Recombinant Nocardia farcinica 50S ribosomal protein L28 1 (rpmB1)

What is the 50S ribosomal protein L28 1 (rpmB1) in Nocardia farcinica?

The 50S ribosomal protein L28 1 (rpmB1) is a component of the large ribosomal subunit in Nocardia farcinica, a pathogenic actinomycete. This protein plays a crucial role in ribosomal assembly and function. The complete genomic sequence of N. farcinica IFM 10152 revealed a single circular chromosome of 6,021,225 bp with approximately 5,674 putative protein-coding sequences, including those involved in ribosomal structure and function . Like other ribosomal proteins, L28 contributes to the structural integrity of the ribosome and facilitates efficient protein synthesis. Mass spectrometry analysis has shown that L28 is one of several proteins (along with L16, L33, L36, and L35) that are significantly underrepresented in 45S ribosomal precursor particles, suggesting its importance in late-stage ribosomal assembly .

How does L28 contribute to bacterial ribosome assembly?

L28 plays a critical role in the late-stage assembly of the 50S ribosomal subunit. Structural studies using cryo-electron microscopy have demonstrated that L28 is among the proteins dramatically underrepresented in 45S precursor particles during ribosomal assembly . This underrepresentation indicates that L28 incorporation represents a rate-limiting step in ribosome maturation. The absence of L28 and other specific proteins (L16, L33, L36, and L35) in assembly intermediates suggests their sequential incorporation is essential for proper ribosomal formation. Research has revealed two major conformational states in 45S assembly intermediates that differ in the stability of the central protuberance (CP) and the orientation of helix 38 (H38) of the 23S rRNA . The incorporation of L28 appears to coincide with these critical conformational changes, suggesting it contributes to the stabilization of key ribosomal structures.

What are the structural characteristics of L28 in pathogenic bacteria?

The L28 protein in Nocardia farcinica and other pathogenic bacteria has distinct structural features that enable its interaction with ribosomal RNA and other ribosomal proteins. While specific structural data for N. farcinica L28 is limited in the provided search results, comparative analyses with L28 from other bacterial species can provide valuable insights. In bacterial ribosome assembly studies, researchers have constructed atomic models using crystal structures from related organisms like Escherichia coli and Thermus thermophilus as templates . These models have been refined through techniques such as Molecular Dynamics Flexible Fitting to better understand protein-RNA interactions within the ribosome. L28 typically interacts with specific regions of the 23S rRNA and is positioned within the ribosomal structure to contribute to the stability of the mature 50S subunit. The protein's strategic positioning in the ribosome makes it potentially important for both structural integrity and functional aspects of protein synthesis.

What mechanisms explain L28's role in late-stage ribosomal assembly?

The incorporation of L28 into the ribosomal structure appears to be linked to critical conformational changes in the 50S subunit assembly. Cryo-EM structural analysis of ribosomal assembly intermediates has revealed that L28 incorporation coincides with the reorientation of helix 38 (H38) of the 23S rRNA to its native-like position . This reorientation leads to global stabilization of the rRNA structure at the central protuberance (CP). Detailed structural analysis indicates that H38's orientation accounts for global conformational differences in intermediate structures, suggesting that the reorientation of H38 to its native position is rate-limiting during late-stage assembly . The interaction between the 5S rRNA and H38 is particularly important, with 5S rRNA serving as a bridge in the mutual stabilization between CP rRNA components and H38. The binding of L30 at the interface between 5S rRNA and the stem base of H38 may partially contribute to H38 reorientation, which could subsequently facilitate L28 incorporation . This complex interplay suggests that L28's role in ribosomal assembly is part of a highly coordinated process involving both RNA and protein components.

How does L28 from Nocardia farcinica compare to homologs in other bacterial species?

While the search results don't provide direct comparative data for N. farcinica L28, researchers typically approach this question through sequence alignment and structural modeling. The atomic model of the bacterial 50S subunit can be constructed using crystal structures from well-studied organisms like E. coli (PDB ID: 2AW4) and T. thermophilus (PDB ID: 2J01) as templates . Sequence alignments of ribosomal components can be constructed using software like S2S (Sequence to Structure), with RNA models built using tools like ModeRNA . For protein components like L28, models can be generated using MODELLER or similar tools, based on templates from related species. These comparative approaches would likely reveal conserved domains important for RNA interactions and structural positions within the ribosome, as well as species-specific variations that might relate to pathogenicity or environmental adaptations of N. farcinica.

What is the significance of L28 in antibiotic resistance mechanisms?

While the search results don't directly address L28's role in antibiotic resistance, this question is relevant given that N. farcinica is emerging as a serious pathogen with multidrug resistance that extends treatment periods for months or even years . The ribosome is a common target for antibiotics, and variations in ribosomal proteins can contribute to resistance mechanisms. The genomic analysis of N. farcinica IFM 10152 revealed many candidate genes for virulence and multidrug resistance , suggesting that components of the protein synthesis machinery, including ribosomal proteins like L28, might play roles in these resistance mechanisms. Research into the specific contributions of L28 to antibiotic resistance would involve comparative genomics, structural analysis of antibiotic binding sites, and functional studies of mutant strains with modified L28 sequences or expression levels. Such studies could provide insights into novel therapeutic approaches for combating resistant Nocardia infections.

What expression systems are optimal for producing recombinant N. farcinica L28?

Based on successful approaches with other Nocardia proteins, E. coli-based expression systems typically offer the most straightforward and efficient method for producing recombinant proteins from N. farcinica. For example, in studies with the NFA49590 protein from N. farcinica, researchers successfully cloned and expressed the recombinant protein in E. coli (DE3) and purified it using a Ni-NTA column . A similar approach would likely be effective for L28.

The experimental workflow would typically include:

• Gene cloning: PCR amplification of the rpmB1 gene from N. farcinica genomic DNA

• Vector construction: Insertion into an expression vector with an appropriate tag (e.g., His-tag) for purification

• Transformation: Introduction of the construct into a suitable E. coli strain (BL21(DE3) or similar)

• Expression optimization: Testing various induction conditions (IPTG concentration, temperature, duration)

• Cell lysis: Breaking cells to release the recombinant protein

• Purification: Affinity chromatography using the introduced tag

For difficult-to-express proteins, alternative approaches might include:

• Codon optimization for E. coli

• Fusion with solubility enhancers like SUMO or MBP

• Testing alternative expression hosts such as Mycobacterium smegmatis for mycobacterial proteins

What purification strategies yield the highest purity for recombinant L28?

A multi-step purification strategy typically yields the highest purity for ribosomal proteins like L28. Based on successful approaches with other bacterial proteins, the following protocol would be recommended:

• Initial capture: Affinity chromatography using Ni-NTA for His-tagged L28

• Intermediate purification: Ion exchange chromatography (typically cation exchange for basic ribosomal proteins)

• Polishing step: Size exclusion chromatography to remove aggregates and degradation products

Table 1: Recommended Purification Protocol for Recombinant L28

Protein purity should be assessed after each step using SDS-PAGE, with expected yields in the range of 5-10 mg per liter of bacterial culture for a well-expressed ribosomal protein. Western blotting using antibodies against the His-tag or L28 itself can confirm the identity of the purified protein.

How can researchers verify the correct folding and function of recombinant L28?

Verifying the correct folding and function of recombinant L28 requires multiple complementary approaches:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to analyze secondary structure content

  • Thermal shift assays to evaluate protein stability

  • Limited proteolysis to assess compact folding

• Functional verification:

  • RNA binding assays using specific 23S rRNA fragments

  • In vitro ribosomal assembly assays

  • Complementation studies in L28-deficient bacterial strains

• Interaction analysis:

  • Pull-down assays with other ribosomal components

  • Surface plasmon resonance (SPR) to measure binding kinetics

  • Cryo-EM analysis of L28 incorporation into ribosomal particles

The most definitive functional verification would involve demonstrating that the recombinant L28 can be incorporated into 45S intermediate particles and contribute to their maturation into 50S subunits, following approaches similar to those used in structural studies of ribosomal assembly .

Can L28 serve as a target for novel antibiotics against Nocardia infections?

L28 represents a potential target for novel antibiotics against Nocardia infections, particularly given the challenges of multidrug resistance in clinical isolates that can extend treatment periods for months or years . Several factors make L28 an intriguing antibiotic target:

• Essential function: As a component of the bacterial ribosome, L28 is essential for protein synthesis and bacterial survival.

• Role in ribosomal assembly: L28's importance in late-stage ribosomal assembly suggests that inhibiting its incorporation could disrupt ribosome biogenesis.

• Structural uniqueness: If structural differences exist between bacterial and human ribosomal components, these could be exploited for selective targeting.

Research approaches to explore L28 as an antibiotic target would include:

• High-throughput screening for compounds that disrupt L28 incorporation into ribosomes

• Structure-based drug design targeting the L28-RNA interface

• Peptide inhibitors designed to mimic L28 interaction surfaces

The discovery of compounds that specifically inhibit L28 function or incorporation could lead to novel antibiotics effective against Nocardia species, including multidrug-resistant strains. These could potentially be developed as standalone treatments or used in combination with existing antibiotics like sulfonamides, amikacin, or carbapenems that are currently used to treat Nocardia infections .

What potential does recombinant L28 have for vaccine development against Nocardia infections?

The potential of recombinant L28 as a vaccine candidate against Nocardia infections should be considered in the context of other Nocardia proteins that have shown immunoprotective effects. While the search results don't directly address L28's immunogenic properties, the approach taken with other Nocardia proteins provides a methodological framework:

Research with the NFA49590 protein from N. farcinica demonstrated that recombinant proteins from this pathogen can elicit protective immune responses . The NFA49590 protein was identified through LC-MS/MS analysis of secreted proteins, evaluated for antigenicity in silico, and then tested experimentally. Similar approaches could be applied to assess L28's vaccine potential:

• Antigenicity assessment:

  • In silico prediction of antigenic epitopes within the L28 sequence

  • Experimental verification using sera from patients recovered from Nocardia infections

• Immunological characterization:

  • Testing the ability of recombinant L28 to activate innate immunity

  • Examining cytokine responses (IL-6, TNF-α, IL-10) to L28 exposure

  • Assessing antibody production in immunized animal models

• Protection studies:

  • Challenging immunized animals with virulent N. farcinica

  • Measuring bacterial clearance and survival rates

How do structural studies of L28 contribute to understanding ribosomal assembly in pathogenic bacteria?

Structural studies of L28 provide critical insights into ribosomal assembly pathways in pathogenic bacteria like Nocardia farcinica. The cryo-EM analysis of 45S intermediates has demonstrated that L28, along with several other proteins (L16, L33, L36, and L35), is underrepresented in these particles, indicating its importance in the late stages of 50S subunit assembly .

These structural investigations reveal:

• Assembly checkpoints: The incorporation of L28 appears to represent a critical checkpoint in ribosomal maturation, potentially serving as a quality control mechanism.

• Conformational changes: The absence of L28 in 45S intermediates coincides with distinct conformational states that differ in the stability of the central protuberance and orientation of helix 38 (H38) .

• Coordinated assembly: The data suggests that L28 incorporation is part of a coordinated process involving both RNA remodeling (particularly H38 reorientation) and sequential protein addition.

The reorientation of H38 to its native position appears to be rate-limiting during late-stage assembly, with H38 playing an essential role in stabilizing the central protuberance through interaction with the 5S rRNA . Understanding the precise timing and mechanism of L28 incorporation could reveal fundamental principles of ribosomal assembly that might be conserved across diverse bacterial species, including pathogenic organisms like N. farcinica.

How does L28 structure and function relate to Nocardia farcinica pathogenicity?

The relationship between L28 and N. farcinica pathogenicity must be considered within the broader context of this organism's clinical significance. N. farcinica is an opportunistic pathogen that often infects immunocompromised individuals, with the ability to invade through the respiratory tract or skin wounds, cause local infection, and disseminate to other organs hematogenously .

The genomic sequence of N. farcinica IFM 10152 revealed many candidate genes for virulence and multidrug resistance , suggesting complex mechanisms underlying its pathogenicity. While the direct role of L28 in virulence hasn't been explicitly established in the search results, ribosomal proteins can influence pathogenicity through several mechanisms:

• Growth rate modulation: Efficient ribosomal assembly and function, facilitated by proteins like L28, may enable rapid adaptation to host environments.

• Stress response: Alterations in ribosomal composition during stress conditions might help the pathogen survive host defense mechanisms.

• Moonlighting functions: Some ribosomal proteins have secondary roles outside the ribosome that could directly impact virulence.

Research approaches to investigate L28's potential role in pathogenicity would include comparative genomics and transcriptomics between virulent and avirulent strains, construction of L28 mutants with altered expression or sequence, and infection models to assess the impact of these modifications on virulence.

What challenges exist in targeting ribosomal proteins for therapeutic development?

Developing therapeutics targeting ribosomal proteins like L28 presents several significant challenges that researchers must address:

• Structural conservation: Ribosomal proteins often show conservation across bacterial species and potentially share structural similarities with eukaryotic counterparts, raising specificity concerns.

• Accessibility: Ribosomal proteins are often buried within the complex ribosomal structure, potentially limiting drug accessibility.

• Resistance development: Bacteria can develop resistance through mutations in target proteins or upregulation of efflux pumps.

• In vivo efficacy: Compounds effective in vitro may not achieve sufficient concentrations at infection sites or may be metabolized too rapidly.

Methodological approaches to overcome these challenges include:

• Structure-based design focusing on bacterial-specific regions of L28

• Targeting L28-rRNA interactions rather than the protein itself

• Developing combination therapies to reduce resistance emergence

• Using advanced delivery systems to improve pharmacokinetics