Recombinant Bordetella bronchiseptica Ribosome maturation factor RimM (rimM)

What is Bordetella bronchiseptica Ribosome Maturation Factor RimM?

Ribosome maturation factor RimM (rimM) is a conserved bacterial protein that plays a critical role in the maturation process of the 30S ribosomal subunit. In Bordetella bronchiseptica, RimM functions similarly to its homologs in other bacterial species, participating in the assembly of functional ribosomes. Structurally, the protein is encoded in the trmD operon of bacterial genomes and demonstrates evolutionarily conserved features across prokaryotes .

The protein is particularly notable for its specific interaction with ribosomal protein S19, which is located in the head domain of the 30S subunit . This interaction appears central to its function in ribosome assembly. RimM has been observed to associate exclusively with the free 30S subunit and not with 30S subunits incorporated into complete 70S ribosomes, indicating its role is specifically in the maturation process rather than in translation itself .

B. bronchiseptica is a gram-negative respiratory pathogen affecting various animals, and studying its ribosome assembly factors like RimM contributes to understanding both fundamental bacterial physiology and potential targets for intervention against infections .

What is the structural organization of the RimM protein?

The RimM protein exhibits a two-domain structure with distinct N-terminal and C-terminal regions, as confirmed through multiple sequence alignments and structural characterization . Detailed structural analyses using nuclear magnetic resonance (NMR) spectroscopy on Thermus thermophilus RimM have provided significant insights into this organization.

The N-terminal domain of RimM (approximately residues 1-80) folds into a well-defined structural domain characterized by a six-stranded β-barrel fold . This domain appears to be highly conserved across bacterial species and possesses a distinctive hydrophobic patch that plays a significant role in protein-protein interactions, particularly with ribosomal protein S19 .

In contrast, the C-terminal region (approximately residues 81-162 in T. thermophilus RimM) demonstrates a partially folded state in solution . This region also participates in interactions with ribosomal protein S19, as evidenced by significant changes in NMR spectra upon complex formation .

For B. bronchiseptica specifically, the recombinant RimM protein typically referenced spans amino acids 1-207, suggesting a potentially longer C-terminal region compared to the T. thermophilus homolog . This structural organization, with two functional domains working cooperatively, appears essential for the protein's role in ribosome assembly.

How does RimM contribute to ribosome maturation?

RimM contributes to ribosome maturation through several interconnected mechanisms centered around its interaction with the 30S ribosomal subunit head domain. The primary role of RimM appears to be facilitating the incorporation of late-binding ribosomal proteins, particularly S19, into the maturing 30S subunit .

Genetic approaches have revealed that RimM is specifically involved in the maturation of regions composed of helices 31 and 33b of 16S rRNA, as well as ribosomal proteins S13 and S19 in the head domain of the 30S subunit . According to the assembly and kinetics order maps for 30S particle maturation, proteins S2, S13, and S19 are classified as late binders for the assembly of the head region .

The binding of S19 with helix 33b of 16S rRNA induces conformational changes in the 3' major domain of 16S rRNA, suggesting that RimM's interaction with S19 may indirectly influence rRNA folding and structure . This is supported by the observation that in E. coli rimM disruptants, an unprocessed precursor of 16S rRNA (17S rRNA) accumulates, indicating a defect in the maturation pathway .

NMR studies have demonstrated that both the N-terminal and C-terminal domains of RimM participate in complex formation with S19 . The binding surface involves residues near a hydrophobic patch in the N-terminal domain and a wide range of residues in the C-terminal region, suggesting a multi-point interaction mechanism that may serve to properly position S19 for incorporation into the 30S structure .

What methods are used for recombinant expression of B. bronchiseptica RimM?

Recombinant expression of B. bronchiseptica RimM can be achieved through several expression systems, with the choice depending on research objectives and downstream applications. Based on available research, the following methodological approaches have proven effective:

• Bacterial Expression Systems: E. coli represents the most commonly utilized host for recombinant RimM expression . Researchers have successfully produced soluble RimM protein by cloning the rimM gene into appropriate expression vectors under the control of inducible promoters (typically T7 or tac promoters). For structural studies of RimM from Thermus thermophilus, researchers have utilized E. coli to produce both full-length RimM and N-terminal fragments (residues 1-85) . The same approach can be adapted for B. bronchiseptica RimM.

• Alternative Expression Systems: While E. coli remains predominant, recombinant B. bronchiseptica RimM can also be produced using yeast, baculovirus, or mammalian cell expression systems, particularly when post-translational modifications or improved solubility are required .

• Expression Constructs: For structural and functional studies, researchers frequently utilize:

  • Full-length constructs (amino acids 1-207 for B. bronchiseptica RimM)

  • Domain-specific constructs (e.g., N-terminal domain constructs)

  • Tagged constructs (typically incorporating His6, GST, or MBP tags) to facilitate purification

• Isotopic Labeling: For NMR spectroscopy studies, expression in minimal media supplemented with 15N-labeled ammonium chloride and/or 13C-labeled glucose enables production of isotopically labeled RimM for structural investigations .

Optimization of expression conditions (temperature, induction timing, media composition) is typically necessary to maximize yield and solubility of the recombinant protein. For B. bronchiseptica RimM specifically, lower induction temperatures (16-25°C) often improve solubility compared to standard 37°C induction protocols.

How can researchers purify recombinant RimM protein for structural studies?

Purification of recombinant B. bronchiseptica RimM for structural studies requires a systematic approach to achieve high purity and homogeneity. Based on protocols used for RimM from other bacterial species, the following methodological workflow is recommended:

• Affinity Chromatography (Primary Purification):

  • For His-tagged RimM: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-based resins. Typical buffers contain 20-50 mM Tris-HCl or phosphate buffer (pH 7.5-8.0), 300-500 mM NaCl, and increasing imidazole concentrations (10-20 mM for washing, 250-300 mM for elution) .

  • For GST-tagged RimM: Glutathione-Sepharose chromatography with elution using reduced glutathione (typically 10 mM).

  • For MBP-tagged RimM: Amylose resin with elution using maltose (typically 10 mM).

• Tag Removal (Optional):

  • Proteolytic cleavage using site-specific proteases (TEV, thrombin, or PreScission protease) followed by a second affinity step to remove the cleaved tag and protease.

  • Optimized conditions typically involve overnight incubation at 4°C with a protease:protein ratio of 1:50 to 1:100.

• Intermediate Purification:

  • Ion exchange chromatography (IEX) using either anion exchange (e.g., Q-Sepharose) or cation exchange (e.g., SP-Sepharose) depending on the protein's theoretical pI. Typically performed with a salt gradient (0-1 M NaCl) in 20 mM Tris-HCl or phosphate buffer.

• Polishing Step:

  • Size exclusion chromatography (SEC) using appropriate matrices (Superdex 75 or Superdex 200) to achieve final purity and to confirm monodispersity. Typical buffers contain 20 mM Tris-HCl or phosphate buffer (pH 7.5), 150 mM NaCl, and potentially 1-5 mM DTT or 2-mercaptoethanol .

• Quality Assessment:

  • SDS-PAGE and Western blotting to verify purity and identity

  • Dynamic light scattering (DLS) to assess homogeneity

  • Mass spectrometry to confirm molecular weight and integrity

For NMR studies specifically, buffer exchange into appropriate NMR buffers (typically phosphate buffer with reduced salt concentration) and concentration to 0.5-1.0 mM is necessary following purification . Additionally, optimization of buffer components (pH, salt concentration, additives) may be required to ensure protein stability during extended data collection periods.

What spectroscopic techniques are effective for studying RimM structure?

Multiple spectroscopic techniques have proven effective for studying the structure of RimM proteins, with nuclear magnetic resonance (NMR) spectroscopy being particularly informative. Based on successful structural characterization of RimM from T. thermophilus, the following spectroscopic approaches are recommended:

For studying the interaction between RimM and S19, NMR titration experiments using 15N-labeled RimM have proven particularly informative, allowing identification of residues involved in complex formation through chemical shift perturbation analysis . This approach revealed that both the N-terminal domain (specifically residues near a hydrophobic patch) and C-terminal region of RimM participate in S19 binding.

How does RimM interact with ribosomal protein S19?

The interaction between RimM and ribosomal protein S19 represents a key aspect of RimM's function in 30S subunit maturation. Detailed structural and biochemical studies have revealed several important characteristics of this interaction:

• Interaction Surfaces:
  NMR studies using 1H-15N HSQC spectra have demonstrated that RimM interacts with S19 through two distinct regions :

  • The N-terminal domain, particularly residues in the vicinity of a hydrophobic patch on the six-stranded β-barrel structure

  • A wide range of residues in the C-terminal region (residues 81-162 in T. thermophilus RimM)

  The involvement of both domains suggests a multi-point interaction mechanism that may provide specificity and affinity for proper recognition of S19.

• Binding Specificity:
  Previous studies using glutathione S-transferase pull-down assays confirmed that RimM specifically binds to ribosomal protein S19 but not to other ribosomal proteins . This specificity is critical for RimM's role in facilitating the incorporation of S19 into the maturing 30S subunit.

• Functional Context:
  The interaction between RimM and S19 is particularly significant because:

  • S19 is classified as a late binder in the assembly of the head of the 30S subunit according to assembly and kinetics order maps

  • S19 binding to helix 33b of 16S rRNA causes conformational changes in the 3' major domain of 16S rRNA

  • RimM appears to be involved in facilitating this interaction, playing an important role in the maturation of the head domain of the 30S subunit

• Structural Consequences:
  The binding of S19 to RimM induces significant chemical shift perturbations in NMR spectra, indicating conformational adjustments upon complex formation . These changes likely reflect the dynamic nature of the interaction and potentially prepare S19 for proper incorporation into the ribosomal structure.

What experimental approaches can be used to study RimM-S19 binding dynamics?

Several sophisticated experimental approaches can be employed to investigate the binding dynamics between RimM and ribosomal protein S19, providing insights into affinity, kinetics, and structural changes. These methodologies range from biophysical techniques to advanced structural biology approaches:

• Surface Plasmon Resonance (SPR):

  • Provides real-time measurement of association and dissociation rates (kon and koff)

  • Enables determination of equilibrium dissociation constants (KD)

  • Experimental setup typically involves immobilizing either RimM or S19 on a sensor chip (via amine coupling or capture approaches) and flowing the partner protein at various concentrations

  • Temperature dependence studies can provide thermodynamic parameters (ΔH, ΔS, ΔG)

• Isothermal Titration Calorimetry (ITC):

  • Measures heat changes during binding to determine binding stoichiometry, affinity, and thermodynamic parameters in solution

  • Doesn't require protein immobilization or labeling

  • Provides direct measurement of enthalpy changes (ΔH)

  • Typically requires higher protein concentrations than other methods

• Nuclear Magnetic Resonance (NMR) Titration Experiments:

  • 1H-15N HSQC spectra of 15N-labeled RimM can be recorded with increasing concentrations of unlabeled S19

  • Chemical shift perturbations identify residues involved in binding

  • Line-shape analysis can provide information on binding kinetics

  • ZZ-exchange experiments can detect conformational exchange processes

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Maps regions of altered solvent accessibility upon complex formation

  • Identifies stabilized regions within both proteins

  • Provides information on conformational changes without size limitations

  • Requires relatively low protein concentrations (~1 μM)

• Fluorescence-based Methods:

  • Fluorescence anisotropy using labeled proteins to monitor binding

  • Förster resonance energy transfer (FRET) with donor-acceptor pairs to measure distances

  • Microscale thermophoresis (MST) for binding affinity determination using minimal sample amounts

• Analytical Ultracentrifugation (AUC):

  • Sedimentation velocity experiments to characterize complex formation

  • Sedimentation equilibrium for accurate molecular weight determination of complexes

  • Provides information on complex stoichiometry and heterogeneity

• Cryo-Electron Microscopy (Cryo-EM):

  • Single-particle analysis to visualize RimM-S19 complexes

  • Potentially allows visualization of different binding states

  • Can place the interaction in the context of the assembling 30S subunit

For studying the dynamics specific to B. bronchiseptica RimM-S19 interaction, a combination of these approaches would be most informative. Based on previous successful studies, NMR spectroscopy has proven particularly valuable for detailed mapping of interaction surfaces and detecting conformational changes , while SPR or ITC would provide complementary quantitative binding parameters.

How can recombinant RimM be evaluated as a potential vaccine candidate?

Evaluation of recombinant B. bronchiseptica RimM as a potential vaccine candidate requires a systematic approach encompassing immunogenicity assessment, protective efficacy studies, and safety evaluation. Based on methodologies used for other Bordetella recombinant proteins, the following research protocol would be appropriate:

• Immunogenicity Assessment:

  • Antibody Response Measurement:

    • Immunize mice with purified recombinant RimM using appropriate adjuvants

    • Collect sera at various time points post-immunization

    • Measure antibody titers using enzyme-linked immunosorbent assay (ELISA)

    • Determine antibody subclass distribution (IgG1, IgG2a, IgG2b, IgG3) to assess Th1/Th2 balance

  • T-cell Response Characterization:

    • Harvest splenocytes from immunized mice

    • Perform proliferation assays upon restimulation with recombinant RimM

    • Measure cytokine production (IFN-γ, IL-4, IL-17) by ELISA or intracellular cytokine staining

    • Assess cytokine profiles to determine T-helper cell polarization

• Protective Efficacy Studies:

  • Challenge Model Development:

    • Establish reproducible B. bronchiseptica challenge model in appropriate animal species

    • Determine optimal challenge dose and route (intranasal for respiratory infection)

    • Define clear clinical and microbiological endpoints

  • Vaccination-Challenge Experiments:

    • Immunize animals with recombinant RimM using different dosing regimens

    • Challenge with virulent B. bronchiseptica via appropriate route

    • Monitor clinical signs, bacterial loads in respiratory tissues, and survival rates

    • Calculate protection ratio compared to control groups

  • Comparative Studies:

    • Include other recombinant B. bronchiseptica proteins as comparators

    • Based on published data, outer membrane porin protein (PPP) and lipoprotein (PL) showed protection ratios of 62.5% and 50%, respectively, in similar studies

    • Include commercial vaccines as positive controls when available

• Immune Mechanism Investigation:

  • Passive Transfer Studies:

    • Transfer serum from RimM-immunized animals to naïve recipients

    • Challenge with B. bronchiseptica to assess contribution of humoral immunity

  • T-cell Depletion/Transfer:

    • Deplete specific T-cell subsets prior to challenge in immunized animals

    • Alternatively, transfer T-cells from immunized to naïve animals

    • Determine role of cellular immunity in protection

• Formulation Optimization:

  • Adjuvant Screening:

    • Test RimM with different adjuvants (alum, oil-in-water emulsions, TLR agonists)

    • Assess impact on antibody titers, T-cell responses, and protection

  • Delivery System Evaluation:

    • Explore alternative delivery platforms (liposomes, nanoparticles)

    • Assess mucosal delivery options for respiratory pathogen

• Safety Assessment:

  • Local Reactogenicity:

    • Monitor injection site reactions (redness, swelling, pain)

  • Systemic Effects:

    • Monitor body temperature, weight changes, and behavioral alterations

  • Histopathological Examination:

    • Examine tissues at injection sites and key organs

Based on previous studies with other B. bronchiseptica recombinant proteins, a comprehensive evaluation would include analysis of both humoral and cell-mediated immune responses, as proteins like PPP and PL have demonstrated the ability to induce both types of responses, with a predominant Th2-type profile .

What challenges exist in developing RimM-based diagnostic tools?

Developing diagnostic tools based on recombinant B. bronchiseptica RimM presents several technical and practical challenges that researchers must address:

• Protein Localization Constraints:

  • RimM is an intracellular protein involved in ribosome assembly , not surface-exposed like outer membrane proteins

  • This localization means:

    • Natural infections may not induce strong anti-RimM antibody responses

    • RimM antigens would not be readily accessible in whole-cell diagnostic formats

    • Serological assays targeting RimM antibodies might have limited sensitivity for detecting past infections

• Specificity Considerations:

  • RimM is conserved across bacterial species , creating potential cross-reactivity challenges

  • Diagnostic applications would require:

    • Identification of B. bronchiseptica-specific epitopes within RimM

    • Careful assay validation against related Bordetella species (B. pertussis, B. parapertussis, B. holmesii)

    • Evaluation of potential cross-reactivity with other respiratory pathogens

• Technical Assay Development Challenges:

  • For PCR-based Detection:

    • Need to design primers/probes targeting rimM gene regions with sufficient specificity

    • Must differentiate from related Bordetella species, which may have highly similar rimM sequences

    • Current PCR diagnostic methods for Bordetella typically target other genes (IS481, pIS1001, ptxA-Pr, hIS1001)

  • For Protein-based Detection:

    • Identifying sensitive epitopes for antibody development

    • Optimizing recombinant protein production for consistent assay performance

    • Developing standard curves and reference materials for quantitative applications

• Comparative Advantage Assessment:

  • Current diagnostic algorithms for Bordetella species use well-established targets:

    • IS481 for B. pertussis, B. holmesii, and some B. bronchiseptica strains

    • pIS1001 for B. parapertussis

    • ptxA-Pr as a confirmatory target for B. pertussis

    • hIS1001 as a confirmatory target for B. holmesii

  • Any RimM-based diagnostic would need to demonstrate advantages over these established methods in terms of:

    • Sensitivity and specificity

    • Speed and simplicity

    • Cost-effectiveness

    • Potential for multiplexing

• Validation Requirements:

  • Extensive clinical validation would be necessary, including:

    • Testing against diverse B. bronchiseptica isolates

    • Performance evaluation across different host species (since B. bronchiseptica affects various animals)

    • Determination of analytical parameters (limit of detection, linear range, precision)

    • Comparison with gold standard methods

Current diagnostic algorithms for Bordetella species achieve high sensitivity through targeting insertion sequences present in multiple copies in the genome (like IS481) . For comparison, RimM is typically present as a single-copy gene, potentially limiting detection sensitivity compared to multi-copy targets. Nevertheless, if specific regions of B. bronchiseptica RimM could be identified that offer unique diagnostic value, particularly for differentiation from other Bordetella species, this could complement existing diagnostic approaches.

How conserved is RimM structure and function across different bacterial species?

RimM demonstrates significant conservation in both structure and function across diverse bacterial species, though with notable variations that provide insights into evolutionary adaptations. The analysis of this conservation reveals:

• Sequence Conservation:

  • RimM is widely conserved among bacteria, indicating its fundamental importance in ribosome assembly

  • Primary sequence analysis shows higher conservation in the N-terminal domain compared to the C-terminal region

  • Specific functional motifs, particularly those involved in the interaction with ribosomal protein S19, show the highest degree of conservation

• Structural Conservation:

  • The N-terminal domain's six-stranded β-barrel fold appears to be a highly conserved structural feature across bacterial species

  • Studies comparing T. thermophilus RimM with the structure of P. aeruginosa RimM (PDB identifier: 2F1L) confirmed that both N-terminal domains are composed of β-strands in similar arrangements

  • The hydrophobic patch in the N-terminal domain, implicated in S19 binding, is preserved across diverse bacterial species

  • The C-terminal domain shows greater structural variability while maintaining its role in S19 interaction

• Functional Conservation:

  • The core function of RimM in 30S ribosomal subunit maturation is preserved across bacterial species

  • Specific interactions with ribosomal protein S19 appear to be a universal feature of RimM proteins

  • The association of RimM exclusively with free 30S subunits (not with 70S ribosomes) is consistent across species

  • Deletion of rimM genes in different bacteria consistently results in ribosome assembly defects, though the severity may vary

• Taxonomic Distribution:

  • Beyond bacteria, RimM-related proteins have been identified in select eukaryotic species, including:

    • Malaria parasites (Plasmodium falciparum and Plasmodium yoelii)

    • The malaria mosquito (Anopheles gambiae)

    • Chloroplasts of plants (Arabidopsis thaliana)

  • This distribution suggests ancient evolutionary origins and potential specialized roles in organellar ribosome assembly

• Species-specific Variations:

  • Length variations exist, particularly in the C-terminal region

  • B. bronchiseptica RimM (aa 1-207) appears longer than T. thermophilus RimM (162 residues)

  • These variations may reflect adaptations to species-specific ribosome assembly pathways or interactions with additional factors

What can be learned from comparative genomic approaches to RimM research?

Comparative genomic approaches offer powerful insights into RimM biology, evolution, and potential applications. These methodologies can reveal patterns and relationships not apparent from single-species studies:

• Operon Structure and Gene Context Analysis:

  • RimM is encoded within the trmD operon in E. coli and many other bacteria

  • Comparative genomic analysis of the operon structure across diverse bacterial species can reveal:

    • Conservation of gene order and operon composition

    • Co-evolution patterns with functionally related genes

    • Regulatory elements controlling rimM expression

    • Potential species-specific variations in operon organization

• Phylogenetic Profiling:

  • Construction of phylogenetic trees based on RimM sequences can:

    • Trace evolutionary relationships among bacterial RimM proteins

    • Identify potential horizontal gene transfer events

    • Correlate RimM sequence variations with bacterial lifestyle or ecological niches

    • Track co-evolution with ribosomal protein S19

• Structural Prediction and Domain Analysis:

  • Comparative sequence analysis combined with structural modeling can:

    • Predict functional domains and motifs across diverse species

    • Identify conserved surface patches likely involved in protein-protein interactions

    • Map species-specific insertions or deletions onto structural models

    • Guide mutagenesis studies by highlighting evolutionarily constrained residues

• Bordetella-specific Applications:

  • Comparative analysis of rimM genes across Bordetella species (B. bronchiseptica, B. pertussis, B. parapertussis, B. holmesii) can:

    • Identify species-specific sequence signatures potentially useful for diagnostic development

    • Reveal adaptations potentially related to host range differences

    • Correlate sequence variations with pathogenicity differences

• Methodological Approaches:

  • Sequence-based Methods:

    • Multiple sequence alignment of RimM proteins across diverse species

    • Identification of conserved motifs using tools like MEME or GLAM2

    • Calculation of selection pressure (dN/dS ratios) across different domains

  • Structure-based Approaches:

    • Homology modeling of RimM proteins from diverse species

    • Molecular dynamics simulations to assess structural flexibility differences

    • Protein-protein docking with S19 homologs to predict interaction differences

• Integrated -Omics Approaches:

  • Integration of comparative genomics with:

    • Transcriptomics to identify co-expressed genes across species

    • Proteomics to validate expression and potential post-translational modifications

    • Interactomics to map species-specific protein interaction networks

By applying these comparative genomic approaches, researchers can develop a more comprehensive understanding of RimM biology across bacterial species. For B. bronchiseptica specifically, such approaches could identify unique features of its RimM protein that might correlate with its broader host range compared to other Bordetella species, potentially informing both basic science understanding and applied research directions.

How do RimM homologs in eukaryotes compare to bacterial RimM?

The discovery of RimM-related proteins in select eukaryotic species represents an intriguing aspect of ribosome assembly factor evolution. These eukaryotic homologs exhibit both similarities and significant differences compared to their bacterial counterparts:

• Distribution and Evolutionary Origins:

  • RimM-related proteins have been identified in specific eukaryotic species including:

    • Malaria parasites (Plasmodium falciparum and Plasmodium yoelii)

    • The malaria mosquito (Anopheles gambiae)

    • Plant chloroplasts (Arabidopsis thaliana)

  • This limited distribution suggests:

    • Potential acquisition through endosymbiotic events (particularly for chloroplast-associated homologs)

    • Possible horizontal gene transfer in some lineages

    • Loss of RimM homologs in many eukaryotic lineages during evolution

• Structural Comparison:

  • Eukaryotic RimM homologs generally preserve the two-domain architecture observed in bacterial RimM proteins

  • The N-terminal domain's β-barrel fold appears to be conserved, suggesting its fundamental importance

  • Greater sequence divergence is typically observed in the C-terminal regions

  • Eukaryotic homologs often contain additional sequence insertions or terminal extensions not present in bacterial counterparts

• Localization and Function:

  • In plants, RimM homologs are typically nuclear-encoded but contain N-terminal chloroplast transit peptides for targeting to the organelle

  • In Plasmodium species, RimM homologs may be involved in apicoplast ribosome assembly (the apicoplast being a non-photosynthetic plastid of endosymbiotic origin)

  • The function appears to remain centered on ribosome assembly, but within the context of organellar ribosomes rather than cytoplasmic ones

• Interaction Partners:

  • Eukaryotic RimM homologs likely interact with organellar S19 homologs

  • These interaction networks may be simplified compared to bacterial systems

  • Potential acquisition of eukaryote-specific interactions not present in bacterial systems

• Research Significance:

  • Studying these eukaryotic homologs provides insights into:

    • The evolution of ribosome assembly pathways across domains of life

    • Adaptation of bacterial proteins for functioning in eukaryotic cellular environments

    • Potential divergent functions that may have evolved in specific lineages

• Methodological Considerations for Comparative Studies:

  • Sequence Analysis Approaches:

    • Profile hidden Markov models for sensitive detection of distant homologs

    • Position-specific scoring matrices to identify conserved functional motifs

    • Coevolution analysis to identify compensatory mutations with interaction partners

  • Experimental Approaches:

    • Heterologous expression and complementation studies

    • Localization studies using fluorescent protein fusions

    • Interaction studies with organellar ribosomal components

The presence of RimM homologs in eukaryotes, particularly in parasites like Plasmodium species, suggests potential targets for selective intervention. Since these proteins are absent from the human genome but present in pathogens, they represent possible targets for anti-parasitic drug development with potentially limited side effects. Further comparative studies between bacterial RimM (including B. bronchiseptica RimM) and eukaryotic homologs could reveal fundamental insights into ribosome assembly evolution while potentially identifying novel therapeutic approaches.