Recombinant Nitrosomonas europaea 30S ribosomal protein S4 B (rpsD2)

What is the functional significance of ribosomal protein S4 in Nitrosomonas europaea?

Ribosomal protein S4 in N. europaea has dual functionality. First, it serves as a key component in assembling the 30S ribosomal subunit by nucleating the process through association with the 5′ domain of the 16S rRNA . This initiator role is crucial for proper ribosome formation. Second, similar to its function in E. coli, N. europaea S4 likely acts as a transcriptional regulator through autogenous control mechanisms . Research indicates that S4 can function as a transcription factor with properties similar to NusA, facilitating antitermination during transcription, which has been demonstrated to increase terminator read-through by up to 11-fold in in vitro transcription reactions .

What expression systems are recommended for producing recombinant N. europaea rpsD2?

For expression of recombinant N. europaea S4 B (rpsD2), E. coli-based expression systems have been successfully utilized, as evidenced by commercially available recombinant proteins . When designing expression systems:

• Consider using pET-based vectors with T7 promoters for high-level expression

• Optimize codon usage for the host organism (particularly important when expressing N. europaea genes in E. coli)

• Include appropriate tags (His-tag is commonly used) for purification purposes

• Express at lower temperatures (16-20°C) to enhance proper folding and solubility

• Test multiple host strains including BL21(DE3), Rosetta, or Origami to optimize expression

For higher purity requirements (>85% as achieved with commercial preparations), a combination of affinity chromatography followed by size exclusion or ion exchange chromatography is recommended .

How can I assess the RNA-binding properties of recombinant N. europaea S4 B protein?

To evaluate the RNA-binding properties of recombinant N. europaea S4 B protein, several complementary approaches can be employed:

Methodological approach:

• Gel mobility shift assays: Incubate purified recombinant S4 B with labeled RNA substrates (16S rRNA fragments or putative mRNA targets) and analyze complex formation via non-denaturing PAGE. This approach has been successfully used to study S4-RNA interactions in related systems .

• Chemical probing techniques: SHAPE (Selective 2′-hydroxyl acylation analyzed by primer extension) can reveal structural changes in RNA upon S4 binding, as demonstrated in studies with bacterial S4 proteins .

• Surface plasmon resonance (SPR): Determine binding kinetics and affinity constants by immobilizing either the protein or RNA target on a sensor chip and measuring real-time interactions.

• Fluorescence-based assays: Use fluorescently labeled RNA substrates to monitor binding via changes in anisotropy or FRET.

Compare binding parameters with those of S4 A (rpsD1) to determine functional differences between the two proteins. Based on studies with other bacterial S4 proteins, expected KD values typically range from 10⁻⁷ to 10⁻⁹ M for specific RNA targets .

What role does N. europaea S4 protein play in stress response and adaptation?

Research on N. europaea has shown that ribosomal proteins, including S4, may play important roles in stress response and adaptation. Transcript profiling of N. europaea during growth and under nutrient deprivation revealed significant changes in expression patterns of ribosomal proteins .

When N. europaea cells were exposed to chronic stress conditions such as TiO₂ nanoparticle exposure, the expression of genes involved in ribosomal protein biogenesis was upregulated, including ribosomal protein genes such as rpsB, rpsD, rpsU, and others . This suggests that S4 protein may be part of a broader cellular response to environmental stressors.

The dual role of S4 as both a ribosomal structural component and potential transcriptional regulator (similar to findings in E. coli) positions it as a possible coordinator between stress sensing and protein synthesis regulation. This is particularly relevant in N. europaea given its specialized metabolism and environmental niche as an ammonia oxidizer.

How do mutations in rpsD2 affect N. europaea growth and ammonia oxidation capacity?

While specific studies on rpsD2 mutations in N. europaea are not directly referenced in the search results, research on ribosomal protein S4 in related systems provides insights:

How does the expression of rpsD2 in N. europaea change under different environmental conditions and what regulatory mechanisms control it?

Expression of ribosomal proteins in N. europaea, including rpsD genes, is significantly affected by environmental conditions:

Key findings from transcriptome studies:

Regulatory mechanisms controlling rpsD2 expression likely include:

• Autogenous regulation: Based on studies in B. subtilis, S4 protein can bind to the leader region of its own mRNA to regulate translation . In N. europaea, rpsD2 may be similarly regulated, with the protein binding to a pseudoknot-like structure in its mRNA.

• Response to energy status: As an ammonia oxidizer, N. europaea's metabolism and protein synthesis are tightly coupled to energy generation from ammonia. Under energy-limited conditions, expression of ribosomal proteins including rpsD2 is likely downregulated to conserve resources .

• Stress response pathways: During environmental stress, expression patterns shift as part of a coordinated cellular response, with certain ribosomal proteins being upregulated to potentially serve dual roles in stress resistance .

What are the specific interactions between N. europaea S4 B protein and the 16S rRNA, and how do they differ from those of other bacterial S4 proteins?

The specific interactions between N. europaea S4 B protein and 16S rRNA have not been directly characterized in the search results, but insights can be drawn from studies of related systems:

Key structural interactions likely include:

• 5′ domain binding: Similar to other bacterial S4 proteins, N. europaea S4 B likely interacts with the 5′ domain of 16S rRNA, particularly the five-way junction (5WJ) region .

• Pseudoknot stabilization: S4 proteins are known to stabilize a conserved pseudoknot in helix 18 (H18) that orients G530 for proper interactions with A-site tRNAs in the decoding center . This function is likely conserved in N. europaea S4 B.

• N-terminal extension: In E. coli, the N-terminal extension of S4 (approximately 40 residues) makes extensive sequence-specific interactions with 16S helix 16 . The N. europaea S4 sequence suggests a similar extension that might perform comparable functions.

Unique aspects of N. europaea S4 B interactions might include:

• The presence of two S4 proteins (A and B) in N. europaea suggests potential specialization in RNA binding and regulatory functions not found in organisms with a single S4 protein.

• Specific amino acid residues in the N. europaea S4 B sequence might create unique interaction patterns with its 16S rRNA, possibly reflecting adaptations to the organism's specialized metabolism or environmental niche.

• The globular domains of N. europaea S4 B likely contact helices H3, H4, H17, and H18 in the 16S rRNA, but with sequence variations that could affect binding affinities or structural rearrangements during ribosome assembly.

How can recombinant N. europaea S4 B protein be used to study ribosome assembly in vitro, and what insights might this provide about N. europaea translational regulation?

Using recombinant N. europaea S4 B protein to study ribosome assembly in vitro can provide valuable insights through the following methodological approaches:

Experimental design for in vitro ribosome assembly studies:

• Reconstitution experiments: Purify recombinant S4 B protein and 16S rRNA (or its 5′ domain) from N. europaea and monitor assembly kinetics using techniques such as:

  • Sucrose gradient sedimentation to track formation of ribonucleoprotein complexes

  • Time-resolved chemical probing to observe structural changes in RNA during assembly

  • Fluorescence resonance energy transfer (FRET) to monitor real-time conformational changes

• Comparative assembly studies: Compare assembly properties of S4 A versus S4 B with the same rRNA substrate to determine functional differences between the two proteins.

• Temperature-dependent assembly: Based on studies showing structural differences between S4-16S complexes formed at different temperatures (0°C vs. 42°C) , investigate temperature effects on N. europaea ribosome assembly, particularly relevant given its environmental adaptations.

Potential insights into translational regulation:

• Dual roles: Studies on E. coli S4 showed it functions both in ribosome assembly and as a transcription antitermination factor . Similar experiments with N. europaea S4 B could reveal if it also possesses dual functionality.

• Regulatory networks: In E. coli, S4 regulates translation of the α-operon and can act as a general transcription anti-terminator . Investigation of N. europaea S4 B binding to potential regulatory RNA targets could uncover organism-specific regulatory networks.

• Stress adaptation mechanisms: Given N. europaea's specialized lifestyle and the observed changes in ribosomal protein expression under stress conditions , studying how S4 B functions under different environmental conditions could reveal specialized translational control mechanisms.

What techniques are most effective for detecting interactions between N. europaea S4 B protein and RNA polymerase, and how might these interactions affect transcription regulation?

Based on findings that E. coli S4 protein can interact with RNA polymerase and function as a transcription antitermination factor , similar interactions might exist for N. europaea S4 B. The following techniques would be most effective for detecting and characterizing such interactions:

Methodological approaches:

• Co-immunoprecipitation (Co-IP): Using antibodies against either S4 B or RNA polymerase subunits to pull down protein complexes from N. europaea cell lysates. The presence of interaction partners can be confirmed by western blotting or mass spectrometry.

• His-tag affinity purification: Similar to the approach used with E. coli , express His-tagged RNA polymerase in N. europaea and use nickel beads to isolate the polymerase along with bound proteins, then detect S4 B by western blotting.

• Surface plasmon resonance (SPR): Immobilize purified RNA polymerase on a sensor chip and measure binding of recombinant S4 B protein to determine binding kinetics and affinity constants.

• Bacterial two-hybrid assays: Adapt two-hybrid systems to detect protein-protein interactions between S4 B and RNA polymerase subunits in vivo.

• In vitro transcription assays: Assess the effect of purified S4 B protein on transcription termination using templates with known terminators, similar to studies showing S4 from E. coli increased terminator read-through by 11-fold .

Potential regulatory significance:

• Antitermination activity: If N. europaea S4 B shows antitermination activity similar to E. coli S4, it could regulate expression of specific genes by promoting read-through at Rho-dependent terminators.

• Metabolic coordination: Such interactions might coordinate ribosome assembly with transcription rates, helping N. europaea adjust to changing environmental conditions.

• Competition with other factors: In E. coli, high concentrations of NusG showed reduced antitermination by S4 . Similar interactions in N. europaea could reveal regulatory networks specific to its specialized metabolism.

What are the main challenges in purifying active recombinant N. europaea S4 B protein, and how can they be overcome?

Purifying active recombinant N. europaea S4 B protein presents several challenges that researchers should anticipate:

Common challenges and solutions:

Based on commercial product information, achieving >85% purity by SDS-PAGE is a realistic target for recombinant N. europaea S4 B protein .

How can I design experiments to distinguish between the functions of S4 A (rpsD1) and S4 B (rpsD2) in N. europaea?

To differentiate between the functions of the two S4 proteins in N. europaea, a multi-faceted experimental approach is recommended:

Experimental design strategies:

• Genetic manipulation approaches:

  • Create knockout mutants of either rpsD1 or rpsD2 in N. europaea using CRISPR-Cas9 or traditional homologous recombination techniques

  • Develop complementation strains expressing either protein to rescue phenotypes

  • Use gene tagging to create fusion proteins for localization studies

• Expression analysis:

  • Perform qRT-PCR to measure differential expression of rpsD1 and rpsD2 under various growth conditions

  • Use ribosome profiling to determine association of each protein with actively translating ribosomes

  • Conduct proteomics analysis to determine relative abundance of each protein

• Functional assays:

  • Compare binding affinities of each purified protein to 16S rRNA using gel shift assays

  • Assess effects on in vitro transcription termination, similar to studies with E. coli S4

  • Analyze impacts on ribosome assembly and translation fidelity

• Structural studies:

  • Perform comparative structural analysis through X-ray crystallography or cryo-EM

  • Use hydrogen-deuterium exchange mass spectrometry to identify differential RNA binding regions

Expected outcomes:
Given that E. coli S4 has dual roles in ribosome assembly and transcription regulation , one hypothesis is that N. europaea has evolved specialized versions of S4, with one protein (perhaps S4 A) primarily involved in ribosome assembly and the other (S4 B) more specialized for regulatory functions or adaptation to specific environmental conditions.

How should recombinant N. europaea S4 B protein storage conditions be optimized for maximum stability and activity retention?

Optimal storage conditions for recombinant N. europaea S4 B protein should be carefully determined to maintain stability and functional activity:

Storage recommendations based on empirical data:

• Buffer composition:

  • pH 7.4-8.0 phosphate or Tris buffer

  • 100-150 mM NaCl to maintain ionic strength

  • 1-5 mM DTT or 2-5 mM β-mercaptoethanol as reducing agents

  • Optional: 0.1 mM EDTA to chelate metal ions that could promote oxidation

• Cryoprotectants:

  • Add 50% glycerol for storage at -20°C or -80°C

  • Alternative: 5-10% sucrose or trehalose for lyophilization

• Concentration considerations:

  • Optimal concentration range: 0.1-1.0 mg/mL

  • For long-term storage, higher concentrations (1-5 mg/mL) may improve stability

• Storage temperature:

  • Working aliquots: 4°C for up to one week

  • Medium-term storage: -20°C (with 50% glycerol)

  • Long-term storage: -80°C or lyophilized

• Handling protocols:

  • Avoid repeated freeze-thaw cycles; prepare single-use aliquots

  • Centrifuge briefly before opening tubes to collect any condensate

  • When thawing, keep on ice and use immediately for critical applications

Stability testing protocol:
To verify retained activity after storage, periodic testing should include:

• SDS-PAGE to check for degradation

• RNA binding assays using gel shift with 16S rRNA fragments

• When applicable, functional tests for antitermination activity in in vitro transcription assays

The shelf life for liquid preparations is typically 6 months at -20°C/-80°C, while lyophilized preparations can remain stable for up to 12 months at -20°C/-80°C .

What potential roles might N. europaea S4 B protein play in environmental adaptation and bioremediation applications?

N. europaea's unique metabolic capabilities make it valuable for environmental applications, and understanding S4 B protein's role could enhance these applications:

Potential environmental adaptation roles:

• Stress response coordination: Given that ribosomal proteins show altered expression during stress responses , S4 B may contribute to N. europaea's adaptation to environmental stressors such as pollutants, pH changes, or temperature fluctuations.

• Metabolic regulation: As a potential transcription factor (based on E. coli S4 properties) , S4 B could regulate genes involved in ammonia oxidation or detoxification pathways.

• Energy conservation: During nutrient limitation, S4 B might contribute to regulating translation efficiency, helping N. europaea survive periods of scarcity.

Bioremediation applications:

• Enhanced pollutant degradation: N. europaea can degrade various halogenated organic compounds including trichloroethylene, benzene, and vinyl chloride . Understanding and potentially modifying S4 B's regulatory functions could enhance expression of degradative enzymes.

• Wastewater treatment optimization: As key players in biological nitrogen removal processes , engineered strains with modified S4 B could potentially improve nitrification efficiency in wastewater treatment plants.

• Biosensor development: Knowledge of S4 B's role in stress responses could inform development of whole-cell biosensors using N. europaea to detect environmental contaminants.

• Co-culture systems: Understanding S4 B's role in N. europaea metabolism could improve design of co-culture systems like those with Paracoccus denitrificans for complete nitrogen removal , potentially through engineering strains with altered S4 B function.

How might comparative analysis of S4 proteins across different ammonia-oxidizing bacteria inform our understanding of the evolution of specialized ribosomal protein functions?

Comparative analysis of S4 proteins across ammonia-oxidizing bacteria (AOB) could reveal evolutionary patterns and functional specializations:

Evolutionary insights from comparative analysis:

• Duplication and divergence patterns: The presence of two S4 proteins (A and B) in N. europaea suggests gene duplication followed by functional divergence. Determining whether this pattern exists across other AOB would reveal whether this is a common adaptive strategy.

• Selection pressures: Analyzing selective pressures on S4 sequences across AOB could identify:

  • Conserved regions critical for core ribosomal function

  • Variable regions that might confer specialized regulatory capabilities

  • Correlation between S4 sequence variation and environmental niches of different AOB

• Domain architecture evolution: Comparing the N-terminal extensions and globular domains of S4 proteins across AOB could reveal adaptations specific to ammonia oxidation or particular environmental conditions.

• Structural adaptations: Crystal structures or models of S4 proteins from different AOB could reveal structural adaptations that correlate with functional specialization.

Functional significance:

• Translational regulation: Variations in S4 sequences might reflect differences in translational regulation mechanisms across AOB with different metabolic capabilities or environmental niches.

• Transcriptional control: If the transcription antitermination function observed in E. coli S4 is present in some AOB but not others, this could reveal evolutionary patterns in regulatory network development.

• Environmental adaptation: Correlation of S4 sequence features with the preferred habitats of different AOB (pH ranges, temperature optima, salt tolerance) could identify adaptations of the translation machinery to specific environmental conditions.

This comparative approach would extend beyond the current focus on model organisms like E. coli and B. subtilis to understand ribosomal protein evolution in environmentally and biotechnologically important bacterial groups.

What potential applications exist for recombinant N. europaea S4 B protein in synthetic biology and protein engineering?

Recombinant N. europaea S4 B protein offers several innovative applications in synthetic biology and protein engineering:

Synthetic biology applications:

• Translational control modules: Engineering S4 B binding sites into synthetic mRNAs could create translational switches responsive to S4 B levels, potentially useful for creating genetic circuits in bacteria.

• Transcriptional antitermination tools: If N. europaea S4 B demonstrates antitermination activity similar to E. coli S4 , it could be engineered as a tool to regulate transcription termination in synthetic gene networks.

• Environmental sensing circuits: S4 B could potentially be incorporated into synthetic circuits designed to respond to environmental conditions relevant to nitrogen cycling or bioremediation.

Protein engineering approaches:

• RNA-binding domain engineering: The RNA-binding domains of S4 B could be isolated and modified to create novel RNA-binding proteins with altered specificity.

• Fusion proteins for targeted ribosome engineering: S4 B domains could be fused with other functional domains to create chimeric proteins that modify ribosome function in predictable ways.

• Structural stability enhancements: Understanding the structural features that allow S4 B to function in N. europaea's specialized metabolism could inform the design of proteins with enhanced stability under similar conditions.

Biotechnological implications:

• Improved expression systems: Insights from N. europaea S4 B structure and function could inform the design of expression systems specifically optimized for proteins involved in nitrogen cycling or pollutant degradation.

• Biosensor components: Engineered variants of S4 B with altered RNA binding properties could serve as components in biosensors for environmental monitoring applications.

• Targeted protein evolution: Directed evolution approaches applied to S4 B could yield variants with enhanced properties for specific biotechnological applications in nitrogen removal or bioremediation contexts.