Recombinant Shigella dysenteriae serotype 1 Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is the functional role of UbiB in Shigella dysenteriae?

UbiB functions as a probable ubiquinone biosynthesis protein in S. dysenteriae. It is an essential component of the ubiquinone (coenzyme Q or UQ) biosynthetic pathway, which is critical for aerobic respiration in this bacterial pathogen. Ubiquinone serves as an electron carrier in the respiratory chain, making UbiB indirectly essential for energy production under aerobic conditions. Research indicates that UbiB appears to interact with several other Ubi proteins (including UbiE, UbiF, UbiG, UbiH, UbiI, UbiJ, and UbiX) as part of a potential multi-protein complex involved in ubiquinone biosynthesis . This suggests that UbiB likely contributes to the global UQ biosynthesis process rather than catalyzing a single specific step in the pathway.

How does UbiB expression in S. dysenteriae compare between different infection sites?

S. dysenteriae shows varying levels of virulence gene expression depending on its site of infection. While specific data on UbiB expression across infection sites is limited in the provided search results, transcriptomic analysis of S. dysenteriae isolated from both stool and blood samples of the same patient revealed differential gene expression patterns . Genes involved in invasion were highly expressed in strains isolated from the primary site of infection (intestinal tract). By extension, since ubiquinone biosynthesis is critical for pathogen fitness during infection, UbiB expression may similarly be regulated in a site-specific manner to optimize bacterial adaptation to diverse host environments. Comprehensive transcriptomic studies would be required to precisely quantify UbiB expression levels across different infection sites.

What is the relationship between UbiB and antibiotic resistance in S. dysenteriae?

While UbiB itself is not directly implicated in antibiotic resistance, S. dysenteriae strains commonly carry multiple antimicrobial resistance genes, including dhfr1A, sulII, blaOXA, blaCTX-M-1, and qnrS . The UbiB-dependent ubiquinone biosynthesis pathway contributes to bacterial fitness and survival during infection, potentially influencing persistence during antibiotic treatment. Ubiquinone's role in electron transport chain function means that UbiB indirectly impacts cellular energy production, which could affect efflux pump efficiency and other energy-dependent resistance mechanisms. Mutations in UbiB or alterations in its expression could theoretically modify bacterial metabolism in ways that influence susceptibility to certain antibiotics, though this relationship requires further investigation through targeted gene deletion and complementation studies.

How does UbiB interact with other proteins in the ubiquinone biosynthesis pathway?

UbiB interacts with multiple other Ubi proteins as part of the ubiquinone biosynthesis pathway. Bacterial two-hybrid assays have demonstrated that UbiB forms direct or indirect interactions with UbiE, UbiF, UbiG, UbiH, UbiI, UbiJ, and UbiX . Most notably, UbiB appears to participate in protein-protein interactions with UbiK, which itself forms a complex with UbiJ in an UbiK2-UbiJ1 stoichiometry. This interaction network suggests that UbiB may function within a larger multi-protein complex, potentially as part of a metabolon dedicated to ubiquinone biosynthesis .

The following table summarizes the known protein interactions involving UbiB in the ubiquinone biosynthesis pathway:

Further co-immunoprecipitation and structural studies would be necessary to fully characterize the stoichiometry and functional significance of these interactions.

What are the effects of ubiB gene deletion on S. dysenteriae virulence and fitness?

While specific data on ubiB deletion in S. dysenteriae is not directly provided in the search results, research on related ubiquinone biosynthesis genes offers valuable insights. Studies on ubiK mutants in Salmonella (which interacts with UbiB) showed that deletion resulted in growth defects under aerobic conditions, especially at higher temperatures . The ubiK mutant showed significantly reduced ubiquinone (UQ8) levels (down to 18% compared to wild-type) .

By extension, ubiB deletion in S. dysenteriae would likely:

• Severely impair ubiquinone biosynthesis

• Reduce aerobic respiration capacity

• Decrease growth rates under aerobic conditions

• Compromise fitness during infection, particularly in oxygen-rich environments

• Potentially result in the accumulation of biosynthetic intermediates such as octaprenylphenol

How does the UbiB structure relate to its function in ubiquinone biosynthesis?

The structural features of UbiB that enable its function in ubiquinone biosynthesis remain partially characterized. UbiB belongs to a family of proteins with an α/β-fold domain structure. Current understanding suggests that UbiB likely contains:

• Nucleotide-binding domains - potentially for ATP binding and hydrolysis

• Membrane-association motifs - enabling interaction with the inner membrane where ubiquinone biosynthesis occurs

• Protein-protein interaction domains - facilitating its interactions with other Ubi proteins

Recent research on UbiK, which interacts with UbiB, has shown that UbiK forms a complex with UbiJ that can interact with palmitoleic acid, a major lipid in E. coli . This suggests that lipid interaction may be important for the function of the ubiquinone biosynthesis machinery. UbiB might similarly interact with lipids or play a role in organizing the spatial arrangement of the ubiquinone biosynthetic complex at the membrane interface.

More detailed structural information would require X-ray crystallography, cryo-electron microscopy, or NMR studies of purified UbiB protein, which have not yet been reported in the literature.

What are the optimal conditions for expressing recombinant S. dysenteriae UbiB protein?

For optimal expression of recombinant S. dysenteriae UbiB protein, the following methodological approach is recommended:

Expression System Selection:

• E. coli BL21(DE3) or its derivatives are preferred host strains due to compatibility with Shigella proteins

• Consider using Rosetta or Rosetta2 strains to account for codon usage differences

Vector Construction:

• Use pET-series vectors (pET28a or pET32a) incorporating a His6-tag for purification

• Alternatively, employ a dual-tag system (His-tag and S-tag) to facilitate interaction studies with other Ubi proteins, as successfully used for UbiK-UbiJ interaction studies

Expression Conditions:

• Culture in LB media supplemented with appropriate antibiotics

• Induce at OD600 of 0.6-0.8 with 0.1-0.5 mM IPTG

• Lower induction temperature to 18-25°C for 16-18 hours to enhance solubility

• Consider supplementing media with cofactors like ATP or lipids that might stabilize the protein

Purification Strategy:

• Lyse cells with gentle detergents (0.5-1% Triton X-100) as UbiB may associate with membranes

• Purify using nickel-nitrilotriacetic acid resin for His-tagged protein

• Include ATP (1-5 mM) in purification buffers if UbiB binds nucleotides

• Consider co-expression with UbiJ and UbiK to improve stability and solubility

This approach should yield functionally active UbiB protein suitable for biochemical and structural characterization studies.

What techniques are most effective for studying UbiB interactions with other proteins in the ubiquinone biosynthesis pathway?

Multiple complementary techniques are effective for studying UbiB interactions with other proteins in the ubiquinone biosynthesis pathway:

1. Bacterial Two-Hybrid System (BACTH)
This system has proven effective for detecting both direct and indirect interactions among Ubi proteins. It involves fusion of proteins of interest to adenylate cyclase fragments (T18 and T25) expressed from compatible plasmids . The BACTH system successfully demonstrated interactions between UbiK and multiple Ubi proteins including UbiB, UbiE, UbiF, UbiG, UbiH, UbiI, UbiJ, and UbiX .

2. Co-immunoprecipitation and Pull-down Assays
For confirmation of direct physical interactions:

• Express UbiB with an S-tag and potential interacting partners with His6-tags

• Purify His-tagged proteins on nickel-nitrilotriacetic acid resin

• Detect co-purified UbiB using anti-S-tag antibodies

• This approach successfully identified the UbiK-UbiJ interaction

3. Surface Plasmon Resonance (SPR)
SPR can determine binding kinetics and affinity constants between UbiB and its partners:

• Immobilize purified UbiB on a sensor chip

• Flow various Ubi proteins over the surface

• Measure real-time association and dissociation

4. Cross-linking Mass Spectrometry
This technique can identify interaction interfaces:

• Treat protein complexes with chemical cross-linkers

• Digest cross-linked proteins and analyze by mass spectrometry

• Identify cross-linked peptides to map interaction sites

5. Microscale Thermophoresis (MST)
MST can detect interactions in solution using minimal protein amounts:

• Label UbiB with a fluorescent dye

• Mix with increasing concentrations of unlabeled partner proteins

• Measure changes in thermophoretic mobility to determine binding

A comprehensive interaction study would employ multiple techniques to provide complementary evidence for the composition and dynamics of the UbiB-containing protein complex.

What genomic and transcriptomic approaches can be used to study ubiB regulation in S. dysenteriae during infection?

Several genomic and transcriptomic approaches can be employed to study ubiB regulation in S. dysenteriae during infection:

RNA-Seq Analysis
RNA-Seq provides a comprehensive view of the transcriptome:

• Extract RNA from S. dysenteriae isolated from different infection sites (as demonstrated with stool and blood samples)

• Perform rRNA depletion and construct cDNA libraries

• Sequence using high-throughput platforms (Illumina)

• Analyze differential expression of ubiB and related genes

• This approach has successfully revealed differential expression patterns of virulence genes in S. dysenteriae from different infection sites

RT-qPCR Validation
For targeted quantification of ubiB expression:

• Design primers specific to ubiB and reference genes

• Extract RNA from bacteria under various conditions

• Perform reverse transcription and qPCR

• Calculate relative expression using the 2^(-ΔΔCT) method

ChIP-Seq for Transcription Factor Binding
To identify regulators of ubiB:

• Cross-link S. dysenteriae cells isolated from infection sites

• Immunoprecipitate DNA bound to suspected transcriptional regulators

• Sequence precipitated DNA

• Identify enriched regions corresponding to the ubiB promoter

Reporter Gene Constructs
To study promoter activity:

• Clone the ubiB promoter region upstream of a reporter gene (e.g., GFP or luciferase)

• Transform into S. dysenteriae

• Measure reporter expression under various conditions mimicking different infection sites

In vivo Expression Technology (IVET)
To assess ubiB expression during infection:

• Create a promoter trap library including the ubiB promoter

• Infect animal models

• Recover bacteria and identify promoters active during infection

These approaches can be combined to develop a comprehensive understanding of how ubiB expression is regulated in response to different host environments during S. dysenteriae infection.

How can discrepancies in UbiB function between different bacterial species be resolved?

Resolving discrepancies in UbiB function between different bacterial species requires a systematic comparative approach:

Systematic Ortholog Analysis
Create a table comparing UbiB orthologs across species:

• Perform sequence alignment of UbiB proteins from multiple species

• Calculate sequence identity and similarity percentages

• Identify conserved domains and species-specific variations

Complementation Studies
Test functional conservation across species:

• Express UbiB from different species in a ubiB deletion mutant

• Measure restoration of ubiquinone biosynthesis

• Quantify growth under aerobic conditions

• Compare complementation efficiency across orthologs

This approach has been used successfully to study RibA and RibB function across bacterial species, revealing important insights about gene essentiality despite contradictory annotations .

Biochemical Characterization
Compare biochemical properties of UbiB proteins:

• Express and purify UbiB from different species

• Analyze enzymatic activities under standardized conditions

• Determine cofactor requirements

• Measure interaction affinities with partner proteins

Structural Comparisons
Analyze structural differences:

• Determine structures of UbiB proteins from different species

• Compare active sites and binding pockets

• Identify structural elements that might explain functional differences

Genomic Context Analysis
Examine the genomic neighborhood of ubiB:

• Compare gene organization around ubiB

• Identify species-specific operon structures

• Analyze potential co-regulated genes

By integrating these approaches, researchers can develop a unified model of UbiB function that accounts for species-specific variations while identifying conserved core functions.

What is the relationship between UbiB function and antimicrobial resistance in clinical S. dysenteriae isolates?

The relationship between UbiB function and antimicrobial resistance in clinical S. dysenteriae isolates involves several potential mechanisms:

Metabolic Fitness and Persistence
UbiB's role in ubiquinone biosynthesis affects bacterial energy metabolism:

• Functional ubiquinone biosynthesis enhances aerobic respiration efficiency

• Improved energy production may support energy-dependent resistance mechanisms such as efflux pumps

• Enhanced metabolic fitness could promote bacterial persistence during antibiotic treatment

Co-selection of Resistance Determinants
Analysis of clinical S. dysenteriae isolates has identified multiple antimicrobial resistance genes:

• dhfr1A (trimethoprim resistance)

• sulII (sulfonamide resistance)

• blaOXA and blaCTX-M-1 (β-lactam resistance)

• qnrS (quinolone resistance)

While there is no direct evidence linking ubiB to these resistance determinants, genomic analysis could reveal potential genetic linkages or co-selection patterns.

Stress Response Coordination
Ubiquinone biosynthesis may be coordinated with stress responses:

• Antibiotic exposure often triggers oxidative stress

• Ubiquinone has antioxidant properties

• UbiB function might indirectly contribute to oxidative stress tolerance

Potential Research Approach
To investigate this relationship:

• Compare ubiB expression in resistant versus susceptible isolates

• Analyze correlations between ubiB sequence variations and resistance profiles

• Examine effects of ubiB deletion on minimum inhibitory concentrations (MICs)

• Investigate whether antimicrobial treatment affects ubiB expression

This multi-faceted approach would help clarify whether UbiB function contributes to antimicrobial resistance phenotypes in clinical S. dysenteriae isolates.

How can genomic typing methods improve our understanding of UbiB variation in global S. dysenteriae strains?

Modern genomic typing methods can significantly enhance our understanding of UbiB variation in global S. dysenteriae strains:

Whole Genome Sequencing (WGS) Analysis
Recent advances in Shigella strain typing have moved toward genomic sequencing:

• The Institut Pasteur and international collaborators have validated a new method using bacterial genome sequencing to improve identification and typing of Shigella strains

• This approach has been validated on more than 4,000 reference strains

• WGS data can be mined specifically for ubiB sequence variations across global isolates

Phylogenomic Analysis of UbiB
Constructing evolutionary relationships based on ubiB sequences:

• Extract ubiB coding sequences from WGS data of global isolates

• Perform multiple sequence alignment

• Construct phylogenetic trees to identify lineage-specific patterns

• Correlate ubiB variants with geographic distribution and clinical outcomes

Population Genomics Approach
Analyzing ubiB in the context of core and accessory genomes:

• Determine if ubiB is part of the core genome or shows strain-specific variations

• Identify single nucleotide polymorphisms (SNPs) within ubiB across populations

• Calculate selection pressures (dN/dS ratios) to determine evolutionary constraints

• Detect potential horizontal gene transfer events affecting ubiB

Functional Genomics Integration
Correlating sequence variations with functional differences:

• Express and characterize different ubiB variants

• Measure ubiquinone production efficiency

• Assess growth rates under aerobic conditions

• Evaluate competitive fitness in mixed cultures

This comprehensive approach would provide insights into how UbiB has evolved across global S. dysenteriae populations and whether functional variations contribute to differences in pathogenicity or metabolic adaptation.

What novel approaches could enhance our understanding of UbiB's role in S. dysenteriae pathogenesis?

Several innovative approaches could advance our understanding of UbiB's role in S. dysenteriae pathogenesis:

CRISPR-Cas9 Gene Editing
Precise genetic manipulation:

• Create conditional ubiB mutants using inducible promoters

• Generate point mutations in key functional domains

• Introduce tagged versions of UbiB for in vivo tracking

• Develop CRISPR interference systems for temporal regulation of ubiB expression

Single-Cell Techniques
Analyzing heterogeneity in bacterial populations:

• Apply single-cell RNA-seq to bacteria recovered from infection sites

• Use fluorescent reporters to track ubiB expression in individual bacteria during infection

• Employ microfluidics to analyze growth and division of ubiB mutants

Host-Pathogen Interaction Models
Advanced infection models:

• Utilize intestinal organoids to study S. dysenteriae in a more physiologically relevant context

• Develop humanized mouse models for studying ubiB's role during in vivo infection

• Apply dual RNA-seq to simultaneously analyze host and bacterial transcriptomes

Systems Biology Integration
Multi-omics approaches:

• Combine transcriptomics, proteomics, and metabolomics data

• Construct mathematical models of ubiquinone biosynthesis

• Simulate the impact of UbiB perturbations on cellular metabolism

• Identify potential metabolic vulnerabilities for drug targeting

Structural Biology Advances
Detailed molecular characterization:

• Apply cryo-electron microscopy to visualize the entire ubiquinone biosynthesis complex

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

• Employ computational modeling to predict interaction interfaces

These approaches would collectively provide a more comprehensive understanding of UbiB's role in S. dysenteriae pathogenesis and potentially identify new therapeutic strategies targeting this pathway.