Recombinant Bordetella bronchiseptica 30S ribosomal protein S13 (rpsM)

What is Bordetella bronchiseptica 30S ribosomal protein S13 (rpsM) and what are its primary functions?

Bordetella bronchiseptica 30S ribosomal protein S13 (rpsM) is a component of the small (30S) ribosomal subunit in B. bronchiseptica, a highly contagious bacterial respiratory pathogen with a broad host range including domestic and wild mammals . This protein shares homology with prokaryotic ribosomal protein S15, which is known to bind to the central domain of 16S rRNA and promote the binding of neighboring proteins in the 30S ribosomal subunit . The primary function of rpsM is to participate in ribosome assembly and protein synthesis, playing a critical role in bacterial survival and replication. Based on studies of homologous ribosomal proteins, rpsM likely contributes to the structural integrity of the ribosome and participates in the translation process by helping to ensure proper mRNA positioning during protein synthesis. In B. bronchiseptica specifically, rpsM function is essential for bacterial growth, making it a potential target for therapeutic intervention or research into bacterial pathogenicity.

How does rpsM differ from ribosomal protein S13 in other organisms?

While specific comparative data for B. bronchiseptica rpsM is limited in the provided search results, insights can be drawn from studies of ribosomal protein S13 in other organisms. Human ribosomal protein S13 (rpS13), for instance, is known to regulate its own gene expression through a feedback mechanism involving pre-mRNA splicing . Human rpS13 can bind to its own pre-mRNA at specific sites near splice junctions, inhibiting intron excision and thereby reducing expression . This autoregulatory mechanism appears to be an important way for maintaining appropriate levels of each ribosomal protein in eukaryotic cells. The prokaryotic B. bronchiseptica rpsM likely shares structural similarities with its eukaryotic counterparts but functions within the context of prokaryotic translation without the splicing-based regulation seen in eukaryotes. Additionally, bacterial ribosomal proteins often have specialized roles in ribosome assembly that may differ from their eukaryotic homologs, reflecting the fundamental differences in translation machinery between prokaryotes and eukaryotes.

What expression systems are most effective for producing recombinant B. bronchiseptica rpsM?

Recombinant B. bronchiseptica rpsM protein can be produced using various expression systems including Escherichia coli, yeast, baculovirus, or mammalian cell cultures . The choice of expression system depends on several factors including the desired protein yield, post-translational modifications required, downstream applications, and available laboratory resources. E. coli expression systems are most commonly used for bacterial ribosomal proteins due to their high yield, cost-effectiveness, and simplicity of culture conditions. For experimental protocols requiring specific modifications or folding assistance, yeast or mammalian expression systems might be preferable despite their increased complexity and cost. When designing an expression construct, researchers should consider including affinity tags (such as His-tag or GST) to facilitate purification while ensuring these tags do not interfere with the protein's structure or function. Expression optimization typically requires testing multiple growth conditions (temperature, induction timing, media composition) to maximize soluble protein yield while minimizing inclusion body formation.

What methods are most effective for assessing the structural integrity and functionality of purified recombinant rpsM?

Assessment of structural integrity and functionality of purified recombinant rpsM requires a multi-faceted approach combining biophysical and biochemical techniques. Circular dichroism (CD) spectroscopy can provide information about secondary structure content and proper folding, while thermal shift assays can assess protein stability under various conditions. Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) offers insights into oligomeric state and homogeneity. For functional assessment, RNA binding assays similar to those described for human rpS13 can be adapted, including nitrocellulose filter binding assays performed under physiologically relevant salt concentrations (e.g., 250 mM KCl) to reduce non-specific electrostatic interactions . Competition assays using labeled RNA and various unlabeled competitors can help establish binding specificity . Additionally, researchers should consider ribosome assembly assays to determine whether the recombinant protein can incorporate into bacterial ribosomes, which would confirm its structural and functional integrity. Ribonuclease protection assays may also reveal specific RNA regions protected by protein binding, providing valuable structural insights.

How can researchers investigate potential autoregulatory mechanisms of rpsM in B. bronchiseptica?

Investigating potential autoregulatory mechanisms of rpsM in B. bronchiseptica would require adapting approaches used for studying similar phenomena in other organisms. Based on the autoregulatory mechanism observed with human rpS13, researchers should first determine whether B. bronchiseptica rpsM can bind to its own mRNA or regulatory regions . This could be accomplished through RNA electrophoretic mobility shift assays (EMSA) using purified recombinant rpsM and in vitro transcribed fragments of the rpsM gene locus. Nitrocellulose filter binding assays could quantify binding affinity, ideally testing multiple RNA fragments to identify specific binding regions . In vivo studies might include reporter gene constructs containing the rpsM promoter and potential regulatory regions fused to fluorescent or enzymatic reporters, allowing measurement of expression levels under conditions of rpsM overexpression or depletion. RNA immunoprecipitation followed by sequencing (RIP-seq) could identify RNA targets of rpsM in B. bronchiseptica cells. For mechanistic understanding, researchers should also investigate potential rpsM binding partners using co-immunoprecipitation or bacterial two-hybrid systems, as autoregulation might involve protein-protein interactions in addition to protein-RNA interactions.

What experimental controls are critical when studying RNA-protein interactions involving rpsM?

When studying RNA-protein interactions involving rpsM, several critical controls must be implemented to ensure reliable and interpretable results. First, researchers should include non-rpsM ribosomal proteins (such as S10 or S16, which showed lower binding affinity to S13INT RNA fragment in human studies) as negative controls to demonstrate binding specificity . Second, multiple non-specific RNA competitors (such as poly(AU) or unrelated pre-mRNA fragments) should be included in competition assays to distinguish specific from non-specific binding . Third, experiments should be performed across a range of salt concentrations (from physiological to higher stringency) to differentiate specific interactions from electrostatic associations, particularly important when working with positively charged ribosomal proteins and negatively charged RNA . Fourth, binding studies should include both wild-type rpsM and mutated versions with alterations in putative RNA-binding domains to map the interaction interface. Fifth, experiments should include both full-length and truncated RNA constructs to identify minimal binding regions. Lastly, when performing in vivo studies, appropriate controls for protein expression levels are essential, as overexpression artifacts could lead to non-physiological interactions or cellular responses that do not reflect normal regulatory mechanisms.

How does the BvgAS regulatory system potentially interact with rpsM expression or function?

The BvgAS two-component sensory transduction system is a master regulator of virulence gene expression in B. bronchiseptica, controlling the transition between virulent (Bvg+) and non-virulent (Bvg-) phenotypic phases in response to environmental cues . While the search results do not specifically indicate that rpsM is directly regulated by the BvgAS system, several hypotheses can be formed regarding potential interactions. First, as bacterial ribosomal proteins are often differentially expressed under various growth conditions, the BvgAS system might indirectly influence rpsM expression through its effects on global gene regulation networks. Second, during the virulent Bvg+ phase when virulence-activated genes are highly expressed, increased demand for protein synthesis might alter requirements for ribosomal components including rpsM. Third, some ribosomal proteins in other bacteria have been shown to interact with regulatory proteins, raising the possibility that rpsM might interact with components of the BvgAS pathway or other regulatory systems. To investigate these possibilities, researchers could analyze rpsM expression levels in wild-type B. bronchiseptica compared to BvgAS mutants under various environmental conditions. Chromatin immunoprecipitation (ChIP) experiments with BvgA could determine whether this regulator directly binds to the rpsM promoter region. Additionally, protein-protein interaction studies might reveal whether rpsM physically interacts with BvgA, BvgS, or other regulatory proteins in B. bronchiseptica.

How can recombinant rpsM be utilized in studies of antimicrobial resistance mechanisms in B. bronchiseptica?

Recombinant rpsM offers valuable opportunities for studying antimicrobial resistance mechanisms in B. bronchiseptica, particularly since ribosomal proteins are targets for several antibiotic classes. Researchers can use purified recombinant rpsM in binding studies with aminoglycoside antibiotics, which target the 30S ribosomal subunit, to assess direct interactions and potential binding site mutations that might confer resistance. In vitro translation systems incorporating recombinant rpsM (wild-type or mutant versions) could help determine how specific modifications affect antibiotic susceptibility at the molecular level. The recombinant protein could also be used to generate antibodies for immunoprecipitation studies to identify interaction partners that might be involved in resistance mechanisms. Given that B. bronchiseptica isolates typically exhibit resistance to β-lactam antibiotics (both penicillins and cephalosporins) and macrolides , investigating whether rpsM plays any direct or indirect role in these resistance phenotypes could yield valuable insights. While the search results identified specific resistance genes in B. bronchiseptica (blaBOR, sul2, and aph(3′′)-Ib/strA) , determining whether rpsM expression levels correlate with expression of these genes could reveal regulatory connections relevant to antimicrobial resistance.

What methodological approaches can assess the impact of rpsM mutations on antibiotic susceptibility?

Assessing the impact of rpsM mutations on antibiotic susceptibility requires a multi-layered methodological approach combining genetic, biochemical, and microbiological techniques. Site-directed mutagenesis can be used to generate specific rpsM variants carrying mutations in regions predicted to interact with antibiotics or involved in ribosome assembly. These mutant alleles can then be introduced into B. bronchiseptica strains using allelic exchange techniques to create isogenic strains differing only in their rpsM sequence. Standard antimicrobial susceptibility testing methods, including broth microdilution and disk diffusion assays, can quantify changes in minimum inhibitory concentrations (MICs) for various antibiotics. For mechanistic understanding, ribosome profiling can determine whether rpsM mutations alter ribosome assembly or function. In vitro translation assays using purified components can directly assess how specific mutations affect protein synthesis in the presence of antibiotics. Structural studies, including cryo-electron microscopy of ribosomes containing mutant rpsM, can visualize changes in antibiotic binding sites. Additionally, fitness assays comparing growth rates of wild-type and mutant strains in the presence of sub-inhibitory antibiotic concentrations can reveal whether mutations confer fitness advantages or disadvantages, providing insights into the evolutionary dynamics of resistance development.

What genomic analysis techniques can reveal evolutionary conservation and variation of rpsM across Bordetella species?

Comprehensive genomic analysis of rpsM across Bordetella species requires a multi-faceted approach leveraging various computational and experimental techniques. Whole-genome sequencing (WGS) analysis, as employed for B. bronchiseptica isolates in previous studies , provides the foundation for identifying and comparing rpsM sequences across different strains and species. Multiple sequence alignment tools can quantify conservation at both nucleotide and amino acid levels, identifying highly conserved functional domains and variable regions that might reflect species-specific adaptations. Phylogenetic analysis can establish evolutionary relationships between rpsM sequences from different Bordetella species, potentially revealing horizontal gene transfer events or convergent evolution. Selection pressure analysis using metrics such as dN/dS ratios (non-synonymous to synonymous substitution rates) can identify regions under positive selection, which often indicate functionally important adaptation. Comparative analysis of the genomic context surrounding the rpsM gene might reveal differences in operon structure or regulatory elements across species. RNA-seq data from different Bordetella species can provide insights into expression patterns and potential species-specific regulation. Additionally, structural modeling based on sequence data can predict how sequence variations might impact protein structure and function across species.

How do sequence variations in rpsM correlate with host specificity in Bordetella species?

Understanding how rpsM sequence variations correlate with host specificity in Bordetella species requires integrating genomic data with host range information and functional studies. While the search results indicate that B. bronchiseptica has a broad host range including wild and domesticated mammals , different strains show varying degrees of host adaptation. Researchers should first compile comprehensive data on rpsM sequences from Bordetella isolates with well-documented host origins, creating a database that links sequence variations to host species. Statistical association studies can then identify specific amino acid residues or motifs that correlate with particular host ranges. Comparative analysis with closely related species that have narrower host ranges (such as B. pertussis, which primarily infects humans) could be particularly revealing. Functional validation of putative host-specificity determinants would require expressing recombinant rpsM variants in heterologous systems and assessing their interaction with host-derived factors. Since ribosomal function is central to bacterial adaptation to different growth environments, researchers should investigate whether rpsM variations affect translation efficiency under conditions mimicking different host environments (temperature, pH, nutrient availability). Additionally, experimental evolution studies exposing B. bronchiseptica to different host-derived growth conditions might reveal adaptive changes in rpsM that emerge under specific selective pressures.

How can recombinant rpsM be utilized in developing novel vaccine strategies against B. bronchiseptica?

Recombinant rpsM presents several promising avenues for vaccine development against B. bronchiseptica infections, particularly in swine populations where this pathogen causes significant respiratory disease . As a highly conserved ribosomal protein, rpsM could potentially elicit immune responses effective against multiple B. bronchiseptica strains, addressing the challenge of strain variation. Researchers could assess the immunogenicity of purified recombinant rpsM in animal models, measuring both antibody and cell-mediated immune responses. Epitope mapping studies would identify specific regions of the protein that elicit protective immunity, allowing for the design of peptide-based vaccines focusing on these immunodominant regions. The recombinant protein could be incorporated into various vaccine platforms including adjuvanted subunit vaccines, virus-like particles (VLPs), or as an antigen expressed by live attenuated bacterial or viral vectors. Given that B. bronchiseptica infections increase susceptibility to co-infections with other respiratory pathogens , combination vaccines incorporating rpsM along with antigens from common co-infecting pathogens could provide broader protection. Safety assessment would be critical, ensuring that immune responses against rpsM do not cross-react with host ribosomal proteins and cause autoimmune reactions.

What potential exists for rpsM to serve as a diagnostic marker for B. bronchiseptica infections?

The potential of rpsM as a diagnostic marker for B. bronchiseptica infections stems from several characteristics that make it suitable for detection systems. Being an abundant ribosomal protein produced during active bacterial growth, rpsM would likely be present at detectable levels during infection. Researchers could develop rpsM-specific monoclonal antibodies for use in enzyme-linked immunosorbent assays (ELISA) or lateral flow assays for rapid detection of B. bronchiseptica in clinical samples. PCR-based detection targeting the rpsM gene could offer high sensitivity, especially important for detecting carrier animals that may have low bacterial loads but serve as reservoirs for transmission. Comparative genomic analysis would be necessary to identify regions of the rpsM gene that are unique to B. bronchiseptica, allowing for the design of species-specific primers or probes that avoid cross-reactivity with related Bordetella species or other respiratory pathogens. Reverse transcription PCR (RT-PCR) targeting rpsM mRNA could specifically detect metabolically active bacteria, distinguishing live infections from the presence of dead bacterial cells. Multiplex detection systems incorporating rpsM along with markers for other common respiratory pathogens could facilitate comprehensive diagnostic panels for respiratory disease, particularly valuable in swine herds where complex co-infections are common . Validation studies would need to establish the sensitivity and specificity of rpsM-based diagnostics using diverse clinical samples and bacterial strains.

What are the most promising future research directions for understanding and utilizing B. bronchiseptica rpsM?

The most promising future research directions for B. bronchiseptica rpsM span fundamental biology, applied diagnostics, and therapeutic development. Structural studies using cryo-electron microscopy or X-ray crystallography would provide atomic-level insights into rpsM's interactions within the ribosome and with potential binding partners. Comprehensive characterization of rpsM's potential extraribosomal functions could reveal unexpected roles in bacterial physiology or pathogenesis, similar to the moonlighting functions discovered for ribosomal proteins in other organisms . Investigation of rpsM autoregulation mechanisms would contribute to understanding bacterial gene expression control, potentially revealing novel regulatory paradigms. From an applied perspective, development and validation of rpsM-based vaccines represents a promising direction, particularly for protecting livestock from respiratory disease. High-throughput screening for small molecules that specifically bind rpsM could identify novel antibiotic candidates targeting this essential protein. Additionally, the potential use of rpsM as a species-specific diagnostic marker deserves thorough exploration, especially for rapid point-of-care testing in veterinary settings. These diverse research avenues collectively offer opportunities to both advance fundamental understanding of bacterial biology and develop practical solutions to combat B. bronchiseptica infections.