Recombinant Tropheryma whipplei Recombination protein RecR (recR)

What is the function of RecR in T. whipplei?

RecR in T. whipplei likely functions as part of the RecFOR pathway involved in DNA recombination and repair. This pathway plays a critical role in the bacterium's genome maintenance and rearrangements. Given that T. whipplei exhibits frequent genome rearrangements, particularly involving cell-surface protein families, RecR likely contributes to these processes by facilitating homologous recombination . The genome rearrangements appear to be mediated through highly conserved nucleotide sequences, such as those found in the WND-domain repeats, which show up to 99% identity over 800-nucleotide spans .

How does RecR contribute to T. whipplei's genome plasticity?

T. whipplei's genome displays remarkable plasticity, as evidenced by the large chromosomal inversion observed between the Twist and TW08/27 strains. This inversion occurs within two paralogous genes belonging to a cell-surface protein family . RecR likely facilitates these rearrangements by mediating homologous recombination between identical or nearly identical repeat sequences. This recombination process may result in the expression of different subsets of cell surface proteins, potentially representing a mechanism for evading host defenses .

What genomic context surrounds the recR gene in T. whipplei?

While the search results don't specifically detail the genomic context of recR, T. whipplei possesses genes for site-specific integrase/recombinase (xerC and xerD) that may work in conjunction with RecR in genome rearrangements . The bacterium also contains competence-related genes (comEA, comEC, and comFC) homologous to those in Bacillus subtilis, suggesting potential natural competence mechanisms that might interact with recombination pathways .

What expression systems are most effective for producing recombinant T. whipplei RecR?

For recombinant expression of T. whipplei proteins, researchers should consider the following methodological approach:

• Clone the recR gene into an expression vector with a purification tag

• Transform into an E. coli expression system

• Optimize expression conditions considering T. whipplei's unique codon usage

• Purify using affinity chromatography followed by size exclusion

• Validate protein activity through functional assays

Researchers should note that T. whipplei has only recently been successfully cultured in human fibroblasts , which may complicate native protein studies.

What DNA substrates should be used when assessing T. whipplei RecR activity in vitro?

When designing in vitro assays for T. whipplei RecR activity, consider using:

• Single-stranded DNA gaps similar to those occurring during DNA repair

• Constructs containing the WND-domain repeats found in WiSP proteins

• DNA structures mimicking replication forks

• Homologous sequences that reflect the repeat regions identified in the T. whipplei genome

These substrates should reflect the unique genomic features of T. whipplei, particularly the repeated sequences that appear to trigger frequent genome rearrangements .

How can researchers isolate and analyze RecR-mediated recombination products from T. whipplei?

Isolation and analysis of RecR-mediated recombination products requires:

• PCR amplification of genomic regions containing known inversion sites

• Next-generation sequencing to identify novel junction regions

• Validation of inversion events using primers defining the boundaries of inversions

• Quantification of different genomic arrangements within bacterial populations

This approach follows the methodology used to validate genome organization in the Twist isolate of T. whipplei, which confirmed both the primary genome organization and the presence of minor populations with alternative arrangements .

How does thermal stress affect RecR expression in T. whipplei?

While specific data on RecR expression is not provided in the search results, T. whipplei exhibits distinct transcriptomic responses to thermal stresses. Under heat shock (43°C), few genes are differentially transcribed, primarily affecting the dnaK regulon and putative virulence factors like RibC and IspDF proteins . In contrast, cold shock (4°C) causes extensive transcriptomic modifications affecting 149 genes across eight regulons . RecR may be part of these stress responses, particularly given its potential role in DNA repair following stress-induced damage.

What relationship exists between RecR function and T. whipplei's adaptation to intracellular survival?

As an intracellular pathogen that replicates in macrophages, T. whipplei must adapt to various stresses. Recent research indicates that T. whipplei escapes from the LAPosome (LC3-associated phagocytosis) into the cytosol before being recaptured by xenophagy . The bacterium then blocks autophagic flux to establish its replicative compartment . RecR-mediated DNA repair and recombination likely play roles in maintaining genomic integrity during these processes, particularly when facing oxidative damage within macrophages.

Could RecR be involved in T. whipplei's quinolone resistance?

T. whipplei possesses a mutation in DNA gyrase that predicts resistance to quinolone antibiotics, which has been confirmed experimentally . RecR, as part of DNA repair pathways, might interact with this resistance mechanism. DNA repair proteins often work in concert with DNA gyrase during replication and recombination processes. Therefore, understanding RecR function could provide insights into antibiotic resistance mechanisms and potential therapeutic approaches.

What methodological challenges exist when studying RecR in the context of T. whipplei's reduced genome?

Studying RecR in T. whipplei presents several challenges:

• Limited cultivation options, as T. whipplei was only successfully cultured in 2000 in human fibroblasts

• Small genome size (927,303 bp) with 808 predicted protein-coding genes

• Potential functional adaptations in RecR due to genome reduction

• Interactions with T. whipplei-specific proteins lacking in model organisms

• Need for specialized molecular tools for a relatively understudied organism

These challenges necessitate creative experimental approaches that account for T. whipplei's unique biology.

How can researchers distinguish between RecR-dependent and independent recombination events in T. whipplei?

To distinguish RecR-dependent recombination events:

• Develop conditional knockdown systems targeting recR

• Compare recombination frequencies at WND-domain repeats with and without functional RecR

• Analyze the spectrum of recombination products using next-generation sequencing

• Perform in vitro reconstitution experiments with purified RecR and partner proteins

• Use comparative genomics to identify signature recombination patterns

These approaches would help determine RecR's specific contribution to the genomic plasticity observed in T. whipplei strains.

What are the optimal PCR conditions for amplifying and sequencing the recR gene from clinical T. whipplei isolates?

Based on PCR protocols used for T. whipplei detection, researchers should consider:

• Extraction using QIAamp DNA kit (Qiagen) or equivalent

• Real-time quantitative PCR using appropriate detection systems (LightCycler or CFX96 Touch)

• Targeting conserved regions flanking the recR gene

• Including appropriate controls and standards

• Validating amplicons through sequencing

For increased sensitivity, researchers might design primers based on repeated sequences similar to those used for T. whipplei detection, which have shown 10-100 times more sensitivity than rpoB-based primers .

How does T. whipplei RecR compare to RecR proteins in other reduced-genome bacteria?

Comparative analysis would reveal:

This comparison would highlight how RecR has adapted during genome reduction processes while maintaining essential functions.

What evolutionary patterns are observed in RecR proteins across the Actinobacteria phylum?

T. whipplei is the only known reduced-genome species (<1 Mb) within Actinobacteria, which typically have high G+C content . Evolutionary analysis of RecR across this phylum would likely reveal:

• Conservation of core functional domains

• Potential gene loss or modification during T. whipplei's genome reduction

• Adaptations specific to T. whipplei's intracellular lifestyle

• Comparison with other actinobacteria like Streptomyces griseus, which also uses intra-ORF genome inversions to modulate surface protein expression

This evolutionary perspective would provide context for understanding T. whipplei RecR's unique features and functions.

How might RecR-mediated genomic rearrangements contribute to T. whipplei's virulence?

The genomic inversions facilitated by RecR likely contribute to T. whipplei virulence through:

• Modulation of cell-surface protein expression, potentially aiding immune evasion

• Adaptation to different host environments during infection

• Generation of genetic diversity within bacterial populations

• Potential regulation of virulence factor expression

The WiSP membrane protein family, involved in these inversions, may play a direct role in host-pathogen interactions, with RecR enabling the dynamic expression of different variants .

Could RecR activity be linked to the different clinical manifestations of T. whipplei infection?

T. whipplei causes classic Whipple's disease as well as localized infections, and asymptomatic carriage is also reported . RecR-mediated genomic rearrangements might contribute to these diverse clinical presentations by:

• Generating strain-specific genomic configurations

• Altering expression patterns of virulence determinants

• Adapting the bacterium to different host tissues

• Enabling persistent infection through immune evasion

Molecular epidemiology studies have shown increasing detection of T. whipplei over a 12-year period, with the bacterium recovered from various sample types including stool (43%), saliva (15%), duodenal biopsies (12.5%), blood (5%), and CSF (6%) .

How does the macrophage environment influence RecR function during T. whipplei infection?

Recent research shows that T. whipplei is taken up by macrophages through LC3-associated phagocytosis (LAP), escapes into the cytosol, and is recaptured by xenophagy before blocking autophagic flux to establish its replicative niche . During this complex intracellular lifecycle, RecR likely:

• Responds to DNA damage caused by host defense mechanisms

• Facilitates genomic adaptations to the intracellular environment

• Contributes to bacterial persistence by maintaining genomic integrity

• Potentially interacts with stress response pathways

Understanding these interactions could provide insights into T. whipplei's ability to establish chronic infections.

What novel methodologies could advance our understanding of RecR's role in T. whipplei biology?

Emerging technologies that could enhance RecR research include:

• CRISPR-based approaches for targeted genetic manipulation

• Single-cell genomics to track heterogeneity in T. whipplei populations

• Advanced imaging techniques to visualize RecR activity during infection

• Systems biology approaches integrating transcriptomics, proteomics, and genomics data

• Structural biology studies of the T. whipplei RecFOR complex

These approaches would build upon the existing molecular diagnostic tools developed based on T. whipplei genome analysis .

How might targeting RecR or RecR-dependent processes inform new therapeutic strategies for Whipple's disease?

Therapeutic targeting of RecR could offer novel approaches for treating Whipple's disease:

• Development of RecR inhibitors as potential antimicrobials

• Combination therapies targeting both RecR and other essential processes

• Strategies to prevent genomic adaptations that contribute to persistence

• Approaches to sensitize T. whipplei to existing antibiotics

Given that current diagnosis often relies on PCR-based detection and the bacterium's resistance to quinolone antibiotics , new therapeutic targets like RecR could significantly advance treatment options.

What is the potential role of RecR in environmental survival of T. whipplei?

T. whipplei is suspected to have an environmental origin and shows adaptive responses to thermal stresses consistent with this hypothesis . RecR likely contributes to environmental survival through:

• DNA repair following exposure to environmental stressors

• Genomic adaptations during transitions between hosts and environment

• Maintaining DNA integrity during cold conditions, where T. whipplei exhibits strong transcriptomic responses

• Potential contributions to dormancy or persistence mechanisms