Recombinant Pseudomonas syringae pv. tomato NADH-quinone oxidoreductase subunit G (nuoG), partial

What is the molecular structure and functional role of NADH-quinone oxidoreductase subunit G (nuoG) in Pseudomonas syringae pv. tomato?

nuoG is a critical subunit of the NADH-quinone oxidoreductase complex (Complex I) in the respiratory chain of Pseudomonas syringae pv. tomato. Based on genomic analysis, nuoG in P. syringae pv. tomato DC3000 is encoded at locus PSPTO_3370 and has the following biochemical properties:

The nuoG subunit contains several iron-sulfur clusters that participate in electron transfer from NADH to ubiquinone . As part of the bacterial respiratory chain, this complex plays a fundamental role in energy conservation by coupling electron transfer to proton translocation across the membrane.

The bacterial NADH-quinone oxidoreductase is structurally simpler than its mitochondrial counterpart but maintains similar functionality. In prokaryotes like P. denitrificans and T. thermophilus, the complex contains 14 subunits compared to over 40 in the mammalian enzyme, while maintaining the same number of prosthetic groups .

Methodologically, researchers can study nuoG function through enzyme activity assays measuring NADH oxidation and quinone reduction rates, spectroscopic analysis of iron-sulfur clusters, and site-directed mutagenesis of conserved residues.

How does nuoG contribute to electron transfer within the NADH-quinone oxidoreductase complex?

nuoG functions as part of the electron transport pathway within Complex I, likely housing several iron-sulfur clusters that mediate electron transfer from NADH to quinone. Experimental evidence shows that electron transfer within the complex follows a specific pathway:

• Initial electron acceptance from NADH by a flavin mononucleotide (FMN) cofactor

• Transfer through a series of iron-sulfur clusters of increasing redox potential

• Final transfer to quinone, which occurs at a specific region of the complex

A major unresolved question in the field concerns "the location and mechanism of the terminal electron transfer step from iron–sulfur cluster N2 to quinone" . While research on mammalian and some bacterial systems has identified the PSST subunit as crucial for coupling electron transfer from cluster N2 to quinone, the precise role of nuoG in this process in P. syringae requires further investigation.

To experimentally assess nuoG's contribution to electron transfer, researchers can:

• Generate site-directed mutants targeting conserved cysteine residues that coordinate iron-sulfur clusters

• Measure electron transfer rates using stopped-flow spectroscopy

• Employ EPR (electron paramagnetic resonance) spectroscopy to characterize the redox properties of individual iron-sulfur clusters

• Use inhibitors that target specific steps in the electron transfer pathway, such as rotenone, piericidin A, or pyridaben

The high conservation of nuoG across 494 bacterial genera suggests its fundamental importance in respiratory metabolism , with variations likely reflecting adaptive changes to specific ecological niches.

What are the optimal strategies for expressing and purifying functional recombinant nuoG from P. syringae pv. tomato?

Expressing and purifying membrane-associated proteins like nuoG presents several challenges that require optimization at multiple steps:

Expression System Selection:

For prokaryotic expression, E. coli BL21(DE3) or C41/C43(DE3) strains specifically designed for membrane protein expression are recommended. Consider these vector design elements:

• Inducible promoter systems (T7 or tac) with tunable expression levels

• Fusion tags to improve solubility and facilitate purification:

  • N-terminal His6 or His10 tags for IMAC purification

  • Solubility-enhancing partners (MBP, SUMO, or Trx)

  • Protease cleavage sites (TEV or PreScission) for tag removal

• Codon optimization for E. coli expression

Expression Optimization Parameters:

Purification Strategy:

• Cell lysis using either French press or sonication in buffer containing protease inhibitors

• Membrane fraction isolation by differential centrifugation

• Membrane protein solubilization using mild detergents:

  • n-Dodecyl β-D-maltoside (DDM) at 1-2%

  • Lauryl maltose neopentyl glycol (LMNG) at 0.5-1%

  • Digitonin at 0.5-1%

• Metal affinity chromatography (IMAC) for initial capture

• Ion exchange chromatography to remove contaminants

• Size exclusion chromatography for final polishing and buffer exchange

Functional Validation:

• NADH oxidation activity assay (monitoring A340nm decrease)

• Quinone reduction assay

• Iron-sulfur cluster content analysis by UV-Vis spectroscopy and EPR

• Thermal stability assessment using differential scanning fluorimetry

This systematic approach, combined with iterative optimization, maximizes the likelihood of obtaining functionally active recombinant nuoG suitable for biochemical and structural studies.

What structural biology techniques are most appropriate for studying nuoG structure and interactions?

Multiple complementary structural biology approaches should be employed to fully elucidate nuoG's structure and interactions within the NADH-quinone oxidoreductase complex:

Cryo-Electron Microscopy (Cryo-EM):

Cryo-EM has revolutionized the structural analysis of large membrane protein complexes like NADH-quinone oxidoreductase. Recent studies have successfully employed this technique for similar complexes :

• Purify the intact complex in detergent micelles or reconstituted into nanodiscs

• Apply samples to grids and vitrify in liquid ethane

• Collect images using a high-end transmission electron microscope with direct electron detector

• Process data using software packages like RELION, cryoSPARC, or EMAN2

• Generate 3D reconstructions at near-atomic resolution

• Fit atomic models or homology models into electron density maps

X-ray Crystallography:

Despite challenges with membrane proteins, X-ray crystallography remains valuable, particularly for individual domains or stable subcomplexes:

• Purify nuoG to high homogeneity (>95%)

• Screen crystallization conditions using sparse matrix screens

• Optimize promising conditions for crystal growth

• Consider crystallizing with substrate analogs, inhibitors, or antibody fragments

• Use synchrotron radiation for high-resolution data collection

A precedent exists for crystallizing oxidoreductases from P. syringae, as demonstrated with the successful crystallization of zeta-crystallin-like quinone oxidoreductase .

Cross-linking Mass Spectrometry (XL-MS):

This approach is particularly valuable for mapping protein-protein interactions within the complex:

• Treat purified complex with chemical cross-linkers (e.g., BS3, DSS, or EDC)

• Digest with proteases and analyze by LC-MS/MS

• Identify cross-linked peptides using specialized software

• Map interaction interfaces based on spatial constraints imposed by cross-links

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

HDX-MS provides information about protein dynamics and solvent accessibility:

• Expose protein to deuterated buffer for various time periods

• Quench exchange and perform proteolytic digestion

• Analyze deuterium incorporation by mass spectrometry

• Identify regions with differential exchange rates, indicating structural dynamics

Integrative Modeling:

Combine data from multiple experimental approaches with computational modeling:

• Generate homology models based on related structures

• Refine models using experimental constraints from cryo-EM, XL-MS, and other methods

• Perform molecular dynamics simulations to study conformational dynamics

• Validate models against additional experimental data

This multi-technique approach provides complementary information about nuoG structure, dynamics, and interactions, leading to a comprehensive understanding of its role within the NADH-quinone oxidoreductase complex.

How does nuoG and NADH-quinone oxidoreductase activity contribute to the virulence and fitness of P. syringae pv. tomato during plant infection?

While nuoG is not classified as a direct virulence factor like Type III secretion system (T3SS) effectors, its role in energy metabolism significantly impacts P. syringae pathogenicity through several mechanisms:

Energy Production for Virulence Factor Expression:

The NADH-quinone oxidoreductase complex is central to bacterial respiration, generating the proton motive force necessary for ATP synthesis. This energy is critical for:

• Synthesis and assembly of the Type III secretion system (T3SS), which P. syringae uses to deliver effector proteins into plant cells

• Production of virulence factors, including effector proteins and phytotoxins

• Bacterial growth and multiplication in planta

• Motility and chemotaxis toward favorable infection sites

Adaptation to Plant Environment:

P. syringae encounters various challenging conditions during infection that necessitate metabolic adaptations:

• Nutrient limitation in the apoplastic space

• Varying oxygen availability in different plant tissues

• Plant defense responses including oxidative burst

• Exposure to plant antimicrobial compounds

Transcriptional profiling studies of P. syringae under plant-mimicking conditions have shown significant changes in gene expression when bacteria are exposed to plant extracts, apoplastic fluid, or bean pod extracts . Although nuoG wasn't specifically highlighted, respiratory chain components likely need to adapt to these changing conditions.

Experimental Approaches to Study nuoG's Role in Pathogenicity:

• Gene Knockout/Knockdown Studies:

  • Generate nuoG deletion or conditional expression mutants

  • Assess impact on bacterial growth in planta

  • Measure T3SS function and effector translocation efficiency

  • Evaluate virulence in plant infection assays

• Expression Analysis:

  • Monitor nuoG expression during different infection stages using qRT-PCR

  • Employ transcriptomics to identify co-regulated genes

  • Use reporter fusions (nuoG promoter::GFP) to visualize expression patterns in planta

• Metabolic Analysis:

  • Compare ATP levels in wild-type and nuoG mutant strains

  • Measure NADH/NAD+ ratios during infection

  • Assess respiration rates in the presence of plant extracts

• Interaction with Plant Defenses:

  • Test sensitivity to reactive oxygen species and other plant defense molecules

  • Evaluate potential recognition by plant pattern recognition receptors

Understanding how respiratory metabolism supports virulence will provide new insights into P. syringae pathogenicity mechanisms and potential targets for disease control strategies.

What experimental approaches can reveal how nuoG expression is regulated during different stages of plant infection?

Understanding the regulation of nuoG expression during plant infection requires integrated approaches that capture both regulatory mechanisms and environmental influences:

Transcriptional Analysis Methods:

• RNA-Seq Analysis:

  • Isolate RNA from bacteria grown under various conditions (minimal media, plant extracts, in planta)

  • Perform RNA-seq to identify differentially expressed genes

  • Use computational approaches to identify co-regulated genes and potential regulatory networks

• Quantitative RT-PCR:

  • Design primers specific to nuoG and reference genes

  • Measure expression levels under different conditions

  • Validate RNA-seq findings with targeted analysis

• Promoter Reporter Fusions:

  • Clone the nuoG promoter region upstream of reporter genes (GFP, LUX)

  • Transform constructs into P. syringae

  • Monitor reporter activity during infection using fluorescence microscopy or luminometry

P. syringae pv. phaseolicola gene expression has been successfully analyzed in response to plant extracts using microarray technology , providing a methodological framework for similar studies with nuoG in P. syringae pv. tomato.

Promoter Analysis and Transcription Factor Identification:

• Promoter Mapping:

  • Use 5' RACE to identify transcription start sites

  • Perform DNase I footprinting to identify protected regions

  • Create promoter deletion series to identify critical regulatory elements

• Transcription Factor Identification:

  • Perform DNA affinity chromatography using nuoG promoter fragments

  • Identify bound proteins by mass spectrometry

  • Validate interactions using electrophoretic mobility shift assays (EMSA)

• Chromatin Immunoprecipitation (ChIP):

  • Perform ChIP with antibodies against suspected transcriptional regulators

  • Use ChIP-seq to identify genome-wide binding sites

  • Compare binding profiles under different conditions

Environmental Sensing and Regulation:

• Two-Component System Analysis:

  • Investigate the role of known sensor kinases in nuoG regulation

  • Test nuoG expression in response to specific environmental stimuli

  • Analyze sensor kinase mutants for altered nuoG expression

The search results mention the sensor kinases RetS and LadS in regulating P. syringae virulence , which could be investigated for potential roles in nuoG regulation.

• Quorum Sensing Analysis:

  • Examine nuoG expression in quorum sensing mutants

  • Test expression in response to synthetic autoinducers

  • Investigate population density effects on expression

• In Planta Expression Analysis:

  • Use leaf apoplastic fluid isolation to extract bacteria from infected tissues

  • Employ laser capture microdissection to isolate bacteria from specific infection sites

  • Perform single-cell RNA-seq to capture expression heterogeneity

These methods will provide a comprehensive understanding of nuoG regulation during plant infection, potentially revealing new therapeutic targets and insights into P. syringae pathogenicity mechanisms.

How does nuoG from P. syringae pv. tomato compare to homologous proteins in other bacterial species?

Comparative analysis of nuoG across different bacterial species provides insights into both conserved functions and species-specific adaptations:

Sequence Homology Analysis:

nuoG in P. syringae pv. tomato DC3000 shows varying degrees of homology with equivalent proteins in different organisms:

The nuoG protein is highly conserved across bacterial species, with homologs found in 494 different genera . It belongs to the Pseudomonas Ortholog Group POG002912, which contains 517 members. This high conservation indicates nuoG's essential role in bacterial metabolism.

Domain Architecture and Functional Elements:

nuoG typically contains multiple iron-sulfur cluster binding motifs characterized by conserved cysteine residues that coordinate iron-sulfur clusters. Key functional elements include:

• NADH binding domains

• Iron-sulfur cluster coordination sites (typically CxxCxxC motifs)

• Subunit interaction interfaces

• Electron transfer pathways

In bacterial systems like P. denitrificans and T. thermophilus, NADH-quinone oxidoreductase contains the same number of prosthetic groups as the mammalian enzyme despite having fewer subunits , suggesting functional conservation of electron transfer mechanisms.

Methodological Approaches for Comparative Analysis:

• Multiple Sequence Alignment:

  • Align nuoG sequences from diverse bacteria using tools like MUSCLE or CLUSTALW

  • Identify conserved residues and motifs

  • Map conservation onto structural models

• Phylogenetic Analysis:

  • Construct phylogenetic trees to understand evolutionary relationships

  • Correlate sequence divergence with ecological niches or pathogenicity

  • Identify lineage-specific adaptations

• Structural Comparison:

  • Generate homology models based on available structures

  • Superimpose models to identify structural conservation and divergence

  • Analyze conservation of catalytic and binding sites

• Functional Complementation:

  • Express P. syringae nuoG in heterologous systems

  • Test ability to complement nuoG mutants in other bacterial species

  • Identify species-specific functional constraints

This comparative approach reveals evolutionary adaptations of nuoG in P. syringae that may relate to its lifestyle as a plant pathogen, potentially identifying unique features that could be targeted for antimicrobial development.

What insights can specific nuoG mutations provide about structure-function relationships in NADH-quinone oxidoreductase?

Site-directed mutagenesis of nuoG offers a powerful approach to dissect structure-function relationships within the NADH-quinone oxidoreductase complex. The following methodological framework outlines how to design, implement, and analyze mutagenesis studies:

Target Selection for Mutagenesis:

• Conserved Residues:

  • Identify highly conserved amino acids through multiple sequence alignment

  • Focus on cysteine residues in potential iron-sulfur cluster binding motifs

  • Target acidic and basic residues that may participate in proton transport

• Structural Elements:

  • Target residues at predicted subunit interfaces

  • Identify residues in potential quinone binding regions

  • Select residues in predicted NADH binding domains

• Homology-Based Targets:

  • Identify residues equivalent to functionally important positions in well-characterized homologs

  • Focus on residues implicated in inhibitor binding in other systems

Mutagenesis Design Strategy:

Experimental Implementation:

• Generate mutations using PCR-based methods:

  • QuikChange site-directed mutagenesis

  • Gibson Assembly for larger modifications

  • Golden Gate Assembly for multiple simultaneous mutations

• Expression and purification:

  • Express mutant proteins under identical conditions

  • Purify using standardized protocols

  • Verify protein integrity by SDS-PAGE and western blotting

• Functional characterization:

  • Measure NADH oxidation activity

  • Assess quinone reduction capability

  • Determine electron transfer rates

  • Analyze iron-sulfur cluster content by spectroscopic methods

Data Analysis and Interpretation:

• Activity Profiling:

  • Compare kinetic parameters (Km, Vmax, kcat) between wild-type and mutants

  • Analyze effects on substrate specificity

  • Determine changes in inhibitor sensitivity

• Structural Analysis:

  • Perform thermal stability assays to assess structural integrity

  • Use circular dichroism to detect secondary structure changes

  • Apply HDX-MS to identify altered dynamics

• In vivo Phenotypic Analysis:

  • Introduce mutations into genomic nuoG using allelic exchange methods similar to those described for other nuo genes

  • Assess growth rates under different conditions

  • Measure membrane potential in whole cells

  • Evaluate pathogenicity in plant infection models

Genetic analysis methods similar to those described for other nuo locus mutations can be applied to introduce nuoG mutations into the chromosome through homologous recombination, allowing assessment of phenotypic effects in the native genetic context.

This comprehensive mutagenesis approach provides mechanistic insights into nuoG function and its role within the complex, potentially identifying targets for antimicrobial development or genetic engineering of bacterial metabolism.

What methodologies can determine how nuoG contributes to energy coupling in NADH-quinone oxidoreductase?

Investigating nuoG's contribution to energy coupling requires sophisticated methodologies that can link electron transfer events to proton translocation or other energy conservation mechanisms:

Proton Translocation Measurements:

• Reconstituted Liposome Assays:

  • Purify NADH-quinone oxidoreductase complex

  • Reconstitute into liposomes with controlled lipid composition

  • Monitor pH changes using:

    • pH-sensitive fluorescent dyes (ACMA, pyranine)

    • pH electrodes

    • pH-sensitive protein probes

  • Calculate H+/e- stoichiometry

  • Compare wild-type complex with nuoG mutants

• Inverted Membrane Vesicle Studies:

  • Prepare inside-out membrane vesicles from bacterial cells

  • Measure NADH-driven proton pumping

  • Quantify effects of specific inhibitors

  • Assess proton pumping efficiency in nuoG mutants

Membrane Potential Analysis:

• Fluorescence-Based Methods:

  • Use voltage-sensitive dyes (DiSC3(5), Oxonol VI)

  • Measure fluorescence changes during NADH oxidation

  • Calibrate using ionophores and known potential differences

  • Compare membrane potential generation in wild-type and mutant strains

• Patch-Clamp Electrophysiology:

  • Apply patch-clamp techniques to bacterial spheroplasts or reconstituted systems

  • Measure currents associated with complex I activity

  • Characterize conductance properties and ion selectivity

Conformational Change Analysis:

• FRET (Förster Resonance Energy Transfer):

  • Introduce fluorescent protein pairs at strategic locations in nuoG

  • Measure energy transfer during catalysis

  • Correlate conformational changes with catalytic events

• EPR Spectroscopy:

  • Analyze iron-sulfur cluster redox states during turnover

  • Identify clusters specifically associated with nuoG

  • Use spin labels to detect conformational changes

  • Apply techniques similar to those that identified roles of other subunits

• Time-Resolved Structural Methods:

  • Use time-resolved cryo-EM to capture different conformational states

  • Apply hydrogen-deuterium exchange mass spectrometry to identify dynamic regions

  • Correlate structural changes with catalytic events

Computational Approaches:

• Molecular Dynamics Simulations:

  • Build models of nuoG within the complete complex

  • Perform extended simulations to identify conformational changes

  • Model proton pathways and energy transduction mechanisms

• Quantum Mechanics/Molecular Mechanics (QM/MM):

  • Apply QM calculations to active sites and iron-sulfur clusters

  • Use MM for the surrounding protein environment

  • Calculate energy profiles for electron and proton transfer

These methodologies, applied systematically, can elucidate nuoG's specific contribution to energy coupling in NADH-quinone oxidoreductase, providing insights into this fundamental bioenergetic process.

How can researchers determine the quinone binding sites and electron transfer pathways within nuoG and the NADH-quinone oxidoreductase complex?

Identifying quinone binding sites and mapping electron transfer pathways through nuoG requires an integrated approach combining biochemical, biophysical, and computational methods:

Quinone Binding Site Identification:

• Photoaffinity Labeling:
  Search result describes using "(trifluoromethyl)diazirinyl[³H]pyridaben ([³H]TDP) as a photoaffinity ligand because it combines outstanding inhibitor potency, a suitable photoreactive group, and tritium at high specific activity." Similar approaches can be applied to P. syringae Complex I:

  • Synthesize photoaffinity analogs of quinones or known inhibitors

  • Incubate with purified complex and activate by UV irradiation

  • Identify labeled residues by mass spectrometry

  • Map binding sites on structural models

• Site-Directed Mutagenesis:

  • Target conserved residues predicted to interact with quinones

  • Measure altered binding affinities or inhibitor sensitivity

  • Assess impact on quinone reduction activity

  • Create a map of functionally important residues

• Inhibitor Binding Studies:

  • Test sensitivity to known Complex I inhibitors (rotenone, piericidin A, pyridaben)

  • Perform competition assays between different inhibitors

  • Determine structure-activity relationships with different inhibitors

  • Use differential scanning fluorimetry to measure stabilization by inhibitors

Electron Transfer Pathway Mapping:

• Time-Resolved Spectroscopy:

  • Employ ultrafast spectroscopic techniques to follow electron transfer events

  • Use specific wavelengths to monitor different redox centers

  • Determine electron transfer rates between centers

  • Construct a kinetic model of the electron transfer sequence

• EPR Spectroscopy:

  • Perform power saturation studies to determine distances between paramagnetic centers

  • Use DEER (double electron-electron resonance) to measure distances between spin centers

  • Identify clusters specifically associated with nuoG

  • Determine midpoint potentials of individual iron-sulfur clusters

• Redox Potential Gradient Analysis:

  • Determine redox potentials of individual electron transfer components

  • Map the energetic landscape of electron transfer

  • Identify thermodynamically favorable and unfavorable steps

  • Correlate with structural information

Structural Approaches for Pathway Identification:

• Cryo-EM Analysis:

  • Obtain high-resolution structures of the complex in different redox states

  • Identify quinone binding sites and access channels

  • Map the spatial arrangement of redox cofactors

  • Measure edge-to-edge distances between cofactors

• Computational Pathway Prediction:

  • Apply electron tunneling pathway algorithms

  • Calculate electronic coupling between redox centers

  • Identify key residues mediating electron transfer

  • Simulate electron transfer events using quantum mechanical methods

What genetic engineering approaches can be used to study nuoG function in P. syringae pv. tomato?

Genetic manipulation of nuoG provides powerful insights into its function within NADH-quinone oxidoreductase and its broader role in P. syringae physiology and pathogenicity. The following methodological approaches enable comprehensive genetic analysis:

Gene Knockout and Complementation:

• Allelic Exchange Methods:
  Search result describes genetic manipulation of the nuo locus: "Finally, alleles ΔnuoG1 and nuoG2 were introduced into the chromosome by means of homologous recombination following transformation." Similar approaches can be applied specifically to nuoG:

  • Design constructs with upstream and downstream homology regions flanking an antibiotic resistance marker

  • Transform into P. syringae using electroporation

  • Select for double recombinants using positive and negative selection

  • Confirm deletion by PCR and sequencing

• Complementation Analysis:

  • Clone wild-type nuoG into a broad-host-range vector

  • Transform into the nuoG knockout strain

  • Assess restoration of function

  • Introduce site-directed mutations to test specific hypotheses

• Campbell-Type Integration:
  Search result mentions: "A strain carrying both the wild-type and ΔnuoG1 alleles on its chromosome was isolated following an integrative, homologous recombination event by the Campbell-type mechanism." This approach can create partial duplications for genetic analysis.

Conditional Expression Systems:

• Inducible Promoters:

  • Replace the native nuoG promoter with an inducible system (e.g., arabinose, rhamnose, or IPTG-inducible)

  • Titrate expression levels by varying inducer concentration

  • Study effects of nuoG depletion on cellular functions

  • Assess minimum expression levels required for viability

• Degron-Based Systems:

  • Fuse nuoG to degron tags for conditional protein degradation

  • Trigger degradation using small molecules or temperature shifts

  • Monitor rapid depletion effects on cellular physiology

  • Study dynamic responses to nuoG loss

Reporter Fusions and Localization Studies:

• Translational Fusions:

  • Create C-terminal fusions with fluorescent proteins or epitope tags

  • Visualize subcellular localization using microscopy

  • Track expression levels under different conditions

  • Study interactions with other complex components

Search result describes using YFP fusions to study subcellular localization of P. syringae effector proteins. Similar approaches could be applied to nuoG.

• Split Protein Complementation:

  • Fuse nuoG and potential interaction partners to complementary fragments of a reporter protein

  • Reconstitute reporter activity when proteins interact

  • Map interaction domains using truncated constructs

  • Visualize interactions in living cells

Genome-Wide Approaches:

• Synthetic Genetic Array Analysis:

  • Cross nuoG mutants with libraries of other bacterial mutants

  • Identify genetic interactions through growth phenotypes

  • Map functional relationships with other metabolic pathways

• Transposon Mutagenesis Screens:

  • Perform transposon mutagenesis in nuoG mutant background

  • Screen for suppressors or synthetic lethal interactions

  • Identify genes with functional relationships to nuoG

• CRISPRi-Based Studies:

  • Implement CRISPR interference to partially repress nuoG

  • Create libraries targeting different genes in combination with nuoG manipulation

  • Identify genetic interactions through growth phenotypes

These genetic approaches, complemented with biochemical and physiological analyses, provide a comprehensive toolkit for investigating nuoG function in P. syringae.

How can transcriptomic and proteomic approaches inform our understanding of nuoG function and regulation?

High-throughput 'omics approaches provide systems-level insights into nuoG function and regulation within the broader context of P. syringae metabolism and pathogenicity:

Transcriptomic Approaches:

• RNA-Seq Analysis:
  Search result describes transcriptional profiling of P. syringae in response to plant extracts. Similar approaches can be applied to study nuoG regulation:

  • Compare transcriptomes between wild-type and nuoG mutant strains

  • Profile expression under different growth conditions (minimal media, plant extracts, apoplastic fluid)

  • Analyze expression during different stages of plant infection

  • Identify co-regulated genes that may function with nuoG

• Differential Expression Analysis:

  • Identify genes up or down-regulated in nuoG mutants

  • Perform pathway enrichment analysis to identify affected processes

  • Construct regulatory networks associated with nuoG function

  • Compare with other respiratory mutants to identify common responses

• Transcription Start Site Mapping:

  • Use 5' RACE or RNA-seq variants to identify transcription start sites

  • Map operon structure of the nuo gene cluster

  • Identify potential regulatory elements in promoter regions

  • Characterize alternative transcripts under different conditions

Proteomic Approaches:

• Quantitative Proteomics:

  • Compare protein abundance between wild-type and nuoG mutants using:

    • iTRAQ or TMT labeling

    • Label-free quantification

    • SILAC in appropriate organisms

  • Identify post-translational modifications affecting nuoG function

  • Measure changes in other complex I subunits

  Search result contains proteomic data for several NADH dehydrogenase subunits from P. syringae pv. tomato DC3000, which could serve as a foundation for more detailed studies.

• Protein-Protein Interaction Analysis:

  • Perform immunoprecipitation followed by mass spectrometry (IP-MS)

  • Use proximity labeling methods (BioID, APEX) to identify neighboring proteins

  • Apply crosslinking mass spectrometry to map interaction interfaces

  • Construct protein interaction networks centered on nuoG

• Membrane Proteomics:

  • Employ specialized protocols for membrane protein extraction

  • Use blue native PAGE to preserve native complexes

  • Identify complex I assembly intermediates in nuoG mutants

  • Compare membrane proteomes under different growth conditions

Metabolomic Approaches:

• Central Metabolism Analysis:

  • Measure levels of key metabolites (NADH/NAD+, ATP/ADP, etc.)

  • Analyze carbon flux using 13C-labeled substrates

  • Compare metabolic profiles between wild-type and nuoG mutants

  • Identify metabolic adaptations to nuoG disruption

• Respiratory Chain Analysis:

  • Measure quinone/quinol ratios

  • Analyze respiratory chain component levels

  • Determine electron flow through alternative pathways

  • Assess impact on pmf (proton motive force) generation

Integrative Data Analysis:

• Multi-Omics Integration:

  • Correlate transcriptomic, proteomic, and metabolomic datasets

  • Identify causal relationships between different levels of regulation

  • Construct predictive models of nuoG function and regulation

  • Apply machine learning approaches to identify subtle patterns

• Comparative Analysis Across Conditions:

  • Identify condition-specific regulation of nuoG

  • Compare responses to different plant extracts or host species

  • Analyze evolutionary conservation of regulatory mechanisms

  • Develop predictive models of nuoG function under different conditions

These multi-omics approaches provide a comprehensive view of how nuoG functions within the larger context of bacterial physiology and pathogenicity, revealing both direct effects of nuoG activity and broader cellular adaptations to changes in respiratory metabolism.