Recombinant Nitrosomonas europaea 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (ubiD)

What is Nitrosomonas europaea and why is it significant for recombinant studies?

Nitrosomonas europaea is a well-characterized ammonia-oxidizing bacterium that plays a crucial role in nitrogen cycling. It oxidizes ammonia to nitrite as its primary energy source and has been whole-genome sequenced. This organism is particularly valuable for recombinant studies because:

• It serves as a model organism for studying ammonia oxidation and environmental nitrogen cycling

• It demonstrates unique metabolic capabilities, including the ability to co-oxidize various compounds

• It has practical applications in wastewater treatment and potential bioremediation of halogenated compounds

• It has established transformation protocols allowing for genetic manipulation

N. europaea plays a central role in the availability of nitrogen to plants and contributes to CO2 fixation limitations. These bacteria are important in industrial and sewage waste treatment through the oxidation of ammonia to nitrate. Additionally, N. europaea can degrade various halogenated organic compounds, including trichloroethylene, benzene, and vinyl chloride, making it potentially valuable for bioremediation applications .

What is 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (ubiD) and its role in bacterial metabolism?

The ubiD gene encodes 3-octaprenyl-4-hydroxybenzoate carboxy-lyase, an enzyme that catalyzes the third step in ubiquinone (coenzyme Q) biosynthesis. This enzyme specifically:

• Catalyzes the decarboxylation of 3-octaprenyl-4-hydroxybenzoate to 2-octaprenylphenol

• Functions as part of the electron transport chain components synthesis pathway

• Is typically membrane-associated but can be found in cytoplasmic fractions during cell disruption

• Has a molecular weight of approximately 340,000 Da by gel filtration, suggesting a hexameric structure in vivo

The reaction catalyzed by this enzyme is:
3-octaprenyl-4-hydroxybenzoate + H+ → 2-octaprenylphenol + CO2

While the ubiD gene has been well-characterized in Escherichia coli, expressing this gene in N. europaea presents unique research opportunities for studying ubiquinone biosynthesis in ammonia-oxidizing bacteria and potentially enhancing their metabolic capabilities.

What transformation methods are available for creating recombinant N. europaea?

Several transformation methods have been successfully employed for N. europaea, with electroporation being the most widely reported:

• Electroporation: Most commonly used method, as demonstrated in studies with luciferase and GFP reporter genes. Standard protocols typically use field strengths of 5-15 kV/cm with specialized buffers to maximize transformation efficiency .

• DNA delivery parameters: Successful transformation has been achieved using plasmid vectors with appropriate promoters, such as the hydroxylamine oxidoreductase (hao) gene promoter region, which has shown effective expression of heterologous genes .

When performing electroporation of N. europaea:

• Prepare cells in mid-log growth phase (OD600 0.2-0.5)

• Wash cells multiple times with ice-cold electroporation buffer

• Mix cells with purified plasmid DNA (typically 0.5-2 μg)

• Apply electric pulse according to optimized parameters

• Immediately transfer cells to recovery medium

• Incubate for 6-24 hours before plating on selective media

The choice of promoter significantly impacts expression levels. The hao gene promoter has proven effective for expressing reporter genes like luxAB, resulting in consistent bioluminescence during growth phases (8-10 RLU/ml/unit of OD600) .

How do environmental stressors affect recombinant protein expression in N. europaea?

Environmental stressors significantly impact recombinant protein expression in N. europaea through multiple mechanisms. Research has shown that:

Salinity stress effects:

• At 30 mS cm-1 (200 mM NaCl), N. europaea exhibits distinct proteomic changes including production of osmolytes, regulation of cell permeability, and oxidative stress responses

• Carbon metabolism shifts to produce additional reducing power, potentially affecting energy availability for recombinant protein synthesis

• Gene expression patterns change to prioritize stress response over normal metabolic functions

Oxygen limitation effects:

• Oxygen-limited conditions reduce growth yield and alter ammonia-to-nitrite conversion stoichiometry

• Transcriptomic analysis reveals upregulation of cytochrome c oxidases, particularly B-type heme-copper oxidase

• These changes in energy metabolism likely affect recombinant protein expression levels and functionality

These findings suggest that researchers should carefully consider and control environmental conditions when working with recombinant N. europaea, especially when expressing energy-demanding heterologous proteins like ubiD. Maintaining consistent osmotic conditions and oxygen availability is crucial for reproducible expression levels.

What molecular mechanisms help stabilize recombinant ubiD expression in N. europaea under stress conditions?

Based on research findings, several molecular mechanisms could be exploited to stabilize recombinant ubiD expression in N. europaea under stress conditions:

• Co-expression of stress response proteins: Proteomic analysis of N. europaea under salt stress revealed upregulation of specific stress response proteins. Co-expressing these proteins with ubiD could enhance stability:

  • Osmolyte production enzymes

  • Cell permeability regulators

  • Oxidative stress response proteins

• Carbon metabolism engineering: N. europaea shows specific metabolic responses to stress that involve carbon metabolism for producing reducing power. Engineering these pathways could provide the energy needed for stable recombinant expression:

  • Enhancing pathways that generate NADH/NADPH

  • Optimizing electron transport chain components

  • Modifying carbon fixation pathways to increase energy availability

• Membrane association optimization: Since ubiD naturally functions as a membrane-associated protein in E. coli, proper membrane targeting in N. europaea would be critical:

  • The enzyme requires Mn2+, phospholipids, and a heat-stable factor for optimal activity

  • Addition of dithiothreitol and methanol strongly stimulates enzyme activity

  • Ensuring proper folding and assembly of the hexameric structure (MW ~340,000 Da)

Understanding these mechanisms will allow researchers to design expression systems that maintain stable recombinant ubiD activity even under variable environmental conditions.

How can synthetic communities containing recombinant N. europaea be optimized for enhanced stability and function?

Research on synthetic microbial communities containing N. europaea provides insights into optimizing communities with recombinant strains:

Community composition effects:
Synthetic communities of N. europaea with other nitrifying bacteria show different stability patterns and functional characteristics as demonstrated in this experimental matrix:

The combination of N. europaea with N. winogradskyi (EW) showed significantly higher ammonia oxidation rates than other synthetic communities across all tested conditions, doubling the rate compared to pure cultures .

Optimization strategies for synthetic communities with recombinant N. europaea:

• Partner species selection: Choose companion species that enhance ammonium oxidation rates of recombinant N. europaea. N. winogradskyi has been shown to significantly improve N. europaea performance .

• Stress response balancing: Engineer the community to distribute stress response burdens. Under salt stress, different community members show distinct responses:

  • N. europaea produces osmolytes and regulates cell permeability

  • N. winogradskyi exhibits primarily oxidative stress responses

• Metabolic complementation: Design communities where metabolic byproducts of one organism benefit others, creating positive feedback loops that enhance community stability.

• Quorum sensing considerations: Account for intercellular signaling that might affect recombinant gene expression in community settings.

These strategies would be particularly important when working with recombinant N. europaea expressing energy-demanding enzymes like ubiD.

What are the most effective promoters for expressing recombinant ubiD in N. europaea?

Based on experimental evidence with reporter genes in N. europaea, several promoter options show promise for recombinant ubiD expression:

1. Hydroxylamine oxidoreductase (hao) promoter:

• Successfully used to drive expression of luxAB genes in N. europaea

• Provides consistent expression during early and mid-logarithmic growth phases

• Maintains stable expression levels (8-10 RLU/ml/unit of OD600) until nitrite reaches ~10 mM

• Native to N. europaea, reducing potential regulatory incompatibilities

2. Stress-responsive promoters:

• mbla (NE2571) promoter: Shows 3-18 fold induction in response to chloroform stress and 8-10 fold induction with hydrogen peroxide

• clpB (NE2402) promoter: Demonstrates 6-10 fold induction with chloroform exposure

• These promoters could be valuable for conditional expression of ubiD under specific environmental conditions

3. Constitutive promoters from related bacteria:

• Promoters from other betaproteobacteria have shown functionality in N. europaea

• Selection should consider codon usage compatibility and GC content matching

When selecting a promoter for ubiD expression, researchers should consider:

• Desired expression timing and level

• Metabolic burden on the host

• Potential interaction with native regulatory networks

• Stability under experimental conditions

The hao promoter appears particularly promising for stable, constitutive expression, while stress-responsive promoters offer interesting options for conditional expression systems.

How can enzyme activity of recombinant ubiD be accurately measured in N. europaea?

Measuring ubiD enzyme activity in recombinant N. europaea requires specialized techniques that account for both the unique metabolism of the host and the specific requirements of the enzyme:

Assay components for optimal ubiD activity measurement:

Based on studies of the enzyme in E. coli, an optimized assay for ubiD activity measurement should include:

• Mn2+ (required cofactor)

• Washed membranes or phospholipid extract

• Heat-stable factor (MW <10,000)

• Dithiothreitol (strong stimulator)

• Methanol (strong stimulator)

Methodological approach:

• Cell fractionation optimization:

  • French press disruption to partially separate enzyme from membranes

  • Careful separation of cytoplasmic and membrane fractions

  • Analysis of both fractions as ubiD distributes between them

• Substrate preparation:

  • Synthesize 3-octaprenyl-4-hydroxybenzoate substrate

  • Incorporate into membrane-like structures for better enzyme access

• Activity quantification:

  • Measure decarboxylation rate by monitoring CO2 release

  • Analyze 2-octaprenylphenol formation via HPLC or LC-MS

  • Consider radioisotope-labeled substrate for increased sensitivity

• Controls and normalization:

  • Include wild-type N. europaea as negative control

  • Use E. coli expressing ubiD as positive control

  • Normalize activity to total protein or specific membrane protein markers

This methodological approach acknowledges the complex requirements of ubiD and the challenges of expressing and measuring a membrane-associated enzyme in a non-native host.

What molecular biology techniques can be used to improve the stability of recombinant ubiD in N. europaea?

Several molecular biology approaches can enhance the stability and functionality of recombinant ubiD in N. europaea:

1. Codon optimization strategies:

• Analyze N. europaea codon usage patterns

• Redesign the ubiD coding sequence to match host preferences

• Remove rare codons that might cause translational pausing

• Adjust GC content to match host genome (approximately 50.7%)

2. Protein engineering approaches:

• Create fusion proteins with well-expressed N. europaea native proteins

• Add stabilizing domains like maltose-binding protein (MBP)

• Incorporate specific membrane-targeting sequences for proper localization

• Consider hexameric structure requirements in design (native MW ~340,000 Da)

3. Expression regulation optimization:

• Use inducible systems to control expression timing and level

• Consider stress-responsive promoters like mbla (NE2571) that show 3-18 fold induction under defined conditions

• Engineer ribosome binding sites with appropriate strength

• Include transcriptional terminators to prevent read-through

4. Genetic context considerations:

• Identify genomic integration sites with high stability

• Avoid integration near essential genes

• Consider compatibility with native regulatory networks

• Use genome editing tools to remove competing pathways

For researchers using plasmid-based expression systems, stability can be enhanced by:

• Incorporating plasmid addiction systems

• Using compatible antibiotic resistance markers

• Ensuring plasmid copy number is appropriate for metabolic burden

• Including elements that promote plasmid maintenance under non-selective conditions

How does recombinant expression of ubiD potentially impact ammonia oxidation pathways in N. europaea?

The integration of recombinant ubiD into N. europaea's metabolism presents complex interactions with native ammonia oxidation pathways that researchers should consider:

Potential metabolic interactions:

• Electron transport chain effects:

  • ubiD catalyzes a step in ubiquinone biosynthesis, potentially altering electron carrier availability

  • N. europaea relies on electron transport for energy generation during ammonia oxidation

  • Ammonia oxidation in N. europaea follows this pathway: NH3 + O2 + 2H+ + 2e− → NH2OH + H2O (catalyzed by ammonia monooxygenase)

  • Subsequent oxidation: NH2OH + H2O → NO2− + 5H+ + 4e− (catalyzed by hydroxylamine oxidoreductase)

• Energy allocation trade-offs:

  • Recombinant protein production requires energy

  • Under oxygen-limited conditions, N. europaea shows reduced growth yield and altered nitrogen conversion

  • Expression of heterologous proteins may further stress energy availability

• Regulatory cross-talk:

  • Native gene regulation might be affected by heterologous expression

  • Stress responses induced by recombinant expression could alter ammonia oxidation gene expression

  • Potential competition for transcription/translation machinery

Experimental approaches to assess impacts:

• Measure ammonia oxidation rates in recombinant versus wild-type strains

• Compare growth yields under various substrate concentrations

• Conduct transcriptomic analysis to identify regulatory changes

• Perform metabolic flux analysis to quantify changes in carbon and nitrogen flow

• Examine nitrite production stoichiometry for evidence of altered electron flow

Understanding these interactions is crucial for both fundamental research on N. europaea metabolism and applications using recombinant strains for environmental or industrial purposes.

What are the potential applications of recombinant N. europaea expressing ubiD for environmental remediation?

Recombinant N. europaea expressing ubiD offers several promising applications for environmental remediation based on the unique metabolic capabilities of both the host organism and the introduced enzyme:

Potential applications:

• Enhanced co-metabolic degradation of pollutants:

  • N. europaea naturally co-oxidizes various halogenated compounds including trichloroethylene, benzene, and vinyl chloride

  • Recombinant ubiD could enhance electron transfer efficiency, potentially improving degradation rates

  • The ubiquinone pathway modification might expand the range of compounds that can be co-metabolized

• Biosensor development for environmental monitoring:

  • Building on successful biosensor construction in N. europaea using reporter genes like GFP

  • ubiD activity could be coupled with sensing elements for specific pollutants

  • Integration with stress-responsive promoters like mbla and clpB that show 3-18 fold induction under specific conditions

• Improved wastewater treatment systems:

  • N. europaea is naturally involved in nitrogen removal from wastewater

  • Enhanced metabolic capabilities through ubiD expression could improve treatment efficiency

  • Potential for dual-function systems addressing both nitrogen cycling and organic pollutant degradation

Research gaps to address:

• Stability of recombinant N. europaea in environmental conditions

• Metabolic burden of ubiD expression and its effect on ammonia oxidation

• Regulatory approval pathways for environmental release of recombinant organisms

• Development of containment strategies for field applications

These applications would need to be developed with careful attention to ecological impacts and regulatory requirements for recombinant organisms in environmental settings.

How might CRISPR-Cas9 gene editing advance recombinant N. europaea ubiD research?

CRISPR-Cas9 gene editing technologies offer transformative potential for recombinant N. europaea ubiD research through several key applications:

Precise genomic integration strategies:

• Site-specific integration of ubiD into the N. europaea genome

• Targeted disruption of competing metabolic pathways

• Precise promoter replacements to optimize expression levels

• Introduction of regulatory elements for controlled expression

Host optimization approaches:

• Modification of native ubiquinone biosynthesis pathways to accommodate heterologous ubiD

• Enhancement of stress response mechanisms identified in proteomic studies

• Engineering of membrane composition to improve ubiD function (since it's membrane-associated)

• Disruption of native carboxy-lyases that might compete with ubiD

Multiplexed engineering opportunities:

• Simultaneous modification of multiple genes involved in electron transport

• Creation of synthetic operons combining ubiD with supporting enzymes

• Introduction of complete heterologous pathways from other organisms

• Development of conditional expression systems based on environmental sensing

Methodological considerations for N. europaea CRISPR applications:

• Delivery methods for Cas9 and guide RNAs suited to N. europaea

• Optimization of homology-directed repair templates

• Selection strategies for identifying successful edits

• Screening approaches for phenotype verification

While CRISPR-Cas9 has revolutionized genetic engineering in many organisms, its application in N. europaea remains challenging due to the organism's unique physiology and limited genetic tools. Future research should focus on adapting CRISPR systems specifically for this ammonia-oxidizing bacterium.

What insights could systems biology provide about integrating ubiD into N. europaea metabolism?

Systems biology approaches offer powerful frameworks for understanding the complex integration of recombinant ubiD into N. europaea metabolism:

Multi-omics integration strategies:

• Transcriptomics insights:

  • Global gene expression changes in response to ubiD introduction

  • Regulatory network perturbations affecting ammonia oxidation pathways

  • Stress response activation patterns similar to those observed under oxygen limitation or salinity stress

• Proteomics applications:

  • Quantification of ubiD protein levels and subcellular localization

  • Changes in membrane protein composition affecting enzyme function

  • Post-translational modifications affecting activity

  • Protein-protein interaction networks involving recombinant ubiD

  • Building on existing proteomic studies of N. europaea under stress conditions

• Metabolomics approaches:

  • Alterations in ubiquinone/ubiquinol pools

  • Changes in central carbon metabolism supporting recombinant protein production

  • Shifts in nitrogen metabolism and ammonia oxidation intermediates

  • Identification of unexpected metabolic byproducts

• Genome-scale metabolic modeling:

  • Prediction of flux changes resulting from ubiD introduction

  • Identification of potential metabolic bottlenecks

  • Optimization of growth conditions for recombinant strains

  • Exploration of genetic modifications to improve performance

These systems biology approaches would be particularly valuable given the complex metabolic interactions expected between recombinant ubiD (involved in ubiquinone biosynthesis) and N. europaea's core energy metabolism, which relies heavily on electron transport processes for ammonia oxidation.

What are common challenges in expressing active ubiD in recombinant N. europaea and how can they be addressed?

Researchers working with recombinant N. europaea expressing ubiD commonly encounter several challenges that require specific troubleshooting approaches:

Challenge 1: Low expression levels

• Possible causes: Promoter incompatibility, codon usage bias, mRNA instability

• Solutions:

  • Test alternative promoters (hao promoter shows strong, consistent expression)

  • Perform codon optimization specific to N. europaea

  • Include stabilizing RNA elements in the construct

  • Optimize cultivation conditions based on promoter characteristics

Challenge 2: Improper enzyme folding/inactive protein

• Possible causes: Missing cofactors, inappropriate cellular localization, incorrect oligomerization

• Solutions:

  • Supplement media with Mn2+ (required cofactor)

  • Include proper membrane-targeting sequences (ubiD is membrane-associated)

  • Consider hexameric structure requirements (native MW ~340,000 Da)

  • Co-express chaperones or folding assistants

Challenge 3: Metabolic burden and toxicity

• Possible causes: Energy drain, interference with native metabolism, accumulation of intermediate metabolites

• Solutions:

  • Use inducible promoters for controlled expression

  • Optimize growth conditions (temperature, media composition)

  • Consider oxygen supply carefully (N. europaea shows distinct responses to oxygen limitation)

  • Balance expression levels with host metabolic capacity

Challenge 4: Recombinant strain instability

• Possible causes: Plasmid loss, genetic rearrangements, selection against expression

• Solutions:

  • Use genomic integration rather than plasmid-based expression

  • Include selection pressure during cultivation

  • Monitor strain stability over generations

  • Create frozen stocks from verified expression-positive colonies

When troubleshooting, systematic approaches comparing multiple conditions simultaneously will yield the most informative results, as demonstrated in studies of synthetic microbial communities with N. europaea .

How can researchers distinguish between effects caused by ubiD expression versus general recombinant protein stress in N. europaea?

Distinguishing between specific ubiD-related effects and general stress responses to recombinant protein expression requires careful experimental design and multiple control conditions:

Essential control strategies:

• Multiple control strains:

  • Wild-type N. europaea (no recombinant protein)

  • N. europaea expressing an irrelevant protein of similar size (general expression control)

  • N. europaea expressing inactive ubiD mutant (specific protein control)

  • These controls help isolate effects specific to ubiD activity versus expression burden

• Activity-specific measurements:

  • Quantify ubiquinone/ubiquinol ratios and pool sizes

  • Measure 3-octaprenyl-4-hydroxybenzoate levels (ubiD substrate)

  • Assess 2-octaprenylphenol production (ubiD product)

  • Compare with control strains to identify metabolic signatures of ubiD activity

• Transcriptomics/proteomics approach:

  • Compare global expression patterns between controls and ubiD-expressing strains

  • Identify genes specifically responding to ubiD activity versus general stress

  • Look for patterns similar to known stress responses in N. europaea

• Physiological characterization:

  • Assess growth rates, substrate consumption, and product formation

  • Compare oxygen uptake rates across strains

  • Measure ammonia oxidation kinetics under standardized conditions

  • Evaluate stress tolerance (e.g., to salinity, chlorinated compounds)