Recombinant Nitrosomonas europaea S-adenosylmethionine synthase (metK)

What is S-adenosylmethionine synthase (metK) and what is its function in Nitrosomonas europaea?

S-adenosylmethionine synthase, encoded by the metK gene, catalyzes the formation of S-adenosylmethionine (SAM) from methionine and ATP. In Nitrosomonas europaea, as in other bacteria, SAM serves as the primary methyl donor for various methylation reactions, including DNA methylation, protein methylation, and metabolite methylation. These methylation processes are critical for cellular function, gene regulation, and metabolic adaptation in this ammonia-oxidizing bacterium.

The reaction catalyzed by MetK can be represented as:
Methionine + ATP → S-adenosylmethionine + Triphosphate + Pi

N. europaea has a single circular chromosome of 2,812,094 bp containing approximately 2,460 protein-encoding genes . Unlike some duplicated genes in N. europaea (such as those encoding ammonia monooxygenase and hydroxylamine oxidoreductase), metK appears to exist as a single copy, highlighting its essential and conserved nature in bacterial metabolism .

How is metK expression regulated in Nitrosomonas europaea?

The regulation of metK expression in N. europaea likely involves multiple mechanisms that coordinate SAM production with cellular methylation needs. While specific regulatory mechanisms for N. europaea metK have not been extensively characterized, research on related bacteria suggests that metK expression is regulated by:

• Feedback inhibition by SAM: High SAM concentrations typically repress metK expression through several potential mechanisms, including SAM-responsive riboswitches that can regulate transcription or translation.

• Methionine availability: As a direct substrate for MetK, methionine levels influence metK expression through metabolic sensing mechanisms.

• Environmental stress conditions: Various stressors (oxidative stress, pH changes, nutrient limitation) may alter metK expression to adjust methylation capacity during adaptation.

• Growth phase-dependent regulation: MetK expression often varies with growth phase to match the changing methylation demands during different cellular states.

Experimental approaches to study metK regulation in N. europaea include constructing promoter-reporter fusions, quantitative RT-PCR analysis under various conditions, and implementing conditional expression systems similar to those used in other bacteria .

What genomic insights have been gained about metK in Nitrosomonas europaea?

Genomic analysis of N. europaea has revealed several important features regarding metK:

Understanding the genomic context of metK provides valuable insights into its evolutionary conservation and functional importance in the specialized metabolism of this ammonia-oxidizing bacterium.

What are the recommended methods for cloning and expressing recombinant N. europaea metK?

For successful cloning and expression of recombinant N. europaea metK, researchers should consider the following methodological approach:

• Gene amplification: Design primers based on the annotated metK sequence from the N. europaea genome (ATCC 19718), including appropriate restriction enzyme sites for directional cloning. Consider codon optimization if expressing in a heterologous host with different codon bias.

• Vector selection: Choose an expression vector suitable for bacterial expression:

  • pET series vectors for E. coli expression (pET28a for N-terminal His-tag)

  • pMAL vectors for expression as maltose-binding protein fusions to increase solubility

  • pSK vectors with IPTG-inducible promoters for conditional expression systems

• Expression conditions optimization:

  • Host strain: E. coli BL21(DE3) or Rosetta for rare codon supplementation

  • Induction temperature: 16-25°C to enhance protein solubility

  • IPTG concentration: 0.1-0.5 mM typically sufficient

  • Expression duration: 4-16 hours depending on temperature

  • Media composition: LB or TB supplemented with appropriate antibiotics

• Purification strategy:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged proteins

  • Follow with size exclusion chromatography to obtain highly pure enzyme

  • Consider ion exchange chromatography as an additional purification step

• Activity verification:

  • Enzymatic assays measuring ATP consumption or SAM formation

  • Western blot analysis using anti-His antibodies or MetK-specific antibodies

  • Mass spectrometry to confirm protein identity

This systematic approach ensures production of pure, active recombinant MetK suitable for subsequent biochemical and functional studies.

What assays can be used to measure the enzymatic activity of recombinant N. europaea metK?

Several robust assays can be employed to measure MetK enzymatic activity:

When reporting activity, standardize to specific activity (nmol SAM produced per minute per mg of enzyme) under defined reaction conditions:

• pH (typically 7.5-8.5)

• Temperature (25-37°C)

• Substrate concentrations (ATP and methionine)

• Mg2+ concentration (typically 5-10 mM)

This multi-assay approach provides comprehensive characterization of the catalytic properties of recombinant N. europaea MetK under various conditions.

How can RNA-binding properties of N. europaea MetK be investigated?

Based on the recently discovered RNA-binding capability of MetK in Sinorhizobium meliloti , investigating similar properties in N. europaea MetK requires specialized methodological approaches:

• In vitro RNA binding assays:

  • Filter binding assays using purified recombinant MetK and radiolabeled RNA transcripts

  • Electrophoretic mobility shift assays (EMSA) to visualize MetK-RNA complexes

  • Fluorescence anisotropy measurements using fluorescently labeled RNA

  • Surface plasmon resonance (SPR) for real-time binding kinetics

• RNA target identification:

  • RNA immunoprecipitation (RIP) using FLAG-tagged MetK expressed in N. europaea

  • RIP-seq to identify binding targets genome-wide

  • CLIP-seq (cross-linking immunoprecipitation) for higher resolution of binding sites

• Binding specificity determination:

  • Test various RNA species (sRNAs, tRNAs, mRNAs) for differential binding

  • Competition assays with unlabeled RNA to determine relative affinities

  • Mutational analysis of RNA structures to identify binding determinants

• Functional analysis:

  • Effect of MetK binding on RNA stability using pulse-chase experiments

  • Influence on translation efficiency using reporter constructs

  • Potential effects on RNA structure using chemical probing methods

The dot-blot filtration assay method described for S. meliloti MetK is particularly useful, where increasing concentrations of purified MetK protein are incubated with radiolabeled RNA, and the mixture is applied to nitrocellulose and PVDF membranes to capture protein-RNA complexes and free RNA, respectively. This allows calculation of dissociation constants (KD values) for different RNA-MetK interactions .

How does MetK contribute to methylation-dependent processes in Nitrosomonas europaea?

MetK plays a central role in numerous methylation-dependent processes in N. europaea through SAM production:

• DNA methylation:

  • Methylation of specific DNA sequences influencing gene expression

  • Protection against restriction endonucleases

  • Potential role in DNA replication timing and chromosome organization

• Protein methylation:

  • Post-translational modification of enzymes involved in ammonia oxidation

  • Methylation of transcription factors affecting regulatory networks

  • Modification of structural proteins impacting cellular architecture

• Metabolite methylation:

  • Biosynthesis of secondary metabolites

  • Production of compatible solutes for stress protection

  • Modification of cell wall components affecting permeability

• RNA modification:

  • Methylation of rRNA affecting ribosome assembly and function

  • tRNA modifications influencing translation efficiency and fidelity

  • Potential regulatory RNA modifications

Research approaches to investigate these processes include:

• Comparative genomics to identify methyltransferase repertoire in N. europaea

• Metabolomic profiling of methylated compounds under varying MetK expression

• Proteomic analysis of methylated proteins using specific antibodies and mass spectrometry

• Genome-wide methylation profiling using bisulfite sequencing

These methylation-dependent processes likely influence how N. europaea adapts to environmental changes and maintains its specialized metabolism centered on ammonia oxidation.

What is the potential role of MetK in the RNA biology of Nitrosomonas europaea?

Recent discoveries regarding MetK's RNA-binding capability in S. meliloti suggest potential novel functions in N. europaea RNA biology:

• RNA binding capacity:

  • N. europaea MetK may share the unexpected ability to bind various RNA species despite lacking conventional RNA-binding domains

  • Binding is likely structure-dependent rather than sequence-specific

  • Estimated binding affinities (KD values) for different RNA targets in S. meliloti range from 1.3 nM for tRNA to 45.6 nM for sRNAs

• Potential target diversity:

  • May interact with regulatory sRNAs involved in post-transcriptional regulation

  • Strong affinity for tRNAs suggests potential role in translation

  • Could bind mRNAs encoding key metabolic enzymes

  • May recognize specific RNA structural motifs common across different transcripts

• Functional implications:

  • Potential role as an RNA chaperone, similar to Hfq protein

  • May protect certain RNAs from degradation

  • Could influence translation efficiency of specific transcripts

  • Might coordinate cellular metabolism (via SAM levels) with gene expression

• Integration with methylation function:

  • Dual functionality may allow coordination between SAM synthesis and RNA regulation

  • RNA binding could potentially regulate MetK's own enzymatic activity

  • May represent a moonlighting function evolved in response to metabolic constraints

This RNA-binding capability represents an exciting frontier in understanding MetK beyond its canonical enzymatic function, potentially revealing new layers of metabolic integration in N. europaea .

How does N. europaea MetK function compare with MetK from other organisms?

Comparative analysis of MetK across different organisms reveals important evolutionary adaptations:

N. europaea MetK likely shows specialized adaptations related to:

• Function in an obligate chemolithoautotroph with limited carbon sources

• Integration with ammonia oxidation pathways

• Adaptation to environments with fluctuating ammonia concentrations

• Operation within the constraints of a specialized metabolic network

Comparative biochemical studies would reveal how N. europaea MetK has evolved to support the unique metabolic requirements of ammonia-oxidizing bacteria while maintaining its core enzymatic function.

What is the relationship between MetK activity and the nitrification process in N. europaea?

The relationship between MetK activity and nitrification in N. europaea likely involves multiple interconnected pathways:

• Enzyme regulation:

  • SAM-dependent methylation may modify key nitrification enzymes:

    • Ammonia monooxygenase (AMO)

    • Hydroxylamine oxidoreductase (HAO)

    • Nitrite reductase (NirK)

  • Methylation could alter enzyme activity, stability, or subcellular localization

• Transcriptional control:

  • DNA methylation patterns may regulate expression of nitrification genes

  • Methylation of transcription factors could influence their binding to promoters

  • SAM availability may serve as a metabolic sensor linking methylation to nitrogen status

• Metabolic integration:

  • The methionine cycle (including MetK) intersects with nitrogen assimilation pathways

  • SAM is required for synthesis of polyamines that protect against stress conditions

  • MetK activity may influence intracellular pH through byproducts of the methionine cycle

• Post-transcriptional regulation:

  • The newly discovered RNA-binding function of MetK may affect expression of nitrification genes

  • SAM-dependent RNA methylation could regulate transcript stability or translation

Research approaches to investigate these connections include:

• Metabolic flux analysis using stable isotope labeling

• Manipulation of metK expression combined with nitrification rate measurements

• Identification of methylated proteins in the nitrification pathway

• Transcriptomic and proteomic analyses under varying SAM availability

Understanding these relationships would provide insights into how methylation processes contribute to the specialized metabolism of this ammonia-oxidizing bacterium.

How does N. europaea MetK contribute to environmental stress responses?

N. europaea MetK likely plays multiple roles in environmental stress adaptation:

• Osmotic stress response:

  • SAM-dependent synthesis of osmoprotectants (glycine betaine, ectoine)

  • Methylation of membrane proteins affecting cell permeability

  • Regulation of compatible solute transporters

• Oxidative stress management:

  • SAM-dependent synthesis of antioxidant compounds

  • Regulation of enzymes involved in reactive oxygen species detoxification

  • Protection of proteins through specific methylation patterns

• pH adaptation:

  • Production of pH-buffering compounds

  • Modification of cell surface components affecting proton permeability

  • Regulation of pH-responsive genes through methylation-dependent mechanisms

• Heavy metal resistance:

  • Methylation of metals as a detoxification mechanism

  • Regulation of metal efflux systems

  • Production of protective compounds requiring SAM as methyl donor

• Temperature stress adaptation:

  • Methylation of proteins to maintain structure at temperature extremes

  • Production of thermoprotective compounds

  • Regulation of chaperone expression through methylation-dependent processes

These adaptive processes would allow N. europaea to maintain nitrification activity under varying environmental conditions encountered in both natural and engineered systems, with MetK serving as a key connection between environmental sensing and cellular response mechanisms.

How can recombinant N. europaea MetK be used to study methylation patterns in environmental samples?

Recombinant N. europaea MetK provides a valuable tool for investigating methylation patterns in environmental samples:

• Environmental epigenetics studies:

  • Use purified MetK to generate isotopically labeled SAM as a tracer

  • Apply to environmental samples to identify actively methylated DNA and proteins

  • Compare methylation patterns across different environments

• Community-level analysis:

  • Combine MetK-dependent SAM synthesis with metagenomics approaches

  • Identify methylation patterns in uncultivated ammonia-oxidizing microorganisms

  • Assess methylation as an adaptation mechanism across ecological niches

• Wastewater treatment applications:

  • Study methylation patterns in nitrifying biofilms

  • Correlate methylation status with nitrification efficiency

  • Manipulate methylation to potentially enhance treatment performance

• Agricultural soil studies:

  • Investigate methylation in response to fertilizer application

  • Analyze adaptation of ammonia oxidizers to changing nitrogen loads

  • Develop molecular markers based on methylation patterns

• Methodology development:

  • Use recombinant MetK to produce SAM for in vitro methylation reactions

  • Develop targeted methylation profiling techniques for environmental samples

  • Create methylation-specific biosensors using MetK-dependent systems

This approach connects fundamental enzymatic studies with applied environmental research, potentially revealing how methylation processes influence microbial community function in natural and engineered systems where nitrification is a key process.

What structural and mechanistic studies could advance our understanding of N. europaea MetK?

Several advanced structural and mechanistic studies could significantly enhance our understanding of N. europaea MetK:

• High-resolution structure determination:

  • X-ray crystallography of purified recombinant N. europaea MetK

  • Cryo-electron microscopy of MetK in different conformational states

  • Structural comparison with MetK from other bacteria to identify adaptations

• Mechanistic investigations:

  • Pre-steady-state kinetics to identify rate-limiting steps

  • Isotope effect studies to probe transition state structures

  • Computational modeling of the reaction coordinate

• RNA-binding interface mapping:

  • Hydrogen-deuterium exchange mass spectrometry to identify RNA-binding regions

  • Cross-linking studies to capture RNA-protein interaction sites

  • Structure determination of MetK-RNA complexes

• Structure-function analysis:

  • Site-directed mutagenesis of catalytic and RNA-binding residues

  • Domain swapping with MetK from other bacteria

  • Construction of enzymes with separated catalytic and RNA-binding functions

• Interaction networks:

  • Identification of protein interaction partners through co-immunoprecipitation

  • Analysis of higher-order complexes by native mass spectrometry

  • Investigation of subcellular localization and potential compartmentalization

These studies would provide mechanistic insights into how this dual-function enzyme operates in the specialized metabolic context of an ammonia-oxidizing bacterium, potentially revealing unique adaptations that support N. europaea's ecological niche.

What systems biology approaches could integrate MetK function into the broader metabolic network of N. europaea?

Systems biology approaches offer powerful tools to integrate MetK function into N. europaea's metabolic network:

• Multi-omics integration:

  • Combining transcriptomics, proteomics, and metabolomics data

  • Correlating MetK expression with global methylation patterns

  • Mapping the effects of MetK perturbation across multiple cellular processes

• Genome-scale metabolic modeling:

  • Incorporation of SAM-dependent reactions into metabolic models

  • Flux balance analysis to predict effects of altered MetK activity

  • Identification of metabolic bottlenecks involving methylation reactions

• Network analysis:

  • Construction of methylation-dependent regulatory networks

  • Identification of hub points connecting methylation to other cellular processes

  • Comparison with metabolic networks of other ammonia oxidizers

• Synthetic biology approaches:

  • Engineering MetK variants with altered properties

  • Creating conditional expression systems for controlled perturbation

  • Developing biosensors for intracellular SAM levels

• Mathematical modeling:

  • Dynamic models of the methionine cycle and connected pathways

  • Stochastic modeling of MetK's dual enzymatic and RNA-binding functions

  • Multi-scale models linking molecular events to cellular phenotypes

Such integrated approaches would provide a comprehensive understanding of how MetK connects methylation processes to the core metabolism of N. europaea, potentially revealing new principles of metabolic integration in specialized bacteria.

How might studying N. europaea MetK contribute to biotechnological applications?

Research on N. europaea MetK has several potential biotechnological applications:

• Bioremediation enhancement:

  • Understanding methylation-dependent adaptation could improve nitrification in contaminated environments

  • Engineering MetK to enhance stress resistance in bioremediation applications

  • Developing biosensors for monitoring nitrification efficiency based on methylation status

• Wastewater treatment optimization:

  • Knowledge of MetK function could lead to improved nitrification in treatment systems

  • Manipulation of methylation patterns to enhance ammonia removal efficiency

  • Development of molecular markers for monitoring nitrifier population health

• Enzyme engineering:

  • Creating MetK variants with improved catalytic properties for SAM production

  • Developing MetK-based biosynthetic systems for methylated compounds

  • Engineering the RNA-binding function for specific biotechnological applications

• Agriculture applications:

  • Understanding nitrogen cycle methylation patterns could inform fertilizer management

  • Developing soil amendments that optimize nitrifier activity through methylation effects

  • Creating diagnostic tools for monitoring soil nitrification potential

• Analytical tools:

  • Using recombinant MetK for producing labeled SAM for analytical applications

  • Developing MetK-based assays for environmental monitoring

  • Creating methylation-specific detection systems for environmental samples