Recombinant Lactobacillus johnsonii 50S ribosomal protein L29 (rpmC)

What is the genomic context of the rpmC gene in Lactobacillus johnsonii?

The rpmC gene in L. johnsonii encodes the 50S ribosomal protein L29, a critical component of the bacterial ribosome large subunit. Based on genomic analyses of L. johnsonii strains such as MT4, the genome is approximately 1.88 Mbp with a GC content of 34.4% . While the search results don't specifically detail the rpmC gene location, ribosomal protein genes in bacteria typically cluster in operons. For L. johnsonii, the rpmC gene would be expected within the conserved str operon that includes other ribosomal proteins and translation factors.

Whole genome sequencing has revealed that L. johnsonii strain MT4 shares over 99.96% genome identity with strain NCK2677, both isolated from mouse GI tracts, with strain NCC 533 (La1) being their closest relative . This genomic conservation suggests rpmC sequence and location are likely highly conserved among L. johnsonii strains.

How does L. johnsonii rpmC compare structurally to rpmC proteins in other bacterial species?

The L29 ribosomal protein (encoded by rpmC) is typically highly conserved across bacterial species due to its essential role in ribosome structure and function. While the search results don't provide specific structural information about L. johnsonii rpmC, comparative genomics would predict significant structural conservation.

Based on general ribosomal protein characteristics, L29 typically features:

• A small, basic protein (~7-9 kDa)

• RNA-binding domains for interaction with ribosomal RNA

• Interface regions for interaction with other ribosomal proteins

• Relatively high lysine and arginine content

Researchers investigating L. johnsonii rpmC should consider performing multiple sequence alignments with rpmC from closely related Lactobacillus species to identify conserved and variable regions, which can inform recombinant protein design strategies.

What are the primary functions of the 50S ribosomal protein L29 in L. johnsonii?

The L29 protein in L. johnsonii, like in other bacteria, serves several critical functions:

• Structural role in the 50S ribosomal subunit assembly

• Contribution to mRNA binding and positioning

• Stabilization of tRNA interactions during translation

• Potential role in antibiotic susceptibility (many ribosomal proteins are targets for antimicrobials)

Given L. johnsonii's probiotic properties and anticandidal activities , studying its ribosomal components may provide insights into its growth characteristics, stress responses, and antimicrobial mechanisms. The production of various bioactive compounds by L. johnsonii, including bacteriocins like lactacin-F and helveticin J , relies on functional protein synthesis machinery where rpmC plays an integral role.

What are the optimal expression systems for producing recombinant L. johnsonii rpmC protein?

For recombinant expression of L. johnsonii rpmC, researchers should consider multiple expression systems based on research objectives:

Table 1: Comparison of Expression Systems for Recombinant L. johnsonii rpmC

For expression in E. coli, codon optimization may be necessary due to the low GC content (34.4%) of L. johnsonii . Using a pET vector system with a His-tag for purification often provides good results for ribosomal proteins. Temperature modulation (expression at 18-25°C rather than 37°C) can improve solubility for L. johnsonii proteins.

What purification strategies are most effective for isolating recombinant L. johnsonii rpmC?

Purification of recombinant L. johnsonii rpmC typically requires a multi-step approach:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged rpmC protein

• Intermediate Purification: Ion exchange chromatography (typically cation exchange given the basic nature of ribosomal proteins)

• Polishing: Size exclusion chromatography to remove aggregates and achieve high purity

Critical Considerations:

• Buffer optimization is essential, typically using pH 7.5-8.0 with 150-300 mM NaCl to maintain stability

• Addition of reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) to prevent oxidation

• Testing various detergents (0.05-0.1% Triton X-100 or 0.01-0.05% DDM) if aggregation occurs

• Inclusion of RNase treatment steps to remove bound RNA that may co-purify with ribosomal proteins

For analytical purposes, SDS-PAGE combined with western blotting using anti-His antibodies provides confirmation of protein identity and purity assessment.

How can recombinant L. johnsonii rpmC be used to study ribosome assembly and function?

Recombinant L. johnsonii rpmC offers valuable opportunities for ribosome assembly studies:

• In vitro Reconstitution Assays: Purified recombinant rpmC can be used in reconstitution experiments with other ribosomal components to study assembly pathways specific to L. johnsonii ribosomes.

• Binding Studies: Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) with recombinant rpmC can characterize interactions with rRNA and other ribosomal proteins.

• Cryo-EM Analysis: Labeled recombinant rpmC can aid in structural determination of L. johnsonii ribosomes, potentially revealing strain-specific features.

• Translation Assays: In vitro translation systems reconstituted with recombinant L. johnsonii ribosomal proteins, including rpmC, can help understand the impact of specific mutations or modifications on protein synthesis.

Given the role of L. johnsonii in producing antimicrobial compounds like bacteriocins , understanding ribosome function may provide insights into optimizing production of these bioactive compounds.

What approaches can determine if L. johnsonii rpmC contributes to its probiotic and antimicrobial properties?

To investigate potential roles of rpmC in L. johnsonii's probiotic and antimicrobial functions:

• Gene Knockout/Knockdown Studies: CRISPR-Cas9 or RNA interference approaches targeting rpmC can be used to assess impacts on:

  • Growth characteristics

  • Stress responses

  • Production of antimicrobial compounds like the bacteriocins identified in strain MT4

  • Anticandidal activities observed against C. albicans

• Complementation Experiments: Reintroducing wild-type or modified rpmC into knockout strains to confirm phenotypes

• Comparative Proteomics: Comparing proteome profiles between wild-type and rpmC-modified strains

• Functional Assays: Testing modified strains in:

  • Anticandidal assays against C. albicans (similar to those described for L. johnsonii MT4 )

  • Immunomodulatory studies to assess impact on dendritic cell function and cytokine production (as described for L. johnsonii supplementation )

L. johnsonii has demonstrated significant anticandidal properties, reducing C. albicans growth and inhibiting hyphal transition and biofilm formation . Investigating whether ribosomal proteins like rpmC contribute to these properties through their roles in protein synthesis or potentially through moonlighting functions could yield valuable insights.

What structural analysis techniques are most informative for characterizing L. johnsonii rpmC?

For comprehensive structural characterization of recombinant L. johnsonii rpmC:

Table 2: Structural Analysis Techniques for L. johnsonii rpmC

Combining these methods provides complementary structural information. For instance, CD spectroscopy can quickly assess whether recombinant rpmC has the expected secondary structure composition, while more resource-intensive techniques like X-ray crystallography provide atomic-level details necessary for understanding specific interactions within the ribosome.

How can transcriptomic approaches be integrated with rpmC research to understand L. johnsonii function?

Integrating transcriptomics with rpmC research offers deeper insights into L. johnsonii biology:

• Expression Correlation Analysis: RNA-seq data can reveal genes co-expressed with rpmC under various conditions, potentially identifying functional relationships.

• Ribosome Profiling: This technique can determine how rpmC mutations or modifications affect translation efficiency and mRNA selection across the transcriptome.

• Differential Expression Analysis: Comparing transcriptomes between wild-type and rpmC-modified strains can identify downstream effects on gene expression.

• Condition-Specific Expression: Analyzing rpmC expression changes during:

  • Growth in different nutrient environments

  • Exposure to stress conditions

  • Co-culture with pathogens like C. albicans

  • Interaction with host immune cells

Studies have shown that L. johnsonii supplementation reduces airway Th2 cytokines and viral clearance during RSV infection . Transcriptomic approaches could determine whether these effects involve altered translation dynamics dependent on ribosomal proteins like rpmC.

What are common challenges in expressing recombinant L. johnsonii rpmC and how can they be addressed?

Researchers often encounter several challenges when expressing recombinant L. johnsonii rpmC:

Table 3: Troubleshooting Recombinant L. johnsonii rpmC Expression

When expressing ribosomal proteins like rpmC, it's worth noting that their natural role involves interaction with RNA and other proteins within the ribosome complex. Therefore, when expressed recombinantly, they may exhibit non-specific binding or aggregation tendencies that require optimization steps beyond those needed for typical cytoplasmic proteins.

How can researchers validate the functional activity of recombinant L. johnsonii rpmC?

Validating functional activity of recombinant rpmC is crucial before downstream applications:

• RNA Binding Assays:

  • Electrophoretic mobility shift assays (EMSA) with labeled rRNA fragments

  • Filter binding assays to quantify RNA affinity

  • Fluorescence anisotropy to measure binding kinetics

• Ribosome Incorporation:

  • In vitro reconstitution assays with L. johnsonii ribosomal components

  • Complementation of L29-depleted ribosomes to restore translation activity

  • Sucrose gradient ultracentrifugation to assess incorporation into ribosomal subunits

• Translation Activity:

  • In vitro translation systems to assess functionality

  • Polysome profiling to evaluate impact on translation

  • Peptidyl transferase activity assays

• Structural Validation:

  • Circular dichroism to confirm secondary structure

  • Limited proteolysis to assess proper folding

  • Thermal shift assays to evaluate stability

For L. johnsonii specifically, given its documented role in modulating immune responses and anticandidal activities , functional validation might extend to testing whether recombinant rpmC affects these properties when added to cellular systems, potentially revealing moonlighting functions beyond its canonical ribosomal role.

How might rpmC research contribute to understanding L. johnsonii's immunomodulatory properties?

Research on L. johnsonii rpmC could provide novel insights into its immunomodulatory mechanisms:

• Potential Moonlighting Functions: Many ribosomal proteins have been found to have secondary functions beyond protein synthesis. Recombinant rpmC could be tested for:

  • Direct interaction with immune cell receptors

  • Modulation of dendritic cell function, which has been observed with L. johnsonii supplementation

  • Influence on T-regulatory cell development

• Role in Metabolite Production: L. johnsonii supplementation has been associated with altered metabolic profiles, including increased docosahexanoic acid (DHA) . Investigating whether rpmC mutations affect these metabolite profiles could reveal linkages between ribosomal function and immunomodulatory metabolite production.

• Translation-Dependent Immunomodulation: L. johnsonii reduces Th2 cytokines (IL-4, IL-5, IL-13) during respiratory infections . Studying how rpmC variants affect translation of specific mRNAs encoding immunomodulatory factors could provide mechanistic insights.

The research by Cufney et al. on characterization and interpretation methods may provide analytical frameworks applicable to interpreting complex datasets from immunomodulation studies involving rpmC variants.

What potential biotechnological applications exist for recombinant L. johnsonii rpmC?

Several promising biotechnological applications emerge from L. johnsonii rpmC research:

• Antimicrobial Development: Given L. johnsonii's documented anticandidal properties , engineered rpmC variants could potentially enhance:

  • Production of antifungal compounds like bacteriocins

  • Stress resistance to improve probiotic viability

  • Targeted protein synthesis regulation

• Biomarkers and Diagnostics:

  • Antibodies against L. johnsonii-specific rpmC epitopes could serve as strain-specific detection tools

  • rpmC sequence variations could provide taxonomic markers for Lactobacillus strain identification

• Vaccine Adjuvants:

  • Ribosomal proteins from probiotic bacteria have shown immunostimulatory properties

  • L. johnsonii rpmC could be investigated as a potential adjuvant, particularly given the strain's documented immunomodulatory effects

• Synthetic Biology Platforms:

  • Engineering ribosomes with modified L29 proteins could create specialized translation systems in L. johnsonii

  • Such systems could enhance production of therapeutic proteins or antimicrobial compounds

Research has shown that L. johnsonii supplementation affects dendritic cell function and T-regulatory cells , suggesting complex interactions with host immunity that could be leveraged through engineered ribosomal components.