Recombinant Vibrio vulnificus Chromosome partition protein mukE (mukE)

Advanced Research Questions

• How does MukE function within the context of the MukBEF complex?

  MukE is thought to serve as a mediator within the MukBEF complex, potentially helping to regulate the ATPase activity of MukB and the organization of higher-order protein-DNA structures. In many bacterial systems, MukE enhances MukB-MukF interactions and promotes the proper assembly of the chromosome segregation machinery.

  To study these interactions, researchers should consider:

  • Yeast two-hybrid or bacterial two-hybrid assays to map interaction domains

  • In vitro reconstitution of the MukBEF complex using purified components

  • Förster resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) to study protein interactions in real-time

  • Analytical ultracentrifugation or size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to determine complex stoichiometry

• How does MukE expression vary between clinical and environmental strains of Vibrio vulnificus?

  Vibrio vulnificus demonstrates significant genetic differentiation between clinical and environmental strains . While specific information about MukE expression differences is not provided in the search results, this represents an important research question.

  To investigate this question:

  1. Perform comparative genomic analysis of mukE sequences from multiple clinical and environmental isolates

  2. Use quantitative PCR to measure mukE expression levels in different strains under various growth conditions

  3. Conduct Western blot analysis with anti-MukE antibodies to compare protein levels

  4. Perform RNA-seq to identify potential differences in transcriptional regulation

  5. Investigate if any single nucleotide polymorphisms (SNPs) in the mukE gene correlate with clinical vs. environmental isolates

• What is the relationship between MukE function and environmental adaptation in Vibrio vulnificus?

  Vibrio vulnificus is expanding its geographical range due to climate change and rising sea temperatures . The role of chromosome partition proteins like MukE in environmental adaptation represents an intriguing research question.

  Methodological approaches could include:

  • Comparing MukE function at different temperatures representing various marine environments

  • Assessing chromosome segregation efficiency under stress conditions such as changing salinity or pH

  • Creating temperature-sensitive MukE mutants to determine how chromosome partitioning affects adaptation

  • Investigating potential horizontal gene transfer of mukE variants between strains in aquaculture settings where different Vibrio vulnificus clusters co-exist

• How might inhibiting MukE function affect Vibrio vulnificus pathogenicity?

  As a chromosome partition protein essential for bacterial cell division, MukE could potentially serve as a target for antimicrobial development. Approaches to investigate this include:

  • High-throughput screening of small molecule libraries to identify MukE inhibitors

  • Structure-based drug design targeting key functional domains of MukE

  • Testing identified inhibitors in cell-based assays to evaluate effects on growth and virulence

  • In vivo testing of promising compounds in infection models

  • Analysis of potential resistance mechanisms that might develop

  The efficacy of such approaches would need to be compared with targeting established virulence factors like VVH (hemolysin) or MARTX toxin, which have been directly implicated in tissue damage and bacterial dissemination .

• What experimental models are appropriate for studying MukE function in Vibrio vulnificus?

  Several experimental systems can be employed to study MukE function:

  In vitro systems:

  • Purified protein biochemical assays

  • DNA binding and condensation assays

  • ATPase activity measurements (in conjunction with MukB)

  Cellular systems:

  • Gene deletion or depletion studies in Vibrio vulnificus

  • Fluorescence microscopy with labeled chromosomes to visualize segregation defects

  • Time-lapse microscopy to observe cell division abnormalities

  • Complementation studies with mutant versions of MukE

  Advanced model systems:

  • Human intestinal epithelial cell lines (e.g., Caco-2, INT-407) to study bacterial interactions

  • Mouse models of infection to assess virulence of MukE mutants

  • Zebrafish embryo models for visualizing infection dynamics

• How does MukE interact with other chromosome organization systems in Vibrio vulnificus?

  Bacteria typically possess multiple systems for chromosome organization beyond the MukBEF complex. These may include nucleoid-associated proteins (NAPs), SMC-ScpAB complexes, and topoisomerases.

  Research methodologies to investigate these interactions include:

  • ChIP-seq to identify genomic binding sites of MukE and potential overlap with other systems

  • Genetic interaction screens (e.g., synthetic lethality) to identify functional relationships

  • Co-immunoprecipitation coupled with mass spectrometry to identify protein-protein interactions

  • Hi-C or other chromosome conformation capture techniques to analyze global chromosome organization

  • Super-resolution microscopy to visualize the spatial organization of different chromosome management systems

• What technical challenges are associated with studying MukE in Vibrio vulnificus?

  Researchers face several technical challenges when investigating MukE:

  1. Genetic manipulation: Developing efficient genetic tools for Vibrio vulnificus can be challenging compared to model organisms like E. coli

  2. Protein solubility: Chromosome partition proteins often form large complexes that can be difficult to maintain in solution during purification

  3. Safety considerations: Working with Vibrio vulnificus requires appropriate biosafety measures as it is a BSL-2 pathogen that can cause serious infections

  4. Functional redundancy: Potential overlap with other chromosome organization systems may complicate phenotypic analysis

  5. Strain variation: The significant genetic diversity between Vibrio vulnificus strains means findings from one strain may not be universally applicable

  Addressing these challenges requires careful experimental design, appropriate controls, and validation across multiple strains when possible.

Methodological Considerations

• What expression systems are optimal for producing recombinant MukE for structural studies?

  The choice of expression system depends on the specific requirements of the structural studies:

  Expression System	Advantages	Disadvantages	Best For
  E. coli BL21(DE3)	High yield, simple, cost-effective	Limited post-translational modifications	Basic structural studies, crystallography
  E. coli Arctic Express	Better folding at low temperatures	Lower yield	Proteins prone to misfolding
  Cell-free expression	Rapid, avoids toxicity issues	Expensive, lower yield	Toxic proteins, quick screening
  Insect cells	Enhanced folding, post-translational modifications	Complex, expensive	Proteins requiring specific modifications

  For MukE from Vibrio vulnificus, an E. coli-based system with careful optimization of induction conditions (temperature, inducer concentration, time) is typically sufficient, especially when using solubility-enhancing fusion partners like MBP or SUMO.

• How can researchers effectively study MukE-DNA interactions?

  Understanding how MukE contributes to chromosome condensation requires specialized techniques:

  1. Electrophoretic Mobility Shift Assays (EMSA): To detect direct binding of MukE (likely in complex with MukB and MukF) to DNA fragments

  2. Microscale Thermophoresis (MST): For quantitative measurement of binding affinities

  3. Atomic Force Microscopy (AFM): To visualize MukBEF-DNA complexes and DNA condensation

  4. Magnetic tweezers or optical tweezers: To measure the mechanical forces exerted during DNA condensation

  5. DNA curtains: For single-molecule visualization of protein-DNA interactions

  6. Fluorescence microscopy with reconstituted systems: Using fluorescently labeled components to observe dynamics in real-time

  These methods should be employed with careful controls, including mutant versions of MukE lacking key functional domains.

• What bioinformatic approaches can help understand MukE evolution in Vibrio species?

  Several computational methods can provide insights into MukE evolution:

  1. Phylogenetic analysis: Constructing phylogenetic trees of MukE sequences across Vibrio species and correlating with pathogenicity

  2. Protein structure prediction: Using AlphaFold2 or RoseTTAFold to predict MukE structural features where crystal structures are unavailable

  3. Molecular dynamics simulations: To investigate the dynamics of MukE-MukB-MukF interactions

  4. Coevolution analysis: Identifying co-evolving residues within MukE or between MukE and its binding partners

  5. Selection pressure analysis: Calculating dN/dS ratios to identify residues under positive or purifying selection

  6. Comparative genomic context analysis: Examining the conservation of gene neighborhoods around mukE across different Vibrio species

  These analyses can provide valuable insights into functional constraints and adaptations of MukE across the evolutionary history of Vibrio species.

• How can researchers investigate the potential role of MukE in Vibrio vulnificus virulence?

  While MukE is primarily known for its role in chromosome segregation rather than direct virulence, investigating its potential contribution to pathogenicity requires:

  1. Conditional knockdown systems: To reduce MukE levels without completely eliminating chromosome segregation

  2. Tissue culture infection models: Using human intestinal epithelial cells to assess the impact of MukE alterations on bacterial invasion and cytotoxicity

  3. Animal infection models: Comparing wild-type and MukE-modified strains in appropriate animal models

  4. Transcriptomic analysis: Identifying downstream effects of MukE alteration on expression of known virulence factors

  5. Growth curve analysis under infection-relevant conditions: Testing whether MukE alterations affect bacterial fitness under conditions mimicking the host environment

  6. Competition assays: Mixed infections with wild-type and MukE-modified strains to assess relative fitness during infection

This research direction may reveal whether chromosome partition proteins like MukE could serve as indirect virulence factors by enabling efficient bacterial replication during infection, particularly in the context of the rapid proliferation observed in fatal Vibrio vulnificus septicemia .