Recombinant Bordetella bronchiseptica Ribosome-recycling factor (frr)

What is the ribosome-recycling factor and why is it important in bacterial systems?

Ribosome-recycling factor (RRF), encoded by the frr gene, is essential for bacterial survival as it mediates the dissociation of ribosomes from mRNA after translation termination. This process is critical for "recycling" ribosomes for subsequent rounds of protein synthesis. Studies in Escherichia coli have established that frr is an essential gene, as bacterial strains with disrupted frr cannot sustain growth without complementation . In the absence of proper RRF function, post-termination 70S ribosomal complexes accumulate in 3′-UTRs, and elongating ribosomes become blocked by non-recycled ribosomes at stop codons, severely compromising protein synthesis .
For Bordetella bronchiseptica, a respiratory pathogen in various animals, the ribosome-recycling factor would play a similarly critical role in maintaining protein synthesis efficiency. Understanding its function is particularly relevant given B. bronchiseptica's complex lifestyle as both a persistent colonizer and an acute pathogen.

How does Bordetella bronchiseptica frr differ from other bacterial species?

While the search results don't provide specific sequence or structural comparisons of B. bronchiseptica frr with other bacterial species, we can infer from comparative genomics principles that:

• The frr gene is likely highly conserved across bacterial species due to its essential function

• Any species-specific variations would primarily occur in non-catalytic regions

• B. bronchiseptica, as a member of the Bordetella genus which includes the human pathogen B. pertussis, may have specific adaptations in its translation machinery related to its host interaction strategies
  Researchers should conduct sequence alignment analyses to identify B. bronchiseptica-specific features of the frr gene and protein that might reflect adaptation to its ecological niche as a respiratory pathogen.

What cellular processes are affected by frr depletion in bacteria?

Based on ribosome profiling studies in E. coli, frr depletion has multiple downstream effects on bacterial physiology:

• Accumulation of ribosomes in 3′-UTRs

• Blockage of elongating ribosomes at stop codons

• Changes in gene expression profiles, particularly affecting ribosome rescue factors

• Altered activity of rescue factors including tmRNA and ArfA
  The effects of frr depletion specifically in B. bronchiseptica have not been directly documented in the provided search results, but would likely include similar disruptions to translation termination and ribosome availability, with potentially species-specific consequences related to pathogenesis.

What expression systems are optimal for producing recombinant B. bronchiseptica frr protein?

Based on successful approaches with other B. bronchiseptica proteins, the following expression system considerations are recommended:

What purification challenges are specific to recombinant B. bronchiseptica frr?

While the search results don't specifically address purification challenges for B. bronchiseptica frr, common issues with bacterial ribosomal proteins include:

• Maintaining proper folding and stability during purification

• Preventing non-specific RNA binding

• Optimizing buffer conditions to maintain functionality
  Based on successful purification of other B. bronchiseptica recombinant proteins, researchers should consider:

• Using SDS-PAGE to confirm protein expression and purity

• Employing buffer systems that maintain protein stability

• Considering chromatography methods beyond initial affinity purification, such as ion exchange or size exclusion, to achieve high purity

• Validation of protein functionality through activity assays specific to ribosome-recycling function

How can the biological activity of recombinant B. bronchiseptica frr be verified?

To verify that recombinant B. bronchiseptica frr retains biological activity, researchers can utilize several approaches:

• In vitro ribosome dissociation assays: Using purified B. bronchiseptica ribosomes or heterologous systems to measure the ability of recombinant frr to dissociate post-termination ribosomal complexes

• Complementation studies: Testing whether recombinant B. bronchiseptica frr can functionally complement frr-deficient E. coli strains (such as the temperature-sensitive MC1061-2 strain described)

• Binding assays with ribosomal components: Investigating interactions between recombinant frr and B. bronchiseptica ribosomal components using techniques like surface plasmon resonance or pull-down assays (similar to methods used for studying Bcr4-BscI interactions)

• Structural integrity assessment: Using circular dichroism or thermal shift assays to confirm that the recombinant protein has proper folding

What strategies can be used to study frr essentiality in B. bronchiseptica?

The essential nature of frr makes traditional knockout approaches challenging. Based on strategies used in E. coli and approaches for studying other essential genes, researchers can:

• Construct conditional mutants: Create a B. bronchiseptica strain carrying both a chromosomal frame-shifted frr and a wild-type frr on a temperature-sensitive plasmid (similar to the E. coli MC1061-2 strain)

• Deploy controlled depletion systems: Use inducible promoters to control frr expression levels and monitor phenotypic consequences

• Implement CRISPR interference: Use CRISPRi approaches to partially repress frr expression without complete gene deletion

• Perform temperature-sensitivity studies: Analyze growth patterns at permissive and non-permissive temperatures with conditional mutants

• Monitor plasmid segregation: Under incompatibility pressure, essential genes like frr cannot be deleted as evidenced by inability to segregate plasmids carrying wild-type frr

How does frr function integrate with the pathogenesis mechanisms of B. bronchiseptica?

While direct evidence linking frr to B. bronchiseptica pathogenesis is not provided in the search results, we can infer potential connections based on bacterial physiology principles:

• Translation efficiency during host colonization: As B. bronchiseptica adapts to different microenvironments within the host respiratory tract, efficient ribosome recycling would be critical for rapid protein synthesis adaptation

• Stress response during infection: Under host-induced stress conditions, efficient translation termination and ribosome recycling may become even more crucial for bacterial survival

• Potential interaction with virulence regulation: The translation machinery often interfaces with regulatory networks controlling virulence factor expression, including the type III secretion system components discussed in the search results
  Researchers could investigate these connections through:

• Transcriptomic analysis of frr expression during different stages of infection

• Proteomic studies of translation efficiency under conditions mimicking the host environment

• Examining potential interactions between frr and known virulence regulators

What can ribosome profiling reveal about frr function in B. bronchiseptica?

Ribosome profiling, a technique that provides genome-wide information on ribosome positions on mRNAs, could yield valuable insights about frr function in B. bronchiseptica, similar to findings in E. coli :

• Mapping translation dynamics: Identify ribosome positioning patterns specific to B. bronchiseptica, particularly at termination sites

• Consequences of frr depletion: Establish how ribosome distribution changes across the B. bronchiseptica transcriptome when frr levels are reduced, identifying genes most sensitive to ribosome recycling deficiencies

• Species-specific termination characteristics: Determine whether B. bronchiseptica exhibits unique patterns of ribosome behavior at stop codons compared to model organisms

• Translational coupling effects: Assess whether frr affects expression of operons containing virulence factors, potentially linking translation efficiency to pathogenesis

• Stress response integration: Examine how environmental conditions relevant to infection alter the frr-dependent ribosome distribution

How conserved is the frr gene across Bordetella species?

The search results don't directly address frr conservation within Bordetella species, but the available genomic data resources for Bordetella would enable comparative analysis. Researchers should:

• Extract frr gene sequences from the comprehensive Bordetella genome database mentioned in search result

• Perform multiple sequence alignments to identify:

  • Core conserved regions likely essential for function

  • Variable regions that might reflect species-specific adaptations

  • Selection pressures acting on different regions of the gene

• Compare evolutionary rates of frr with other translation-related genes and with virulence factors to understand its relative evolutionary constraints

• Analyze genomic context conservation of the frr locus across species to identify potential co-evolutionary patterns with functionally related genes

What can structural modeling reveal about B. bronchiseptica frr compared to other bacterial species?

Structural biology approaches would provide insight into the specific characteristics of B. bronchiseptica frr:

• Homology modeling: Using solved structures of RRF from other bacteria as templates to predict the B. bronchiseptica frr structure

• Comparative structural analysis: Identifying B. bronchiseptica-specific structural features that might influence:

  • Interaction with ribosomal components

  • Stability under different environmental conditions

  • Potential as a drug target with species selectivity

• Molecular dynamics simulations: Examining the dynamic behavior of B. bronchiseptica frr under conditions mimicking the host environment

• Structure-function correlations: Mapping sequence conservation onto structural models to identify functionally critical regions versus potentially adaptable regions

How can recombinant B. bronchiseptica frr be evaluated as a potential vaccine candidate?

While frr is not among the B. bronchiseptica proteins specifically evaluated as vaccine candidates in the search results, the methodological approach used for other recombinant proteins can be applied :

• Immunogenicity assessment:

  • Measure antibody titers in mice immunized with recombinant frr

  • Determine IgG subtype profiles to characterize the type of immune response (Th1 vs. Th2)

  • Assess stimulation index in lymphocyte proliferation assays

• Protection studies:

  • Challenge immunized animals with virulent B. bronchiseptica

  • Monitor protection ratio compared to control groups

  • Evaluate bacterial clearance from respiratory tissues

• Immune response characterization:

  • Cytokine profiling to determine whether frr induces appropriate protective responses

  • Analysis of both humoral and cell-mediated immunity components
    Based on the evaluation framework used for other B. bronchiseptica proteins, a successful candidate would need to demonstrate both strong antibody responses and protection against challenge, ideally with protection ratios exceeding 50% as observed with the outer membrane porin protein (PPP) and lipoprotein (PL) .

What approaches can identify interactions between frr and other B. bronchiseptica proteins?

To characterize the protein-protein interaction network involving B. bronchiseptica frr, researchers could employ methods similar to those used to study Bcr4 interactions :

• Pull-down assays: Using tagged recombinant frr to identify interacting partners from B. bronchiseptica lysates, followed by mass spectrometry identification

• Bacterial two-hybrid screening: Systematically testing potential interactions with translation-related factors and regulatory proteins

• Co-immunoprecipitation with specific antibodies: Validating interactions in the native context

• Crosslinking studies: Capturing transient interactions during the ribosome recycling process

• Truncation analysis: Creating deletion variants of frr to map interaction domains, similar to the approach used with Bcr4
  These approaches would help establish whether B. bronchiseptica frr has species-specific interaction partners that might represent novel therapeutic targets.

How might frr contribute to stress adaptation in B. bronchiseptica during host colonization?

Translation regulation plays a crucial role in bacterial adaptation to changing environments, including stresses encountered during infection. Research approaches to investigate frr's role in this process could include:

• Controlled expression studies: Manipulating frr levels and assessing survival under various stress conditions relevant to host colonization:

  • Oxidative stress

  • Nutrient limitation

  • pH fluctuations

  • Immune effector exposure

• Transcriptome-ribosome profiling integration: Comparing transcriptional and translational responses to stress conditions with normal versus reduced frr levels

• Infection model comparisons: Assessing whether frr expression/activity varies between acute infection and persistent colonization states of B. bronchiseptica

• Regulatory network mapping: Identifying how frr activity interfaces with stress response regulators in B. bronchiseptica
  Understanding these connections could reveal why translation efficiency maintenance through proper ribosome recycling might be particularly important during certain stages of B. bronchiseptica's interaction with its host.

What are the critical parameters for optimizing recombinant B. bronchiseptica frr expression?

Based on experience with other B. bronchiseptica recombinant proteins, researchers should consider:

How can structural and functional integrity of purified recombinant frr be validated?

Comprehensive validation of recombinant B. bronchiseptica frr should include:

• Biochemical characterization:

  • Size exclusion chromatography to confirm monomeric state

  • Circular dichroism to verify secondary structure elements

  • Thermal shift assays to assess stability

  • Mass spectrometry for exact mass confirmation

• Functional assays:

  • Ribosome binding capacity

  • Ribosome-splitting activity measurement

  • ATP-dependent recycling efficiency with translation factors

• Comparative analysis:

  • Side-by-side comparison with E. coli frr in functional assays

  • Evaluation of species-specific functional characteristics
    This multi-faceted validation approach ensures that the recombinant protein accurately represents the native B. bronchiseptica frr in both structure and function.

What considerations are important when designing ribosome profiling experiments for B. bronchiseptica?

Ribosome profiling for B. bronchiseptica would require careful optimization based on approaches developed for E. coli :

• Growth conditions: Standardize conditions that are physiologically relevant to B. bronchiseptica lifestyle

• Antibiotic treatment: Optimize chloramphenicol or other translation inhibitors for rapid ribosome fixation

• Nuclease digestion parameters: Determine optimal conditions for B. bronchiseptica-specific rRNA content and membrane composition

• Fractionation protocols: Adjust for B. bronchiseptica-specific ribosome properties

• Computational analysis pipeline: Account for B. bronchiseptica genome features:

  • Multiple operons

  • Leaderless mRNAs

  • Species-specific translation initiation sites

• Controls for frr depletion studies: Establish rapid and conditional frr depletion systems
  Researchers should also consider comparative ribosome profiling between wild-type and frr-depleted conditions at multiple time points to capture the progression of translation defects, similar to the approach used in E. coli studies .