Recombinant Lactobacillus acidophilus Chaperone protein DnaK (dnaK), partial

What is the genomic organization of the dnaK operon in Lactobacillus acidophilus?

The dnaK gene in Lactobacillus acidophilus is organized in an operon structure that includes dnaJ, unlike some other lactobacilli where dnaJ is found elsewhere. Preliminary research indicates that in L. acidophilus CRL 639, the dnaJ gene is encoded in the same operon as dnaK . This genomic arrangement helps explain why dnaK and dnaJ are induced together under acid adaptation conditions, while in other lactobacilli, dnaJ has been found to be constitutively expressed . This organizational difference may contribute to the unique stress response mechanisms in L. acidophilus.

The co-expression pattern of these genes suggests a tightly regulated stress response system that may be advantageous for survival in acidic environments, which is particularly relevant for this organism's probiotic capabilities.

How does acid adaptation affect DnaK expression in L. acidophilus?

Acid adaptation in L. acidophilus induces the expression of both dnaK and dnaJ genes simultaneously . Low pH serves as an important signal for the induction of mechanisms that protect L. acidophilus against several environmental stresses . The acid-induced expression of DnaK contributes to the organism's ability to survive in acidic environments like the gastrointestinal tract.

Research methodologies to study this phenomenon typically involve:

• Exposing L. acidophilus cultures to sublethal acidic conditions (pH 5.0-5.5)

• Monitoring gene expression using qRT-PCR

• Performing Western blot analysis to quantify protein levels

• Comparing acid-adapted cells with non-adapted controls

What functional domains characterize the DnaK protein in L. acidophilus?

Like other HSP70 family members, L. acidophilus DnaK typically contains:

• An N-terminal nucleotide-binding domain (NBD) with ATPase activity

• A substrate-binding domain (SBD) that recognizes hydrophobic peptide segments

• A C-terminal lid domain that regulates substrate binding and release

These domains work together in an ATP-dependent cycle of substrate binding and release. Researchers investigating these domains should consider employing:

• Multiple sequence alignment with characterized HSP70 family proteins

• Homology modeling based on crystallized DnaK structures

• Limited proteolysis experiments to identify domain boundaries

• Mutational analysis of conserved residues in each domain

What experimental approaches can be used to study the chaperone activity of recombinant L. acidophilus DnaK?

To effectively study the chaperone activity of recombinant L. acidophilus DnaK, researchers should consider:

In vitro assays:

• Prevention of aggregation assay: Measure the ability of DnaK to prevent thermal aggregation of model substrates (e.g., luciferase, citrate synthase)

• ATPase activity assay: Monitor ATP hydrolysis rates using colorimetric methods

• Substrate binding assay: Use fluorescently labeled peptides to measure binding affinity

• Protein refolding assay: Assess refolding of denatured proteins in the presence of DnaK

In vivo approaches:

• Complementation studies in DnaK-deficient strains

• Analysis of α-complementation of β-galactosidase, as demonstrated in E. coli DnaK studies

Example protocol for aggregation prevention assay:

• Incubate substrate protein (0.5 μM) at 43°C in buffer containing ATP

• Add various concentrations of purified DnaK (0-5 μM)

• Monitor light scattering at 320 nm over 60 minutes

• Calculate the percentage of aggregation prevention

Note: Values represented are hypothetical examples based on typical chaperone assays

How does temperature affect the function of L. acidophilus DnaK, and what methods can detect these changes?

Temperature significantly impacts DnaK function, as evidenced by studies of DnaK in other bacteria. Drawing from E. coli research, we can infer that L. acidophilus DnaK likely exhibits temperature-dependent functional changes .

Methodological approaches:

• Temperature-dependent ATPase activity assays (25-45°C range)

• Circular dichroism spectroscopy to monitor structural changes

• Differential scanning calorimetry to determine thermal stability

• Binding affinity measurements at various temperatures

• In vivo functional assays at different growth temperatures

Research on E. coli DnaK shows that in the dnaK756(Ts) mutant, α-complementation of β-galactosidase occurs at 30°C but not at higher temperatures (37°C or 40°C), even though these temperatures permit bacterial growth . Similar temperature-sensitive phenotypes could be investigated in L. acidophilus DnaK mutants.

What strategies are effective for optimizing recombinant expression of L. acidophilus DnaK?

Optimizing recombinant expression of L. acidophilus DnaK requires consideration of:

Expression systems:

• Homologous expression in L. acidophilus using inducible promoters

  • Benefits: Proper folding, authentic post-translational modifications

  • Challenges: Lower yield, more complex genetic manipulation

• Heterologous expression in E. coli

  • Benefits: High yield, established protocols

  • Challenges: Potential improper folding, inclusion body formation

Experimental conditions to optimize:

• Temperature during induction (typically lower temperatures improve solubility)

• Induction duration

• Inducer concentration

• Media composition

• Co-expression with co-chaperones (DnaJ, GrpE)

Purification strategy:

• Affinity chromatography using His-tag or other fusion tags

• Ion exchange chromatography

• Size exclusion chromatography for final polishing

Practical methodology:

• Clone the L. acidophilus dnaK gene with appropriate tags

• Transform into expression host

• Screen multiple expression conditions using small-scale cultures

• Analyze protein solubility and activity

• Scale up production using optimized conditions

• Implement multi-step purification protocol

What approaches can identify DnaK-substrate interactions in L. acidophilus?

Identifying DnaK-substrate interactions requires multiple complementary approaches:

In vitro methods:

• Pull-down assays with immobilized DnaK

• Cross-linking followed by mass spectrometry (XL-MS)

• Surface plasmon resonance (SPR) for binding kinetics

• Isothermal titration calorimetry (ITC) for thermodynamic parameters

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS)

In vivo approaches:

• Co-immunoprecipitation followed by mass spectrometry

• Bacterial two-hybrid assays

• In vivo cross-linking and identification of complexes

• Proximity-dependent biotin labeling (BioID)

Data analysis workflow:

• Perform experiments under both normal and stress conditions

• Identify enriched proteins by mass spectrometry

• Validate key interactions with secondary methods

• Categorize substrates by functional classification

• Compare with known DnaK substrates from other organisms

How can targeted mutagenesis be used to study L. acidophilus DnaK functional domains?

Systematic mutagenesis approaches provide valuable insights into DnaK structure-function relationships:

Target selection strategies:

• Conserved residues identified by multiple sequence alignment

• Known functional residues from E. coli DnaK studies

• Predicted active site and substrate-binding residues

• Interface residues for co-chaperone interactions

Recommended mutation types:

• Conservative substitutions (e.g., K→R) to probe electrostatic interactions

• Non-conservative substitutions to disrupt function

• Alanine scanning of binding interfaces

• Domain deletion or swapping to assess domain autonomy

Functional assessment:

• ATPase activity assays

• Substrate binding assays

• In vivo complementation of dnaK-deficient strains

• Co-chaperone interaction studies

• Thermotolerance tests

What role does DnaK play in the probiotic functionality of L. acidophilus?

DnaK likely contributes significantly to L. acidophilus probiotic properties, particularly:

Stress resistance mechanisms:

• Acid tolerance enabling gastric transit

• Bile salt resistance

• Oxidative stress protection

• Temperature fluctuation adaptation

Host interaction aspects:

• Potential immunomodulatory effects

• Cellular adhesion to intestinal epithelium

• Biofilm formation capabilities

• Competition with pathogens

Research approaches to investigate:

• Construction of DnaK-deficient or overexpression strains

• Assessment of survival under simulated gastrointestinal conditions

• Adhesion assays with intestinal cell lines

• Immunomodulation studies with immune cells

• Competition assays with pathogenic bacteria

• In vivo colonization studies in animal models

The genome sequence of L. acidophilus NCFM revealed features that likely contribute to gastric survival and interactions with intestinal mucosa and microbiota , and DnaK may play a role in these functions.

What protocols are optimal for studying DnaK-mediated protein folding in L. acidophilus?

In vitro protein folding assay protocol:

• Denature substrate protein (e.g., luciferase) in 6M guanidinium-HCl

• Dilute denatured protein into refolding buffer containing:

  • Purified L. acidophilus DnaK (0.5-5 μM)

  • DnaJ co-chaperone (0.1-1 μM)

  • GrpE nucleotide exchange factor (0.1-1 μM)

  • ATP regeneration system (phosphoenolpyruvate + pyruvate kinase)

  • ATP (2 mM)

• Incubate at 30°C for defined time periods

• Measure recovery of substrate protein activity

• Compare with spontaneous refolding and with E. coli DnaK system

Data representation:

Note: Values represented are hypothetical based on typical chaperone-assisted protein folding assays

How can comparative studies between L. acidophilus DnaK and E. coli DnaK inform functional conservation?

Comparative studies between L. acidophilus and E. coli DnaK can reveal:

• Evolutionary conservation of chaperone mechanisms

• Host-specific adaptations

• Substrate specificity differences

• Co-chaperone interaction preferences

Methodological approach:

• Sequence and structural comparison

  • Multiple sequence alignment

  • Homology modeling

  • Conservation analysis of functional domains

• Functional comparison

  • Substrate binding preferences

  • ATPase activity under various conditions

  • Complementation studies in dnaK-deficient strains

• Chimeric protein analysis

  • Domain swapping between L. acidophilus and E. coli DnaK

  • Assessment of hybrid functionality

Evidence from E. coli suggests DnaK is necessary for α-complementation of β-galactosidase , providing a phenotypic assay that could be used to test the functional conservation of L. acidophilus DnaK.

What considerations are important when designing expression vectors for recombinant L. acidophilus DnaK?

Key design elements:

• Promoter selection

  • Constitutive vs. inducible

  • Strength (high expression may lead to aggregation)

  • Host compatibility

• Fusion tags

  • N-terminal vs. C-terminal placement

  • Tag size impact on folding and function

  • Cleavable vs. permanent tags

  • Common options: His6, GST, MBP, SUMO

• Codon optimization

  • Match to expression host

  • Avoid rare codons

  • Consider mRNA secondary structure

• Signal sequences

  • For secretion or surface display if needed

  • Native vs. heterologous signals

• Vector backbone considerations

  • Copy number

  • Selection markers

  • Origin of replication

When creating recombinant L. acidophilus strains for surface display applications, selection of appropriate anchoring motifs significantly affects protein expression and localization, as seen in studies with other recombinant L. acidophilus proteins .

How can L. acidophilus DnaK be utilized in vaccine development research?

L. acidophilus DnaK has potential applications in vaccine development through several approaches:

As an adjuvant:

• DnaK can enhance immune responses to co-delivered antigens

• May promote both humoral and cellular immunity

• Can be co-expressed with vaccine antigens in L. acidophilus

As a carrier protein:

• Fusion of antigens to DnaK may increase immunogenicity

• Can improve solubility and stability of problematic antigens

• May facilitate antigen processing and presentation

As a vaccine delivery vehicle:

• Recombinant L. acidophilus expressing DnaK-antigen fusions

• Mucosal delivery to induce local and systemic immunity

• Potential for oral vaccine formulations

Research on recombinant L. acidophilus strains has demonstrated the feasibility of displaying heterologous proteins on the cell surface using different anchoring motifs , a technology that could be adapted for DnaK-based vaccine applications.

What methods can assess the role of DnaK in acid tolerance of L. acidophilus?

Experimental approaches:

• Growth curve analysis at various pH values

  • Compare wild-type, dnaK mutant, and complemented strains

  • Monitor growth kinetics using automated plate readers

  • Determine minimum pH for growth

• Acid challenge survival assays

  • Expose cultures to lethal acid challenge (e.g., pH 2.0)

  • Enumerate survivors by plate counting

  • Calculate survival percentages

• Gene expression analysis

  • qRT-PCR to quantify dnaK expression under acid stress

  • RNA-seq for global transcriptional response

  • Western blot for protein level confirmation

• Acid adaptation studies

  • Pre-adapt cultures to mild acid stress

  • Challenge with severe acid stress

  • Assess protective effect of adaptation

Studies have shown that dnaK and dnaJ are induced together under acid adaptation in L. acidophilus , suggesting DnaK plays a crucial role in the acid tolerance response.

How can computational approaches predict DnaK substrate specificity in L. acidophilus?

Computational methods:

• Sequence-based prediction

  • Machine learning algorithms trained on known DnaK binding motifs

  • Scanning proteome for hydrophobic binding motifs

  • Considering protein disorder and accessibility

• Structural bioinformatics

  • Homology modeling of L. acidophilus DnaK

  • Molecular docking with potential substrates

  • Molecular dynamics simulations of DnaK-substrate complexes

• Network analysis

  • Protein-protein interaction predictions

  • Functional association networks

  • Co-expression data integration

• Comparative genomics

  • Analysis across lactobacilli species

  • Identification of conserved DnaK-dependent pathways

  • Evolutionary analysis of substrate recognition

Implementation workflow:

• Generate or obtain L. acidophilus DnaK structure

• Identify binding pocket characteristics

• Screen proteome for compatible binding motifs

• Rank potential substrates by binding energy

• Validate top predictions experimentally