Recombinant Nitrosomonas europaea 50S ribosomal protein L1 (rplA)

What is the role of the rplA gene in Nitrosomonas europaea compared to other bacteria?

The rplA gene in Nitrosomonas europaea encodes the 50S ribosomal protein L1, which is a crucial component of the bacterial ribosome. This protein participates in the assembly of the large ribosomal subunit and is involved in protein synthesis. In the Nitrosomonas europaea genome, which consists of a single circular chromosome of 2,812,094 bp, rplA is one of the 2,460 protein-encoding genes identified .

Research has demonstrated that mutations in rplA result in marked attenuation of virulence in certain bacterial species, with an LD50 of >10³ CFU . While N. europaea is not pathogenic, the conservation of rplA across bacterial species suggests its fundamental importance for bacterial growth and metabolism.

The comparison of ribosomal proteins across different bacterial species can provide insights into evolutionary relationships. The table below compares key characteristics of rplA in different bacterial species:

What expression systems are most effective for producing recombinant Nitrosomonas europaea rplA?

For expressing recombinant N. europaea rplA, several expression systems can be considered, with Escherichia coli being the most commonly used for bacterial proteins. Based on recombinant protein expression studies, the following systems have proven effective:

• E. coli expression system: Most suitable for bacterial ribosomal proteins due to similar codon usage and post-translational modifications. Typically, strains like BL21(DE3) or Rosetta are used with pET vector systems to control expression levels .

• Yeast expression systems: These can be employed when proper folding is challenging in prokaryotic systems. Both Pichia pastoris and Saccharomyces cerevisiae have been used for expressing bacterial proteins .

• Baculovirus/insect cell systems: These may be considered for complex bacterial proteins that require specific folding environments .

The methodological approach should include:

• Codon optimization for the selected expression system

• Inclusion of appropriate affinity tags (His-tag or GST-tag) for purification

• Design of suitable promoters (IPTG-inducible for E. coli)

• Optimization of growth conditions using Design of Experiments (DoE) approach

What are the optimal conditions for expressing soluble recombinant Nitrosomonas europaea rplA in E. coli?

Optimization of expression conditions is crucial for obtaining soluble recombinant N. europaea rplA. Based on similar recombinant protein expression studies, the following conditions are recommended:

Growth and induction parameters:

• Cell density at induction: OD600 of 0.8

• IPTG concentration: 0.1 mM

• Post-induction temperature: 25°C

• Induction time: 4 hours

• Medium composition: 5 g/L yeast extract, 5 g/L tryptone, 10 g/L NaCl, 1 g/L glucose

• Antibiotic: 30 μg/mL kanamycin

These conditions have been validated to produce high levels (approximately 250 mg/L) of soluble recombinant protein with appropriate functional characteristics . For N. europaea rplA specifically, lower temperatures during induction (16-25°C) are likely beneficial to enhance proper folding.

Experimental design methodology is highly recommended for optimization, as demonstrated in recombinant protein expression studies where a 2^8-4 factorial design was applied to evaluate eight variables related to medium composition and induction conditions .

How can Design of Experiments (DoE) methodology be applied to optimize the expression and purification of recombinant Nitrosomonas europaea rplA?

Design of Experiments (DoE) is a powerful approach for optimizing recombinant protein expression, particularly for challenging proteins like rplA from N. europaea. A systematic DoE methodology includes:

• Factor Identification: For N. europaea rplA expression, critical factors include:

  • Temperature (16-37°C)

  • IPTG concentration (0.1-1.0 mM)

  • Cell density at induction (OD600 0.4-1.0)

  • Post-induction time (2-16 hours)

  • Media composition (minimal vs. rich)

  • pH (6.8-8.0)

  • Oxygen levels

  • Additives (glycerol, glucose, etc.)

• Experimental Design: A fractional factorial design (e.g., 2^8-4) can be employed to screen these factors with a minimal number of experiments .

• Response Measurement: Key responses include:

  • Protein yield (mg/L)

  • Solubility percentage

  • Functional activity (if applicable)

• Statistical Analysis: Analysis of variance (ANOVA) to determine significant factors and interactions.

• Optimization: Response surface methodology (RSM) to fine-tune the significant factors identified in screening.

This approach has been successfully used for other recombinant proteins, resulting in up to 75% homogeneity and retention of functional characteristics .

What structural characteristics of Nitrosomonas europaea rplA contribute to ribosome assembly, and how can they be studied?

The structural characteristics of N. europaea rplA that contribute to ribosome assembly can be studied using various methodological approaches:

• Sequence Analysis and Structural Prediction:

  • Comparative sequence analysis with other bacterial L1 proteins

  • Secondary structure prediction using bioinformatic tools

  • Identification of RNA-binding domains and protein-protein interaction sites

• Experimental Structure Determination:

  • X-ray crystallography of purified recombinant rplA

  • Cryo-electron microscopy of ribosomal assemblies containing rplA

  • NMR spectroscopy for dynamic regions

• Functional Characterization:

  • RNA binding assays to examine interaction with 23S rRNA

  • Ribosome assembly assays in vitro

  • Site-directed mutagenesis of conserved residues

The L1 protein typically contains an N-terminal domain involved in binding to 23S rRNA and a C-terminal domain involved in interactions with other ribosomal proteins. Specific conserved residues are crucial for these interactions and can be identified through comparative genomic analysis.

How do different growth conditions affect the expression levels of native rplA in Nitrosomonas europaea, and how can this information improve recombinant production?

The expression of native rplA in N. europaea is influenced by various growth conditions, which can provide insights for optimizing recombinant production. Studies on N. europaea transcriptomics reveal:

• Oxygen Limitation Effects:
  Transcriptomic studies of N. europaea under oxygen-limited conditions showed differential expression of various genes . While specific data on rplA expression is limited, ribosomal proteins generally show altered expression under stress conditions.

• Ammonia Availability Impact:
  As an ammonia-oxidizing bacterium, N. europaea's metabolism is significantly affected by ammonia availability. Under ammonia-limited conditions, N. europaea consumed NH₃ at a rate (qNH₃) of 24.73 ± 0.53 mmol g (dry cell weight)⁻¹ h⁻¹ with an apparent growth yield (Y) of 0.40 ± 0.01 g (dry cell weight) mol⁻¹ NH₃ . These conditions likely influence ribosomal protein expression.

• Biofilm vs. Planktonic Growth:
  N. europaea forms biofilms differently in single-species cultures versus dual-species cultures with P. aeruginosa . In single-species biofilms, N. europaea formed thin, dispersed layers, while in dual-species biofilms, it developed much greater biovolume (2.78 μm³/μm² at day 3) . These growth modes may differentially affect ribosomal protein expression.

Understanding these native expression patterns can inform recombinant production strategies by:

• Selecting growth conditions that naturally upregulate ribosomal proteins

• Designing expression constructs with promoters responsive to specific environmental cues

• Engineering host strains to mimic favorable conditions for ribosomal protein expression

What methods can be used to verify the structural integrity and functionality of purified recombinant Nitrosomonas europaea rplA?

Verifying the structural integrity and functionality of purified recombinant N. europaea rplA requires multiple analytical approaches:

• Structural Integrity Assessment:

  • Circular Dichroism (CD) spectroscopy to analyze secondary structure composition

  • Differential Scanning Calorimetry (DSC) to assess thermal stability

  • Size-Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS) to determine oligomeric state and homogeneity

  • Limited proteolysis to probe folding and domain structure

• Functional Characterization:

  • In vitro binding assays with 23S rRNA fragments

  • Ribosome reconstitution assays to test incorporation into 50S subunits

  • Poly(U)-directed poly(Phe) synthesis to assess contribution to translation

  • Sucrose gradient ultracentrifugation to assess incorporation into ribosomal subunits

• Molecular Dynamics Simulations:

  • Computational analysis of protein stability

  • Prediction of RNA-binding interfaces

  • Simulation of interactions with other ribosomal components

Each of these methods contributes complementary information about the protein's structure and function, allowing for comprehensive characterization of the recombinant product.

How can activity-based protein profiling be adapted to study the interactions of recombinant Nitrosomonas europaea rplA within the ribosomal complex?

Activity-based protein profiling (ABPP) methodologies, similar to those used for studying ammonia monooxygenase in N. europaea , can be adapted to investigate rplA interactions:

• Probe Design for rplA Interactions:

  • Synthesis of RNA oligonucleotides corresponding to the rplA binding site on 23S rRNA

  • Incorporation of reactive groups (photoactivatable crosslinkers) and reporter tags (biotin, fluorophores)

  • Design of protein-based probes representing interaction partners within the ribosome

• Crosslinking Methodology:

  • In vitro reconstitution of ribosomal subunits with recombinant rplA

  • UV-induced crosslinking of interacting components

  • Chemical crosslinking using bifunctional reagents

• Interaction Analysis:

  • Mass spectrometry identification of crosslinked peptides

  • Competition assays with unlabeled rplA to validate specificity

  • Mutational analysis of interaction interfaces

• Visualization Techniques:

  • Fluorescence microscopy to track labeled rplA localization

  • In-gel detection of crosslinked complexes

  • Structural analysis of crosslinked complexes by cryo-EM

This approach would provide detailed insights into the dynamic interactions of rplA within the ribosomal complex, potentially revealing novel aspects of ribosome assembly and function in N. europaea.

What impact does rplA mutation have on Nitrosomonas europaea metabolism and ammonia oxidation, and how can this be studied with recombinant proteins?

The impact of rplA mutation on N. europaea metabolism can be studied using a combination of genomic, proteomic, and biochemical approaches:

• Genetic Modification Strategies:

  • Creation of rplA knockout or knockdown strains

  • Point mutations in conserved residues

  • Complementation with recombinant wild-type or mutant rplA

• Phenotypic Characterization:

  • Growth rate analysis under various conditions

  • Ammonia oxidation rates measured by NO₂⁻ production

  • Oxygen consumption rates

  • Cell morphology and ultrastructure analysis

• Metabolic Impact Assessment:

  • Transcriptomic analysis to identify differentially expressed genes

  • Proteomic analysis focusing on ammonia oxidation enzymes (AMO, HAO)

  • Metabolomic profiling

• Recombinant Protein Applications:

  • In vitro translation assays comparing wild-type and mutant rplA

  • Structure-function studies of mutant rplA variants

  • Protein-protein interaction studies with other ribosomal components

Based on studies of N. europaea's metabolic pathways, key processes to monitor would include:

• Ammonia oxidation by ammonia monooxygenase (AMO)

• Hydroxylamine oxidation by hydroxylamine dehydrogenase (HAO)

• Expression of nitrite reductase (NirK) and nitric oxide reductase (NorB)

• Carbon fixation pathways

How can recombinant Nitrosomonas europaea rplA be incorporated into synthetic biology applications for environmental monitoring?

Recombinant N. europaea rplA can be engineered for novel synthetic biology applications, particularly in environmental monitoring:

• Biosensor Development:

  • Creation of fusion proteins between rplA and reporter systems (GFP, luciferase)

  • Development of riboswitch-based sensors using rplA binding domains

  • Design of cell-free translation systems incorporating modified rplA

• Environmental Monitoring Applications:

  • Detection of ribosome-targeting antibiotics in water samples

  • Assessment of protein synthesis inhibitors in environmental samples

  • Monitoring of factors affecting ammonia oxidation

• Methodological Approaches:

  • Bioluminescence assays similar to those developed for N. europaea

  • Cell-based reporter systems in E. coli expressing recombinant rplA

  • Paper-based dipstick tests with immobilized recombinant proteins

• Integration with Existing Systems:

  • Incorporation into microbial fuel cells for power generation coupled with sensing

  • Development of microfluidic devices for continuous monitoring

  • Creation of whole-cell biosensors using genetic circuits involving rplA

These applications build on existing knowledge of N. europaea's biology, including its ammonia oxidation pathway and response to environmental stressors, while leveraging the fundamental role of rplA in protein synthesis to create novel detection systems.