Recombinant Lactobacillus johnsonii Proline--tRNA ligase (proS), partial

What is the functional role of Proline-tRNA ligase (proS) in Lactobacillus johnsonii?

Proline-tRNA ligase (proS) in L. johnsonii is a critical enzyme involved in translation processes. It specifically catalyzes the attachment of proline to its cognate tRNA molecule (tRNA^Pro), which is essential for protein synthesis. According to proteomics data, proS functions in "Translation; tRNA aminoacylation for protein translation; prolyl tRNA aminoacylation; aminoacyl-tRNA metabolism involved in translational fidelity" . This enzyme plays a particularly significant role in L. johnsonii's physiology because genome analysis has revealed that this bacterium appears incapable of de novo synthesis of most amino acids, making efficient tRNA charging mechanisms crucial for protein synthesis .

How is proS structurally and functionally conserved across different L. johnsonii strains?

The proS gene appears to be highly conserved across L. johnsonii strains isolated from different environments, similar to other essential housekeeping genes. While the search results don't specifically address sequence conservation of proS, we can infer its importance from comparative genomic studies of L. johnsonii strains. The enzyme likely maintains high sequence conservation due to its essential role in translation, though strain-specific variations might exist that could affect enzyme efficiency or regulation.

What are the key structural domains of L. johnsonii Proline-tRNA ligase and how do they contribute to its function?

While the search results don't provide specific structural information about L. johnsonii proS, aminoacyl-tRNA synthetases typically contain several functional domains:

• A catalytic domain with conserved motifs for ATP binding and amino acid activation

• An anticodon-binding domain that recognizes the specific tRNA

• Editing domains in some synthetases that ensure translational fidelity

In L. johnsonii, the proS enzyme likely follows this general architecture, with specific adaptations that may relate to its function in the metabolic context of this organism that depends heavily on amino acid transport rather than synthesis .

What expression systems are most effective for producing recombinant L. johnsonii Proline-tRNA ligase?

The optimal expression system for recombinant L. johnsonii proS should consider several factors:

For functional studies requiring properly folded protein, expression in Gram-positive hosts may be advantageous. When using E. coli systems, researchers should consider that L. johnsonii has a relatively low G+C content genome (34.6%) , which may necessitate codon optimization for efficient expression.

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

A multi-step purification approach is typically necessary to obtain pure, active proS enzyme:

• Initial capture using affinity chromatography (if a tag is incorporated)

• Ion-exchange chromatography to separate based on charge properties

• Size-exclusion chromatography for final polishing and buffer exchange

Critical considerations include maintaining appropriate cofactors (such as Mg²⁺) throughout purification and preventing enzymatic inactivation. Purification buffers should be optimized to maintain enzyme stability, typically including components that mimic the physiological environment of L. johnsonii.

How can researchers accurately assess the enzymatic activity of purified recombinant L. johnsonii proS?

Several complementary approaches can be used to assess proS activity:

• Aminoacylation assay: Measuring the formation of prolyl-tRNA using radioisotope-labeled proline or indirect detection methods

• ATP-PPi exchange assay: Quantifying the first step of the aminoacylation reaction

• Thermal shift assays: Assessing protein stability and cofactor binding

• Kinetic characterization: Determining Km and kcat values for substrates (proline, ATP, and tRNA)

For comprehensive characterization, researchers should determine the enzyme's optimal reaction conditions including pH, temperature, and ionic requirements, which likely reflect L. johnsonii's natural intestinal environment .

How does the genomic context of proS in L. johnsonii provide insights into its regulation and potential interactions?

Analysis of the genomic neighborhood surrounding the proS gene can reveal potential functional relationships and regulatory mechanisms. In bacteria, genes involved in related processes are often clustered. Examining the genome sequence of L. johnsonii NCC 533 (1.99-Mb) would allow researchers to identify whether proS is part of an operon or positioned near genes involved in translation, amino acid metabolism, or stress response.

Transcriptomic studies under various growth conditions could further elucidate how proS expression is regulated in response to environmental changes or stress conditions relevant to the intestinal environment where L. johnsonii naturally resides.

How might the function of proS in L. johnsonii be affected by the organism's limited biosynthetic capabilities?

L. johnsonii appears incapable of de novo synthesis of most amino acids, which has significant implications for proS function . This metabolic constraint likely means that L. johnsonii has evolved specialized regulatory mechanisms to coordinate amino acid availability with protein synthesis.

Several research questions emerge from this characteristic:

• How does proS activity respond to proline limitation?

• Is there coordination between proS expression and the abundant amino acid transporters found in L. johnsonii?

• Does proS play a role in sensing amino acid availability and triggering appropriate metabolic responses?

These questions could be addressed through transcriptomic and proteomic analyses comparing L. johnsonii grown under different amino acid availability conditions.

What role might post-translational modifications play in regulating L. johnsonii proS activity?

Post-translational modifications (PTMs) of aminoacyl-tRNA synthetases can significantly affect their activity, specificity, and cellular localization. While the search results don't specifically mention PTMs of proS, this represents an important research direction.

Potential PTMs to investigate include:

• Phosphorylation, which might regulate activity in response to cellular energy status

• Acetylation, which could coordinate proS activity with metabolic state

• Proteolytic processing, which might generate alternative forms with distinct functions

Mass spectrometry-based proteomic approaches would be the method of choice for identifying and characterizing potential PTMs of L. johnsonii proS.

How does the proS sequence and structure in L. johnsonii compare to those from related Lactobacillus species and potential pathogens?

Comparative sequence analysis of proS across multiple bacterial species could reveal:

• Conserved catalytic residues essential for function

• Species-specific variations that might confer unique properties

• Potential structural differences that could be exploited for selective targeting

What are the critical parameters for optimizing in vitro aminoacylation assays with recombinant L. johnsonii proS?

Optimizing aminoacylation assays requires careful consideration of several parameters:

Researchers should establish the reaction kinetics and determine whether the assay is affected by product inhibition or substrate depletion. Control reactions lacking essential components (ATP, proline, or tRNA) should be included to verify specificity.

What approaches can be used to investigate potential moonlighting functions of L. johnsonii proS beyond its canonical role in translation?

Aminoacyl-tRNA synthetases in various organisms have been found to perform secondary "moonlighting" functions. To investigate such possibilities for L. johnsonii proS:

• Protein interaction studies using pull-down assays or bacterial two-hybrid systems to identify binding partners

• Phenotypic analysis of proS mutants beyond effects on protein synthesis

• Localization studies to determine if proS distributes to unexpected cellular compartments

• Binding assays with non-canonical substrates or regulatory molecules

These approaches could reveal unexpected roles for proS in L. johnsonii physiology or host interactions, potentially related to its probiotic properties .

How can researchers effectively distinguish between direct and indirect effects when manipulating proS expression in L. johnsonii?

Given the essential nature of proS for protein synthesis, manipulating its expression will likely have pleiotropic effects. Strategies to distinguish direct from indirect effects include:

• Using conditional expression systems rather than complete gene knockouts

• Employing active site mutants that specifically affect catalytic function without disrupting potential protein-protein interactions

• Complementation studies with heterologous tRNA synthetases that provide the essential function but lack species-specific interactions

• Time-course studies to differentiate primary effects from secondary responses

These approaches would be particularly valuable when investigating potential roles of proS in L. johnsonii's probiotic activities such as immunomodulation, pathogen inhibition, and epithelial cell attachment .

How might proS function relate to the ability of L. johnsonii to colonize and persist in the gastrointestinal tract?

L. johnsonii has been extensively studied for its probiotic activities , and proS may play both direct and indirect roles in these functions:

• Adaptation to nutrient availability: The intestinal environment has fluctuating nutrient levels, and efficient proS function might be crucial for rapid adaptation to changing amino acid availability

• Stress response: Intestinal conditions include various stressors (acid, bile, oxidative stress), and proS might participate in stress response mechanisms

• Host interaction: Proline-rich proteins often mediate bacterial adhesion to host tissues, and efficient proline incorporation via proS might be important for producing adhesins

Research approaches to investigate these potential connections could include comparative expression analysis of proS during in vitro growth versus intestinal colonization, and phenotypic characterization of proS mutants in colonization models.

Could variations in proS contribute to strain-specific differences in L. johnsonii probiotic properties?

Different L. johnsonii strains isolated from various hosts and environments show variation in their probiotic properties . While specific information about proS variation is not provided in the search results, it represents an interesting research question. Strain-specific variations in proS sequence, expression, or regulation could potentially influence:

• Growth rates and competitive fitness in the intestinal environment

• Production of proline-rich proteins involved in host interactions

• Stress tolerance and adaptation to intestinal conditions

Comparative genomic and functional analyses across multiple L. johnsonii strains could reveal whether proS variations correlate with differences in probiotic properties.

How does the efficiency of proS function compare between free-living L. johnsonii and bacteria in biofilms or microbial communities?

L. johnsonii exists in complex microbial communities in the intestinal environment, potentially forming biofilms or participating in multi-species aggregates. The function of proS might differ between planktonic cells and those in biofilms:

• Expression levels might change due to altered growth rates in biofilms

• Substrate availability might differ in the structured environment of a biofilm

• Potential moonlighting functions of proS might be more prominent in community settings

Research approaches could include transcriptomic and proteomic comparisons of planktonic versus biofilm growth, and visualization techniques to examine proS localization in different growth modes.