Recombinant Bartonella tribocorum Ribosome-recycling factor (frr)

What is Bartonella tribocorum and what is its significance in research?

Bartonella tribocorum is a Gram-negative facultative intracellular bacterium that primarily infects rodents worldwide. It was first isolated from wild rats in France and has since been identified in various rodent species across different geographical regions. Recent research has expanded our understanding with genome sequences of B. tribocorum strains isolated from a rodent (Mus cookii) in Laos and a shrew (Suncus murinus) in Cambodia . This bacterium has gained significance in research not only for understanding vector-borne zoonotic infections but also because it has been considered a potential zoonotic species that could cause undifferentiated chronic illness in humans following tick bites . B. tribocorum serves as an important model for studying host-pathogen interactions, bacterial adaptation mechanisms, and translation processes specific to intracellular bacteria.

What is the function of the ribosome-recycling factor (frr) in bacterial translation?

The ribosome-recycling factor (encoded by the frr gene) plays a critical role in the final stage of protein synthesis by disassembling the post-termination complex. When translation terminates, the ribosome remains bound to the mRNA with a deacylated tRNA in the P-site. The RRF works in conjunction with elongation factor G (EF-G) or release factor 3 (RF3) to dissociate this complex, thereby freeing the ribosomal subunits for a new round of translation . Without functional RRF, ribosomes become sequestered in inactive complexes, drastically reducing the pool of available ribosomes for protein synthesis. This makes RRF essential for bacterial survival, as it ensures efficient recycling of translation components.

How conserved is the frr gene across bacterial species compared to B. tribocorum?

The frr gene is highly conserved across bacterial species due to its essential function in protein synthesis. Research on homologous proteins, such as the plant chloroplast RRF homologue (RRFHCP) isolated from spinach, demonstrates significant sequence conservation. The spinach chloroplast RRFHCP shows 46% sequence identity and 66% sequence homology with the ribosome recycling factor of Escherichia coli .

Based on general patterns of conservation in essential genes, we can infer that B. tribocorum frr would show significant homology with other alpha-proteobacteria, particularly within the Bartonella genus. Analysis of the B. tribocorum genome indicates that genes involved in protein metabolism constitute approximately 17.6% of its genome, highlighting the importance of translation-related functions .

What expression systems are most effective for producing recombinant B. tribocorum RRF?

For expressing recombinant B. tribocorum RRF, several expression systems can be employed, with methodological considerations for each:

For optimal results, the recombinant protein should include a purification tag (His, GST, or MBP) positioned to minimize interference with RRF function, preferably at the C-terminus since the N-terminus is often involved in functional interactions.

What purification strategies yield functional B. tribocorum RRF protein?

A multi-step purification strategy is recommended to obtain functional B. tribocorum RRF:

• Initial capture: Affinity chromatography using the appropriate resin for the fusion tag (Ni-NTA for His-tagged proteins, glutathione resin for GST fusion proteins).

• Intermediate purification: Ion-exchange chromatography, exploiting the predicted isoelectric point of the protein.

• Polishing step: Size-exclusion chromatography to remove aggregates and ensure homogeneity.

Throughout purification, it's crucial to maintain conditions that preserve the native structure of RRF. Based on information from related bacterial proteins, a buffer system containing 20-50 mM Tris-HCl (pH 7.5), 100-200 mM NaCl, 5-10% glycerol, and 1-5 mM DTT or β-mercaptoethanol would likely be suitable. Since RRF functions with ribosomes, avoid the use of chelating agents like EDTA, which can affect ribosome integrity if the protein is to be used in functional assays.

How can the activity of recombinant B. tribocorum RRF be assessed in vitro?

To verify the functionality of purified recombinant B. tribocorum RRF, several complementary assays can be employed:

• Ribosome recycling assay: This assay measures the ability of RRF to disassemble post-termination complexes in conjunction with EF-G and GTP. The reaction typically includes:

  • Purified ribosomes (70S)

  • mRNA with a stop codon

  • Deacylated tRNA

  • Recombinant RRF

  • EF-G and GTP

  Disassembly can be monitored by light scattering, centrifugation, or fluorescence techniques if labeled components are used.

• Complementation assay: Using E. coli strains with temperature-sensitive RRF, assess whether B. tribocorum RRF can restore growth at non-permissive temperatures. Care must be taken with this approach as heterologous RRF can exhibit toxicity, as observed with the plant chloroplast RRFHCP .

• Binding assays: Analyze the binding affinity of the recombinant RRF to ribosomes using techniques such as surface plasmon resonance (SPR) or microscale thermophoresis (MST).

These assays provide complementary information about different aspects of RRF functionality and should ideally be used in combination.

How do recombination events influence the evolution of the frr gene in Bartonella species?

Recombination plays a significant role in the evolution of Bartonella species, potentially affecting functional genes including frr. Studies on Bartonella isolates from wild rodents have revealed extensive recombination among housekeeping genes, creating complex genetic networks . While specific recombination events in the frr gene have not been directly reported in the provided literature, the pattern observed in other housekeeping genes suggests similar dynamics may affect frr.

Analysis of various housekeeping genes (gltA, rpoB, ribC, ftsZ, groEl) and the 16S RNA gene in Bartonella isolates has revealed clear evidence of numerous recombinant events . For example, recombination within the gltA gene has generated hybrid variants between B. taylorii and B. doshiae, and between B. taylorii and B. grahamii . These recombination events appear to have played a dominant role in the evolution of Bartonella diversity.

The implications for the frr gene are significant, as recombination could:

• Introduce adaptive variations that optimize ribosome recycling efficiency in different host environments

• Generate novel functional properties through domain swapping between species

• Potentially lead to mosaic genes with segments derived from different Bartonella species

Researchers studying B. tribocorum frr should be aware that single-gene phylogenies may not accurately reflect evolutionary relationships, as housekeeping gene phylogenies are not robust indicators of Bartonella diversity when only a single gene is analyzed .

What genetic approaches are recommended for characterizing the frr gene in clinical or environmental B. tribocorum isolates?

For comprehensive characterization of the frr gene in B. tribocorum isolates, a multi-faceted genetic approach is recommended:

• Multi-locus sequence typing (MLST): Include frr alongside other housekeeping genes (gltA, rpoB, ribC, ftsZ, groEl) to establish genetic relationships and detect potential recombination events. This approach has proven valuable in identifying complex genetic relationships in Bartonella species .

• Full gene sequencing: Rather than analyzing fragments, sequence the entire frr gene to detect potential breakpoints for recombination events. Partial gene analysis can miss important recombination signals, as demonstrated in studies of gltA .

• Recombination analysis: Use specialized software packages like RDP-2 that incorporate multiple algorithms (GENECONV, MaxChi, Chimaera, 3Seq) to detect potential recombination events in the frr sequence .

• Comparative genomics: Position the frr gene analysis in the context of whole-genome sequencing data when available. The draft genome sequences of B. tribocorum strains (like L103 and C635) provide valuable reference points .

• Host association analysis: Correlate frr variants with host species, as nested clade analysis has shown that Bartonella isolates can be assigned to clades that correlate with host identity .

These approaches should be applied to isolates from diverse geographical regions and host species to capture the full genetic diversity of the frr gene in B. tribocorum populations.

What animal models are appropriate for studying B. tribocorum RRF in vivo?

Laboratory mice represent an appropriate model system for studying B. tribocorum infection and potentially the in vivo role of RRF. Experimental infection studies have demonstrated that laboratory mice can be successfully infected with B. tribocorum strains isolated from wild Mus species, providing a homologous host-bacteria model system at the genus level . This model reproduces key characteristics of naturally acquired Bartonella infections in rodents.

When designing experiments to study B. tribocorum RRF in vivo, researchers should consider:

• Mouse strain selection: Outbred CD1 female mice have been successfully used in previous studies . Different mouse strains may exhibit varying susceptibility to infection.

• Inoculation method: Subcutaneous inoculation with low doses (10-1000 bacteria/mouse) has been effective in establishing infection .

• Monitoring parameters: Regular blood collection (e.g., weekly) allows for evaluation of bacteremia kinetics in infected mice over extended periods (up to 27 weeks in published studies) .

• Transmission assessment: Collection and testing of biological samples such as urine during the post-inoculation period can help determine potential sources of bacterial transmission .

This model system offers advantages for studying the role of specific bacterial factors, including RRF, in the establishment and maintenance of B. tribocorum infection. Genetic manipulation of the frr gene followed by in vivo infection studies could reveal its importance in bacterial fitness and persistence.

How can genetic manipulation techniques be applied to study B. tribocorum RRF function?

Several genetic approaches can be employed to study the function of RRF in B. tribocorum:

• Conditional knockdown systems: Since RRF is likely essential for viability (as in other bacteria), conditional expression systems such as tetracycline-regulated promoters can be used to control frr expression levels. This allows for studying the effects of RRF depletion on bacterial growth, translation, and infection dynamics.

• Site-directed mutagenesis: Introducing specific mutations in the frr gene can help identify critical residues for RRF function. Mutant variants can be expressed in recombinant systems and tested for activity using the in vitro assays described in section 2.3.

• Domain swapping: Creating chimeric proteins with domains from RRF of different Bartonella species or other bacteria can help identify species-specific functions and adaptations. This approach is particularly relevant given the evidence of recombination in other Bartonella genes .

• Reporter systems: Fusing the frr promoter to reporter genes can reveal regulation patterns of RRF expression under different conditions or during different stages of infection.

• Complementation studies: Testing whether B. tribocorum RRF can complement RRF deficiency in other bacterial species can provide insights into functional conservation and specificity.

When implementing these approaches, researchers should be mindful of the potential essential nature of RRF and design experiments that allow for controlled manipulation without completely abolishing function.

How does B. tribocorum RRF contribute to bacterial adaptation to different host environments?

The ribosome recycling factor likely plays a crucial role in B. tribocorum's adaptation to different host environments, though this has not been directly studied in the available literature. Based on our understanding of bacterial physiology and the information from the search results, several hypotheses can be proposed:

• Translation efficiency in different hosts: B. tribocorum infects various rodent species and potentially humans . Different host environments may require adjustments in translation efficiency, which could be mediated in part by adaptation of the RRF to function optimally under varying conditions.

• Stress response during host transition: When transitioning between hosts or host cell types, bacteria often encounter stress conditions. RRF may be particularly important during these transitions, ensuring efficient protein synthesis when the bacterium needs to rapidly adapt to new environments.

• Interaction with host-specific factors: Though primarily interacting with bacterial components, RRF function could be indirectly affected by host-specific conditions that alter bacterial physiology.

• Recombination-driven adaptation: Given the significant role of recombination in Bartonella evolution , the frr gene might undergo recombination events that optimize its function for specific host environments. This could parallel the recombination observed in other housekeeping genes like gltA, which has generated hybrid variants between different Bartonella species .

Research examining the expression levels and sequence variations of the frr gene across B. tribocorum isolates from different host species could provide valuable insights into its role in host adaptation.

What are the structural and functional differences between B. tribocorum RRF and RRFs from other bacterial pathogens?

While specific structural information about B. tribocorum RRF is not provided in the search results, comparative analysis with RRFs from other bacteria can highlight potential differences of functional significance. Based on general principles of bacterial RRF structure and function:

• Domain organization: Bacterial RRFs typically consist of two domains: a three-helix bundle domain and a domain resembling tRNA. Variations in the interdomain angle and flexibility could affect interactions with species-specific ribosomes and translation factors.

• Surface electrostatics: Differences in surface charge distribution could influence interactions with ribosomes and other factors like EF-G. These differences might reflect adaptations to the intracellular environment of B. tribocorum.

• Species-specific interactions: B. tribocorum RRF likely has evolved specific interaction interfaces with its cognate EF-G. Comparative sequence analysis focusing on surface-exposed residues could identify potential species-specific interaction sites.

• Stability adaptations: As an intracellular pathogen, B. tribocorum faces different physiological conditions compared to extracellular bacteria. Its RRF might show adaptations in thermostability or pH optimum reflecting these conditions.

• Regulatory elements: Differences in promoter regions and regulatory elements of the frr gene could lead to different expression patterns compared to other bacteria, potentially reflecting the infection cycle of B. tribocorum.

Research comparing the structure and function of B. tribocorum RRF with those from other bacterial pathogens could provide insights into bacterial adaptation and potentially identify species-specific features that could be targeted for therapeutic development.

Can B. tribocorum RRF serve as a target for developing novel antimicrobial approaches?

The ribosome recycling factor represents a potentially valuable target for antimicrobial development due to several characteristics:

Research approaches for developing RRF-targeted antimicrobials could include:

• Structure-based drug design: Determining the crystal structure of B. tribocorum RRF would enable rational design of inhibitors targeting specific functional sites.

• High-throughput screening: Developing assays to screen compound libraries for molecules that specifically inhibit B. tribocorum RRF activity.

• Peptide inhibitors: Designing peptides that mimic the interaction interfaces between RRF and its binding partners (ribosomes or EF-G) to competitively inhibit its function.

• Antisense strategies: Developing antisense oligonucleotides specifically targeting B. tribocorum frr mRNA to reduce RRF expression.

These approaches would need to address the challenge of delivering inhibitors to the intracellular location where B. tribocorum resides within host cells.