Recombinant Lactococcus lactis subsp. cremoris Queuine tRNA-ribosyltransferase (tgt)

What is Queuine tRNA-ribosyltransferase (tgt) and what is its basic function in Lactococcus lactis?

Gene expression analysis shows that tgt is significantly upregulated (expression ratio of 1.68, Bayesian p-value of 1.6×10^-3) when L. lactis is induced to overproduce membrane proteins, suggesting its potential role in cellular adaptation to protein production stress .

How is the genomic organization of tgt characterized in L. lactis subsp. cremoris?

The tgt gene (llmg_0164) in L. lactis subsp. cremoris is genomically positioned adjacent to genes encoding predicted integral membrane proteins. Specifically, llmg_0165, which encodes a predicted integral membrane protein, shows an expression ratio of 1.96 (Bayesian p-value of 3.7×10^-4) in response to membrane protein overproduction, suggesting potential co-regulation with tgt .

The genomic context of tgt is particularly important as it appears to be part of a gene cluster involved in cellular responses to membrane protein production stress. Complete genome sequencing of various L. lactis strains, including subsp. cremoris strains like SK11 and MG1363, has provided detailed information about the genetic organization surrounding the tgt gene .

What role does tgt play in the stress response of L. lactis?

Transcriptome analysis reveals that tgt is part of the cellular response when L. lactis is subjected to the stress of membrane protein overproduction. When L. lactis is induced to overproduce membrane proteins like BcaP (a branched-chain amino acid permease), tgt shows significant upregulation, suggesting its involvement in helping the cell cope with this stress .

The data indicates that tgt may be part of a broader response system that includes other upregulated genes related to protein translocation (secE), ribosomal function (rpmGC), and membrane integrity. This coordinated response appears to be critical for maintaining cellular viability under conditions of recombinant protein production stress .

What are the optimal conditions for recombinant expression of tgt in L. lactis subsp. cremoris?

For recombinant expression of tgt in L. lactis, the nisin-inducible expression system (NICE) is frequently employed due to its tight regulation and dose-dependent induction capabilities. Based on protocols established for other recombinant proteins in L. lactis, the following conditions are recommended:

• Growth medium: M17 supplemented with 0.5% glucose (GM17)

• Growth temperature: 30°C without aeration

• Induction: 5 ng/ml of nisin A at mid-exponential phase (OD600 of 0.5)

• Expression time: 1-3 hours post-induction

These parameters are derived from successful expression protocols used for membrane proteins like BcaP in L. lactis, which demonstrated significant tgt upregulation . When designing expression constructs, including a C-terminal tag (such as hexa-histidine) is advisable for purification and detection purposes.

How does co-expression of tgt with membrane proteins affect protein production yields?

Transcriptome analysis has revealed an interesting correlation between tgt expression and membrane protein production in L. lactis. The tgt gene shows significant upregulation (expression ratio of 1.68) during membrane protein overproduction stress . This suggests that tgt may play a role in the cellular adaptation to this stress.

Based on this correlation, a potential strategy would be to co-express tgt with the target membrane protein. This approach may help mitigate the stress response and potentially improve production yields of difficult-to-express membrane proteins. Similar approaches have been successfully employed with other stress-response genes in L. lactis, such as the CesSR two-component system, which improved production yields of membrane proteins when overexpressed .

What vector systems are most suitable for recombinant tgt expression in L. lactis?

For recombinant expression of tgt in L. lactis, several vector systems have proven effective:

The pNZ8048-based vectors, which utilize the nisin-inducible promoter system, are particularly well-suited for tgt expression as they allow precise control over expression levels . This system has been successfully used for the expression of membrane proteins in L. lactis, with tgt upregulation observed as part of the cellular response.

For complementation studies or when stable expression is required, pNZ44 with its constitutive promoter may be advantageous, as demonstrated in studies with other bacterial species like S. thermophilus .

What purification strategies yield the highest purity and activity for recombinant tgt from L. lactis?

For optimal purification of recombinant tgt from L. lactis, a multi-step approach is recommended:

• Cell lysis optimization: Since L. lactis has a thick peptidoglycan layer, efficient lysis requires a combination of lysozyme treatment (10 mg/ml) and mechanical disruption.

• Initial capture: If the recombinant tgt includes a hexa-histidine tag, immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins serves as an effective initial purification step.

• Secondary purification: Ion exchange chromatography (typically Q-Sepharose) can further separate tgt from contaminating proteins.

• Polishing step: Size exclusion chromatography provides final purification and allows buffer exchange into an optimal storage buffer (typically 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM β-mercaptoethanol, 10% glycerol).

This protocol is adapted from successful purification strategies for recombinant proteins from L. lactis and should maintain enzymatic activity while achieving high purity.

How can researchers accurately measure tgt enzymatic activity in vitro?

The enzymatic activity of purified recombinant tgt can be assessed using the following methodological approach:

• Substrate preparation: Purify tRNAAsn, tRNAAsp, tRNAHis, or tRNATyr from either in vitro transcription or cellular extraction.

• Guanine exchange assay: Measure the exchange of radiolabeled guanine ([14C]guanine or [3H]guanine) with the guanine at the wobble position of the substrate tRNA.

• HPLC-based assay: An alternative non-radioactive method involves using HPLC to detect the replacement of guanine with queuine at the wobble position.

Activity can be expressed as pmol of guanine incorporated per minute per mg of enzyme under standardized conditions (typically 37°C, pH 7.5, in the presence of magnesium ions). For accurate measurements, it's essential to establish the linear range of the assay and include appropriate controls.

What techniques are most effective for studying the relationship between tgt and membrane protein biogenesis?

To investigate the relationship between tgt and membrane protein biogenesis in L. lactis, the following experimental approaches are recommended:

• Transcriptome analysis: Compare gene expression profiles in wild-type vs. tgt knockout/overexpression strains during membrane protein production. Previous studies have shown significant changes in expression of membrane protein-related genes, including tgt itself, during membrane protein overproduction stress .

• Fluorescence microscopy: Express membrane proteins fused to GFP to visualize proper folding and localization. This approach was successfully used with BcaP-GFP-H6 to monitor correct protein folding .

• Growth phenotype analysis: Monitor growth rates of tgt-manipulated strains during membrane protein overproduction. Significant growth defects can indicate stress responses related to protein production challenges .

• Membrane fraction analysis: Isolate membrane fractions and quantify target protein incorporation using Western blotting.

• Proteomics analysis: Use quantitative proteomics to identify changes in membrane protein composition when tgt expression is altered.

These approaches, combined with genetic manipulation of tgt expression levels, can provide comprehensive insights into the role of tgt in membrane protein biogenesis.

What are the most efficient methods for generating tgt knockout mutants in L. lactis?

For generating tgt knockout mutants in L. lactis, several approaches have proven effective:

• Homologous recombination with temperature-sensitive plasmids:

  • Clone homologous regions flanking the tgt gene into a temperature-sensitive plasmid (like pG+host)

  • Transform into L. lactis and select for integration at non-permissive temperature

  • Return to permissive temperature to force plasmid excision, screening for mutants that have lost the tgt gene

• Double-crossover replacement:

  • Replace tgt with an antibiotic resistance marker through double homologous recombination

  • This method yields stable deletion mutants useful for long-term studies

• CRISPR-Cas9 based deletion:

  • Design guide RNAs targeting the tgt gene

  • Provide a repair template containing the desired deletion

  • Select transformants and verify deletion by PCR and sequencing

When creating tgt knockouts, it's important to verify that any observed phenotypes are directly attributable to the loss of tgt function. This can be confirmed through complementation studies, reintroducing the tgt gene on a plasmid like pNZ8048 under nisin-inducible control .

How can researchers design effective complementation experiments for tgt mutants?

Effective complementation experiments for tgt mutants should follow these methodological principles:

• Expression vector selection: For L. lactis, the nisin-inducible pNZ8048 vector system is recommended due to its tight regulation and wide expression range . For constitutive expression, pNZ44 can be used as demonstrated in similar complementation studies .

• Construct design considerations:

  • Include the native tgt gene with its ribosome binding site

  • Consider adding a C-terminal tag (e.g., hexa-histidine) for detection without interfering with function

  • For controlled expression studies, use the nisin-inducible promoter with varying inducer concentrations

• Transformation protocol optimization:

  • For L. lactis, electroporation in the presence of glycine (0.25%) improves transformation efficiency

  • Use growth media supplemented with appropriate selection agents (e.g., chloramphenicol at 5 μg/ml)

  • Incubate transformants at optimal temperature (30°C for L. lactis)

• Phenotypic validation:

  • Compare growth curves of wild-type, mutant, and complemented strains

  • Assess membrane protein production capacity

  • Measure tgt enzymatic activity in cellular extracts

Success in complementation is indicated by restoration of wild-type phenotypes, including growth rate, protein production capacity, and specific enzymatic activities .

What site-directed mutagenesis strategies can elucidate critical residues in tgt function?

To identify critical functional residues in L. lactis tgt, the following site-directed mutagenesis strategy is recommended:

• Residue identification:

  • Use multiple sequence alignment with characterized tgt enzymes from other organisms

  • Apply structural prediction software to identify potential catalytic and substrate-binding residues

  • Focus on conserved motifs in the tgt protein family

• Mutagenesis protocol:

  • Employ PCR-based site-directed mutagenesis using the QuikChange method or overlap extension PCR

  • Design primers with the desired mutations flanked by 15-20 nucleotides of homologous sequence

  • Verify mutations by sequencing before functional testing

• Functional characterization of mutants:

  • Express and purify mutant proteins using the same methods as for wild-type

  • Assess enzymatic activity using established assays

  • Evaluate protein stability through thermal shift assays

  • Determine substrate binding affinity through isothermal titration calorimetry or similar methods

• Data analysis approach:

  • Compare kinetic parameters (Km, kcat, kcat/Km) between wild-type and mutant enzymes

  • Analyze the correlation between structural predictions and functional outcomes

  • Create structure-function relationship models based on mutagenesis results

This systematic approach allows for comprehensive mapping of functional domains within the tgt enzyme and provides insights into mechanisms of action.

How does tgt expression interact with the CesSR stress response system in L. lactis?

The interaction between tgt expression and the CesSR stress response system in L. lactis represents an important area for systems biology investigation. Transcriptome analysis has revealed that tgt (llmg_0164) is upregulated (expression ratio of 1.68) during membrane protein overproduction stress, which also activates the CesSR two-component system .

The CesSR system senses cell envelope stresses and regulates genes involved in maintaining membrane integrity. Key genes in the CesSR regulon include ftsH, oxaA2, llmg_2163, and rmaB, which are critical for membrane protein production in L. lactis . The upregulation of tgt alongside these genes suggests a potential functional relationship.

Experimental approaches to investigate this interaction should include:

• Analyzing tgt expression in CesSR knockout strains

• Determining if CesSR directly regulates tgt expression through DNA-binding studies

• Assessing whether tgt overexpression can compensate for CesSR deficiency during membrane protein production stress

Understanding this interaction could provide valuable insights for engineering improved L. lactis strains for recombinant protein production.

What omics approaches are most informative for studying the role of tgt in global cellular metabolism?

To comprehensively understand the role of tgt in global cellular metabolism, an integrated multi-omics approach is recommended:

• Transcriptomics: Full-genome DNA microarrays or RNA-seq to compare wild-type and tgt-manipulated strains under various conditions (e.g., normal growth, membrane protein overproduction). Previous studies have successfully used microarray analysis to identify expression changes in response to membrane protein overproduction in L. lactis .

• Proteomics: Quantitative proteomics using techniques such as iTRAQ or SILAC to detect changes in protein abundance in response to tgt manipulation.

• Metabolomics: Targeted and untargeted metabolomic approaches to identify metabolic shifts associated with altered tgt expression.

• Translatomics: Ribosome profiling to assess the impact of tgt-mediated tRNA modifications on translation efficiency and accuracy.

Integration of these datasets using computational approaches allows for:

• Identification of metabolic pathways affected by tgt activity

• Mapping of regulatory networks connecting tgt to cellular responses

• Development of predictive models for optimizing recombinant protein production

This systems biology approach provides a comprehensive view of tgt's role beyond its direct enzymatic function.

How can tgt expression data be integrated into metabolic models of L. lactis?

Integrating tgt expression data into metabolic models of L. lactis requires a systematic approach:

• Model foundation: Begin with existing genome-scale metabolic models (GSMMs) of L. lactis, such as those derived from the sequenced genomes of L. lactis subsp. cremoris MG1363 or SK11 .

• Data integration protocol:

  • Map transcriptomic data of tgt and related genes to reactions in the metabolic model

  • Use algorithms like GIMME (Gene Inactivity Moderated by Metabolism and Expression) or E-Flux to constrain flux bounds based on expression levels

  • Incorporate regulatory constraints based on known or predicted interactions

• Model validation experiments:

  • Measure specific growth rates and substrate uptake rates in wild-type vs. tgt-manipulated strains

  • Quantify production of key metabolites

  • Compare predicted vs. observed phenotypes to refine model parameters

• Prediction and experimental design:

  • Use the refined model to predict optimal conditions for recombinant protein production

  • Design validation experiments to test model predictions

  • Iterate between prediction, experimentation, and model refinement

This approach enables the development of increasingly accurate predictive models that can guide strain engineering efforts for improved protein production.

How can manipulation of tgt expression improve recombinant protein yields in L. lactis?

Based on the observation that tgt is upregulated during membrane protein overproduction , strategic manipulation of tgt expression may improve recombinant protein yields in L. lactis through the following approaches:

• Co-expression strategy: Express tgt alongside the target recombinant protein, particularly for difficult-to-express membrane proteins. Similar approaches with the CesSR system have shown success, improving the production yield of the presenilin variant PS1Δ9-H6 more than 4-fold .

• Promoter engineering: Design expression constructs with calibrated promoter strengths for both tgt and the target protein to maintain optimal expression ratios.

• Induction timing optimization: Induce tgt expression prior to target protein induction to prepare the cellular machinery for the upcoming production stress.

• Strain engineering: Develop L. lactis strains with genomically integrated, moderately upregulated tgt expression as specialized production hosts.

These strategies should be evaluated through systematic testing, measuring protein yield, quality, and cellular stress responses to determine the most effective approach for specific recombinant proteins.

What is the impact of tgt on the quality and folding of recombinant membrane proteins?

The impact of tgt on recombinant membrane protein quality and folding can be evaluated through several experimental approaches:

• Folding assessment: Utilize fluorescence-based reporters, such as GFP fusion proteins, to monitor proper folding. Correctly folded BcaP-GFP-H6 fusion proteins display fluorescence, serving as an indicator of proper folding .

• Functional assays: Assess the functionality of produced membrane proteins through specific activity assays. For example, BcaP functionality was confirmed by its ability to restore growth of L. lactis mutants lacking essential branched-chain amino acid transporters .

• Membrane integration analysis: Fractionate cells and analyze the distribution of target proteins between membrane and cytoplasmic fractions using Western blotting.

• Structural integrity evaluation: Use circular dichroism or limited proteolysis to assess the structural integrity of purified membrane proteins.

Current evidence suggests that tgt upregulation occurs as part of a cellular response to membrane protein overproduction stress . This correlation indicates that tgt may play a role in maintaining translational fidelity during stress conditions, potentially improving the quality of synthesized proteins by ensuring accurate tRNA modification and codon reading.

How does tgt function compare between L. lactis and other expression hosts used for recombinant protein production?

Comparing tgt function across different expression hosts reveals important considerations for recombinant protein production:

L. lactis offers distinct advantages as an expression host, particularly for membrane proteins. Its tgt upregulation during membrane protein overproduction stress suggests an adaptive response that may facilitate protein production . This response appears to be coordinated with other cellular systems, including the CesSR two-component system and various protein translocation components like SecE and OxaA2 .

The connection between tgt and successful membrane protein production in L. lactis contrasts with E. coli, where different stress response systems predominate. Understanding these host-specific differences allows researchers to select the most appropriate expression system for specific recombinant proteins and to develop optimized expression strategies.