Recombinant Tropheryma whipplei UvrABC system protein B (uvrB), partial

What is Tropheryma whipplei and what makes it significant for UvrB studies?

Tropheryma whipplei is the bacterial agent of Whipple's disease, a chronic infectious disease characterized by intestinal malabsorption that can lead to cachexia and death without appropriate antibiotic treatment. T. whipplei is particularly significant in microbiology research because it represents the only known reduced genome species (less than 1 Mb) within the Actinobacteria phylum (high G+C Gram-positive bacteria) . The organism has a small 927,303 bp circular genome encoding 808 predicted protein-coding genes and 54 RNA genes . Its compact genome makes it an excellent model for studying essential bacterial DNA repair mechanisms, including the UvrABC nucleotide excision repair system, as these functions must be preserved even in a highly reduced genome.

What is the UvrABC system and what role does UvrB play within it?

The UvrABC system constitutes the nucleotide excision repair (NER) pathway in bacteria, responsible for removing a wide range of DNA helix-distorting lesions from the genome. This system operates across all domains of life and plays a crucial role in maintaining genomic integrity .

Within this system, UvrB serves as a central component with the following functions:

• Damage verification after initial recognition by UvrA

• Formation of a pre-incision complex at the damage site

• Recruitment of UvrC endonuclease to execute the dual incisions around the lesion

• Orchestration of downstream NER reactions after UvrA dissociates from the lesion complex

UvrB's critical role is evidenced by experiments showing that deletion of the uvrB gene prevents proper recruitment of repair machinery to DNA damage sites, rendering cells non-functional in nucleotide excision repair .

How are UvrA and UvrB recruited to DNA damage sites?

Recent single-molecule fluorescence imaging studies have revealed that NER initiation involves a two-step "scan and recruit" mechanism:

• UvrA independently scans the genome and locates DNA damage without requiring UvrB. In studies with UvrB-deleted cells, UvrA still successfully binds to DNA, demonstrating its independent DNA scanning ability .

• Once UvrA identifies potential damage, it recruits UvrB directly from solution to verify the lesion. This is supported by observations that exposure to ultraviolet light causes a significant increase in immobile UvrB molecules (59%) with a concomitant decrease in fast-diffusing molecules .

This process is coordinated by ATP binding and hydrolysis in UvrA's "proximal" and "distal" ATP-binding sites. Initial damage recognition by UvrA requires ATPase activity in the distal site only, while subsequent UvrB recruitment requires ATP hydrolysis in the proximal site .

What methods are used to detect Tropheryma whipplei in clinical and research settings?

The primary method for detecting T. whipplei is PCR amplification targeting its DNA. This approach is particularly important because:

• The bacterium resisted reproducible culture until it was grown in human fibroblasts in 2000

• Untreated Whipple's disease leads to death

• Appropriate antimicrobial therapy differs significantly from treatments for diseases with similar clinical presentations

PCR-based detection typically targets the 16S ribosomal DNA sequence of T. whipplei, which allows classification of this organism as a member of the gram-positive bacteria with high G+C content .

What are the kinetics of UvrA and UvrB binding during DNA damage recognition and verification?

Single-molecule imaging studies have provided detailed insights into the binding kinetics of UvrA and UvrB during damage recognition and verification:

These measurements demonstrate that the initial steps of NER (damage recognition and verification) are significantly slower than the final repair stages. The longer dwell time of UvrB compared to UvrA after UV exposure supports the model in which UvrA dissociates from the lesion complex before UvrB recruits UvrC .

How does the methodology for studying UvrB loading onto damaged DNA in vitro compare with in vivo observations?

In vitro UvrB loading assays provide valuable complementary evidence to in vivo single-molecule imaging studies:

In vitro methodology:

• UvrA (60 nM dimer) is incubated with biotinylated damaged DNA (50 bp with fluorescein lesion) in buffer containing ATP

• Magnetic beads are added to isolate the DNA-protein complexes

• UvrB (120 nM) is added and incubated for specific time intervals

• The reaction is stopped by increasing NaCl concentration to 1M

• After washing, bound proteins are analyzed by SDS-PAGE gel electrophoresis

Key findings comparing preloaded UvrA versus solution-formed UvrA-UvrB complexes:
When UvrB was added to DNA preloaded with UvrA, the efficiency of UvrB recruitment was higher than when premixed UvrA and UvrB were added to damaged DNA simultaneously. This in vitro result strongly supports the in vivo observation that UvrA first locates and verifies DNA damage independently, then recruits UvrB directly from solution .

How do mutations in the UvrB gene affect the DNA repair capacity of Tropheryma whipplei?

While specific mutations in T. whipplei UvrB have not been extensively characterized in the provided search results, we can infer the potential impacts based on the functional importance of UvrB in the NER pathway:

• Deletion mutations: Complete loss of UvrB function would severely compromise the NER pathway as demonstrated in UvrB deletion studies where no UvrB recruitment was observed after ultraviolet light exposure .

• ATPase domain mutations: UvrB contains ATPase activity essential for DNA unwinding and damage verification. Mutations affecting the ATPase domain would likely impair damage verification and formation of the pre-incision complex.

• UvrA interaction domain mutations: Given that UvrB recruitment depends on UvrA, mutations affecting the UvrA-interaction interface would disrupt the coordination between damage recognition and verification steps.

• DNA binding domain mutations: These would likely affect UvrB's ability to form stable complexes with damaged DNA, compromising subsequent repair steps.

Research on specific T. whipplei UvrB mutations would be particularly valuable for understanding any adaptations in the DNA repair mechanisms of this organism with its reduced genome.

What experimental approaches can be used to study the differential recruitment of UvrB to various types of DNA lesions?

Several sophisticated experimental approaches can be employed to study UvrB recruitment to different DNA lesions:

• Single-molecule fluorescence imaging:

  • Label UvrB with photoactivatable fluorescent proteins like PAmCherry

  • Track single-molecule movements in living cells after exposure to different DNA-damaging agents

  • Analyze diffusion coefficients and dwell times to determine recruitment efficiency and binding stability

• In vitro reconstitution assays:

  • Create DNA substrates with specific lesions (UV-induced cyclobutane pyrimidine dimers, chemical adducts, mismatches)

  • Measure UvrB loading efficiency using purified components

  • Quantify using gel-shift assays, fluorescence anisotropy, or surface plasmon resonance

• Competition assays:

  • Design experiments with competing damaged DNA substrates

  • Assess preferential recruitment of UvrB to different lesion types

  • Measure relative affinities and repair efficiencies

• ATP hydrolysis coupling:

  • Monitor ATP consumption rates during UvrB recruitment to different lesions

  • Correlate ATP hydrolysis with verification efficiency

  • Use non-hydrolyzable ATP analogs to trap specific intermediates

How does the genome structure of Tropheryma whipplei influence its DNA repair mechanisms?

T. whipplei's reduced genome (927,303 bp) exhibits several features that potentially influence its DNA repair mechanisms:

• Genomic rearrangements and WiSP protein family:

  • T. whipplei contains a large chromosomal inversion with boundaries located within paralogous genes belonging to a cell-surface protein family (WiSP)

  • These WiSP genes contain highly conserved WND-domain sequences (up to 99% identical over 800 nucleotides) that can trigger frequent genome rearrangements

  • These rearrangements may affect the expression of different subsets of cell surface proteins, potentially representing a mechanism for evading host defenses

• Limited repair pathways:

  • The reduced genome likely retains only essential DNA repair mechanisms

  • The UvrABC pathway's preservation indicates its critical importance for survival

  • T. whipplei lacks clear thioredoxin and thioredoxin reductase homologs, which may impact its oxidative stress response and indirectly affect DNA repair needs

• DNA gyrase mutation:

  • T. whipplei contains a mutation in DNA gyrase that predicts resistance to quinolone antibiotics

  • This may affect DNA topology and potentially influence repair protein access to damage sites

• Competence genes:

  • The genome contains three competence-related genes homologous to B. subtilis comEA, comEC, and comFC

  • This suggests T. whipplei might naturally take up DNA from its environment, potentially providing an alternative mechanism for DNA repair through recombination

What are the best approaches to produce recombinant Tropheryma whipplei UvrB protein for structural and functional studies?

Producing recombinant T. whipplei UvrB requires specialized approaches due to the unique characteristics of this organism:

• Expression system selection:

  • E. coli is typically the first-choice expression system for bacterial proteins

  • BL21(DE3) or similar strains designed for recombinant protein expression are recommended

  • Consider using T7 promoter-based vectors (pET series) for high-level expression

• Construct optimization:

  • Include a polyhistidine (His6) tag for affinity purification

  • Consider expressing the protein as a fusion with solubility enhancers like MBP (maltose-binding protein) or SUMO

  • For structural studies, design constructs that remove flexible regions while maintaining functional domains

  • Optimize codon usage for the expression host, as T. whipplei has an unusually low G+C content (46%) for an Actinobacteria

• Purification strategy:

  • Multi-step purification typically including:

    • Immobilized metal affinity chromatography (IMAC)

    • Ion exchange chromatography

    • Size exclusion chromatography

  • Include ATP in buffers to stabilize the protein

  • Consider tag removal using specific proteases for structural studies

• Functional verification:

  • ATPase activity assays using colorimetric methods

  • DNA binding assays using electrophoretic mobility shift assays (EMSA)

  • In vitro reconstitution of UvrB loading onto damaged DNA as described in the search results

How can the UvrA-UvrB interaction in Tropheryma whipplei be studied at the molecular level?

Studying the UvrA-UvrB interaction at the molecular level requires a combination of structural, biochemical, and biophysical approaches:

• Co-immunoprecipitation studies:

  • Express tagged versions of UvrA and UvrB

  • Use antibodies against one protein to pull down the complex

  • Detect interaction by Western blotting

  • Identify domains essential for interaction using truncated constructs

• Förster Resonance Energy Transfer (FRET):

  • Label UvrA and UvrB with appropriate fluorophore pairs

  • Monitor energy transfer as a measure of protein proximity

  • Conduct FRET analyses with various damaged DNA substrates to assess complex formation dynamics

• Surface Plasmon Resonance (SPR):

  • Immobilize one protein on a sensor chip

  • Flow the partner protein over the surface

  • Determine binding kinetics (kon and koff) and affinity (KD)

  • Assess how different nucleotides (ATP, ADP, non-hydrolyzable analogs) affect interaction

• X-ray crystallography or Cryo-EM:

  • Attempt to crystallize the UvrA-UvrB complex

  • Alternatively, use cryo-electron microscopy for structural determination

  • Focus on capturing different states by using ATP analogs or damaged DNA

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Identify regions of altered solvent accessibility upon complex formation

  • Map interaction interfaces and conformational changes

• Two-step UvrB loading assay:

  • Utilize the in vitro UvrB loading assay described in the search results

  • Compare efficiency of UvrB recruitment using preloaded UvrA versus premixed UvrA-UvrB

  • Quantitatively assess loading efficiency via SDS-PAGE analysis

What experimental strategies can resolve contradictory data about UvrB recruitment mechanisms?

Resolving contradictory data about UvrB recruitment mechanisms requires multifaceted experimental approaches:

• Combined in vivo and in vitro approaches:

  • Verify findings across multiple experimental systems

  • Use in vitro reconstitution with purified components to control variables

  • Validate with in vivo single-molecule tracking to ensure biological relevance

• Genetic approaches:

  • Create specific point mutations in UvrA and UvrB

  • Assess the impact on complex formation and recruitment

  • Use complementation studies to determine functional significance of specific domains

• Time-resolution studies:

  • Implement time-course experiments with precise temporal control

  • Compare UvrB recruitment efficiency under different experimental conditions

  • Use stopped-flow techniques for rapid kinetic measurements

• Concentration dependence:

  • Systematically vary UvrA and UvrB concentrations

  • Determine if contradictory results stem from concentration-dependent mechanisms

  • Establish physiologically relevant concentration ranges

• Substrate variation:

  • Test different DNA damage types

  • Assess whether contradictory data reflects substrate-specific mechanisms

  • Include undamaged DNA controls to evaluate specificity

A particularly informative experimental setup would be the time-course assay described in the search results, where two parallel reactions are compared:

• Reaction A: UvrB added to preloaded UvrA on damaged DNA

• Reaction B: Premixed UvrA and UvrB added to damaged DNA

This direct comparison can resolve whether UvrB is more efficiently recruited via preformed UvrA-UvrB complexes or through a two-step mechanism where UvrA binds first and then recruits UvrB.

How can knowledge of the T. whipplei UvrABC system be applied to develop new diagnostic tools for Whipple's disease?

Understanding the T. whipplei UvrABC system offers several avenues for improved diagnostic tool development:

• PCR-based detection targeting UvrB:

  • Design primers specific to conserved regions of the T. whipplei UvrB gene

  • Develop multiplex PCR assays targeting UvrB alongside established targets like 16S rDNA

  • This approach can increase specificity and reduce false positives compared to single-target assays

• UvrB as a serological marker:

  • Produce recombinant UvrB protein as an antigen for antibody detection

  • Develop ELISA or Western blot assays to detect anti-UvrB antibodies in patient samples

  • This approach could complement DNA-based detection methods

• UvrABC activity-based detection:

  • Design DNA substrates containing specific damage recognized by the UvrABC system

  • Develop assays measuring repair activity in clinical samples

  • This functional approach could help assess bacterial viability in patient samples

• Structural information for targeted therapies:

  • Detailed structural analysis of T. whipplei UvrB could identify unique features

  • These features could serve as targets for developing selective antibiotics

  • Structure-based drug design could lead to T. whipplei-specific treatments

The search results indicate that the analysis of T. whipplei's genome sequence is already guiding the development of molecular diagnostic tools and more convenient culture conditions , suggesting that further characterization of the UvrABC system could contribute to these efforts.

What insights does the UvrB protein provide about T. whipplei evolution and adaptation to its human host?

The UvrB protein and broader UvrABC system offer important insights into T. whipplei evolution and host adaptation:

• Genome reduction while preserving DNA repair:

  • Despite having a highly reduced genome (927,303 bp), T. whipplei has maintained the UvrABC repair system

  • This preservation indicates the critical importance of DNA repair for survival, even in an organism that has undergone extensive genome reduction

  • Suggests strong selective pressure to maintain genomic integrity during host adaptation

• Genomic rearrangements and WiSP proteins:

  • The genome contains large chromosomal inversions with boundaries in WiSP cell-surface protein genes

  • These rearrangements may represent a mechanism for evading host defenses by altering surface protein expression

  • DNA repair systems like UvrABC may play a role in managing potential damage during these genomic rearrangements

• Low G+C content:

  • T. whipplei has a G+C content of 46%, which is unusually low for an Actinobacteria

  • This suggests adaptation to a specific niche within the human host

  • UvrB may have evolved to efficiently repair damage specific to this genomic composition

• Antibiotic resistance:

  • T. whipplei contains a mutation in DNA gyrase predicting resistance to quinolone antibiotics

  • This could influence treatment approaches and suggests ongoing adaptation to antibiotic pressure

  • The UvrABC system may complement this resistance by repairing DNA damage caused by other antibiotics

Understanding T. whipplei UvrB evolution could provide insights into how essential DNA repair mechanisms adapt during genome reduction and host specialization.

What emerging technologies could advance our understanding of UvrB function in T. whipplei?

Several cutting-edge technologies hold promise for advancing our understanding of UvrB function in T. whipplei:

• CRISPR-based gene editing:

  • Though challenging in T. whipplei due to its difficult cultivation, CRISPR systems could enable precise genetic manipulation

  • Creation of point mutations in UvrB to assess functional domains

  • Introduction of fluorescent tags for live-cell imaging without disrupting function

• Cryo-electron microscopy (Cryo-EM):

  • High-resolution structural analysis of UvrB alone and in complex with UvrA and damaged DNA

  • Visualization of conformational changes during the damage verification process

  • Potential to capture previously unseen repair intermediates

• Single-molecule real-time sequencing:

  • Direct detection of DNA damage and repair events at single-nucleotide resolution

  • Mapping of UvrABC repair hotspots across the T. whipplei genome

  • Correlation of repair efficiency with genomic features

• AlphaFold and machine learning approaches:

  • Prediction of UvrB structure and interaction interfaces

  • Modeling of conformational changes during repair process

  • Identification of potential drug-binding sites

• Organ-on-a-chip and advanced cell culture systems:

  • Development of improved cultivation methods for T. whipplei

  • Creation of more physiologically relevant environments to study infection

  • Assessment of DNA damage and repair during actual host-pathogen interactions

• Super-resolution microscopy:

  • Building on the single-molecule fluorescence imaging techniques described in the search results

  • Visualization of UvrB localization and dynamics at unprecedented resolution

  • Tracking repair complex assembly in real-time within the cellular context

How might understanding T. whipplei UvrB contribute to broader research on bacterial DNA repair mechanisms?

Research on T. whipplei UvrB has the potential to make significant contributions to our understanding of bacterial DNA repair:

• Minimal functional requirements:

  • As a bacterium with a highly reduced genome, T. whipplei likely retains only essential components of the NER pathway

  • Studying its UvrB could reveal the minimal functional requirements for effective DNA repair

  • This knowledge could guide the design of synthetic minimal repair systems

• Adaptation to specialized niches:

  • T. whipplei is an obligate intracellular pathogen, facing unique DNA damage challenges

  • Understanding how its UvrB functions could reveal adaptations to the intracellular environment

  • This could provide insights into how DNA repair systems evolve in response to specific ecological niches

• Coordination of repair pathways:

  • With limited DNA repair genes, T. whipplei must efficiently coordinate available pathways

  • Studying how UvrB interfaces with other repair proteins could reveal principles of repair pathway coordination

  • This knowledge could be applicable to understanding repair networks in more complex bacteria

• Antimicrobial development:

  • DNA repair is essential for bacterial survival during infection

  • Targeting UvrB or the broader NER pathway could represent a novel antimicrobial strategy

  • Understanding T. whipplei UvrB could guide development of inhibitors with potential broad-spectrum applications

• Evolution of repair mechanisms:

  • Comparing T. whipplei UvrB with homologs from bacteria with larger genomes

  • Identifying conserved features versus specialized adaptations

  • Contributing to our understanding of how essential cellular processes evolve during genome reduction