Recombinant Nitrosomonas europaea 50S ribosomal protein L19 (rplS)

Advanced Research Questions

• How might rplS expression patterns change under oxygen-limited conditions in N. europaea?

While specific data on rplS expression under oxygen limitation is not directly available, we can infer potential patterns based on related research:

Under oxygen-limited conditions, N. europaea shows significant transcriptional changes in respiratory proteins and terminal oxidases. All three subunits of the cytochrome c aa3 HCO and the cytochrome c oxidase assembly gene ctaG are upregulated 1.7-3.0 fold during O₂-limited growth .

To study rplS expression under oxygen limitation:

• Experimental design: Culture N. europaea under controlled oxygen tensions using bioreactors with dissolved oxygen monitoring.

• Methodological approach:

  • Transcriptomics: RNA extraction followed by RT-qPCR targeting rplS or RNA-seq for global expression profiles

  • Proteomics: Western blotting or LC-MS/MS to quantify rplS protein levels

  • Polysome profiling to assess translational activity

• Expected outcomes: Based on other bacterial systems, ribosomal proteins often show coordinated regulation during stress. Since N. europaea upregulates specific respiratory components under O₂ limitation, rplS might show altered expression to support metabolic adaptation .

• How can researchers investigate potential protein-protein interactions involving rplS in N. europaea?

Several methodological approaches can be employed to study rplS interactions:

• Co-immunoprecipitation (Co-IP):

  • Generate antibodies against recombinant rplS or use tagged versions

  • Perform pull-down experiments using N. europaea cell lysates

  • Identify co-precipitated proteins via mass spectrometry

• Bacterial two-hybrid (B2H) system:

  • Create fusion constructs between rplS and one domain of a split reporter

  • Screen a library of N. europaea proteins fused to the complementary domain

  • Positive interactions restore reporter activity

• Cross-linking coupled with mass spectrometry (XL-MS):

  • Treat intact ribosomes or cellular fractions with cross-linking agents

  • Digest and analyze cross-linked peptides by MS/MS

  • Identify spatial relationships between rplS and neighboring proteins

• Cryo-electron microscopy:

  • Isolate intact ribosomes from N. europaea

  • Determine structure through cryo-EM

  • Map rplS position and its contact points with other ribosomal components

These approaches would be particularly valuable given N. europaea's unique metabolism as an ammonia-oxidizing bacterium with distinctive energy generation systems .

• What role might rplS play in stress response and adaptation mechanisms in N. europaea?

The role of rplS in stress responses can be explored through several research avenues:

• Salinity stress: N. europaea exposed to elevated salinity (30 mS cm⁻¹) shows significant proteomic changes. While rplS was not specifically mentioned in stress studies, the regulation of translation machinery is often critical during osmotic stress. Research could examine if rplS is differentially expressed alongside the observed changes in transporters, outer membrane proteins, and osmolyte production enzymes .

• Oxidative stress: During oxygen limitation, N. europaea activates several stress response mechanisms. For instance, in N. winogradskyi (which often co-exists with N. europaea), alkyl hydroperoxide reductases were upregulated 1.7-2.3 fold under stress conditions. Investigation of translational machinery components like rplS during oxidative stress could reveal adaptation mechanisms .

• Nutrient deprivation: When deprived of ammonia and carbonate, N. europaea shows dramatic transcriptional changes with 68% of genes downregulated at least two-fold. This response differs from heterotrophic bacteria, suggesting unique starvation strategies that may involve ribosomal proteins like rplS .

• Methodological approach:

  • Gene knockout or knockdown studies to assess rplS essentiality under stress

  • Reporter gene fusions to monitor rplS promoter activity

  • Ribosome profiling to measure translational efficiency during stress

• How can recombinant rplS be used to develop detection methods for N. europaea in environmental samples?

Development of detection methods using rplS would entail:

• Antibody-based detection:

  • Raise specific antibodies against purified recombinant N. europaea rplS

  • Develop ELISA or immunofluorescence assays for environmental samples

  • Optimize for sensitivity and specificity against related ammonia-oxidizing bacteria

• PCR-based quantification:

  • Design primers targeting unique regions of the N. europaea rplS gene

  • Develop quantitative PCR assays similar to those used for other Nitrosomonas species

  • Validate using competitive PCR approaches as demonstrated for N. oligotropha-like bacteria, which were quantified at 0.0033% ± 0.0022% of total bacterial population in wastewater treatment plants

• Mass spectrometry detection:

  • Identify unique peptide markers from rplS

  • Develop targeted MS methods (SRM/MRM) for environmental proteomics

  • Create isotopically labeled standards for absolute quantification

• Data analysis considerations:

  • Account for potential gene copy number (assuming one copy per cell)

  • Consider extraction efficiency from complex matrices

  • Compare with established markers like amoA for validation

• What insights can comparative analysis of rplS provide about the evolution of ammonia-oxidizing bacteria?

Comparative analysis of rplS can provide valuable evolutionary insights:

• Phylogenetic analysis:

  • Align rplS sequences from all Nitrosomonas species (N. europaea, N. eutropha, N. halophila, N. mobilis, N. communis, N. nitrosa, N. ureae, N. oligotropha, N. marina, N. estuarii, and N. cryotolerans)

  • Compare with other ammonia-oxidizing bacteria including comammox Nitrospira

  • Construct phylogenetic trees to infer evolutionary relationships

• Structural conservation analysis:

  • Perform homology modeling of rplS proteins from different lineages

  • Identify conserved domains and variable regions

  • Correlate structural features with ecological niches

• Selective pressure analysis:

  • Calculate Ka/Ks ratios to identify portions of rplS under purifying or positive selection

  • Compare selection patterns between ammonia oxidizers from different environments

• Coevolution with other ribosomal components:

  • Examine co-evolutionary patterns between rplS and other ribosomal proteins

  • Identify lineage-specific adaptations in the translational machinery

• How does the interaction between N. europaea and other bacteria in mixed cultures affect rplS expression?

The effect of mixed cultures on rplS expression would be an intriguing research direction:

• Background information: Mixed cultures significantly affect N. europaea growth. When co-cultured with heterotrophic bacteria, N. europaea shows enhanced nitrite formation and shortened lag phase. This effect can be reproduced by adding sodium pyruvate to the medium .

• Experimental approach:

  • Establish defined co-cultures of N. europaea with heterotrophic partners

  • Monitor growth parameters and metabolic activities

  • Measure rplS expression at transcriptional and translational levels

  • Compare with monoculture controls

• Hypothetical mechanisms:

  • Metabolite exchange (e.g., amino acids from N. europaea, organic carbon from heterotrophs)

  • Signal molecule-mediated gene regulation

  • Alterations in translation efficiency due to changed growth dynamics

• Potential findings:

  • rplS expression might be upregulated during the enhanced growth phase in mixed cultures

  • Different heterotrophic partners may have varying effects on rplS expression

  • The presence of organic carbon (like pyruvate) might alter ribosomal protein synthesis patterns

This research would contribute to understanding how N. europaea adapts its translational machinery in realistic environmental settings where pure cultures rarely exist.

• What role might rplS play in the regulation of denitrification pathways in N. europaea?

While direct evidence linking rplS to denitrification regulation is not available, this research question presents interesting opportunities:

• Denitrification context: N. europaea possesses genes for denitrification including nirK (encoding copper-containing nitrite reductase) and norCBQD (encoding nitric oxide reductase). The norCBQD cluster functions as the main NO reductase under anoxic and hypoxic conditions .

• Research approaches:

  • Create rplS mutants with altered expression levels and assess impact on denitrification

  • Perform ribosome profiling under denitrifying conditions to identify mRNAs preferentially translated

  • Examine co-regulation patterns between rplS and denitrification genes under various oxygen conditions

• Translational regulation hypothesis:

  • Ribosomal proteins can act as regulatory proteins when not incorporated into ribosomes

  • rplS might selectively bind mRNAs of denitrification genes

  • Altered rplS levels could affect translation efficiency of specific transcripts

• Methodological design:

  • Compare wild-type and rplS-modified strains under aerobic vs. anoxic conditions

  • Measure nirK and norCBQD expression and activity

  • Assess NO and N₂O production rates

This research would connect translational machinery to metabolic regulation, potentially revealing novel regulatory mechanisms in ammonia-oxidizing bacteria .