or complete ribosomal assemblies.

• RNA binding studies: Assessing interaction with ribosomal RNA components using techniques such as filter binding assays or electrophoretic mobility shift assays.

• Structure-function relationship studies: Using site-directed mutagenesis to identify critical residues for function, similar to the approach used for studying methyl coenzyme M reductase in M. maripaludis .

What methods can be used to study the interaction of rps9p with other ribosomal components?

To investigate interactions between rps9p and other ribosomal components, researchers can employ:

• Co-immunoprecipitation: To identify protein-protein interactions within the ribosomal complex.

• Cross-linking coupled with mass spectrometry: Similar to techniques used to identify protein complexes in M. maripaludis, such as the approach used for studying the MCM complex .

• Cryo-electron microscopy: For structural characterization of the intact ribosome with its components.

• Proteomics approaches: Similar to the shotgun proteomic studies that have been used to detect peptides from multiple MCM proteins in M. maripaludis .

What genetic manipulation techniques are available for studying rps9p in M. maripaludis?

M. maripaludis has a well-developed genetic toolbox that can be applied to studying rps9p:

• Markerless mutagenesis: This technique allows for clean genetic manipulations without leaving selection markers in the genome .

• CRISPR-Cas9 system: Two distinct CRISPR-mediated genome editing systems have been established in M. maripaludis , which can be used for precise genetic manipulation of rps9p.

• Plasmid invader assay: While initially developed for testing CRISPR functionality, this approach can be adapted to study rps9p function through controlled expression systems .

• Shuttle vectors: Various shuttle vectors with improved transformation efficiency (e.g., pAW42) can be used for complementation studies .

The transformation efficiency of these systems has been significantly improved through optimization, with up to 7,000-fold increases in transformation efficiency for pURB500-based vectors in strain S0001 .

How can researchers create and validate rps9p knockout or modification strains?

To create and validate modifications to the rps9p gene in M. maripaludis:

• In-frame deletion strategy:

  • Design constructs targeting the rps9p gene with appropriate flanking regions

  • Transform using validated vectors for M. maripaludis

  • Confirm deletions using PCR with primers designed to give size-differentiated products in wild-type versus mutant cells

  • Verify by Southern blotting using DIG-labeled probes predicted to hybridize to size-shifted fragments

• Complementation strategy:

  • Clone the rps9p gene into vectors like pWLG40 under the control of a constitutive promoter (such as the hmv promoter)

  • Express the protein in its native form without additional tags to minimize interference with assembly

  • Verify restoration of function through phenotypic assays

• Site-directed mutagenesis:

  • Introduce specific mutations to study structure-function relationships

  • Use complementation to assess the impact of specific amino acid changes

What role does rps9p play in codon usage bias and translational selection in M. maripaludis?

M. maripaludis exhibits specific patterns of codon usage bias that affect translational efficiency and accuracy. Research on translational selection in M. maripaludis has revealed:

• Selected codon usage bias patterns:

  • Stronger bias observed across two-fold degenerate amino acid groups compared to four-fold degenerate groups

  • This pattern is consistent across Archaea and may reflect ancestral patterns of optimal codon divergence

• Strength of selected codon usage bias:

  • Can be estimated through comparison of codon frequencies between highly expressed genes and genome-wide averages

  • Selection parameter S (2Nes) can be calculated to quantify the strength of selection on codon usage

• Accuracy-selected codon usage bias:

  • Higher levels of selected codon usage bias at conserved amino acid positions

  • Plays a role in minimizing mistranslation at functionally important sites

As a component of the 30S ribosomal subunit, rps9p likely influences translation accuracy and efficiency, particularly at these conserved sites.

How does post-translational modification affect rps9p function in M. maripaludis?

While specific post-translational modifications (PTMs) of rps9p in M. maripaludis have not been directly characterized in the search results, research on other M. maripaludis proteins provides a framework for investigation:

• PTM impact on function: PTMs can significantly impact protein function, as demonstrated by the methylation of arginine in methyl coenzyme M reductase, which profoundly affects both methanogenesis and growth .

• Methylation patterns: M. maripaludis proteins can undergo various methylation events with functional consequences. For example, the deletion of mmpX (MMP1554) resulted in complete loss of 5-C-(S)-methylarginine PTM of residue 275 in the McrA subunit .

• Motif recognition: Specific sequence motifs can be recognized by modification enzymes, such as the PXRR275(A/S)R(G/A) signature sequence in McrAs that is targeted for methylation . Identifying similar motifs in rps9p could suggest potential modification sites.

What is the evolutionary significance of archaeal rps9p compared to bacterial and eukaryotic homologs?

The evolutionary relationship between archaeal ribosomal proteins and their bacterial and eukaryotic counterparts provides insights into the evolution of translation machinery:

• Archaea-Eukarya connection: Many archaeal genes encoding fundamental processes in replication, transcription, and translation are homologous to eukaryotic genes, providing direct evidence for a close relationship between Archaea and Eukaryotes in information processing systems .

• Essential gene classification: A genome-wide survey of gene functionality in M. maripaludis classified about 30% of the genome as possibly essential or strongly advantageous for growth in rich medium, including many information processing genes .

• Domain-specific variation: Some genes classified as essential in M. maripaludis are unique to the archaeal or methanococcal lineages, suggesting domain-specific adaptations in translation machinery .

How can researchers overcome challenges in expressing functional archaeal ribosomal proteins in heterologous systems?

Challenges in heterologous expression of archaeal ribosomal proteins can be addressed through:

• Codon optimization: M. maripaludis exhibits specific codon usage patterns that differ from common expression hosts. The strength of selected codon usage bias varies across different amino acid groups , suggesting that codon optimization should take these patterns into account.

• Growth conditions: For optimal expression of archaeal proteins:

  • Temperature: Consider using lower temperatures (e.g., 30°C) to slow protein production and improve folding

  • Induction parameters: Use lower inducer concentrations and longer expression times

  • Media composition: Supplement with rare amino acids or tRNAs that may be limiting in the expression host

• Folding assistance: Co-express with archaeal chaperones or use archaeal-derived in vitro translation systems for improved folding.

What are the challenges in studying ribosomal assembly in M. maripaludis and how can they be overcome?

Studying ribosomal assembly in M. maripaludis presents unique challenges:

• Anaerobic conditions: As a strictly anaerobic organism, M. maripaludis requires special handling. Researchers should:

  • Prepare media in anaerobic chambers (95% N₂-5% H₂)

  • Reduce media prior to autoclaving with Na₂S·9H₂O and L-cysteine-HCl

  • Maintain H₂-CO₂ (80:20) at a pressure of 2×10⁵ Pa as the gas phase

• Scale-up challenges: Methods for large-scale cultivation have been developed:

  • Progress from 0.05 L serum bottles to 0.4 L Schott bottle cultures for regular biomass generation

  • Further scale up to 1.5 L reactor cultures with optimized agitation and harvesting methods

  • A 300-fold scale-up from serum bottles to 22 L stainless steel bioreactors has been achieved

• Ribosome isolation: After growth, cells can be harvested and processed by:

  • Suspending frozen cells in potassium phosphate buffer (pH 7.0)

  • Passing through a French pressure cell at 10⁸ Pa

  • Removing cell debris and membrane fractions by ultracentrifugation

How can proteomics approaches be used to study rps9p expression and incorporation into ribosomes?

Proteomics approaches offer powerful tools for studying rps9p expression and incorporation:

• Shotgun proteomics: This approach has been used to detect peptides from multiple proteins in M. maripaludis , and can be applied to monitor rps9p expression levels under different conditions.

• Quantitative proteomics: Using techniques such as SILAC (Stable Isotope Labeling with Amino acids in Cell culture) or TMT (Tandem Mass Tag) labeling to quantify changes in rps9p abundance under different conditions.

• Protein-protein interaction networks: Network Portal tools have been developed for M. maripaludis that show gene module memberships and functional annotations . For rps9p (MMP1325), these reveal connections to modules 103 and 118, indicating its position within larger functional networks.

What transcriptomic approaches can reveal about rps9p regulation in different growth conditions?

Transcriptomic approaches can provide insights into rps9p regulation:

• Differential expression analysis: Comparing rps9p transcript levels under different growth conditions, such as varying selenium availability which has been shown to affect global gene expression in M. maripaludis .

• Motif identification: Computational approaches have identified DNA motifs that may regulate gene expression in M. maripaludis. For the gene neighborhood containing rps9p, motifs 863 (GGaGGC) and 864 (GcGCcgagggG) have been predicted , which may play a role in coordinating expression of ribosomal components.

• Co-expression analysis: Identifying genes with similar expression patterns to rps9p can reveal functional relationships and regulatory mechanisms. The close genomic proximity of rps9p to RNA polymerase subunits suggests coordinated regulation of transcription and translation machinery .

What are the potential applications of rps9p in archaeal synthetic biology?

M. maripaludis has emerging potential as a synthetic biology platform, with specific applications for ribosomal proteins like rps9p:

• Engineered translation systems: Modifications to ribosomal proteins could create specialized ribosomes for incorporating non-standard amino acids or operating under extreme conditions.

• Biosensor development: The incorporation of modified ribosomal proteins could serve as the basis for biosensors that respond to specific environmental conditions.

• Metabolic engineering applications: M. maripaludis has already been metabolically engineered as a cell factory for high-value products such as geraniol and the bioplastic polymer polyhydroxybutyrate . Understanding and optimizing translation through ribosomal protein engineering could enhance these applications.

How might structural biology approaches advance our understanding of archaeal ribosomal proteins?

Structural biology approaches offer significant potential for advancing our understanding of archaeal ribosomal proteins:

• Cryo-electron microscopy: This technique could provide high-resolution structures of the intact M. maripaludis ribosome, revealing the precise position and interactions of rps9p within the complex.

• X-ray crystallography: Determining the structure of isolated rps9p could reveal specific features that distinguish it from bacterial and eukaryotic homologs.

• Integrative structural biology: Combining multiple techniques (cryo-EM, X-ray crystallography, NMR, mass spectrometry) could provide comprehensive insights into both structure and dynamics of ribosomal assemblies.

• Structure-guided functional studies: Structural information could guide the design of targeted mutations to probe specific functional hypotheses about rps9p's role in translation.