Recombinant Shigella sonnei Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is the role of UbiB in ubiquinone biosynthesis in Shigella sonnei?

UbiB in Shigella sonnei likely functions as a key component in the ubiquinone (UQ) biosynthesis pathway. While specific research on S. sonnei UbiB is limited, studies on related Enterobacteriaceae suggest UbiB participates in the early steps of UQ biosynthesis, potentially functioning as a monooxygenase or hydroxylase. UbiB appears to be involved in the aerobic hydroxylation reactions required for ubiquinone synthesis, though its exact biochemical mechanism requires further characterization. The protein likely works in conjunction with other Ubi proteins to facilitate electron transport in the bacterial respiratory chain, making it essential for cellular bioenergetics .

How does UbiB relate to the virulence mechanisms of Shigella sonnei?

UbiB, as part of the ubiquinone biosynthesis pathway, indirectly contributes to S. sonnei virulence by supporting bacterial metabolism and survival under varying oxygen conditions. S. sonnei exhibits multiple virulence mechanisms, including the production of toxins, resistance to host antimicrobial peptides, and expression of colicins that kill phylogenetically related bacteria . The ubiquinone biosynthesis pathway likely supports these virulence mechanisms by enabling metabolic flexibility, particularly when S. sonnei encounters oxygen-limited environments within the host intestinal tract. While UbiB is not directly linked to the type III secretion system (a primary virulence factor in Shigella), its role in cellular bioenergetics makes it important for sustaining the energy requirements needed for bacterial invasion and survival within host cells .

What is known about the protein structure and conserved domains of S. sonnei UbiB?

The protein structure of S. sonnei UbiB has not been fully characterized, but comparative analysis with homologous proteins suggests it likely contains:

• A conserved kinase-like domain with an ATP-binding site

• Multiple transmembrane regions anchoring it to the cytoplasmic membrane

• Conserved motifs common to the ABC1 family of atypical kinases

A putative protein structural analysis would predict that UbiB contains around 6-7 transmembrane domains and likely forms oligomeric structures in the membrane. Multiple sequence alignments would reveal highly conserved residues across proteobacteria that are essential for function, particularly those involved in ATP binding and potential hydroxylase activity. Researchers should consider these structural elements when designing experiments for protein purification and functional characterization .

How do the O₂-dependent and O₂-independent ubiquinone biosynthesis pathways interact in S. sonnei, and what role might UbiB play in this interaction?

Recent research has uncovered the existence of parallel O₂-dependent and O₂-independent pathways for ubiquinone biosynthesis in proteobacteria, which allows bacteria to synthesize this essential electron carrier under varying oxygen conditions. In the O₂-dependent pathway, molecular oxygen serves as a co-substrate for hydroxylation reactions. In contrast, the O₂-independent pathway utilizes alternative hydroxylases, specifically the UbiU-UbiV complex containing 4Fe-4S clusters, to perform similar reactions without requiring molecular oxygen .

UbiB's role in this dual pathway system likely involves:

• Potential interaction with both pathways as a regulatory protein

• Differential expression under aerobic versus anaerobic conditions

• Possible functional redundancy with components of the O₂-independent pathway

Researchers investigating this interaction should design experiments comparing the expression and activity of UbiB under precisely controlled oxygen concentrations, possibly using continuous culture systems with oxygen monitoring. Co-immunoprecipitation studies could identify protein-protein interactions between UbiB and components of both pathways. Additionally, comparative metabolomic profiling of ubiquinone intermediates in wild-type versus ΔubiB mutants under varying oxygen conditions would provide insight into which specific biosynthetic steps are affected .

What methodological approaches are most effective for expressing and purifying recombinant S. sonnei UbiB for structural and functional studies?

Expressing and purifying membrane proteins like UbiB presents significant challenges. Based on current approaches for similar proteins, a recommended methodology would include:

Expression System Selection:

• E. coli BL21(DE3) strain is often suitable due to its reduced protease activity

• C41(DE3) or C43(DE3) strains are specifically engineered for membrane protein expression

• Codon optimization should be performed based on E. coli usage patterns, targeting a Codon Adaptation Index (CAI) of at least 0.8 to enhance expression levels

Vector Design:

• Incorporate an N-terminal His₆ or His₁₀ tag for purification

• Include a tobacco etch virus (TEV) protease cleavage site for tag removal

• Consider fusion partners like maltose-binding protein (MBP) to enhance solubility

Expression Conditions:

• Induction at lower temperatures (16-20°C) for 16-18 hours

• IPTG concentration optimization between 0.1-0.5 mM

• Addition of 0.5-1% glucose to reduce leaky expression

Purification Protocol:

• Membrane fraction isolation using differential centrifugation

• Solubilization with appropriate detergents (recommended initial screening of DDM, LMNG, and DMNG)

• Immobilized metal affinity chromatography (IMAC)

• Size exclusion chromatography for further purification

Stability Enhancement:

• Addition of lipids (E. coli total lipid extract at 0.1-0.2 mg/ml)

• Buffer optimization containing 10-15% glycerol

• Use of cholesteryl hemisuccinate (CHS) at 0.01-0.05% to stabilize the protein

Protein activity should be assessed using ATP binding assays and functional complementation in ΔubiB strains to confirm that the purified protein retains its native functionality .

What knockout and complementation strategies would be most effective for studying UbiB function in S. sonnei?

A comprehensive genetic approach to studying UbiB function should employ both knockout and controlled complementation strategies:

Knockout Strategy:

• Use lambda Red recombineering or CRISPR-Cas9 to precisely delete the ubiB gene

• Confirm deletion by PCR, sequencing, and Western blot

• Create marker-free deletions to avoid polar effects on adjacent genes

Complementation Approaches:

• Chromosomal Complementation: Integrate the wild-type ubiB gene back into the chromosome at a neutral site using Tn7-based systems

• Plasmid-Based Complementation: Use low-copy plasmids (pWSK29 or pACYC184 derivatives) with native promoters for physiological expression levels

• Controlled Expression: Employ inducible promoters (like arabinose-inducible pBAD) to create an expression gradient for dose-response studies

Advanced Genetic Manipulations:

• Site-directed mutagenesis of conserved residues to identify essential functional domains

• Construction of chimeric proteins with UbiB from related species to determine region-specific functionality

• Creation of fluorescent protein fusions for localization studies (ensuring C-terminal fusions to avoid disrupting membrane insertion)

Phenotypic Analysis:

• Growth curve comparison under aerobic, microaerobic, and anaerobic conditions

• Ubiquinone quantification using HPLC-MS/MS

• Metabolic profiling using 13C-labeled precursors

• Measurement of membrane potential and ATP synthesis rates

This comprehensive genetic approach should be combined with biochemical validation to establish conclusive structure-function relationships for UbiB .

How can researchers effectively analyze the interaction between UbiB and other ubiquinone biosynthesis proteins in S. sonnei?

Investigating protein-protein interactions involving membrane proteins like UbiB requires specialized approaches:

In Vivo Interaction Methods:

• Split-Protein Complementation Assays: Using split-GFP or split-luciferase systems adapted for membrane proteins

• Bacterial Two-Hybrid Systems: Modified for membrane protein analysis using BACTH (Bacterial Adenylate Cyclase Two-Hybrid) system

• In Vivo Crosslinking: Using photo-activatable or chemical crosslinkers followed by pulldown and mass spectrometry

In Vitro Methods:

• Co-Purification Assays: Using tandem affinity purification with different tags on potential interacting partners

• Surface Plasmon Resonance: For quantitative binding kinetics, requiring purified components in appropriate detergent/lipid environments

• Native Mass Spectrometry: To identify intact membrane protein complexes preserved in micelles or nanodiscs

Structural Analysis:

• Cryo-EM: For structural characterization of UbiB-containing complexes

• Hydrogen-Deuterium Exchange Mass Spectrometry: To map interaction interfaces

• FRET-Based Assays: Using site-specific labeling to measure proximity between purified components

Computational Approaches:

• Molecular docking simulations to predict protein-protein interaction interfaces

• Coevolution analysis to identify potentially interacting residues across the ubiquinone biosynthesis pathway

The combination of these complementary approaches would provide robust evidence for specific interactions between UbiB and other components of the ubiquinone biosynthesis machinery, with particular attention to potential interactions with UbiA, UbiX, and components of the O₂-independent pathway (UbiT, UbiU, UbiV) .

How should researchers approach comparative genomic analysis of ubiB across Shigella species and related enterobacteria?

A comprehensive comparative genomic analysis of ubiB should follow this methodological framework:

Sequence Retrieval and Alignment:

• Obtain ubiB sequences from multiple Shigella isolates (all four species), E. coli strains, and other related enterobacteria

• Perform multiple sequence alignment using MUSCLE or MAFFT algorithms

• Calculate sequence conservation scores for each position

Phylogenetic Analysis:

• Construct phylogenetic trees using maximum likelihood methods with appropriate substitution models

• Perform bootstrap analysis (>1000 replicates) to assess branch support

• Compare ubiB phylogeny with species phylogeny to identify potential horizontal gene transfer events

Comparative Genomic Context:

• Analyze gene neighborhood conservation around ubiB

• Identify syntenic regions and gene order conservation

• Detect potential operonic structures using intergenic distance analysis

Selection Pressure Analysis:

• Calculate dN/dS ratios to identify positions under purifying or positive selection

• Perform codon-based Z-test of selection

• Use branch-site models to detect lineage-specific selection patterns

Protein Domain Analysis:

• Identify conserved domains and motifs across species

• Map conservation onto predicted structural models

• Compare with experimental structural data when available

Table 1. Comparative analysis of key features in UbiB proteins across selected enterobacterial species

*Note: Values are approximate based on typical conservation patterns in enterobacterial proteins; exact values would require specific sequence analysis .

This comparative approach will reveal evolutionary patterns and functional constraints acting on ubiB, providing insights into its role across different ecological niches and pathogenic lifestyles .

What techniques are most appropriate for analyzing the impact of ubiB mutations on ubiquinone biosynthesis and S. sonnei virulence?

A comprehensive analysis of ubiB mutations requires an integrated approach combining biochemical, genetic, and phenotypic methods:

Ubiquinone Quantification:

• LC-MS/MS Analysis: For precise quantification of ubiquinone and biosynthetic intermediates

  • Internal standards using isotope-labeled ubiquinone

  • Multiple reaction monitoring (MRM) for specific detection

  • Extraction protocol optimization with 2-propanol:hexane (3:5, v/v)

• HPLC Analysis with Electrochemical Detection:

  • For routine quantification and isolation of intermediates

  • Standard curve ranging from 0.1-100 μM ubiquinone

  • Sample preparation using methanol:hexane extraction

Respiratory Chain Analysis:

• Oxygen Consumption Rates:

  • High-resolution respirometry using Oroboros or similar systems

  • Substrate-dependent respiration with NADH, succinate, and glycerol-3-phosphate

  • Inhibitor profiling with rotenone, antimycin A, and cyanide

• Membrane Potential Measurements:

  • Flow cytometry using DiOC2(3) or JC-1 dyes

  • Microplate fluorimetry for high-throughput screening

  • Real-time monitoring during growth phase transitions

Virulence Assessment:

• Tissue Culture Invasion Assays:

  • HeLa or Caco-2 cell infection models

  • Quantification of invasion efficiency by gentamicin protection assay

  • Immunofluorescence microscopy for actin polymerization visualization

• Animal Models:

  • Guinea pig keratoconjunctivitis (Serény test)

  • Murine pulmonary infection model

  • Quantification of bacterial loads in tissues and histopathological scoring

Gene Expression Analysis:

• RNA-Seq:

  • Differential expression analysis between wild-type and ubiB mutants

  • Pathway enrichment focusing on metabolism and virulence genes

  • Time-course analysis during oxygen transition

• qRT-PCR Validation:

  • Targeted analysis of key genes in ubiquinone biosynthesis

  • Virulence gene expression (esp. T3SS components)

  • Normalization with validated reference genes for S. sonnei

Table 2. Expected phenotypic consequences of ubiB mutations in S. sonnei

This integrated approach provides a comprehensive assessment of how ubiB mutations affect both bacterial physiology and virulence capabilities, establishing clear structure-function relationships for this important protein .

What are the main challenges in differentiating the functions of UbiB from other ubiquinone biosynthesis proteins, and how can researchers address them?

Differentiating UbiB function from other ubiquinone biosynthesis proteins presents several technical challenges that require specialized approaches:

Challenge 1: Functional Redundancy
UbiB may share functional overlap with other proteins in the pathway, particularly those involved in the recently discovered O₂-independent pathway .

Solution Approaches:

• Generate combination knockouts (e.g., ΔubiB/ΔubiU, ΔubiB/ΔubiV) to identify synthetic phenotypes

• Perform metabolic flux analysis using 13C-labeled precursors to identify pathway-specific bottlenecks

• Use complementation assays with heterologous proteins to identify function-specific domains

Challenge 2: Membrane Protein Localization and Interaction
As a membrane protein, traditional interaction assays may not accurately capture UbiB's native interactions.

Solution Approaches:

• Use proximity-labeling approaches (BioID or APEX2) fused to UbiB to identify nearby proteins in their native membrane environment

• Employ nanodiscs or liposomes with defined lipid compositions for in vitro reconstitution

• Develop split-GFP systems specifically optimized for membrane protein topology

Challenge 3: Distinguishing Primary from Secondary Effects
Disruption of ubiquinone biosynthesis has pleiotropic effects, making it difficult to identify UbiB's specific role.

Solution Approaches:

• Generate conditional depletion strains using destabilization domains or degron systems for acute UbiB depletion

• Perform time-course -omics analysis (transcriptomics, proteomics, metabolomics) after UbiB depletion

• Use chemical genetic approaches with small-molecule inhibitors specific to different steps in the pathway

Challenge 4: Separating Enzymatic from Structural Roles
UbiB may have both catalytic and structural/scaffolding functions.

Solution Approaches:

• Design separation-of-function mutations based on structural predictions

• Use chemical crosslinking followed by mass spectrometry to identify structural interactions

• Perform in vitro activity assays with reconstituted components to verify direct enzymatic activity

By systematically addressing these challenges, researchers can more precisely define UbiB's unique contributions to ubiquinone biosynthesis and distinguish its role from other pathway components .

How can researchers effectively integrate structural biology approaches to understand UbiB function in S. sonnei?

Structural biology offers powerful insights into UbiB function, but membrane proteins present unique challenges requiring specialized approaches:

X-ray Crystallography Approaches:

• Construct Optimization:

  • Design multiple constructs with varying N- and C-terminal boundaries

  • Screen detergents (DDM, LMNG, GDN) and lipid additives systematically

  • Consider fusion partners (T4 lysozyme, BRIL) to improve crystal contacts

• Crystallization Strategies:

  • Lipidic cubic phase (LCP) crystallization as primary approach

  • In meso crystallization with monoolein or other host lipids

  • Vapor diffusion with bicelles or facial amphiphiles as alternatives

Cryo-EM Approaches:

• Sample Preparation:

  • Reconstitution in nanodiscs with MSP1D1 or MSP1E3D1 scaffolds

  • Amphipol (A8-35) or SMA copolymer solubilization

  • GraFix method for stabilizing protein complexes

• Data Collection and Processing:

  • Use of Volta phase plates to enhance contrast

  • Energy filters to improve signal-to-noise ratio

  • 3D classification to separate conformational states

Integrated Structural Methods:

• HDX-MS (Hydrogen-Deuterium Exchange Mass Spectrometry):

  • Map conformational dynamics and ligand-binding sites

  • Optimize detergent compatibility and back-exchange minimization

  • Compare exchange patterns with and without substrates/cofactors

• EPR Spectroscopy:

  • Site-directed spin labeling at key residues

  • DEER measurements for distance constraints

  • Analysis of conformational changes upon nucleotide binding

• NMR Approaches:

  • Solution NMR of isolated soluble domains

  • Solid-state NMR of reconstituted samples

  • Selective isotope labeling strategies for large membrane proteins

Computational Integration:

• Homology Modeling:

  • Using related ABC1 kinase structures as templates

  • Refinement with molecular dynamics in explicit membrane environments

  • Validation using evolutionary coupling analysis

• Molecular Dynamics Simulations:

  • All-atom simulations in explicit lipid bilayers

  • Coarse-grained simulations for longer timescales

  • Enhanced sampling techniques to capture conformational transitions

• In Silico Docking:

  • Virtual screening of potential substrates and inhibitors

  • Analysis of cofactor binding sites (ATP, Fe-S clusters)

  • Protein-protein docking with other ubiquinone biosynthesis components

By integrating these complementary structural approaches, researchers can develop a comprehensive understanding of UbiB's structure-function relationships, including its membrane topology, substrate binding sites, and potential interaction interfaces with other components of the ubiquinone biosynthesis machinery .

What are the most promising future research directions for understanding UbiB's role in bacterial adaptation to varying oxygen environments?

Future research on UbiB should focus on its role in helping bacteria adapt to different oxygen conditions, with these promising directions:

Ecological and Evolutionary Studies:

• Investigate UbiB expression and function across oxygen gradients in natural microbial communities

• Compare UbiB sequence evolution between obligate aerobes, facultative anaerobes, and microaerophiles

• Examine horizontal gene transfer patterns of ubiB and related genes across bacterial lineages

Systems Biology Approaches:

• Develop genome-scale metabolic models incorporating both O₂-dependent and O₂-independent ubiquinone biosynthesis pathways

• Perform multi-omics integration (transcriptomics, proteomics, metabolomics) during oxygen transitions

• Apply flux balance analysis to quantify metabolic rewiring during adaptation to oxygen limitation

Advanced Genetic Engineering:

• Create oxygen-responsive UbiB expression systems for biotechnological applications

• Engineer UbiB variants with enhanced activity under specific oxygen conditions

• Develop biosensors based on UbiB activity to monitor cellular energetics

Host-Pathogen Interaction Studies:

• Investigate UbiB's role in Shigella adaptation to varying oxygen levels in different intestinal niches

• Determine how UbiB activity affects bacterial persistence during infection

• Explore UbiB as a potential target for anti-virulence strategies that specifically disrupt adaptation to host environments

Clinical Applications:

• Assess correlations between UbiB sequence variants and clinical outcomes in Shigella infections

• Investigate UbiB's potential role in antibiotic tolerance under oxygen limitation

• Develop screening assays for compounds that specifically target UbiB function

These research directions would significantly advance our understanding of UbiB's role in bacterial adaptation to varying oxygen environments and potentially lead to new strategies for controlling bacterial infections .

How might understanding UbiB function contribute to novel antimicrobial strategies against Shigella sonnei?

Understanding UbiB function could reveal several promising avenues for novel antimicrobial strategies against S. sonnei:

Target-Based Drug Discovery:

• Structure-Based Design:

  • Using solved UbiB structures to design specific inhibitors

  • Focus on unique pockets not present in human homologs

  • Develop allosteric inhibitors that lock UbiB in inactive conformations

• High-Throughput Screening:

  • Develop cell-based assays measuring ubiquinone production

  • Screen for compounds that specifically inhibit UbiB function

  • Prioritize compounds effective under both aerobic and anaerobic conditions

Metabolic Vulnerability Exploitation:

• Combination Therapies:

  • Pair UbiB inhibitors with conventional antibiotics to increase efficacy

  • Design dual-targeting compounds affecting both UbiB and traditional antibiotic targets

  • Develop strategies targeting both O₂-dependent and O₂-independent pathways simultaneously

• Conditional Lethality:

  • Identify synthetic lethal interactions with UbiB under specific host conditions

  • Develop compounds that become activated by bacterial metabolism

  • Create oxygen-responsive prodrugs that specifically target bacteria in low-oxygen niches

Anti-Virulence Approaches:

• Attenuation Strategies:

  • Develop compounds that modify UbiB function without killing bacteria

  • Target UbiB to reduce bacterial fitness during infection

  • Design narrow-spectrum inhibitors specific to Shigella UbiB

• Host-Directed Therapies:

  • Modulate host metabolism to create environments where UbiB inhibition is more effective

  • Combine UbiB inhibitors with immunomodulatory compounds

  • Develop nanoparticle delivery systems targeting Shigella-containing vacuoles

Vaccine Development:

• Attenuated Strains:

  • Engineer S. sonnei with modified UbiB for use as live attenuated vaccines

  • Create strains with oxygen-dependent attenuation for controlled colonization

  • Develop regulatory-approved defined mutations in UbiB for vaccine production

Table 3. Comparison of potential antimicrobial strategies targeting UbiB

This multi-faceted approach to targeting UbiB function represents a promising strategy for developing novel antimicrobials against increasingly antibiotic-resistant S. sonnei strains .

What are the best practices for heterologous expression of S. sonnei UbiB for functional and structural studies?

Successful heterologous expression of S. sonnei UbiB requires careful optimization of multiple parameters:

Expression Host Selection:

• E. coli Strains:

  • BL21(DE3) for standard expression

  • C41(DE3) or C43(DE3) for toxic or membrane proteins

  • Lemo21(DE3) for tunable expression levels

  • SHuffle strains if disulfide bonds are critical

• Alternative Expression Systems:

  • Lactococcus lactis for difficult membrane proteins

  • Cell-free expression systems for toxic proteins

  • Insect cell expression for improved folding

Vector Design Optimization:

• Promoter Selection:

  • T7 for high-level expression

  • trc or tac for moderate expression

  • araBAD for titratable expression

• Fusion Tags:

  • N-terminal His₈-MBP for enhanced solubility and purification

  • C-terminal GFP for expression monitoring and folding assessment

  • SUMO tag for enhanced expression and native N-terminus after cleavage

• Codon Optimization:

  • Harmonization rather than maximization approach

  • Adjust GC content to match expression host

  • Avoid rare codon clusters and RNA secondary structures

Expression Condition Optimization Matrix:

Table 4. Systematic optimization parameters for S. sonnei UbiB expression

Membrane Extraction and Solubilization:

• Cell Lysis Methods:

  • French press for complete membrane extraction

  • Sonication with controlled temperature

  • Enzymatic lysis with lysozyme and DNase

• Detergent Screening:

  • Mild detergents: DDM, LMNG, GDN as primary options

  • Progressive solubilization trials (0.5%, 1%, 2%)

  • Detergent exchange during purification

• Stabilization Strategies:

  • Addition of specific lipids (POPE, cardiolipin)

  • Inclusion of cholesterol hemisuccinate

  • Buffer optimization with glycerol and salt screening

By systematically optimizing these parameters, researchers can significantly improve the yield and quality of recombinant S. sonnei UbiB for both functional and structural studies. The key is to establish a logical experimental matrix, testing multiple conditions in parallel, and using both quantitative (yield) and qualitative (activity, homogeneity) metrics to identify optimal conditions .

How can researchers effectively study the interplay between UbiB and the Type III Secretion System in S. sonnei pathogenesis?

Investigating the potential functional relationship between UbiB and the Type III Secretion System (T3SS) requires specialized methodological approaches that bridge metabolism and virulence:

Genetic Interaction Studies:

• Conditional Depletion Systems:

  • Develop tunable expression systems for both UbiB and key T3SS components

  • Create dual reporter strains monitoring both UbiB activity and T3SS expression

  • Employ CRISPRi for titratable repression of target genes

• Epistasis Analysis:

  • Generate double mutants (ΔubiB with various T3SS component deletions)

  • Perform quantitative phenotyping under various conditions

  • Use statistical interaction models to identify synthetic phenotypes

Metabolic-Virulence Connections:

• Energy Coupling Analysis:

  • Measure ATP levels and proton motive force in wild-type vs. ΔubiB strains during T3SS activation

  • Quantify NADH/NAD+ ratios during infection processes

  • Assess oxygen consumption rates during host cell contact

• Metabolic Flux Analysis:

  • Use 13C-labeled carbon sources to track metabolic rewiring during infection

  • Compare flux distributions between wild-type and UbiB-deficient strains

  • Integrate with quantitative proteomics of T3SS components

Infection Models with Controlled Oxygen Conditions:

• In Vitro Systems:

  • Develop cell culture infection models with defined oxygen gradients

  • Use microfluidic devices to create oxygen-controlled microenvironments

  • Apply live-cell imaging with oxygen-sensitive probes during infection

• Ex Vivo Models:

  • Intestinal organoid infection under controlled oxygen conditions

  • Human intestinal enteroid models with oxygen monitoring

  • Precision-cut tissue slices with defined oxygenation

Molecular Interaction Studies:

• Co-localization Analysis:

  • Fluorescently tag UbiB and T3SS components for co-localization studies

  • Use super-resolution microscopy to examine spatial relationships

  • Perform time-lapse imaging during infection progression

• Proximity-Based Interactomics:

  • Apply BioID or APEX2 proximity labeling with UbiB as bait

  • Identify potential interaction partners related to T3SS regulation

  • Validate specific interactions with targeted approaches

Systems Biology Integration:

• Multi-Omics Integration:

  • Correlate transcriptomics, proteomics, and metabolomics datasets

  • Apply network analysis to identify regulatory connections

  • Develop predictive models of UbiB-T3SS interactions

• Temporal Analysis:

  • Perform time-course studies during infection progression

  • Map metabolic state transitions to virulence gene expression

  • Identify key temporal coordination points