Recombinant Nitrosomonas europaea 50S ribosomal protein L11 (rplK)

What are the optimal storage conditions for maintaining recombinant rplK stability?

Maintaining stability of recombinant Nitrosomonas europaea 50S ribosomal protein L11 requires specific storage conditions that prevent degradation. The protein can be stored in either liquid or lyophilized form. For liquid preparations, the shelf life is approximately 6 months when stored at -20°C to -80°C. Lyophilized preparations demonstrate greater stability with a shelf life of approximately 12 months when stored at the same temperature range .

It is important to note that repeated freeze-thaw cycles significantly decrease protein stability and should be avoided. For short-term use during experiments, working aliquots can be stored at 4°C for up to one week . The following table summarizes the recommended storage conditions:

How does Nitrosomonas europaea utilize ribosomal proteins in its growth and metabolism?

Nitrosomonas europaea is an ammonia-oxidizing bacterium (AOB) that plays a significant role in the nitrogen cycle. Ribosomal proteins like L11 are essential for protein synthesis, which supports N. europaea's specialized metabolism. As a chemolithoautotroph, N. europaea depends on efficient protein synthesis machinery to express enzymes needed for ammonia oxidation and energy production.

In research contexts, N. europaea has been studied for its biofilm formation capabilities, which are influenced by the expression of various proteins. Studies have shown that N. europaea forms thin, dispersed layers of cells when grown in single-species biofilms . The expression and function of ribosomal proteins like L11 may be critical for supporting the protein synthesis needs during biofilm development and maintenance, though this specific relationship requires further investigation.

What methodological approaches can be employed to study interactions between rplK and other ribosomal components?

To investigate interactions between Nitrosomonas europaea 50S ribosomal protein L11 and other ribosomal components, researchers can employ multiple complementary techniques:

• Co-immunoprecipitation (Co-IP): Using antibodies specific to rplK to pull down the protein along with its binding partners from cellular lysates. This technique can be combined with mass spectrometry for identification of interacting proteins.

• Surface Plasmon Resonance (SPR): To measure binding kinetics and affinity constants between rplK and potential interaction partners. The recombinant rplK can be immobilized on a sensor chip, and various ribosomal components can be flowed over to detect interactions.

• Crosslinking Mass Spectrometry: By using chemical crosslinkers to capture transient or weak interactions between rplK and other ribosomal proteins or rRNA, followed by mass spectrometry analysis to identify crosslinked peptides.

• Cryo-Electron Microscopy: For structural characterization of rplK within the context of the assembled ribosome, providing insights into spatial relationships and conformational changes.

When designing these experiments, it is crucial to reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL with the addition of 5-50% glycerol for stability . Proper controls must include analyses of protein-protein interactions in both native and denatured states to distinguish specific from non-specific interactions.

How does the expression and function of rplK in Nitrosomonas europaea compare to other ammonia-oxidizing bacteria?

Comparative analysis of rplK across ammonia-oxidizing bacteria reveals evolutionary adaptations and functional specializations. While direct comparative data for rplK specifically is limited in the provided search results, methodological approaches for such analysis would include:

• Sequence Alignment and Phylogenetic Analysis: Multiple sequence alignment of rplK from various ammonia-oxidizing bacteria can identify conserved and variable regions. Phylogenetic tree construction can reveal evolutionary relationships and potential functional divergence.

• Structural Modeling and Comparison: Homology modeling of rplK from different AOB species based on known structures can highlight structural differences that may impact function.

• Functional Complementation Studies: Genetic experiments where rplK from other AOB species is expressed in N. europaea mutants lacking functional rplK can assess functional conservation.

• Differential Expression Analysis: RNA-seq or proteomics approaches can determine whether rplK expression patterns differ among AOB species under various environmental conditions.

For experimentally testing these comparisons, researchers should consider the growth conditions specific to each AOB species. For instance, N. europaea cultures are typically grown in specialized media such as ATCC medium 2265 with incubation at 30°C in dark conditions , whereas other AOB may require different cultivation parameters.

What are the optimal expression and purification protocols for obtaining high-quality recombinant rplK?

Obtaining high-quality recombinant Nitrosomonas europaea 50S ribosomal protein L11 requires optimization of expression and purification protocols. Based on established practices for similar ribosomal proteins and the information provided in the product datasheet, the following methodological approach is recommended:

Expression System Selection:
E. coli is the preferred expression host for recombinant rplK production . BL21(DE3) or Rosetta strains are commonly used for ribosomal protein expression due to their reduced protease activity and ability to accommodate rare codons that may be present in N. europaea genes.

Expression Vector Design:

• Include a fusion tag (His6, GST, or MBP) to facilitate purification

• Incorporate a precision protease cleavage site for tag removal

• Ensure proper codon optimization for E. coli expression

Culture Conditions:

• Grow transformed E. coli in LB or TB medium supplemented with appropriate antibiotics

• Incubate at 37°C until OD600 reaches 0.6-0.8

• Induce protein expression with 0.1-1.0 mM IPTG

• Reduce temperature to 16-25°C post-induction

• Continue expression for 16-20 hours to maximize yield

Purification Protocol:

• Cell lysis: Sonication or French press in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, and protease inhibitors

• Initial purification: Affinity chromatography (Ni-NTA for His-tagged proteins)

• Tag removal: Treatment with specific protease (e.g., TEV protease)

• Secondary purification: Ion exchange chromatography

• Final polishing: Size exclusion chromatography

Quality Control Metrics:

• Purity assessment: SDS-PAGE (target >85%)

• Western blot: Confirmation of identity

• Mass spectrometry: Verification of intact mass

• Circular dichroism: Assessment of secondary structure

• Activity assays: Functional tests (e.g., RNA binding capacity)

For reconstitution of the purified protein, it is recommended to centrifuge the vial briefly before opening, then reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Adding 5-50% glycerol (with 50% being the default recommendation) is advised for long-term storage at -20°C/-80°C .

How can researchers design experiments to study the impact of environmental factors on rplK expression in Nitrosomonas europaea?

Designing experiments to study environmental influences on rplK expression in N. europaea requires careful consideration of this organism's unique physiology and growth requirements. The following methodological framework is recommended:

Experimental Design Strategy:

• Growth Condition Variables:

  • Ammonia/ammonium concentration (0.5-10 mM)

  • pH range (6.0-8.5, with optimal around 7.5)

  • Oxygen availability (aerobic vs. microaerobic conditions)

  • Temperature variations (20-35°C)

  • Presence of heterotrophic bacteria (e.g., P. aeruginosa)

  • Exposure to environmental stressors (heavy metals, organic pollutants)

• Cultivation Systems:

  • Batch cultures in ATCC medium 2265

  • Continuous culture chemostats (for maintaining steady-state conditions)

  • Flow cell systems (for biofilm studies)

  • Bioreactors with controlled parameters

• Expression Analysis Methods:

  • RT-qPCR for mRNA quantification

  • Western blotting for protein level assessment

  • Proteomics approaches (LC-MS/MS)

  • Reporter gene constructs (e.g., rplK promoter fused to fluorescent protein)

Experimental Protocol Example:

For studying the impact of co-culture with heterotrophic bacteria on rplK expression:

• Establish N. europaea cultures in ATCC medium 2265 with 1% TSB

• Set up parallel experiments:

  • Pure N. europaea cultures

  • Co-cultures with P. aeruginosa (using protocols similar to those described in the literature)

• Incubate at 30°C in dark conditions

• Collect samples at regular intervals (e.g., days 3 and 5)

• Extract RNA and proteins for expression analysis

• Quantify rplK mRNA using RT-qPCR

• Assess protein levels using Western blot with anti-rplK antibodies

• Correlate expression data with biofilm formation metrics

This approach will allow researchers to determine whether environmental factors, particularly the presence of heterotrophic bacteria known to enhance N. europaea biofilm formation , affect rplK expression levels.

What techniques can be employed to investigate the role of rplK in antibiotic resistance mechanisms of Nitrosomonas europaea?

Investigating the role of rplK in antibiotic resistance mechanisms requires a multidisciplinary approach combining molecular genetics, biochemistry, and structural biology techniques:

Fundamental Rationale:
The 50S ribosomal protein L11 is a known target for certain antibiotics, particularly those in the macrolide, lincosamide, and streptogramin B (MLSB) groups. Mutations or modifications in rplK can potentially confer resistance to these antibiotics by altering binding sites or ribosomal conformations.

Methodological Approaches:

• Mutational Analysis:

  • Site-directed mutagenesis of specific residues in rplK

  • Random mutagenesis followed by selection on antibiotic-containing media

  • Whole-genome sequencing of spontaneous antibiotic-resistant mutants

• Structural Studies:

  • X-ray crystallography of wild-type and mutant rplK proteins

  • Cryo-EM analysis of ribosomes containing modified rplK

  • In silico molecular docking of antibiotics with wild-type and mutant rplK models

• Functional Assays:

  • Minimum inhibitory concentration (MIC) determinations

  • Ribosome profiling to assess translational efficiency

  • In vitro translation assays with purified components

• Molecular Dynamics:

  • Simulation of antibiotic binding to wild-type and mutant rplK

  • Analysis of conformational changes induced by antibiotic binding

Experimental Protocol Example:

To test the hypothesis that specific residues in rplK are involved in antibiotic resistance:

• Generate recombinant N. europaea strains expressing rplK variants with mutations at positions predicted to interact with antibiotics

• Determine MIC values for various antibiotics using these strains

• Isolate ribosomes from wild-type and mutant strains

• Perform in vitro translation assays in the presence of increasing antibiotic concentrations

• Analyze structural interactions using purified recombinant rplK (wild-type and mutants) with antibiotics via biophysical techniques

The recombinant protein described in the search results could serve as a control in these experiments, while mutant versions would need to be generated using similar expression and purification protocols.

How should researchers analyze contradictory data in rplK functional studies?

When facing contradictory data in functional studies of Nitrosomonas europaea 50S ribosomal protein L11, researchers should implement a systematic approach to resolve inconsistencies:

Methodological Framework for Resolving Data Contradictions:

• Validation of Experimental Conditions:

  • Verify protein quality: Ensure the recombinant rplK meets purity standards (>85% via SDS-PAGE) and maintains proper folding

  • Confirm antibody specificity: Validate that antibodies used in immunodetection are specific to N. europaea rplK

  • Review buffer compositions: Subtle differences in salt concentration, pH, or additives can significantly impact protein behavior

• Statistical Reassessment:

  • Increase biological replicates: Similar to the biofilm studies where 5-10 independent replicates were used

  • Apply appropriate statistical tests: For comparing conditions with unequal variances, use the Welch t-test as demonstrated in the biofilm studies

  • Consider power analysis: Determine if sample sizes are sufficient to detect biologically meaningful effects

• Cross-Validation with Alternative Techniques:

  • If contradictions exist between binding assays, validate with multiple methods (e.g., SPR, ITC, fluorescence polarization)

  • For structural discrepancies, compare results from different techniques (X-ray crystallography vs. NMR vs. Cryo-EM)

  • When functional data conflicts with structural predictions, perform direct functional assays

• Contextual Considerations:

  • Evaluate whether contradictions arise from differences in experimental context (in vitro vs. in vivo)

  • Consider the influence of heterologous expression systems on protein function

  • Assess whether co-factors or binding partners present in one experimental system but absent in another could explain disparate results

Case Example Resolution Strategy:

For contradictory data regarding rplK's role in biofilm formation:

• Compare experimental conditions between studies, particularly focusing on:

  • Growth media composition (exact formulation of ATCC medium 2265)

  • Flow cell system specifications (flow rate, chamber dimensions)

  • Imaging parameters (microscope settings, staining protocols)

• Re-analyze the data using standardized metrics:

  • Normalize biovolume measurements (μm³/μm²) across studies

  • Apply consistent thresholding in image analysis

  • Use multiple quantification methods (e.g., biovolume, surface coverage, thickness)

• Design reconciliation experiments:

  • Test intermediary conditions that bridge disparate experimental setups

  • Perform side-by-side comparisons with standardized protocols

  • Introduce controlled perturbations to identify sensitivity to specific variables

What are the best practices for quantifying rplK expression levels in different growth phases of Nitrosomonas europaea?

Accurately quantifying rplK expression across different growth phases requires methodological rigor and attention to N. europaea's unique growth characteristics:

Comprehensive Quantification Strategy:

• Sample Collection Protocol:

  • Synchronize cultures by diluting to standard OD600 (e.g., 0.05-0.1) in fresh ATCC medium 2265

  • Collect samples at defined growth phases:

    • Early lag phase (immediately after inoculation)

    • Late lag phase (before detectable growth increase)

    • Early exponential phase (first evidence of growth)

    • Mid-exponential phase (maximum growth rate)

    • Late exponential phase (declining growth rate)

    • Stationary phase (plateau in growth curve)

    • Decline phase (if applicable)

  • Process samples immediately or flash-freeze in liquid nitrogen

• RNA Analysis Methods:

  • Total RNA extraction using specialized protocols for N. europaea

  • DNase treatment to remove genomic DNA contamination

  • Quality control via Bioanalyzer (RIN > 8.0 recommended)

  • RT-qPCR with rplK-specific primers

  • Normalization to multiple reference genes validated for stability across growth phases

• Protein Quantification Approaches:

  • Western blotting with anti-rplK antibodies

  • Absolute quantification via selected reaction monitoring (SRM) mass spectrometry

  • Ribosome profiling to assess translational efficiency

• Data Normalization Considerations:

  • For pure cultures: Normalize to cell number or total protein

  • For biofilms or co-cultures: Normalize to biovolume or species-specific markers

  • Consider normalization to housekeeping genes with stable expression across conditions

Optimization Table for RT-qPCR Analysis of rplK Expression:

For biofilm studies specifically, researchers should coordinate sample collection with imaging timepoints (e.g., days 3 and 5 after inoculation) to correlate expression data with observed biofilm structure and composition .

What emerging technologies could advance our understanding of rplK function in Nitrosomonas europaea?

Several cutting-edge technologies hold promise for expanding our understanding of rplK's role in N. europaea biology:

Transformative Methodological Approaches:

• CRISPR-Cas9 Genome Editing:

  • Development of optimized CRISPR systems for N. europaea

  • Creation of conditional knockdowns of rplK to study essentiality

  • Precise introduction of point mutations to study structure-function relationships

  • Tagging of endogenous rplK with fluorescent proteins for localization studies

• Cryo-Electron Tomography:

  • Visualization of ribosomes containing rplK within intact N. europaea cells

  • Structural analysis of rplK in different functional states

  • Examination of spatial organization of ribosomes in biofilm versus planktonic states

• Ribosome Profiling and Ribo-Seq:

  • Genome-wide analysis of translational efficiency and regulation

  • Identification of rplK-dependent translational programs

  • Comparison between pure cultures and co-cultures with heterotrophs like P. aeruginosa

• Single-Cell Technologies:

  • Single-cell RNA-seq to examine cell-to-cell variability in rplK expression

  • Single-molecule fluorescence in situ hybridization (smFISH) to visualize rplK mRNA

  • Single-cell proteomics to correlate rplK levels with cellular phenotypes

• Microfluidics Combined with Live-Cell Imaging:

  • Real-time monitoring of N. europaea growth and biofilm formation

  • Analysis of ribosome dynamics using fluorescently tagged rplK

  • Manipulation of microenvironments to test responses to gradients of nutrients or signaling molecules

These technologies could be particularly valuable for understanding the role of rplK in the enhanced biofilm formation observed in co-cultures with P. aeruginosa , potentially revealing how ribosomal proteins contribute to interspecies interactions and adaptation to different growth modes.

How might rplK research contribute to applications in environmental biotechnology and wastewater treatment?

Research on Nitrosomonas europaea 50S ribosomal protein L11 has significant potential applications in environmental biotechnology, particularly in improving wastewater treatment processes:

Translational Applications in Environmental Biotechnology:

• Biofilm Enhancement Strategies:
  Understanding the molecular basis of N. europaea biofilm formation, potentially involving rplK, could lead to improved strategies for retaining these slow-growing ammonia-oxidizing bacteria (AOB) in engineered bioreactors . Enhanced retention would improve nitrification efficiency in wastewater treatment.

• Bioreactor Design Optimization:
  Knowledge of how rplK expression responds to environmental conditions could inform the design of bioreactors with optimized parameters for AOB growth and activity. This could include:

  • Temperature control systems based on known effects on rplK expression

  • Oxygen delivery systems tailored to optimal AOB metabolism

  • Carrier material selection based on biofilm formation characteristics

• Mixed Culture Engineering:
  The finding that N. europaea forms substantially greater biovolume in co-culture with heterotrophic P. aeruginosa than in pure culture suggests that engineered microbial consortia could enhance wastewater treatment efficiency. Understanding rplK's role in this interaction could help optimize these consortia.

• Biomonitoring Tools:
  If rplK expression correlates with N. europaea activity, it could serve as a molecular biomarker for monitoring nitrification efficiency in treatment systems. Quantitative assays targeting rplK could provide real-time insights into AOB function.

• Resilience to Inhibitors:
  Research on rplK's role in antibiotic resistance mechanisms could extend to understanding resistance to inhibitory compounds found in wastewater. This knowledge could inform strategies to maintain nitrification efficiency in the presence of inhibitors.

Methodological Framework for Application Development:

• Identify correlations between rplK expression and nitrification efficiency

• Develop engineered strains with optimized rplK expression

• Test performance in laboratory-scale bioreactors using methods similar to the flow cell systems described in the literature

• Scale up to pilot systems with real wastewater

• Monitor long-term stability and performance

These applications align with the observation that favorable associations of N. europaea with heterotrophic biofilms could facilitate development of improved strategies for retention of N. europaea and other slow-growing AOB in engineered bioreactors .