Recombinant Escherichia coli O45:K1 Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is the genomic context of ubiB in E. coli O45:K1 strains?

The ubiB gene is part of the ubiquinone biosynthesis pathway in E. coli. While specific information about ubiB in E. coli O45:K1 is limited in the provided search results, we can understand its context by examining the O-antigen gene cluster and other ubiquinone biosynthesis genes. In E. coli strain S88 (O45:K1:H7), the O-antigen gene cluster is located between galF and gnd genes and contains nine open reading frames with a total length of 8,379 bp . The genes have the same transcriptional direction from galF to gnd and display a low G+C content (30.6 to 46.9%) compared to the E. coli core genome (51%) .

How do you confirm the expression of recombinant ubiB in E. coli O45:K1?

To confirm the expression of recombinant ubiB in E. coli O45:K1, researchers should employ a multi-faceted approach:

• Genetic confirmation: PCR amplification of the ubiB gene using specific primers designed based on the known sequence. This can be performed similar to the O-serogroup-specific PCR assay described for E. coli O45:K1:H7, where template DNA is prepared by mixing bacterial colony with sterile water, heating at 100°C for 10 minutes, centrifuging, and using the supernatant for PCR .

• Transcriptional analysis: RNA extraction followed by RT-PCR or qPCR to quantify ubiB mRNA levels. Transcriptional fusions can be constructed using promoter regions cloned into vectors such as pUA66, similar to how ubiT transcriptional fusions were constructed .

• Protein detection: Western blotting using antibodies specific to UbiB or to an epitope tag if the recombinant protein is tagged. Alternatively, mass spectrometry can be used to confirm protein expression and identity.

• Functional complementation: Testing whether the recombinant ubiB can rescue the phenotype of a ubiB deletion mutant, such as growth defects under specific conditions where ubiquinone is required.

What are the key differences between E. coli O45:K1 and other pathogenic E. coli strains?

E. coli O45:K1:H7 represents a unique pathogenic clone with several distinguishing characteristics:

• Serotypic profile: It possesses the unusual serogroup O45, combined with capsular antigen K1 and flagellar antigen H7 . This contrasts with the archetypal neonatal meningitis clone O18:K1:H7 .

• O-antigen gene cluster: The S88 O-antigen gene cluster sequence differs from that of O45 in the reference strain E. coli 96-3285, suggesting that while they share epitopes, they represent two different antigens . The S88 strain has nine ORFs in its O-antigen gene cluster with specific functions assigned based on protein database similarity searches .

• Evolutionary history: Phylogenetic analysis based on the flanking gene gnd sequences indicates that the S88 antigen O45 gene cluster may have been acquired, at least in part, from another member of the Enterobacteriaceae . This horizontal acquisition of a new O-antigen gene cluster may have been a key event in the emergence and virulence of this clone .

• Virulence factors: E. coli O45:K1:H7 strains like S88 belong to phylogenetic group B2 and harbor ribotype B2 1, with a specific set of virulence determinants including fyuA, papC, papGII, iucC, and iroN .

How can I generate a ubiB knockout mutant in E. coli O45:K1 for functional studies?

Creating a ubiB knockout mutant in E. coli O45:K1 can be accomplished using the method of Datsenko and Wanner, as described for S88 mutants in the search results:

• Plasmid preparation: Obtain plasmid pKD46, which allows homologous recombination with PCR products and carries the bacteriophage λ Red system under an arabinose-inducible promoter .

• PCR amplification: Amplify the chloramphenicol acetyltransferase (cat) gene from plasmid pKD3 using primers bearing 40-nucleotide extensions homologous to the initial and final portions of ubiB . Design primers specific to ubiB sequence flanking regions.

• Transformation: Transform E. coli O45:K1 expressing bacteriophage λ Red functions with the PCR product by electroporation to yield recombinants carrying ubiB fused to the cat gene .

• Verification: Confirm correct introduction of the cat gene by PCR with primers flanking the initial and final portions of ubiB and primers homologous to the cat gene .

• Control testing: Verify the conservation of main virulence determinants in the mutants by multiplex PCR with primers located in the main extraintestinal virulence genes, such as fyuA, papC, papGII, iucC, and iroN .

• Phenotypic confirmation: Test for altered ubiquinone production and associated phenotypes, such as growth defects under aerobic conditions where ubiquinone is critical for respiration.

What growth conditions are optimal for studying UbiB function in E. coli O45:K1?

To effectively study UbiB function in E. coli O45:K1, several growth conditions should be considered:

• Aerobic vs. anaerobic conditions: Since ubiquinone is primarily used under aerobiosis while demethylmenaquinones are used under anaerobiosis, both conditions should be tested . For anaerobic conditions, cultures can be grown in sealed containers with an anaerobic atmosphere generator or in specialized anaerobic chambers.

• Respiratory substrates: Include conditions with different electron acceptors:

  • Oxygen for aerobic respiration

  • Nitrate for anaerobic respiration, as UbiUV-dependent UQ synthesis is essential for nitrate respiration under anaerobiosis

  • Other potential electron acceptors like fumarate or DMSO

• Carbon sources: Test multiple carbon sources (glucose, glycerol, succinate, etc.) to determine if UbiB function is affected by the metabolic state of the cell.

• Growth phase monitoring: Measure growth curves under these varied conditions to determine if UbiB affects specific growth phases.

• Oxygen transition: Include experiments where cultures are shifted from anaerobic to aerobic conditions, as this transition appears to be critical for ubiquinone biosynthesis regulation. The UbiT protein has been shown to be crucial for allowing E. coli to shift efficiently from anaerobic to aerobic conditions .

• Pyrimidine availability: Consider conditions with limited pyrimidine availability, as UbiUV-synthesized UQ has been shown to contribute to anaerobic pyrimidine biosynthesis .

How can I measure ubiquinone production in wild-type versus ubiB mutant E. coli O45:K1?

Quantifying ubiquinone production in wild-type versus ubiB mutant strains requires specific extraction and analytical methods:

• Extraction protocol:

  • Harvest cells at mid-log phase (OD600 ≈ 0.6-0.8)

  • Wash cell pellets with phosphate buffer

  • Extract lipids with a 2:1 mixture of chloroform:methanol

  • Separate the organic phase and concentrate under nitrogen

  • Resuspend in an appropriate solvent (ethanol or hexane)

• Analytical methods:

  • HPLC analysis: Use a C18 reverse-phase column with an isocratic or gradient system of methanol:hexane. Detection can be performed at 275 nm (ubiquinone absorption maximum).

  • LC-MS analysis: For more sensitive detection and confirmation of ubiquinone identity, use liquid chromatography coupled with mass spectrometry.

  • Spectrophotometric detection: Measure the difference in absorbance at 275 nm between oxidized and reduced (with sodium borohydride) samples.

• Isotopic labeling: To specifically study the pathway used for ubiquinone synthesis, consider using 18O2 labeling as mentioned in the search results for studying UbiUV-dependent hydroxylation . This can help determine if UbiB contributes to an O2-dependent or O2-independent process.

• Controls and standards: Include commercially available ubiquinone standards (UQ8 or UQ10) for quantification and identification purposes.

• Comparison conditions: Measure ubiquinone levels under both aerobic and anaerobic conditions, and after shifts between these conditions, to fully understand UbiB's role in each context.

What is the proposed enzymatic function of UbiB in the ubiquinone biosynthesis pathway?

Based on general knowledge about ubiquinone biosynthesis (as specific information about UbiB is limited in the search results):

UbiB is classified as a probable ubiquinone biosynthesis protein and likely functions as a kinase or kinase-like protein involved in the early steps of ubiquinone biosynthesis. While the exact function isn't detailed in the provided search results, we can infer its role based on related ubiquinone biosynthesis proteins:

• Probable enzymatic activity: UbiB likely participates in the hydroxylation or C-methylation steps of ubiquinone precursors. The search results indicate that UbiUV contributes to the hydroxylation of ubiquinone precursors through a unique O2-independent process , and UbiB may function in a similar or complementary manner.

• Relationship to other Ubi proteins: UbiB may work in concert with other ubiquinone biosynthesis proteins like UbiUVT. The search results show that UbiUVT proteins are involved in an anaerobic O2-independent UQ biosynthesis pathway .

• Regulatory context: Like other ubi genes, ubiB may be under the control of oxygen-sensing regulatory systems. The search results indicate that ubiTUV genes are under the control of the O2-sensing Fnr transcriptional regulator .

• Physiological importance: UbiB likely plays a role in allowing E. coli to adapt to changing oxygen levels, similar to UbiT which is crucial for allowing E. coli to shift efficiently from anaerobic to aerobic conditions .

Further experimental work would be needed to definitively characterize the enzymatic function of UbiB, including biochemical assays with purified protein and identification of substrates and products.

How does oxygen availability affect the expression and function of UbiB in E. coli O45:K1?

While specific information about UbiB regulation is not provided in the search results, we can infer potential oxygen-dependent regulation based on what is known about other ubiquinone biosynthesis proteins:

• Transcriptional regulation: The search results indicate that ubiTUV genes are under the control of the O2-sensing Fnr transcriptional regulator . It's likely that ubiB is similarly regulated by oxygen-responsive transcription factors like Fnr, which activates gene expression under anaerobic conditions.

• Dual pathway regulation: E. coli has been shown to possess both aerobic and anaerobic pathways for ubiquinone biosynthesis. The search results mention a "dual anaerobic/aerobic regulation" that allows UbiT to secure a rapid shift from anaerobic UbiUV-dependent UQ synthesis to an aerobic UbiIHF-dependent UQ synthesis . UbiB might be involved in either or both pathways.

• Functional transitions: The search results highlight the crucial role of UbiT in allowing E. coli to shift efficiently from anaerobic to aerobic conditions . UbiB may play a similar role in facilitating metabolic transitions during oxygen availability changes.

• Oxygen-independent biochemistry: The search results indicate that UbiUV acts as O2-independent hydroxylases, representing "a new type of chemistry" . If UbiB is involved in hydroxylation steps, it might employ similar oxygen-independent mechanisms under anaerobic conditions.

To experimentally determine the effect of oxygen availability on UbiB:

• Measure ubiB transcript levels under aerobic, microaerobic, and anaerobic conditions using qRT-PCR

• Construct a ubiB promoter-reporter fusion similar to the ubiUVp plasmids mentioned in the search results

• Perform western blotting to quantify UbiB protein levels under different oxygen conditions

• Test the phenotypes of ubiB mutants under varying oxygen levels and during transitions between aerobic and anaerobic growth

What protein-protein interactions are known or predicted for UbiB with other ubiquinone biosynthesis proteins?

Though specific protein-protein interactions involving UbiB are not detailed in the search results, potential interactions can be inferred based on the functional organization of the ubiquinone biosynthesis pathway:

• Interaction with UbiUV complex: The search results indicate that UbiU and UbiV are involved in an O2-independent hydroxylation process for ubiquinone precursors . Given that these proteins perform hydroxylation steps in the ubiquinone pathway, UbiB may interact with this complex to coordinate sequential enzymatic steps.

• Interaction with UbiT: UbiT is mentioned as being crucial for allowing E. coli to shift efficiently from anaerobic to aerobic conditions . If UbiB functions in this transition as well, it might interact with UbiT as part of a regulatory complex.

• Potential interactions with UbiIHF: The search results mention an "aerobic UbiIHF-dependent UQ synthesis" , suggesting that UbiI, UbiH, and UbiF form a functional complex. UbiB may interact with these proteins, particularly during the shift from anaerobic to aerobic metabolism.

Methods to experimentally investigate UbiB protein-protein interactions:

• Co-immunoprecipitation (Co-IP): Using antibodies against UbiB or an epitope-tagged version to pull down potential interaction partners, followed by mass spectrometry identification.

• Bacterial two-hybrid assay: Construct fusion proteins between UbiB and one domain of a split reporter protein, and potential interaction partners with the other domain, to test for interactions in vivo.

• Pull-down assays: Use purified His-tagged or GST-tagged UbiB as bait to identify interacting proteins from cell lysates.

• Crosslinking studies: Chemical crosslinking followed by mass spectrometry to identify proteins in close proximity to UbiB in vivo.

• Fluorescence microscopy: Use fluorescently tagged UbiB and other ubiquinone biosynthesis proteins to visualize potential co-localization in the cell.

• Surface plasmon resonance (SPR): Measure direct binding between purified UbiB and candidate interaction partners.

What is the role of UbiB-dependent ubiquinone production in E. coli O45:K1 virulence?

While the search results don't specifically address UbiB's role in virulence, we can infer its potential importance based on what is known about ubiquinone biosynthesis and O45:K1 virulence:

• Energy production during infection: Ubiquinone is crucial for aerobic respiration , which is likely important for bacterial growth during certain stages of infection when oxygen is available. Defects in UbiB function could impair energy production and reduce bacterial fitness in host tissues.

• Adaptation to host environments: The search results emphasize the importance of ubiquinone in allowing E. coli to adjust its metabolism based on changing O2 levels and respiratory conditions . This adaptability is likely crucial during infection as bacteria encounter different microenvironments within the host.

• Nitrate respiration: The search results indicate that UbiUV-dependent UQ synthesis is essential for nitrate respiration under anaerobiosis . Nitrate respiration may be important for E. coli O45:K1 survival in oxygen-limited niches within the host, suggesting UbiB might similarly contribute to anaerobic metabolism during infection.

• Pyrimidine biosynthesis: UbiUV-synthesized UQ contributes to anaerobic pyrimidine biosynthesis . If UbiB plays a similar role, it could be important for nucleic acid synthesis during infection, particularly in anaerobic environments.

• O-antigen and virulence: The search results highlight that the O-antigen polysaccharide plays a crucial role in S88 virulence in a neonatal rat meningitis model . If UbiB affects membrane composition or function, it could indirectly influence O-antigen presentation and thereby affect virulence.

To experimentally assess UbiB's role in virulence:

• Compare the virulence of wild-type and ubiB mutant strains in appropriate animal models, such as the neonatal rat meningitis model mentioned in the search results .

• Examine the ability of ubiB mutants to survive in serum, adhere to and invade host cells, and resist phagocytosis.

• Test the competitive fitness of wild-type versus ubiB mutant strains in mixed infections to assess relative fitness in vivo.

How does UbiB contribute to E. coli O45:K1 adaptation to different environmental conditions?

Based on the role of ubiquinone in bacterial physiology as described in the search results, UbiB likely contributes to environmental adaptation in several ways:

Experimental approaches to evaluate UbiB's role in environmental adaptation:

• Compare growth kinetics of wild-type and ubiB mutant strains under various conditions (different oxygen levels, pH values, osmolarities, nutrient limitations).

• Assess the survival of ubiB mutants following exposure to various stressors (oxidative stress, pH shock, osmotic stress).

• Measure competitive fitness of wild-type versus ubiB mutant strains in mixed cultures under changing environmental conditions.

• Monitor colonization efficiency of wild-type and ubiB mutant strains in animal models, particularly during transitions between host niches.

What is the relationship between UbiB function and antibiotic resistance in E. coli O45:K1?

While the search results don't directly address UbiB and antibiotic resistance, several potential connections can be inferred based on the known functions of ubiquinone biosynthesis:

• Membrane integrity: Ubiquinone is a membrane-associated molecule, and alterations in its production could affect membrane composition and permeability, potentially influencing the entry of certain antibiotics into the cell.

• Energy-dependent drug efflux: Many antibiotic efflux pumps require energy in the form of proton motive force, which is generated through respiratory chains involving ubiquinone. Defects in UbiB function could potentially affect energy availability for these efflux systems.

• Oxidative stress management: Some antibiotics induce oxidative stress as part of their killing mechanism. Ubiquinone has antioxidant properties that might help bacteria resist this stress, suggesting UbiB could indirectly affect sensitivity to these antibiotics.

• Metabolic state influence: The search results indicate that ubiquinone biosynthesis proteins like UbiUVT help E. coli adjust its metabolism based on changing O2 levels and respiratory conditions . This metabolic flexibility might affect susceptibility to antibiotics that target specific metabolic states.

• Persister cell formation: Metabolic dormancy is associated with persister cell formation and antibiotic tolerance. If UbiB affects energy metabolism, it might influence the formation of these antibiotic-tolerant persister cells.

Experimental approaches to investigate the relationship between UbiB and antibiotic resistance:

• Determine the minimum inhibitory concentrations (MICs) of various antibiotics for wild-type and ubiB mutant strains under both aerobic and anaerobic conditions.

• Assess the frequency of persister cell formation in wild-type versus ubiB mutant populations following antibiotic treatment.

• Measure the expression of antibiotic resistance genes and efflux pumps in the presence and absence of functional UbiB.

• Evaluate the effect of chemical inhibitors of UbiB on antibiotic efficacy in combination therapy approaches.

• Test whether exposure to sublethal antibiotic concentrations affects ubiB expression, potentially as part of an adaptive response.

What bioinformatic approaches can be used to predict functional domains and evolutionary relationships of UbiB across bacterial species?

To comprehensively analyze UbiB's functional domains and evolutionary relationships:

• Sequence analysis:

  • Multiple sequence alignment of UbiB homologs using MUSCLE or CLUSTAL

  • Identification of conserved residues that may indicate functional importance

  • Motif analysis using tools like MEME or InterPro (mentioned in the search results for analyzing protein motifs and domains )

• Domain prediction:

  • Use of InterPro database to identify protein motifs and domains, as mentioned in the search results

  • Application of TMHMM (version 2.0) to identify transmembrane domains, as mentioned for other proteins in the search results

  • Secondary structure prediction using tools like PSIPRED

  • 3D structure prediction using AlphaFold or similar tools

• Phylogenetic analysis:

  • Construction of phylogenetic trees based on UbiB sequences

  • Comparison with species phylogeny to identify potential horizontal gene transfer events

  • Analysis of selection pressures using dN/dS ratios

  • Comparative analysis with other ubiquinone biosynthesis proteins, similar to the analysis performed for the O-antigen gene cluster in the search results

• Synteny analysis:

  • Examination of the genomic context of ubiB across species

  • Use of "synchronized representation of synteny groups" as mentioned in the search results for analyzing other genes

  • Identification of co-evolved gene clusters

• Function prediction:

  • Computational prediction of enzymatic function based on conserved residues

  • Protein-protein interaction network analysis

  • Integration of transcriptomic data to identify co-expressed genes

For E. coli O45:K1 specifically, perform comparative analysis with other E. coli strains to identify any unique features or adaptations in the UbiB protein that might contribute to the specific properties of this pathogenic clone.

How can structural biology approaches be used to investigate UbiB enzymatic mechanisms?

Structural biology offers powerful tools for elucidating UbiB's enzymatic mechanism:

For UbiB from E. coli O45:K1, special attention should be given to any structural features that might be unique to this strain compared to other E. coli strains, potentially contributing to its specific pathogenic properties.

What synthetic biology approaches can be used to engineer UbiB for enhanced ubiquinone production or novel functions?

Synthetic biology offers several strategies to engineer UbiB for enhanced function or novel applications:

• Directed evolution:

  • Create libraries of ubiB variants through error-prone PCR

  • Develop high-throughput screening assays for ubiquinone production

  • Select variants with enhanced activity under specific conditions

  • Combine beneficial mutations through DNA shuffling

• Rational protein design:

  • Use structural information to identify residues for mutation

  • Design mutations to enhance substrate binding, catalytic efficiency, or stability

  • Engineer altered substrate specificity

  • Create fusion proteins with other enzymes in the ubiquinone pathway for enhanced pathway flux

• Pathway engineering:

  • Optimize expression of ubiB along with other ubiquinone biosynthesis genes

  • Use synthetic promoters with different strengths or regulatory properties

  • Engineer expression systems that respond to specific environmental signals

  • Implement feedback regulation to optimize ubiquinone production

• Heterologous expression systems:

  • Express engineered ubiB variants in non-pathogenic host strains

  • Co-express with other components needed for ubiquinone biosynthesis

  • Test expression in different host organisms for optimal production

• CRISPR-Cas9 genome editing:

  • Precisely modify the endogenous ubiB gene

  • Integrate optimized ubiB variants into the genome

  • Create libraries of ubiB mutants for screening

• Biosensor development:

  • Develop biosensors for ubiquinone or its precursors

  • Use for high-throughput screening of engineered UbiB variants

  • Apply for monitoring ubiquinone production in real-time

• Production optimization:

  • Determine optimal growth conditions for ubiquinone production with engineered UbiB

  • Consider the oxygen-dependent regulation aspects mentioned in the search results

  • Develop fermentation protocols that account for the dual anaerobic/aerobic regulation of ubiquinone biosynthesis

For E. coli O45:K1 specifically, consider how engineering UbiB might affect virulence properties and ensure that any engineered strains have appropriate biosafety features to prevent unintended consequences.

What are the best experimental controls when studying UbiB function in E. coli O45:K1?

When studying UbiB function in E. coli O45:K1, implement the following essential controls:

• Genetic controls:

  • Wild-type strain: The parental E. coli O45:K1 strain with unmodified ubiB

  • Gene deletion mutant: A complete ubiB knockout mutant

  • Complemented strain: The ubiB mutant with a plasmid expressing wild-type ubiB

  • Point mutant controls: Strains with specific amino acid substitutions in conserved residues

  • Empty vector control: The ubiB mutant with an empty expression vector

• Expression controls:

  • Housekeeping gene expression: Measure expression of genes like rpoD or gyrA as internal controls

  • Protein expression verification: Western blotting to confirm expression levels of wild-type and mutant UbiB proteins

  • Promoter activity control: Include a constitutive promoter driving a reporter gene as a reference

• Functional controls:

  • Known ubiquinone pathway mutants: Include mutants in other ubi genes (like ubiU, ubiV, or ubiT mentioned in the search results ) for comparison

  • Chemical inhibition: Include samples treated with specific inhibitors of ubiquinone biosynthesis

  • Exogenous ubiquinone: Test whether adding exogenous ubiquinone rescues phenotypes of ubiB mutants

• Growth condition controls:

  • Aerobic and anaerobic conditions: Test both conditions since ubiquinone functions differ based on oxygen availability

  • Different carbon sources: Include multiple carbon sources to control for metabolic state effects

  • Growth phase consistency: Ensure samples are collected at equivalent growth phases

• Analytical controls:

  • Extraction efficiency control: Include an internal standard in ubiquinone extraction procedures

  • Standard curve: Include a series of known ubiquinone concentrations for quantification

  • Technical replicates: Perform multiple technical replicates for each biological sample

  • Biological replicates: Use independent bacterial cultures for each experimental condition

• Method validation controls:

  • When using PCR-based methods, include controls similar to those mentioned in the search results for O45-specific PCR, such as positive controls (strain S88), negative controls, and internal amplification controls (uidA gene)

What techniques can be used to study the kinetics of UbiB-catalyzed reactions in ubiquinone biosynthesis?

To characterize the kinetics of UbiB-catalyzed reactions, researchers can employ several complementary techniques:

• Enzyme activity assays:

  • Spectrophotometric assays: Monitor changes in absorbance associated with substrate consumption or product formation

  • Coupled enzyme assays: Link UbiB activity to a reaction that produces a detectable signal

  • HPLC-based assays: Quantify reaction products at different time points to determine reaction rates

  • Radiometric assays: Use radiolabeled substrates to track conversion to products

• Steady-state kinetics:

  • Determine Michaelis-Menten parameters (Km, Vmax, kcat) using varying substrate concentrations

  • Analyze the effects of potential inhibitors (competitive, noncompetitive, uncompetitive)

  • Study the effects of pH, temperature, and ionic strength on enzyme activity

• Pre-steady-state kinetics:

  • Stopped-flow spectroscopy: Measure rapid changes in absorbance or fluorescence after mixing enzyme and substrate

  • Quenched-flow techniques: Rapidly quench reactions at defined time points for analysis of early reaction intermediates

  • Single-turnover experiments: Study the first catalytic cycle in isolation

• Binding studies:

  • Isothermal titration calorimetry (ITC): Measure thermodynamic parameters of substrate binding

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

  • Fluorescence-based binding assays: Use changes in intrinsic protein fluorescence or fluorescent probes to monitor binding

• Product analysis:

  • Mass spectrometry: Identify and quantify reaction products with high sensitivity

  • NMR spectroscopy: Track changes in substrate structure during the reaction

  • HPLC with multiple detection methods: Combine UV detection with mass spectrometry for comprehensive analysis

• Oxygen dependence studies:

  • Given the findings that some ubiquinone biosynthesis proteins act as O2-independent hydroxylases , specifically test whether UbiB operates via O2-dependent or O2-independent mechanisms

  • Use 18O2 labeling experiments similar to those mentioned in the search results to track the source of oxygen atoms in hydroxylated products

• In vivo kinetics:

  • Metabolic flux analysis: Trace the flow of labeled precursors through the ubiquinone biosynthesis pathway

  • Temporal profiling: Monitor changes in ubiquinone and precursor levels over time following induction or repression of ubiB

How can systems biology approaches integrate UbiB function into the broader context of E. coli O45:K1 metabolism?

Systems biology approaches can provide a comprehensive understanding of how UbiB functions within the broader metabolic network of E. coli O45:K1:

• Multi-omics integration:

  • Genomics: Compare ubiB sequence and genomic context across E. coli strains

  • Transcriptomics: Analyze global gene expression changes in ubiB mutants versus wild-type

  • Proteomics: Identify changes in protein abundance and post-translational modifications

  • Metabolomics: Profile changes in metabolite levels, particularly in respiratory and related pathways

  • Fluxomics: Measure changes in metabolic flux through central carbon metabolism and respiratory pathways

• Regulatory network analysis:

  • Identify transcription factors regulating ubiB expression

  • Determine if ubiB is part of regulons controlled by oxygen-sensing systems like Fnr, which regulates ubiTUV genes as mentioned in the search results

  • Map the complete regulatory network affecting ubiquinone biosynthesis

• Metabolic modeling:

  • Incorporate UbiB function into genome-scale metabolic models of E. coli

  • Perform flux balance analysis to predict the effects of ubiB perturbations

  • Use metabolic control analysis to quantify the control UbiB exerts on ubiquinone production and related pathways

• Network analysis:

  • Construct protein-protein interaction networks including UbiB

  • Identify metabolic pathways connected to ubiquinone biosynthesis

  • Analyze the topological properties of UbiB in these networks

• Condition-dependent analyses:

  • Compare system-wide responses under aerobic versus anaerobic conditions

  • Analyze transitions between oxygen levels, similar to the studies of UbiT's role in the shift from anaerobic to aerobic conditions

  • Examine responses to different electron acceptors, including nitrate which requires UbiUV-dependent UQ synthesis under anaerobiosis

• Comparative systems analysis:

  • Compare system-wide properties of E. coli O45:K1 with other pathogenic and non-pathogenic E. coli strains

  • Identify unique features of ubiquinone metabolism in E. coli O45:K1 that might contribute to its virulence

• Integrative modeling:

  • Develop mathematical models that integrate transcriptional regulation, protein function, and metabolic outcomes

  • Use these models to predict the effects of genetic or environmental perturbations

  • Validate model predictions with targeted experiments