Recombinant Nitrosomonas europaea Orotate phosphoribosyltransferase (pyrE)

What is Nitrosomonas europaea and why is it significant in nitrogen cycle research?

Nitrosomonas europaea is an ammonia-oxidizing bacterium (AOB) that plays a crucial role in the global nitrogen cycle through nitrification processes. It is widely used as a model organism for studying ammonia oxidation and nitrogen oxide production. This bacterium is particularly important in environmental microbiology as it contributes significantly to nitrogen transformation in both natural and engineered systems such as wastewater treatment plants and drinking water treatment facilities .

N. europaea strain ATCC 19718 was the first AOB to have its genome sequenced, making it an invaluable reference organism for genetic and physiological studies of ammonia oxidation and nitrogen oxide production . Its significance extends beyond basic research to applications in environmental remediation, as it influences fertilizer efficiency and the production of greenhouse gases like nitrous oxide.

What are the optimal laboratory conditions for culturing Nitrosomonas europaea?

Successful cultivation of N. europaea requires specific laboratory conditions:

• Growth Medium: Minimal growth medium (such as ATCC medium 2265) containing NH₄Cl (typically 45-84 mM concentration)

• pH Range: Maintenance between 6.8-8.0, with optimal results at pH 7.8±0.1

• Temperature: 26-30°C

• Agitation: 100 rpm

• Incubation Environment: Dark conditions to prevent photoinhibition

• Growth Duration: Typical culture periods of 72-78 hours for optimal biomass development

For batch experiments, 250 ml Erlenmeyer flasks containing 100 ml of liquid medium are commonly used. The pH of cultures should be monitored daily and readjusted to maintain the optimal range using sterile 1 M NH₄HCO₃ or 1 M NaHCO₃ .

How does the genetic structure of Nitrosomonas europaea influence experimental design?

The genetic structure of N. europaea presents unique considerations for experimental design. Unlike many other bacterial species, N. europaea possesses multiple copies of certain genes involved in nitrification. For instance, hydroxylamine oxidoreductase (HAO) is encoded by three gene copies (haoA, haoB, and haoC), while some other genes like cytochrome P-460 (cyp) exist as single copies .

This genetic redundancy necessitates careful consideration when designing knockout experiments or gene expression studies. When targeting specific gene functions, researchers must account for potential compensatory effects from duplicate genes. The presence of multiple gene copies may represent an evolutionary adaptation that provides metabolic redundancy or allows for differential regulation under varying environmental conditions .

The genomic characteristics of N. europaea require specialized approaches when:

• Designing mutation strategies targeting specific genes

• Interpreting phenotypic results following genetic modifications

• Evaluating gene expression patterns under different environmental conditions

What trace elements are essential for optimal Nitrosomonas europaea growth and enzymatic activity?

A properly formulated trace element solution (TES) is critical for optimal growth and enzymatic activity of N. europaea. Statistical optimization studies have identified TES as one of the significant factors affecting biomass concentration, nitrite yield, and ammonium removal efficiency . The standard trace element solution for N. europaea cultivation contains the following components:

The optimal addition of TES to culture medium has been determined to be approximately 0.74 ml per standard batch culture through integrated statistical design methods . The precise balance of these micronutrients supports essential enzymatic functions, particularly the metalloproteins involved in the ammonia oxidation pathway.

What techniques are effective for genetic transformation of Nitrosomonas europaea?

Genetic transformation of N. europaea can be achieved through electroporation coupled with homologous recombination. The following methodology has been demonstrated to be effective:

Electroporation Protocol for N. europaea Transformation:

• Harvest early-stationary-phase liquid culture cells (A₆₀₀ ≈ 0.1) by centrifugation

• Wash cells three times with sterile H₂O

• Mix 120 μl of cell suspension with 1 μg of plasmid DNA in a prechilled electroporation cuvette

• Apply electric pulse at optimal parameters: 1,200 V, 25 μF, and infinite resistance using a 1-mm-gap cuvette

• Immediately transfer electroporated cells to fresh medium

• Allow 24 hours of growth under non-selective conditions before applying antibiotic selection

• Add appropriate selective antibiotic (e.g., kanamycin at 10 μg/ml)

• Monitor growth through nitrite accumulation measurements

This approach achieves approximately 50% cell survival while enabling efficient transformation. The transformed cultures typically show kanamycin resistance after 10 days, with nitrite accumulation rates comparable to wild-type cultures. Transformed N. europaea can grow in the presence of up to 200 μg/ml of kanamycin, demonstrating stable integration of the resistance marker .

How can researchers perform targeted mutagenesis in Nitrosomonas europaea?

Targeted mutagenesis in N. europaea can be accomplished through homologous recombination following transformation. This approach has been successfully demonstrated by inserting an aminoglycoside 3'-phosphotransferase (kan) gene into specific genomic loci:

Targeted Mutagenesis Protocol:

• Construct plasmids containing:

  • Fragment of the target gene

  • Kanamycin resistance cassette inserted within the gene fragment

  • Sufficient flanking sequences for homologous recombination (>500 bp recommended)

• Transform N. europaea with the constructed plasmid using the electroporation method described above

• Select transformants on solid medium containing kanamycin (10 μg/ml)

• Confirm gene disruption through:

  • Southern hybridization analysis

  • PCR verification of the insertion site

  • Phenotypic characterization if applicable

The isolation of mutant strains can typically be achieved within 7-14 days when grown on solid medium. Importantly, the induced mutations have been shown to be stable even in the absence of kanamycin-selective pressure for extended periods (up to 45 days in culture) .

When targeting genes with multiple copies, such as the hao gene family, it is possible to specifically target individual copies by utilizing unique flanking sequences or restriction sites that differentiate between the copies .

How does oxygen limitation affect the transcriptomic responses and metabolic pathways in Nitrosomonas europaea?

Oxygen limitation significantly alters the transcriptomic profile and metabolic functioning of N. europaea, affecting both growth parameters and nitrogen transformation pathways:

Physiological Changes Under Oxygen Limitation:

• Reduced growth yield (0.35 ± 0.01 g [dry cell weight] mol⁻¹ NH₃ compared to 0.40 ± 0.01 under ammonia limitation)

• Increased ammonia consumption rate (28.51 ± 1.13 mmol g [dry cell weight]⁻¹ h⁻¹ compared to 24.73 ± 0.53 under ammonia limitation)

• Non-stoichiometric ammonia-to-nitrite conversion, suggesting production of nitrogenous gases

Key Transcriptomic Responses:

• Significant upregulation of both heme-copper-containing cytochrome c oxidases

• Particularly notable increase in B-type heme-copper oxidase transcription, proposed to function as a nitric oxide reductase (sNOR)

• Reduced transcription of the nitrite reductase-encoding gene (nirK)

• No significant change in the principal nitric oxide reductase (cNOR) transcription

These transcriptomic changes suggest an adaptive response to oxygen scarcity, with the B-type heme-copper oxidase potentially functioning as a high-affinity terminal oxidase. The enzymatic background of NO and N₂O production in N. europaea involves multiple interconnected processes, and the observed transcriptomic changes provide insights into how this bacterium adapts its nitrogen transformation pathways under oxygen limitation .

What statistical approaches can optimize culture conditions for maximum Nitrosomonas europaea biomass and ammonia oxidation efficiency?

Optimization of N. europaea culture conditions can be achieved through a systematic integrated statistical design approach that enhances biomass concentration (CB), nitrite yield (Y), and ammonium removal (NR) simultaneously:

Integrated Statistical Design Methodology:

• Initial Screening: Apply Plackett-Burman design (PBD) to identify significant factors from multiple variables. From 19 potential factors, four were identified as particularly significant:

  • NH₄Cl concentration (C)

  • Trace element solution (TES)

  • Agitation speed (AS)

  • Fermentation time (T)

• Path of Steepest Ascent: Use experimental design to move rapidly toward the vicinity of optimal conditions for the significant factors identified.

• Response Surface Methodology (RSM): Apply to evaluate the significant factors and obtain optimization conditions for each response variable.

• Multi-objective Optimization (MOO): Employ a combination of weighted coefficient method with entropy measurement methodology to obtain optimal conditions that balance all desired objectives simultaneously.

Optimized Culture Conditions:

Under these optimized conditions, the following performance metrics were achieved:

• Biomass concentration (CB): 3.386×10⁸ cells/ml

• Nitrite yield (Y): 1.98 mg/mg

• Ammonium removal (NR): 97.76%

This statistical approach provides a robust framework for optimizing multiple process objectives simultaneously while minimizing experimental runs.

What are the molecular mechanisms of hydroxylamine oxidoreductase (HAO) in Nitrosomonas europaea and how can researchers study enzyme function through mutagenesis?

Hydroxylamine oxidoreductase (HAO) is a key enzyme in the ammonia oxidation pathway of N. europaea, catalyzing the oxidation of hydroxylamine to nitrite. Understanding its molecular mechanisms is crucial for comprehending nitrification processes.

Molecular Characteristics of HAO:

• Encoded by three gene copies (haoA, haoB, and haoC) in the N. europaea genome

• Multiple gene copies may provide metabolic redundancy or enable differential expression under varying environmental conditions

• Essential for energy generation during ammonia oxidation

Mutagenesis Approach for Studying HAO Function:

• Targeted Gene Disruption: Insert a kanamycin resistance gene (kan) into specific hao gene copies using homologous recombination

• Selective Targeting: Utilize conserved restriction sites (such as the BamHI site at the 5' end of the hao coding region) while exploiting unique upstream flanking sequences to discriminate between hao copies

• Mutant Verification: Confirm successful mutations through:

  • Southern hybridization analysis with probes for hao or kan genes

  • PCR analysis using primers specific to the insertion junctions

  • Restriction enzyme digestion patterns (KpnI or EcoRI)

• Phenotypic Analysis: Assess the impact of individual hao gene disruptions on:

  • Growth rates in liquid culture

  • Ammonia oxidation kinetics

  • Hydroxylamine oxidation capability

  • Expression levels of remaining hao copies (potential compensatory effects)

How can researchers implement chemostat-based approaches to study Nitrosomonas europaea under defined nutrient limitation conditions?

Chemostat-based cultivation provides a powerful approach for studying N. europaea under precisely controlled nutrient limitation conditions, offering valuable insights into physiological adaptations and gene expression patterns:

Chemostat Methodology for Nutrient Limitation Studies:

• System Setup: Establish a continuous culture system with controlled dilution rate (D, typically 0.075 h⁻¹)

• Ammonia-Limited Conditions: Maintain low NH₄⁺ concentration in the feed medium while ensuring excess dissolved oxygen

• Oxygen-Limited Conditions: Supply excess NH₄⁺ while restricting oxygen availability

• Steady-State Establishment: Maintain cultures for at least 5 residence times before sampling to ensure steady-state conditions

• Analytical Monitoring:

  • Monitor ammonia consumption rates

  • Measure nitrite production

  • Determine biomass concentration

  • Calculate growth yield (Y)

Key Parameters Under Different Limitation Conditions:

This chemostat-based approach enables researchers to:

• Collect samples for transcriptomic analysis under steady-state conditions

• Compare physiological parameters across precisely controlled limitation conditions

• Investigate metabolic adaptations to specific nutrient limitations

• Generate reproducible data for mathematical modeling of N. europaea metabolism

The chemostat system provides an ideal platform for studying the complex relationship between environmental conditions, gene expression patterns, and metabolic activities in N. europaea, offering insights that batch cultures cannot provide.

What is the significance of the pyrE gene in Nitrosomonas europaea genetic studies?

While specific information about the pyrE gene in N. europaea is limited in the provided search results, orotate phosphoribosyltransferase (encoded by pyrE) generally plays a crucial role in pyrimidine biosynthesis in bacteria. In genetic studies, pyrE has several important applications:

• Counterselection Marker: The pyrE gene can function as both a positive and negative selection marker, making it valuable for genetic manipulations

• Gene Expression System: pyrE promoters can be utilized for controlled gene expression studies

• Genetic Stability: Understanding pyrE function provides insights into nucleotide metabolism and genetic stability

Researchers interested in utilizing pyrE in N. europaea genetic studies should consider adapting established transformation and recombination techniques described for other genes in this organism, while accounting for the specific characteristics of pyrimidine metabolism pathways.

How can researchers verify successful transformation and genetic stability in recombinant Nitrosomonas europaea strains?

Verification of successful genetic modification and stability assessment in recombinant N. europaea strains requires a multi-faceted approach:

Verification Methods:

• Antibiotic Resistance Testing: Confirm growth in the presence of selective antibiotics (e.g., kanamycin at concentrations up to 200 μg/ml)

• Southern Hybridization: Perform genomic DNA digests with appropriate restriction enzymes followed by hybridization with gene-specific probes

• PCR Analysis: Use primers specific to the insertion junctions to confirm correct genomic integration

• Plasmid Extraction Tests: Verify chromosomal integration rather than plasmid maintenance through negative plasmid isolation results

• Sequence Verification: Confirm the exact location and orientation of the genetic modification

Stability Assessment:

• Maintain cultures in non-selective conditions for extended periods (45+ days)

• Periodically check for retention of the genetic modification using the verification methods above

• Monitor for phenotypic consistency over multiple generations

Proper verification is crucial for ensuring that observed phenotypes are directly attributable to the intended genetic modifications rather than other genetic or physiological adaptations.

What are the key challenges in scaling up Nitrosomonas europaea cultures for biochemical and structural studies?

Scaling up N. europaea cultures for biochemical and structural studies presents several significant challenges that researchers must address:

Major Scaling Challenges:

• Slow Growth Rate: N. europaea has a generation time of approximately 8-12 hours, making large-scale cultivation time-consuming

• Media Optimization: Balancing ammonia concentrations to provide sufficient substrate while avoiding toxicity

• pH Control: Maintaining optimal pH as nitrification produces acidity

• Oxygen Demand: Ensuring adequate oxygenation while avoiding excessive agitation that may damage cells

• Biomass Yield: Achieving sufficient cell density for protein purification studies

Recommended Approaches:

• Implement fed-batch cultivation strategies with controlled ammonia feeding

• Utilize pH-stat systems with automated base addition

• Consider immobilization techniques to increase cell retention

• Optimize harvest timing based on enzymatic activity rather than solely on cell density

• Apply statistical experimental design to identify optimal parameters for scaled-up conditions