Recombinant Legionella pneumophila subsp. pneumophila S-adenosylmethionine synthase (metK)

How does recombinant L. pneumophila metK expression differ from other bacterial expression systems?

Expression of recombinant L. pneumophila metK presents unique challenges compared to model organisms like E. coli due to several factors:

• Genetic accessibility: L. pneumophila has historically been more challenging to manipulate genetically than model organisms. RecA-independent recombination techniques have emerged as valuable tools for genetic engineering in L. pneumophila, utilizing short homologous sequences for efficient gene modification .

• Regulatory elements: The metK gene in bacteria typically contains specific regulatory elements in the 5' region, including symmetrical sequences that function as operators and are homologous to sequences upstream from other met genes sharing the same regulatory mechanism . These must be carefully considered when designing expression constructs.

• SOS response differences: L. pneumophila possesses divergent SOS response machinery compared to E. coli, lacking LexA and SulA but containing two copies of Pol V . These differences may affect the expression of recombinant proteins, especially when stress responses are triggered.

• Growth conditions: L. pneumophila's slower growth rate and specific nutritional requirements necessitate careful optimization of expression conditions to achieve adequate yields of functional recombinant metK protein.

For optimal expression, researchers often employ:

• Shuttle vectors capable of replication in both E. coli and L. pneumophila

• Inducible promoter systems to control expression timing

• Codon optimization strategies to improve translation efficiency

• Fusion tags that facilitate purification while minimizing interference with enzymatic activity

What experimental approaches are most effective for studying the enzymatic activity of recombinant L. pneumophila metK?

Characterizing the enzymatic activity of recombinant L. pneumophila metK requires a multi-faceted approach:

Spectrophotometric assays: The most common method measures either the consumption of ATP or production of inorganic phosphate during the conversion of methionine to S-adenosylmethionine. These assays typically employ:

• Malachite green assay for phosphate detection

• Coupled enzyme systems (pyruvate kinase/lactate dehydrogenase) to monitor ATP consumption through NADH oxidation

• Specific conditions: pH 7.5-8.0, Mg²⁺ as cofactor, and temperatures of 30-37°C

HPLC analysis: For precise quantification of S-adenosylmethionine production, high-performance liquid chromatography offers superior sensitivity and specificity. This approach allows:

• Direct measurement of SAM formation

• Kinetic parameter determination (Km, Vmax, kcat)

• Assessment of product inhibition patterns

Isothermal titration calorimetry (ITC): Provides detailed thermodynamic analysis of substrate binding and catalysis:

• Binding affinities for ATP and methionine

• Thermodynamic parameters (ΔH, ΔS, ΔG)

• Allosteric effects of regulatory molecules

Methyltransferase-coupled assays: Since SAM serves as a methyl donor, coupling metK activity to specific methyltransferases can provide insights into functional relevance within methylation pathways that may be altered during L. pneumophila infection of host cells .

How might DNA methylation patterns mediated by metK-generated SAM influence L. pneumophila virulence?

DNA methylation represents a critical epigenetic mechanism that can dramatically alter gene expression without changing the underlying genetic sequence. In L. pneumophila, methylation patterns likely play important roles in virulence regulation:

• Temporal regulation of virulence factors: Methylation may serve as a mechanism for controlling the expression timing of the over 300 effector proteins that L. pneumophila secretes into host cells during infection . This precise temporal control ensures appropriate deployment of virulence factors at different infection stages.

• Adaptation to host environment: Methylation patterns might change in response to host cell conditions, allowing L. pneumophila to adapt its gene expression profile for optimal intracellular replication within Legionella-containing vacuoles (LCVs) .

• Immune evasion strategies: Recent research has shown that L. pneumophila infection induces methylomic changes in host cells, specifically affecting ten-eleven translocation (TET) genes, which play crucial roles in DNA demethylation processes . This suggests complex interplay between bacterial and host methylation systems.

• Stress response modulation: The unique SOS response machinery in L. pneumophila likely relies on methylation-dependent regulatory mechanisms different from those in model organisms like E. coli .

Experimental evidence indicates that methylation inhibitors like 5-azacytidine (5-AZA) and (-)-epigallocatechin-3-O-gallate (EGCG) significantly reduce L. pneumophila reproduction in infected cells, demonstrating the biological importance of methylation processes in this pathogen's life cycle . These inhibitors were shown to target methionine synthase, which works upstream of metK in the same metabolic pathway, indirectly affecting SAM production and subsequent methylation reactions.

What genetic engineering approaches enable functional studies of metK in L. pneumophila?

Several genetic engineering techniques have proven effective for studying gene function in L. pneumophila:

RecA-independent recombination: This approach, which has been specifically demonstrated in L. pneumophila, relies on short homologous sequences to facilitate genetic modifications without requiring the RecA recombinase . Key features include:

• Utilization of oligonucleotides with 20-50 bp homology arms

• Higher efficiency compared to traditional homologous recombination

• Applicability for creating precise point mutations and small insertions/deletions

Phage recombination coupled with site-specific Flp recombination: This two-step approach allows for the construction of unmarked deletions in L. pneumophila :

• Initial recombination event mediated by phage-derived recombination proteins

• Subsequent removal of selection markers using Flp recombinase

• Generation of scarless mutations for studying gene function without polar effects

CRISPR-Cas9 systems: Adapted for use in L. pneumophila, CRISPR-based approaches offer:

• Precise genome editing capabilities

• Multiplexed targeting of several genes simultaneously

• Both knock-out and knock-in strategies

Shuttle vectors and complementation: For functional validation studies:

• Reintroduction of wild-type or mutant metK variants

• Controlled expression using inducible promoters

• Assessment of phenotype rescue to confirm gene function

These approaches can be applied to study metK through:

• Creation of conditional mutants (temperature-sensitive or inducible)

• Site-directed mutagenesis of key catalytic residues

• Domain swapping with homologs from other species

• Reporter fusions to monitor expression patterns during infection

How do methylomic changes induced by L. pneumophila infection relate to metK function?

Recent research has revealed that L. pneumophila infection induces specific methylomic changes in host cells, particularly affecting the ten-eleven translocation (TET) genes responsible for DNA demethylation processes . These findings suggest complex interactions between bacterial methylation systems and host epigenetic regulation.

The potential relationships between these observed methylomic changes and metK function include:

Bacterial SAM availability influencing host methylation: The SAM produced by L. pneumophila metK may directly or indirectly affect host cell methylation patterns. During intracellular growth, bacterial metabolites can influence host cellular processes. If L. pneumophila exports SAM or depletes methionine from host cells, this could alter the host's own methylation capacity.

Coordinated methylation targeting: Research has demonstrated that L. pneumophila infection leads to specific methylation changes within the promoter regions of TET1 and TET3 genes, located on CpG/397–8 and CpG/385–6, respectively . This precise targeting suggests a coordinated mechanism rather than random methylation effects.

Temporal dynamics of methylation changes: The methylation inhibitors 5-AZA and EGCG significantly decrease L. pneumophila reproduction in infected cells, suggesting that methylation processes are crucial for bacterial replication . The timing of these methylation changes likely corresponds to specific phases of the infection cycle when metK activity might be differentially regulated.

Mechanistic pathways: Methylation inhibitors show potent inhibition of methionine synthase expression, an enzyme that functions upstream of metK in the same metabolic pathway . This indicates that disrupting the methionine cycle at different points can impair L. pneumophila's ability to establish successful infection, highlighting the importance of this metabolic pathway.

A conceptual model integrating these findings would suggest that L. pneumophila utilizes metK-generated SAM not only for its own methylation needs but potentially as part of a broader strategy to manipulate host cell epigenetic regulation, creating a more favorable environment for bacterial replication.

What structural features of L. pneumophila metK might explain its potential role in pathogenesis?

While the specific crystal structure of L. pneumophila metK has not been definitively characterized in the provided research, comparative analysis with homologous enzymes reveals several structural features likely relevant to its function in pathogenesis:

Catalytic domains and active site architecture: S-adenosylmethionine synthases typically contain three key domains:

• N-terminal domain for ATP binding

• Central domain for methionine binding

• C-terminal domain involved in trimerization and catalytic activity

The precise configuration of these domains in L. pneumophila metK may confer unique kinetic properties or substrate specificities adapted to the intracellular lifestyle of this pathogen.

Oligomerization interfaces: Most bacterial metK enzymes function as homo-tetramers, with oligomerization essential for catalytic activity. Any unique features at these interfaces in L. pneumophila metK could affect enzyme stability under the stressful conditions encountered during infection.

Regulatory elements: The metK gene in bacteria typically contains specific regulatory regions including symmetrical sequences suggestive of operator structures . These regions may respond to host-derived signals during infection, allowing L. pneumophila to modulate SAM production based on environmental cues.

Post-translational modification sites: Potential phosphorylation, methylation, or other modification sites on L. pneumophila metK could serve as regulatory switches, integrating the enzyme's activity with broader virulence programs.

Understanding these structural features would require advanced approaches:

• X-ray crystallography or cryo-EM studies of purified recombinant L. pneumophila metK

• Molecular dynamics simulations to analyze conformational changes during catalysis

• Site-directed mutagenesis of predicted key residues followed by functional assays

• Comparative analysis with metK structures from non-pathogenic Legionella species

These structural investigations could reveal unique adaptations that enable L. pneumophila metK to function optimally during intracellular infection cycles.

How might recombinant L. pneumophila metK interact with effector proteins during host infection?

L. pneumophila secretes more than 300 effector proteins into host cells to facilitate intracellular replication . While direct interactions between metK and specific effector proteins haven't been definitively established in the literature provided, several potential mechanisms of interaction can be proposed based on current knowledge:

Methylation of effector proteins: Many bacterial effectors undergo post-translational modifications that regulate their activity, localization, or stability. metK-generated SAM could serve as the methyl donor for methyltransferases that modify these effectors, creating a functional link between methionine metabolism and virulence factor regulation.

Temporal coordination with effector secretion: The expression and activity of metK might be synchronized with the deployment of specific effectors during different stages of infection. For example, the metaeffector LubX, which targets the effector SidH for degradation in a temporal manner during infection , represents an example of such temporal regulation that could potentially be influenced by methylation-dependent processes.

Indirect regulation through methylation-sensitive transcription factors: metK-dependent methylation could influence the expression of effector genes by modifying the activity of transcription factors or other regulatory proteins.

Co-localization at specific cellular sites: metK and certain effector proteins might co-localize at specific subcellular locations during infection, particularly at the Legionella-containing vacuole (LCV) membrane, where many effectors are known to act .

Experimental approaches to investigate these potential interactions include:

• Co-immunoprecipitation studies with tagged recombinant metK

• Protein interaction screens using techniques like bacterial two-hybrid systems

• Metabolic labeling with SAM analogs to identify methylated effector proteins

• Live-cell imaging to track co-localization of fluorescently tagged metK and effector proteins

What role does metK play in L. pneumophila's adaptation to different host environments?

L. pneumophila must adapt to diverse environmental conditions, from natural aquatic habitats to different host cell types, including both amoebae and human macrophages. The metK enzyme likely plays a crucial role in these adaptation processes:

Differential expression in various growth conditions: The expression level and activity of metK may vary depending on the environment L. pneumophila encounters. This adaptation would allow the bacterium to adjust its methylation capacity based on the specific nutritional and stress conditions present in different hosts.

Role in stress response: L. pneumophila has evolved a unique SOS response machinery that differs from model organisms like E. coli, lacking LexA and SulA but containing two copies of Pol V . This divergent stress response system likely involves methylation-dependent regulatory mechanisms in which metK plays a central role.

Contribution to metabolic reprogramming: During transition between environmental reservoirs and mammalian hosts, L. pneumophila undergoes significant metabolic reprogramming. As a key enzyme in central metabolism, metK would be integral to these shifts, particularly in adjusting the balance between growth and virulence.

Involvement in biofilm formation: In natural environments, L. pneumophila often exists within biofilms, which provide protection and access to nutrients. Methylation-dependent processes regulated by metK-generated SAM may influence biofilm formation and maintenance.

Host-specific adaptations: The bacterium's interaction with different host species (from environmental amoebae to human macrophages) may require specific methylation patterns that optimize virulence factor expression for each host type.

Experimental approaches to investigate these roles include:

• Comparative transcriptomics and proteomics of L. pneumophila grown in different conditions

• Analysis of metK expression patterns during different stages of infection

• Measurement of intracellular SAM levels in various growth environments

• Creation of metK variants with altered regulatory properties to assess impact on host adaptation

Understanding these adaptation mechanisms could reveal new targets for therapeutic intervention that specifically disrupt L. pneumophila's ability to transition between different environments.

How do inhibitors of methylation pathways affect L. pneumophila metK function and bacterial virulence?

Recent research has demonstrated that methylation inhibitors can significantly disrupt L. pneumophila replication, suggesting they represent promising targets for therapeutic intervention . The effects of these inhibitors on metK function and bacterial virulence operate through several mechanisms:

Direct inhibition of methylation processes: Compounds like 5-azacytidine (5-AZA) and (-)-epigallocatechin-3-O-gallate (EGCG) have been shown to significantly decrease L. pneumophila reproduction in infected cells . These inhibitors target methylation processes that depend on the SAM produced by metK.

Disruption of methionine synthase activity: Both 5-AZA and EGCG exhibit potent inhibition of methionine synthase (MS) expression, as confirmed by docking analysis of inhibitor ligands and the crystal structure of MS protein . Since methionine synthase functions upstream of metK in the same metabolic pathway, this inhibition indirectly affects metK function by limiting methionine availability.

Reversal of host epigenetic modifications: L. pneumophila infection induces specific methylation changes in host cells, particularly affecting the promoter regions of ten-eleven translocation (TET) genes . Methylation inhibitors may counteract these changes, preventing the bacterium from creating a favorable host environment.

Alteration of virulence factor expression: Disrupting methylation pathways likely affects the expression and function of numerous virulence factors, including the more than 300 effector proteins that L. pneumophila secretes into host cells .

The effectiveness of methylation inhibitors has been experimentally demonstrated:

• Pre-treatment of A549 cells with 5-AZA or EGCG significantly decreased bacterial reproduction

• Inhibition was quantified through the expression of L. pneumophila 16S ribosomal RNA

• Bacterial colony-forming units (CFU/ml) were substantially reduced following inhibitor treatment

These findings suggest that targeting methylation pathways, including those dependent on metK-generated SAM, represents a promising approach for developing new therapeutic strategies against L. pneumophila infections.

What expression systems and purification strategies yield optimal results for recombinant L. pneumophila metK?

Obtaining high-quality recombinant L. pneumophila metK requires careful consideration of expression systems and purification strategies:

Expression Systems:

• E. coli-based expression: Despite physiological differences between E. coli and L. pneumophila, E. coli remains the most common host for initial recombinant protein production due to:

  • Rapid growth and high protein yields

  • Well-established genetic tools and expression vectors

  • Availability of specialized strains (BL21(DE3), Rosetta, Arctic Express) optimized for different aspects of protein expression

  Recommended approaches include:

  • Using tightly regulated promoters (T7, araBAD) to control expression levels

  • Lowering induction temperature (16-25°C) to improve proper folding

  • Codon optimization of the L. pneumophila metK gene for E. coli expression

  • Co-expression with chaperone proteins if solubility issues arise

• Legionella-based expression: For studies requiring native post-translational modifications or authentic folding environment:

  • Development of shuttle vectors compatible with both E. coli and L. pneumophila

  • Utilization of RecA-independent recombination techniques for genetic manipulation

  • Integration of the recombinant gene into the Legionella chromosome under native or controlled promoters

Purification Strategies:

• Affinity tags:

  • His6-tag: Most commonly used, allowing purification by IMAC

  • GST-tag: Enhances solubility but may affect enzymatic activity

  • MBP-tag: Excellent for improving solubility while maintaining activity

  • SUMO-tag: Allows for tag removal that leaves no additional amino acids

• Chromatography sequence:

  • Initial capture: Affinity chromatography (IMAC, GST, etc.)

  • Intermediate purification: Ion exchange chromatography to separate charge variants

  • Polishing: Size exclusion chromatography to ensure homogeneity and remove aggregates

• Stability considerations:

  • Addition of cofactors (Mg²⁺, K⁺) to all buffers

  • Inclusion of reducing agents (DTT, TCEP) to prevent oxidation

  • Addition of glycerol (10-20%) to enhance stability during storage

  • Optimization of pH (typically 7.5-8.0) and ionic strength

What assays best characterize the enzymatic activity and kinetic properties of recombinant L. pneumophila metK?

Comprehensive characterization of recombinant L. pneumophila metK requires multiple complementary assays:

Direct Activity Assays:

• Radioactive assay: Measures the incorporation of ¹⁴C-labeled methionine into SAM

  • Highest sensitivity (detection limit ~1 nM)

  • Direct quantification of product formation

  • Requires radioisotope handling facilities and specialized waste disposal

• HPLC-based assay: Quantifies SAM formation by chromatographic separation

  • Excellent specificity and quantification

  • Can simultaneously monitor substrate depletion and product formation

  • Compatible with high-throughput screening when using 96-well formats

• Fluorescence-based assay: Utilizes SAM-dependent methyltransferases coupled with fluorescent methylation sensors

  • Real-time monitoring capabilities

  • Amenable to high-throughput formats

  • May be affected by interfering compounds

Kinetic Parameter Determination:

• Michaelis-Menten kinetics: Determination of Km, Vmax, and kcat for methionine and ATP

  • Requires varying one substrate concentration while keeping others saturating

  • Multiple replicates at 8-12 substrate concentrations for reliable curve fitting

  • Analysis for potential substrate inhibition at high concentrations

• Product inhibition studies: Assessment of SAM and PPi inhibition patterns

  • Important for understanding regulation during cellular conditions

  • Helps distinguish between ordered and random reaction mechanisms

• pH and temperature profiles: Optimization of reaction conditions

  • Typically performed in buffered solutions ranging from pH 6.0-9.0

  • Temperature range of 25-45°C to determine optimal conditions

Structural and Biophysical Approaches:

• Circular dichroism (CD): Analyzes secondary structure content and stability

  • Thermal denaturation studies to determine melting temperature (Tm)

  • Assessment of structural changes upon substrate binding

• Isothermal titration calorimetry (ITC): Provides thermodynamic binding parameters

  • Direct measurement of binding constants, enthalpy, and entropy

  • Can reveal binding stoichiometry and potential allosteric effects

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS): Determines oligomeric state

  • Confirms expected tetrameric structure of active enzyme

  • Identifies potential dissociation under different conditions

These assays should be performed under physiologically relevant conditions, considering the environment L. pneumophila encounters during infection (pH ~6.5-7.5, varying ionic strength, potential chelating agents).

How can site-directed mutagenesis be applied to study functional domains of L. pneumophila metK?

Site-directed mutagenesis represents a powerful approach for dissecting the structure-function relationships of L. pneumophila metK. Based on knowledge of conserved domains in SAM synthetases, several strategic approaches can be implemented:

Targeting Catalytic Residues:

• ATP binding site mutations: Key residues likely include conserved glycine-rich motifs and lysine residues that coordinate the triphosphate moiety

  • Mutations to alanine should abolish ATP binding and enzymatic activity

  • Conservative substitutions may alter kinetic parameters without eliminating activity

  • Outcomes measured through ATP binding assays and catalytic activity measurements

• Methionine binding site alterations: Targeting residues that coordinate the amino and carboxyl groups of methionine

  • Mutations affecting substrate specificity might allow utilization of methionine analogs

  • Changes in binding pocket size could alter Km values without affecting kcat

  • Analysis through methionine binding studies and competitive inhibition assays

• Metal coordination site mutations: SAM synthetases require divalent cations (typically Mg²⁺)

  • Substitutions of metal-coordinating aspartate or glutamate residues

  • Assessment of activity with different divalent cations (Mg²⁺, Mn²⁺, Ca²⁺)

  • Potential to engineer altered metal specificity

Oligomerization Interface Modifications:

• Disruption of tetramer formation: SAM synthetases typically function as tetramers

  • Mutations at subunit interfaces should affect enzyme stability and activity

  • Size exclusion chromatography to confirm altered oligomeric states

  • Correlation between oligomerization and catalytic efficiency

• Cross-subunit communication: Investigation of allosteric regulation

  • Mutations at interfaces that potentially mediate cooperative behavior

  • Hill coefficient determination to assess cooperativity changes

  • Potential to engineer variants with altered regulatory properties

Regulatory Domain Analysis:

• C-terminal domain modifications: Often involved in regulation of activity

  • Truncation analysis to identify minimal catalytic core

  • Point mutations at potential regulatory sites

  • Assessment of impact on product inhibition patterns

Experimental Workflow:

• Initial design: Based on:

  • Homology modeling using E. coli metK structure as template

  • Sequence conservation analysis across bacterial SAM synthetases

  • Prediction of functionally important residues using computational tools

• Mutagenesis techniques:

  • QuikChange PCR for simple substitutions

  • Overlap extension PCR for multiple mutations

  • RecA-independent recombination for chromosomal integration

• Validation methods:

  • Western blotting to confirm expression of mutant proteins

  • Circular dichroism to verify proper folding

  • Thermal shift assays to assess stability changes

  • Complete kinetic characterization of promising variants

What are the best experimental approaches to study the role of metK in L. pneumophila's intracellular life cycle?

Investigating the role of metK during L. pneumophila's intracellular life cycle requires integrating genetic manipulation, infection models, and molecular analyses:

Genetic Approaches:

• Conditional metK mutants: Since metK is likely essential, conditional approaches are necessary

  • Temperature-sensitive mutants for temporal control

  • Inducible expression systems (tetracycline-responsive)

  • Degradation tag systems for protein-level control

  • CRISPR interference (CRISPRi) for partial knockdown

• Reporter fusions: Monitor metK expression during infection

  • Transcriptional fusions (metK promoter driving fluorescent protein)

  • Translational fusions (if metK function tolerates C-terminal tags)

  • Dual reporters to normalize for bacterial numbers in host cells

• Point mutations: Engineer variants with altered catalytic properties

  • Reduced activity mutants to identify threshold requirements

  • Substrate specificity variants to probe metabolic functions

  • Regulation-insensitive mutants to assess feedback importance

Infection Models:

• Cell culture systems:

  • Human alveolar epithelial cells (A549) to model lung infection

  • Human macrophage-like cells (THP-1) to study primary target cells

  • Amoebae (Acanthamoeba castellanii) as environmental hosts

  • Comparative analysis across cell types to identify host-specific requirements

• Microscopy approaches:

  • Visualization of bacteria within Legionella-containing vacuoles (LCVs)

  • Co-localization studies with markers of different infection stages

  • Live-cell imaging to track dynamics of metK expression

  • Super-resolution microscopy for precise subcellular localization

• Infection parameters to monitor:

  • Bacterial entry efficiency

  • Intracellular replication rates

  • LCV formation and maturation

  • Host cell viability and inflammatory responses

Molecular and Biochemical Analyses:

• Metabolite profiling:

  • Measurement of intracellular SAM levels during infection

  • Quantification of methylated metabolites in bacteria and host cells

  • Isotope labeling to track methionine utilization pathways

• Methylation analyses:

  • Global methylome profiling (DNA, RNA, proteins)

  • Site-specific methylation assessment of key virulence factors

  • Temporal changes in methylation patterns during infection stages

• Inhibitor studies:

  • Treatment with methylation inhibitors like 5-AZA and EGCG at different infection stages

  • Rescue experiments with exogenous SAM supplementation

  • Combination with specific virulence factor inhibitors to identify synergistic targets

• Transcriptomic and proteomic analyses:

  • RNA-seq to identify genes affected by metK modulation

  • Proteomics to detect changes in protein expression and post-translational modifications

  • Comparison between wild-type and metK mutant strains during infection

This integrated approach would provide comprehensive insights into how metK contributes to L. pneumophila's successful intracellular life cycle, potentially revealing new targets for therapeutic intervention.

How can computational approaches complement experimental studies of L. pneumophila metK?

Computational methods provide valuable complementary approaches to experimental studies of L. pneumophila metK, offering insights and predictions that can guide wet-lab investigations:

Structural Bioinformatics:

• Homology modeling: In the absence of a crystal structure, models based on homologous proteins

  • Templates from E. coli and other bacterial SAM synthetases

  • Refinement through molecular dynamics simulations

  • Validation through experimental testing of predicted critical residues

• Molecular docking simulations: Predict binding modes of substrates and inhibitors

  • Virtual screening of potential inhibitor libraries

  • Rational design of selective inhibitors for L. pneumophila metK

  • Predictions of binding affinities for experimental validation

• Molecular dynamics simulations: Analyze conformational dynamics

  • Investigation of protein flexibility and allosteric communication

  • Simulation of oligomerization processes

  • Prediction of stability changes in mutant variants

Genomic and Evolutionary Analyses:

• Comparative genomics: Analysis across Legionella species and strains

  • Identification of metK sequence conservation and variations

  • Correlation of metK variants with virulence phenotypes

  • Discovery of species-specific regulatory elements

• Phylogenetic analysis: Evolutionary relationships of metK across bacteria

  • Identification of pathogen-specific adaptations

  • Detection of horizontal gene transfer events

  • Correlation with host range and virulence potential

• Coevolution analysis: Identification of functionally linked genes

  • Prediction of protein interaction partners

  • Discovery of potential regulatory networks

  • Identification of compensatory mutations

Systems Biology Approaches:

• Metabolic modeling: Integration of metK within L. pneumophila metabolism

  • Flux balance analysis to predict metabolic dependencies

  • Identification of synthetic lethal interactions with metK

  • Prediction of metabolic adaptations during host infection

• Network analysis: Placing metK in the context of cellular pathways

  • Integration with transcriptomic and proteomic data

  • Identification of key regulatory nodes

  • Prediction of system-wide effects of metK perturbation

• Machine learning applications: Pattern recognition in large datasets

  • Classification of metK-dependent gene expression patterns

  • Prediction of environmental conditions affecting metK activity

  • Integration of multi-omics data to identify regulatory principles

These computational approaches can significantly accelerate experimental progress by:

• Generating testable hypotheses to focus wet-lab efforts

• Providing structural insights for rational experimental design

• Integrating diverse datasets to reveal non-obvious connections

• Predicting system-wide effects of metK perturbations

• Identifying the most promising targets for therapeutic intervention