Recombinant Lactobacillus johnsonii UPF0042 nucleotide-binding protein LJ_0866 (LJ_0866)

What methods are most effective for recombinant expression of LJ_0866?

Effective recombinant expression of LJ_0866 protein requires careful consideration of expression systems, purification strategies, and validation methods. Based on approaches used for similar bacterial proteins, the following methodology is recommended:

Expression System Selection:

• E. coli-based systems: BL21(DE3) or Rosetta strains are suitable for initial attempts, especially with codon optimization for E. coli

• Lactobacillus-based expression: For native-like folding, expression in a Lactobacillus host may be preferable, though yields may be lower

• Baculovirus expression: For larger-scale production if bacterial systems prove challenging

Expression Optimization:

• Temperature modulation: Lower temperatures (16-25°C) often improve folding of nucleotide-binding proteins

• Induction conditions: Test various IPTG concentrations (0.1-1.0 mM) and induction times

• Co-expression with chaperones: GroEL/GroES or DnaK/DnaJ/GrpE can improve solubility

Construct Design:

• Affinity tags: N-terminal or C-terminal His₆-tag for purification, with a TEV protease cleavage site

• Solubility enhancers: Consider MBP, SUMO, or GST fusion if solubility is problematic

• Domain identification: Express full-length protein and individual domains separately

The recombinant expression approach should be coupled with appropriate purification strategies, including immobilized metal affinity chromatography followed by size exclusion chromatography. Validation of proper folding can be assessed through nucleotide binding assays and circular dichroism spectroscopy.

How does LJ_0866 potentially relate to Lactobacillus johnsonii's adaptation to different environments?

Lactobacillus johnsonii demonstrates remarkable adaptability to different environmental conditions, including switching from anaerobic to aerobic metabolism. As a nucleotide-binding protein, LJ_0866 may play a significant role in these adaptation processes through several potential mechanisms:

• Metabolic adaptation: L. johnsonii possesses genes for aerobic metabolism, including pyruvate oxidase (POX) and acetate kinase (ACK), which allow for extra ATP generation under aerobic conditions . LJ_0866 may function in regulating these metabolic shifts by sensing nucleotide levels (ATP/ADP ratios) that reflect cellular energy status.

• Oxidative stress response: When L. johnsonii is exposed to oxygen, it must manage oxidative stress. Some strains show catalase-like activity and increased resistance to H₂O₂ under respiratory conditions . LJ_0866 could potentially be involved in sensing oxidative stress or in regulating expression of stress response genes.

• Environmental sensing: As a nucleotide-binding protein, LJ_0866 might function as part of a signal transduction pathway that detects environmental changes and triggers appropriate responses. This could include adaptation to the varying oxygen levels encountered in different regions of the gastrointestinal tract.

• Host interaction: L. johnsonii produces bioactive components, including phospholipids that can modulate dendritic cell responses . If LJ_0866 participates in lipid metabolism regulation, it might indirectly influence these host-microbe interactions.

To investigate these potential roles, researchers could employ gene knockout experiments and observe phenotypic changes under different environmental conditions, complemented by transcriptomic and metabolomic analyses.

How is LJ_0866 potentially involved in oxidative stress response mechanisms?

The involvement of LJ_0866 in oxidative stress response mechanisms represents an important area of investigation, particularly given L. johnsonii's ability to adapt to aerobic environments despite being facultatively anaerobic. From the available research, we know that:

• L. johnsonii possesses genes for oxidative stress response, including NADH oxidase (NOX) and NADH peroxidase (NPR), which are involved in H₂O₂ scavenging .

• Some L. johnsonii strains demonstrate catalase-like activity and increased resistance to H₂O₂ under respiratory conditions .

As a nucleotide-binding protein, LJ_0866 could participate in oxidative stress responses through several mechanisms:

• Energy metabolism regulation: Oxidative stress affects cellular ATP levels, which LJ_0866 might sense and respond to by modulating metabolic pathways.

• Signal transduction: LJ_0866 could function within a signaling cascade that responds to oxidative stress by altering gene expression patterns.

• Direct ROS interaction: The protein might directly interact with reactive oxygen species or with molecules modified by oxidative stress.

To investigate these possibilities, researchers should consider employing the following methodology:

• Generate LJ_0866 knockout strains and assess sensitivity to various oxidative stressors

• Measure expression levels of LJ_0866 under different oxidative conditions

• Conduct pull-down assays to identify interaction partners under normal and oxidative stress conditions

• Perform site-directed mutagenesis of key residues to determine functional domains involved in stress response

Understanding LJ_0866's role in oxidative stress responses could provide valuable insights into how L. johnsonii adapts to the oxygen gradients found within the gastrointestinal tract.

What role might LJ_0866 play in the immunomodulatory properties of Lactobacillus johnsonii?

The potential role of LJ_0866 in the immunomodulatory properties of Lactobacillus johnsonii represents a sophisticated research question that integrates microbiology, immunology, and molecular biology. From the search results, we know that L. johnsonii N6.2 phospholipids can induce an immature-like phenotype in dendritic cells with a migratory-regulatory-like transcriptional signature . While LJ_0866's specific involvement has not been directly established, its nucleotide-binding properties suggest potential regulatory functions.

Methodological approach to investigate this question:

• Genetic manipulation studies:

  • Generate LJ_0866 knockout and overexpression L. johnsonii strains

  • Compare phospholipid profiles between wild-type and mutant strains using LC-MS/MS

  • Fractionate lipids (as described in the research) into total lipids (TLs), phospholipids (PLs), and glycolipids (GLs)

• Dendritic cell stimulation experiments:

  • Isolate bone marrow-derived dendritic cells (BMDCs) from mice

  • Stimulate BMDCs with lipid fractions from wild-type and LJ_0866 mutant strains

  • Assess phenotypic changes including surface markers (ICAM-1, MHC-II, CD86, CD40, CD1d)

  • Measure cytokine production (IL-6, IL-10, TNF-α) using ELISA or multiplex assays

• Transcriptional analysis:

  • Perform RNA-Seq on BMDCs treated with lipids from wild-type vs. LJ_0866 mutant strains

  • Focus on genes identified in previous research: Cd86, Cd40, Ccr7, Icam1, Itgb8, Nfkbiz, Jag1, Adora2a, IL2ra, Arg1, and Cd274

  • Validate key findings using RT-qPCR

• Mechanistic studies:

  • Investigate TLR2 involvement using blocking antibodies (as the research showed TLR2 blocking affected IL-6 regulation)

  • Assess non-canonical NF-κB pathway through IKKα inhibition experiments

  • Determine if LJ_0866 directly interacts with or modifies phospholipids

This comprehensive approach would help determine whether LJ_0866 influences the immunomodulatory properties of L. johnsonii through effects on phospholipid metabolism or composition, ultimately affecting how these bacterial components interact with host immune cells.

How might mutations in LJ_0866 affect the aerobic metabolism of Lactobacillus johnsonii?

Investigating how mutations in LJ_0866 affect L. johnsonii's aerobic metabolism requires a systematic approach combining genetic manipulation, physiological characterization, and biochemical analysis. L. johnsonii can adapt to aerobic environments despite being facultatively anaerobic, with specific pathways for oxygen utilization and management .

Comprehensive methodological framework:

• Mutation design and generation:

  • Create point mutations in predicted nucleotide-binding motifs (Walker A/B)

  • Generate complete gene deletion mutants (ΔLJ_0866)

  • Develop complemented strains to confirm phenotypes

  • Use site-directed mutagenesis to target specific functional residues

• Physiological characterization:

  • Compare growth curves under anaerobic, microaerobic, and aerobic conditions

  • Measure oxygen consumption using Clark-type electrodes

  • Determine survival rates under oxidative stress conditions

  • Assess colony morphology and cell ultrastructure using electron microscopy

• Metabolic analysis:

  • Quantify end-product formation (lactate, acetate, CO₂) using HPLC and GC-MS

  • Measure intracellular ATP levels and NAD⁺/NADH ratios

  • Perform metabolic flux analysis using ¹³C-labeled substrates

  • Analyze activity of key aerobic metabolism enzymes including:

    • Pyruvate oxidase (POX)

    • Acetate kinase (ACK)

    • NADH oxidase (NOX)

    • Lactate oxidase (LOX)

• Transcriptomic and proteomic profiling:

  • Conduct RNA-Seq comparing wild-type and mutant strains under different oxygen conditions

  • Perform quantitative proteomics to identify changes in protein abundance

  • Focus on genes involved in oxygen utilization and stress response

This approach would provide comprehensive insights into whether LJ_0866 influences L. johnsonii's aerobic metabolism through direct involvement in metabolic pathways, regulation of enzyme activities, or participation in stress response mechanisms.

What are the challenges in experimental design when studying protein-protein interactions of LJ_0866?

Studying protein-protein interactions of LJ_0866 presents several experimental challenges that require careful consideration and methodological rigor. These challenges span from protein production to interaction detection and validation within physiologically relevant contexts.

Key experimental challenges and methodological solutions:

• Protein expression and purification challenges:

  • Challenge: Nucleotide-binding proteins often have solubility issues and may form inclusion bodies

  • Solution: Optimize expression conditions (temperature, inducer concentration, media composition); test multiple solubility tags (MBP, SUMO, GST); consider cell-free expression systems

  • Challenge: Ensuring proper folding and nucleotide-binding activity

  • Solution: Validate function through nucleotide binding assays; use circular dichroism to confirm secondary structure; employ thermal shift assays to assess stability

• Interaction detection limitations:

  • Challenge: Transient or weak interactions may be missed by conventional methods

  • Solution: Use chemical crosslinking prior to pull-down experiments; employ more sensitive techniques like surface plasmon resonance or microscale thermophoresis; consider proximity labeling approaches (BioID, APEX)

  • Challenge: False positives in affinity purification approaches

  • Solution: Implement stringent washing conditions; use quantitative proteomics with SILAC or TMT labeling; perform reciprocal pull-downs with prey proteins

• Context-dependent interaction considerations:

  • Challenge: LJ_0866 interactions may depend on nucleotide-bound state

  • Solution: Test multiple conditions (apo-protein, ATP-bound, ADP-bound, non-hydrolyzable analogs)

  • Challenge: Interactions may be influenced by redox state, particularly given L. johnsonii's variable responses to oxygen

  • Solution: Control redox environment during experiments; compare interactions under reducing and oxidizing conditions

• Validation in native context:

  • Challenge: Limited genetic tools for L. johnsonii compared to model organisms

  • Solution: Consider heterologous expression in related organisms with better genetic tractability; develop improved genetic manipulation systems for L. johnsonii

  • Challenge: Correlating in vitro interactions with physiological relevance

  • Solution: Perform co-localization studies; use bacterial two-hybrid systems; assess phenotypic consequences of disrupting interactions

• Specific technical considerations for bacterial proteins:

  • Challenge: Membrane association may complicate purification and interaction studies

  • Solution: Test different detergents for extraction; consider membrane mimetics (nanodiscs, liposomes)

  • Challenge: Post-translational modifications may affect interactions

  • Solution: Use mass spectrometry to identify modifications; compare protein produced in different expression systems

A comprehensive approach would combine multiple complementary techniques, each addressing different limitations, to build a convincing interaction network for LJ_0866 and understand its functional role in L. johnsonii physiology.

What techniques can be used to investigate the potential role of LJ_0866 in phospholipid metabolism?

Investigating LJ_0866's potential role in phospholipid metabolism requires a multi-faceted approach combining genetic, biochemical, and analytical techniques. This question is particularly relevant given that L. johnsonii N6.2 phospholipids have been shown to induce immature-like dendritic cells with immunomodulatory properties .

Comprehensive methodological framework:

• Genetic manipulation and phenotypic analysis:

  • Generate LJ_0866 knockout, knockdown, and overexpression strains

  • Compare growth phenotypes under various conditions

  • Assess membrane integrity using fluorescent dyes and electron microscopy

  • Test sensitivity to membrane-disrupting agents

• Comprehensive phospholipid profiling:

  • Extraction protocols: Use optimized Bligh-Dyer method for bacterial phospholipids

  • Analytical techniques:

    • Thin-layer chromatography (TLC) for initial phospholipid class separation

    • Liquid chromatography-mass spectrometry (LC-MS/MS) for detailed profiling

    • ³¹P NMR spectroscopy for phospholipid headgroup analysis

  • Compare profiles between wild-type and LJ_0866 mutant strains

• Metabolic labeling experiments:

  • Perform pulse-chase experiments with ³²P or ¹⁴C-acetate to track phospholipid synthesis

  • Measure phospholipid turnover rates in wild-type versus mutant strains

  • Quantify incorporation of labeled precursors into specific phospholipid classes

• Protein-lipid interaction studies:

  • Direct binding assays:

    • Lipid overlay assays with purified LJ_0866

    • Liposome flotation assays with defined phospholipid compositions

    • Surface plasmon resonance for binding kinetics

  • Structural approaches:

    • Crystallize LJ_0866 with bound lipids

    • Use hydrogen-deuterium exchange mass spectrometry to identify lipid-binding regions

• Enzymatic activity characterization:

  • Test for phospholipase activity (A₁, A₂, C, D)

  • Assess acyltransferase or flippase activities

  • Develop high-throughput fluorescent assays for activity screening

• Immunomodulatory property assessment:

  • Isolate phospholipids from wild-type and LJ_0866 mutant strains

  • Compare effects on dendritic cell phenotype (surface markers, cytokine production)

  • Assess specific phospholipid fractions for bioactivity

  • Correlate with RNA-Seq data on dendritic cell responses

This comprehensive approach would provide mechanistic insights into whether and how LJ_0866 contributes to phospholipid metabolism in L. johnsonii, potentially uncovering molecular mechanisms behind the bacterium's immunomodulatory properties.

How does the function of LJ_0866 potentially differ when L. johnsonii shifts between fermentative and respiratory metabolism?

The potential functional shift of LJ_0866 during L. johnsonii's transition between fermentative and respiratory metabolism represents an intriguing research question that connects bacterial physiology, adaptation, and regulatory mechanisms. From the search results, we know that L. johnsonii can adapt to aerobic environments and some strains can shift toward respiratory metabolism when heme and menaquinone are supplied .

Methodological approach to investigate this question:

• Comparative expression analysis:

  • Culture L. johnsonii under three conditions:

    • Anaerobic (fermentative)

    • Aerobic (aerobic metabolism)

    • Respiratory (aerobic + heme + menaquinone)

  • Quantify LJ_0866 expression using RT-qPCR and Western blotting

  • Perform temporal analysis during metabolic shifts

  • Use reporter gene fusions (e.g., LJ_0866 promoter-GFP) to visualize expression patterns

• Protein localization and modification studies:

  • Use fluorescent protein fusions or immunofluorescence to track LJ_0866 localization

  • Employ mass spectrometry to identify post-translational modifications

  • Compare modifications across metabolic states

  • Assess protein turnover rates using pulse-chase experiments

• Interaction partner identification:

  • Perform immunoprecipitation coupled with mass spectrometry (IP-MS)

  • Compare interaction partners across metabolic states

  • Validate key interactions using bacterial two-hybrid assays

  • Focus on potential interactions with proteins involved in:

    • Electron transport chain components (NADH dehydrogenase, cytochrome bd oxidase)

    • Oxygen utilization enzymes (POX, ACK, LOX, NOX)

    • Stress response proteins

• Nucleotide binding characterization:

  • Measure nucleotide binding affinities using isothermal titration calorimetry

  • Compare ATP/GTP binding and hydrolysis rates across metabolic states

  • Determine if oxygen or redox state affects nucleotide binding

• Phenotypic analysis of mutants under different metabolic conditions:

  • Generate LJ_0866 knockout and point mutants

  • Compare growth, survival, and metabolic end-products across metabolic states

  • Assess respiratory capacity through oxygen consumption measurements

  • Measure membrane potential and proton motive force

This multifaceted approach would provide mechanistic insights into how LJ_0866 potentially adapts its function during L. johnsonii's transition between metabolic states, potentially revealing novel regulatory mechanisms for bacterial adaptation to changing environmental conditions.