Recombinant Nitrosomonas europaea Homoserine kinase (thrB)

What is the genomic context of the homoserine kinase (thrB) gene in Nitrosomonas europaea?

Homoserine kinase (thrB) in N. europaea is part of the threonine biosynthesis pathway genes. While the search results don't specifically mention thrB, we can infer from the genome organization principles that it likely follows patterns similar to other amino acid biosynthesis operons in this organism. N. europaea's genome consists of a single circular chromosome of 2,812,094 bp with protein-encoding genes averaging 1,011 bp in length and intergenic regions averaging 117 bp . Similar to the histidine biosynthesis genes (his) that show a specific organization pattern in N. europaea, thrB may be part of a gene cluster dedicated to threonine biosynthesis. The genomic context analysis would require examining genes adjacent to thrB to determine if they follow the pattern seen in other amino acid biosynthesis pathways where N. europaea exhibits organizational similarities to other proteobacteria while maintaining some unique features .

What is the predicted role of homoserine kinase in the amino acid biosynthesis pathways of Nitrosomonas europaea?

Homoserine kinase catalyzes the phosphorylation of L-homoserine to O-phospho-L-homoserine, a critical step in threonine biosynthesis. In N. europaea, this enzyme likely plays a significant role in amino acid metabolism, particularly since this bacterium must synthesize most of its amino acids de novo as it is an obligate chemolithoautotroph with limited capacity for organic compound uptake . Unlike heterotrophic bacteria that can acquire amino acids from the environment, N. europaea depends heavily on biosynthetic pathways. The genome analysis of N. europaea reveals genes necessary for biosynthesis and CO₂ and NH₃ assimilation, consistent with its autotrophic lifestyle . The organization of amino acid biosynthesis genes in N. europaea shows similarities to other proteobacteria, as evidenced by the his operon structure discussed in the search results, suggesting thrB would function within a well-conserved threonine biosynthesis pathway but adapted to the organism's unique metabolic constraints .

What are the optimal expression systems for producing recombinant N. europaea homoserine kinase?

For successful recombinant expression of N. europaea homoserine kinase, E. coli-based expression systems are typically most effective, particularly BL21(DE3) strains containing pET-based vectors with T7 promoters for controlled, high-level expression. The methodology should address potential challenges with heterologous expression of proteins from a bacterium with such different codon usage and GC content. N. europaea has a genome with distinct organizational features compared to E. coli , which may necessitate codon optimization of the thrB gene for efficient expression.

The expression protocol should include:

• Temperature optimization (typically 18-25°C post-induction to enhance proper folding)

• IPTG concentration titration (0.1-1.0 mM)

• Expression time optimization (4-24 hours)

A fusion tag approach using His₆, MBP, or SUMO tags can enhance solubility and facilitate purification. When designing expression constructs, researchers should consider that N. europaea proteins may require specialized conditions reflecting the bacterium's unusual physiology as an obligate chemolithoautotroph .

What purification strategy yields the highest enzymatic activity for recombinant N. europaea homoserine kinase?

A multi-step purification protocol optimized for preserving enzymatic activity should include:

• Initial capture using affinity chromatography (Ni-NTA for His-tagged protein)

• Ion exchange chromatography (typically anion exchange using Q-Sepharose)

• Size exclusion chromatography for final polishing

The buffer composition is critical for maintaining activity:

Throughout purification, it's essential to monitor enzyme activity using a coupled spectrophotometric assay measuring ATP consumption or through direct detection of O-phospho-L-homoserine formation by HPLC. Since N. europaea has evolved specialized metabolic systems for its chemolithoautotrophic lifestyle , its enzymes may exhibit distinct stability characteristics compared to those from heterotrophic bacteria.

How can researchers effectively address issues with protein solubility when expressing recombinant N. europaea thrB?

Enhancing solubility of recombinant N. europaea homoserine kinase requires a systematic approach:

• Expression temperature reduction (16-18°C) to slow folding and prevent inclusion body formation

• Co-expression with chaperone proteins (GroEL/GroES, DnaK/DnaJ/GrpE)

• Addition of solubility-enhancing fusion partners:

  • N-terminal MBP tag (significantly increases solubility)

  • SUMO tag (promotes proper folding)

  • Thioredoxin fusion

If these approaches yield insufficient soluble protein, refolding protocols can be employed:

• Gradual dilution refolding from 8M urea or 6M guanidine-HCl

• On-column refolding using decreasing urea gradient

• Pulsed refolding with cyclodextrin as aggregation suppressor

The specialized metabolism of N. europaea as an obligate chemolithoautotroph may result in proteins with unique folding characteristics . Exploring buffer conditions that mimic the cytoplasmic environment of N. europaea might improve solubility of its recombinant proteins.

What kinetic parameters characterize N. europaea homoserine kinase activity and how do these compare to homoserine kinases from heterotrophic bacteria?

Comprehensive kinetic characterization of N. europaea homoserine kinase involves determining several parameters:

N. europaea homoserine kinase may exhibit distinct kinetic properties reflecting adaptation to its chemolithoautotrophic lifestyle, where it derives all energy from ammonia oxidation . Compared to heterotrophic bacteria, N. europaea enzymes often show adaptations to lower energy availability and specialized metabolic requirements. The kinetic characterization should include assessment of metal ion dependencies (Mg²⁺, Mn²⁺) and potential allosteric regulation by amino acid pathway intermediates, similar to regulatory mechanisms documented in other amino acid biosynthesis pathways.

How does the MazF toxin-antitoxin system in N. europaea potentially regulate thrB expression?

The MazF toxin in N. europaea specifically recognizes and cleaves the UGG motif in RNA transcripts . Statistical analysis conducted on N. europaea coding sequences identified transcripts that are prime targets for this endoribonuclease . While the search results don't explicitly mention thrB as a target, this regulatory mechanism could potentially impact threonine biosynthesis.

To determine if thrB might be regulated by MazF:

• Analyze the thrB transcript sequence for prevalence of UGG motifs

• Apply the same statistical analysis methodology used for other genes:

  • Calculate the probability (p) of UGG appearing in thrB

  • Determine the expected number (E) of UGG motifs based on gene length

  • Compare with actual number (K) of UGG sequences in thrB

  • Calculate the probability (P) of thrB containing at least K UGG motifs

If thrB contains a statistically significant number of UGG motifs, it suggests potential regulation by the MazF toxin under stress conditions. This would represent a mechanism by which N. europaea could modulate amino acid biosynthesis during environmental stress, similar to how MazF regulates key enzymes like hydroxylamine dehydrogenase (hao) and ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcL) .

What methods are most effective for investigating the relationship between thrB activity and the central metabolic pathways in N. europaea?

Investigating the integration of thrB with N. europaea's central metabolism requires multiple complementary approaches:

• Metabolic Flux Analysis:

  • Isotope labeling with ¹³C-bicarbonate as the carbon source

  • GC-MS or LC-MS/MS quantification of labeled intermediates

  • Computational modeling to determine flux distributions

• Gene Expression Correlation Studies:

  • RNA-Seq under various growth conditions

  • Determine correlation patterns between thrB and ammonia oxidation genes (amo, hao)

  • Examine co-regulation with carbon fixation genes (rbcL)

• Protein-Protein Interaction Studies:

  • Pull-down assays with tagged thrB

  • Bacterial two-hybrid screening

  • Crosslinking mass spectrometry

• Metabolite Profiling:

  • Targeted analysis of threonine pathway intermediates

  • Untargeted metabolomics to identify unexpected connections

Given N. europaea's unique metabolism as an obligate chemolithoautotroph with an incomplete TCA cycle , thrB activity may be coordinated with ammonia oxidation rates and carbon fixation. The relationship between threonine biosynthesis and energy generation would be particularly important since N. europaea derives all its energy from ammonia oxidation to nitrite .

How can gene editing techniques be optimized for modifying thrB in N. europaea given its specialized metabolism?

Genetic modification of N. europaea presents unique challenges due to its obligate chemolithoautotrophic lifestyle. Optimized protocols should include:

• CRISPR-Cas9 System Adaptation:

  • Codon-optimization of Cas9 for N. europaea

  • Design of guide RNAs targeting thrB with minimal off-target effects

  • Development of inducible expression systems compatible with N. europaea's physiology

• Homologous Recombination Strategy:

  • Construct design with extended homology arms (1-2 kb)

  • Selection markers compatible with N. europaea's antibiotic sensitivities

  • Counter-selection systems for marker removal

• Transformation Protocol Optimization:

  • Electroporation conditions specific to N. europaea's cell envelope characteristics

  • Recovery media supplemented with ammonia as energy source

  • Extended recovery periods accounting for slow growth rate (0.054h⁻¹)

The genetic modification approach must consider N. europaea's growth requirements and metabolic constraints. Since N. europaea obtains all its energy from ammonia oxidation , any genetic manipulation affecting central metabolism must be carefully designed to maintain cell viability. Monitoring expression of key genes involved in ammonia oxidation (amo, hao) is crucial when modifying biosynthetic pathways to ensure primary energy metabolism remains functional .

What computational approaches can predict the impact of thrB mutations on N. europaea's metabolic network?

Computational prediction of thrB mutation effects requires integrated approaches:

• Genome-Scale Metabolic Modeling:

  • Construct N. europaea-specific genome-scale metabolic model

  • Incorporate flux balance analysis (FBA) constraints based on chemolithoautotrophic growth

  • Simulate thrB mutations through reaction constraints

  • Predict growth rate and metabolite production changes

• Protein Structure-Function Analysis:

  • Homology modeling of N. europaea homoserine kinase

  • Molecular dynamics simulations to assess mutation effects on protein stability

  • In silico substrate docking to predict changes in catalytic efficiency

• Regulatory Network Analysis:

  • Integration of transcriptomic data to identify regulatory interactions

  • Predict effects of thrB mutations on downstream gene expression

  • Assess potential impact on stress response systems, including MazEF toxin-antitoxin system

These computational approaches should account for N. europaea's unique metabolic architecture, including its incomplete TCA cycle and specialized energy metabolism. The predictions should be contextually appropriate for an organism that derives all energy from ammonia oxidation and carbon from CO₂ fixation .

How does oxygen limitation affect thrB expression and threonine biosynthesis in N. europaea?

Oxygen limitation significantly impacts N. europaea metabolism as oxygen serves as the terminal electron acceptor for ammonia oxidation. Research approaches should include:

• Gene Expression Analysis:

  • qRT-PCR or RNA-Seq at different dissolved oxygen concentrations (0.5, 1.5, and 3.0 mg O₂/L)

  • Compare thrB transcription patterns with those observed for ammonia oxidation genes (amoA, hao)

  • Examine correlation with nitrite reduction genes (nirK, norB)

• Protein Abundance Quantification:

  • Western blotting or targeted proteomics for thrB

  • Compare with patterns observed for other metabolic enzymes

• Metabolite Profiling:

  • Measure threonine pathway intermediates at varying oxygen concentrations

  • Correlate with ammonia oxidation rates

The experimental design should recognize that N. europaea exhibits differential gene expression responses under oxygen limitation. Search results indicate that amoA and hao mRNA concentrations actually increase under decreasing DO concentrations , suggesting complex regulatory mechanisms. Experimental protocols must account for distinct responses in exponential versus stationary phase cultures, as ammonia availability significantly impacts responses to oxygen limitation .