Recombinant Lactobacillus plantarum ATP-dependent protease subunit HslV (hslV)

What is the HslV protease subunit and what is its function in Lactobacillus plantarum?

HslV is a 19-kDa protein similar to proteasome beta subunits that, together with HslU (a 50-kDa protein related to the ATPase ClpX), forms an ATP-dependent protease complex . In bacterial systems including L. plantarum, the HslV-HslU complex plays a crucial role in degrading aberrant and nonfunctional proteins, contributing to protein quality control and cellular homeostasis . This proteolytic activity is essential for bacterial adaptation to environmental stresses, as it helps eliminate misfolded or damaged proteins that could otherwise accumulate and become toxic to the cell.

The L. plantarum genome encodes this proteolytic system alongside other proteases like Lon, which collectively constitute the cell's protein degradation machinery . Unlike eukaryotic proteasomes, the HslV-HslU protease lacks tryptic-like and peptidyl-glutamyl-peptidase activities, demonstrating unique substrate preferences and functional characteristics .

To investigate HslV function experimentally, researchers typically use approaches including:

• Measuring protease activity with fluorogenic peptide substrates (particularly Z-Gly-Gly-Leu-AMC)

• Conducting ATP-dependent peptidase assays with purified components

• Creating knockout mutants to observe phenotypic changes

• Performing protein accumulation analyses under stress conditions

How is the hslV gene structured and regulated in L. plantarum?

The hslV gene in L. plantarum is part of the heat-shock locus hslVU that encodes both the HslV protease subunit and its associated ATPase partner HslU . This genetic organization is consistent with their functional relationship, as both proteins are required to form the active protease complex. The L. plantarum genome, which has been fully sequenced in strain WCFS1, contains this locus as part of its comprehensive stress response system .

Based on studies of similar systems, hslV expression is typically regulated as part of the heat-shock response, with transcription increasing under conditions of cellular stress. The flexible and adaptive nature of L. plantarum is reflected in its relatively large genome containing numerous regulatory functions that likely influence hslV expression under different environmental conditions .

Methodologically, researchers can study hslV regulation through:

• Promoter analysis to identify regulatory elements

• Quantitative PCR to measure expression changes under different conditions

• Reporter gene fusions to visualize and quantify promoter activity

• Chromatin immunoprecipitation to identify transcription factors binding to the hslV promoter

What techniques are used to create recombinant L. plantarum expressing modified HslV?

Creating recombinant L. plantarum strains expressing modified HslV involves several methodological steps:

• Gene design and synthesis:

  • The hslV gene can be designed with optimized codon usage based on L. plantarum codon preferences

  • If necessary, signal peptides can be added to direct protein localization (similar to approaches using the L. brevis S-layer signal peptide for other recombinant proteins)

  • Synthetic gene services can create the designed construct with desired mutations or tags

• Vector construction:

  • Suitable expression vectors include pNZ8148, an inducible expression plasmid that works in Lactobacillus species

  • The modified hslV gene can be cloned using restriction enzymes (common sites include NcoI and KpnI)

  • Both constitutive and inducible promoter systems can be employed depending on experimental needs

• Transformation into L. plantarum:

  • Competent L. plantarum cells are prepared in media containing specific additives (e.g., 0.3 M sucrose for WXD234 strain)

  • Cells are cultured to specific optical densities (OD600 of 0.3-0.4), collected by centrifugation, and washed with appropriate buffers

  • Electroporation is used to introduce the recombinant plasmid, followed by selection on media containing suitable antibiotics

• Verification of recombinant strains:

  • PCR screening to confirm the presence of the insert

  • DNA sequencing to verify the correct sequence

  • Western blotting to confirm protein expression

  • Activity assays to assess functionality of the recombinant protein

How do researchers assess the enzymatic activity of recombinant HslV?

Assessing the enzymatic activity of recombinant HslV requires specific methodological approaches:

• In vitro peptidase assays:

  • The primary substrate used for HslV-HslU activity assessment is the fluorogenic peptide Z-Gly-Gly-Leu-AMC, which is rapidly hydrolyzed by the complex

  • Activity measurements must be performed in the presence of ATP, as ATP hydrolysis by HslU is essential for peptide hydrolysis by HslV

  • Controls should include assays with ADP, AMP, or non-hydrolyzable ATP analogs, which do not support activity

• ATP dependence characterization:

  • Varying ATP concentrations can determine the degree of stimulation (up to 150-fold increase has been observed)

  • The relationship between ATP hydrolysis rates and peptidase activity can reveal mechanistic insights

• Inhibitor studies:

  • Proteasome inhibitors block HslV-HslU activity, while inhibitors of other protease classes do not affect it

  • Anti-HslV antibodies can be used as specific inhibitors in activity assays

• Structural verification:

  • Electron microscopy can confirm the formation of characteristic ring-shaped particles similar to proteasome structures

  • Co-immunoprecipitation can verify complex formation between HslV and HslU components

What methods are used to generate L. plantarum hslV knockout mutants?

Creating hslV knockout mutants in L. plantarum follows established methodological approaches:

• Target region amplification:

  • The chromosomal regions upstream and downstream of the hslV gene are PCR-amplified using proofreading DNA polymerase and L. plantarum chromosomal DNA as template

  • Typically, fragments of approximately 850-960 bp are generated for each flanking region

• Knockout vector construction:

  • The amplified fragments are digested with appropriate restriction enzymes (e.g., Ecl136II and SwaI)

  • These fragments are cloned into a suicide vector such as pNZ5319

  • The resulting construct contains the flanking regions without the hslV gene itself

• Transformation and selection:

  • The knockout vector is introduced into L. plantarum using electroporation

  • Double-crossover events result in replacement of the wild-type gene with the modified construct

  • Selection markers in the vector allow identification of successful recombinants

• Verification of knockout:

  • PCR analysis confirms the absence of the hslV gene

  • Sequencing verifies the correct genomic arrangement

  • Western blotting confirms the absence of HslV protein

  • Phenotypic analysis assesses functional consequences of gene deletion

How does ATP hydrolysis mechanistically activate the HslV-HslU protease complex?

The ATP-dependence of the HslV-HslU protease complex represents a sophisticated mechanism for regulating proteolytic activity:

ATP binding and hydrolysis by HslU drives significant conformational changes that are essential for activating the HslV protease component. Experimental evidence demonstrates that ATP stimulates peptidase activity up to 150-fold, while other nucleoside triphosphates, non-hydrolyzable ATP analogs, ADP, or AMP have no effect . This strict ATP requirement indicates a highly specific coupling between ATP hydrolysis and proteolytic function.

The proposed mechanistic model involves:

• ATP binding to HslU induces conformational changes

• These changes are transmitted to the associated HslV component

• The HslV active sites become properly aligned and activated

• ATP hydrolysis likely drives substrate translocation into the proteolytic chamber

• ADP release resets the complex for another cycle

Research methodologies to investigate this mechanism include:

• Site-directed mutagenesis of ATP-binding residues in HslU

• Cryo-electron microscopy to capture different conformational states

• FRET-based approaches to measure conformational changes

• Pre-steady-state kinetics to dissect the reaction steps

This ATP-dependence ensures that protein degradation is an energy-consuming process, allowing for tight regulation and preventing indiscriminate proteolysis within the cell.

What is the phenotypic impact of hslV gene knockout on L. plantarum stress response?

The phenotypic analysis of L. plantarum hslV knockout mutants provides insights into the physiological role of this protease in stress adaptation:

Knockout mutants can be generated using established genetic techniques, as demonstrated in studies of other L. plantarum genes. For example, the chromosomal regions upstream and downstream of the target gene can be PCR-amplified and cloned into a suicide vector like pNZ5319 to create a knockout construct . This approach has been successfully applied to generate and phenotypically characterize L. plantarum WCFS1 knockout mutants for other genes, such as small heat shock proteins (hsp1 and hsp3) .

Expected phenotypic changes in hslV knockout mutants would likely include:

• Altered stress tolerance:

  • Reduced survival under heat shock conditions

  • Potential changes in resistance to other stresses (oxidative, pH, osmotic)

  • The degree of these effects might vary depending on the redundancy of proteolytic systems

• Protein homeostasis defects:

  • Accumulation of misfolded or damaged proteins

  • Potential formation of protein aggregates

  • Changes in turnover rates of specific proteins

• Altered growth characteristics:

  • Potential growth defects under stress conditions

  • Possible changes in lag phase duration or growth rate

Comprehensive phenotypic characterization requires multiple methodological approaches:

• Growth curve analysis under various stress conditions

• Microscopic examination for morphological changes

• Proteomics to identify accumulated proteins

• Transcriptomics to detect compensatory gene expression changes

How can structure-function analysis guide engineering of HslV for enhanced specificity?

Structure-function analysis of HslV provides a foundation for rational protein engineering:

While specific structural data for L. plantarum HslV is not detailed in the available literature, insights can be drawn from studies of homologous systems. The E. coli HslV-HslU complex forms characteristic ring-shaped particles similar to proteasome structures, as revealed by electron microscopy . This structural organization creates a proteolytic chamber where substrate degradation occurs.

Key aspects for engineering enhanced specificity include:

• Active site modifications:

  • The catalytic mechanism involves an N-terminal threonine residue, similar to proteasome beta subunits

  • Mutations near this catalytic site can alter substrate preferences

  • Rational design based on homology models can guide mutation selection

• Substrate binding region alterations:

  • Engineering the substrate-binding pockets can modify specificity

  • Introducing charged or hydrophobic residues can favor certain substrates

• Complex assembly engineering:

  • Modifications that affect HslV-HslU interaction can influence activity

  • Changes to the gating mechanism controlling substrate entry can enhance selectivity

Table 1: Structure-Based Engineering Strategies for HslV Modification

Verification of engineered variants requires:

• Expression and purification of modified proteins

• Structural confirmation via circular dichroism or limited proteolysis

• Functional assays with various substrates

• Complex formation analysis with HslU

What are the challenges in maintaining stability of recombinant L. plantarum expressing HslV in experimental settings?

Several challenges exist in maintaining stable recombinant L. plantarum strains expressing HslV:

• Plasmid stability:

  • Expression plasmids may be lost during continuous culture without selection pressure

  • Methodological solutions include chromosomal integration or use of plasmid addiction systems

  • Regular verification through antibiotic resistance testing and PCR is essential

• Expression level consistency:

  • Expression levels may fluctuate due to various factors including growth phase and media composition

  • Inducible systems like those based on pNZ8148 can provide more controlled expression

  • Western blotting should be used to verify consistent expression levels

• Functional protein production:

  • Recombinant HslV must fold correctly and form functional complexes with endogenous HslU

  • Over-expression might lead to formation of inclusion bodies

  • Co-expression of chaperones may improve functional yield

• Metabolic burden:

  • Expression of recombinant proteins imposes metabolic costs on the host

  • This can lead to selective pressure favoring cells with reduced expression

  • Codon optimization and controlled expression levels can mitigate this issue

• Environmental stress factors:

  • Various environmental conditions can affect recombinant protein expression

  • For L. plantarum, considerations include media composition, pH, and growth temperature

  • Strain-specific factors may influence recombinant protein production (e.g., WCFS1 strain characteristics)

Stability assessment protocols should include:

• Regular PCR screening for plasmid retention

• Protein expression verification via Western blotting

• Activity assays to confirm functional protein production

• Growth rate monitoring to assess metabolic burden

How does the composition of the growth medium affect expression of recombinant HslV in L. plantarum?

The growth medium composition significantly influences recombinant protein expression in L. plantarum:

• Base media selection:

  • MRS (de Man, Rogosa and Sharpe) medium is commonly used for Lactobacillus cultivation

  • For specific applications, MRS may be supplemented with additional components

  • When preparing L. plantarum for transformation, specific additives are required (e.g., 0.3 M sucrose for strain WXD234)

• Critical media components:

  • Carbon source type and concentration affect growth rate and protein synthesis

  • Nitrogen sources influence protein expression levels

  • Divalent cations (especially Mg²⁺) are important for plasmid stability and protein folding

  • Buffer capacity affects pH stability during growth

• Induction parameters:

  • For inducible systems like pNZ8148, the concentration and timing of inducer addition are critical

  • The growth phase at induction significantly affects recombinant protein yield

  • Temperature during induction influences protein folding and solubility

• Strain-specific considerations:

  • Different L. plantarum strains may have different media requirements

  • For example, strain WXD234 requires specific washing buffers (0.5 M saccharose, 0.5 mM KH₂PO₄, and 0.5 mM MgCl₂) during competent cell preparation

Table 2: Media Optimization Strategies for Recombinant HslV Expression in L. plantarum

Methodological approach for optimization:

• Factorial design experiments to systematically test multiple variables

• Real-time monitoring of growth and protein expression

• Activity assays to verify functional protein production

• Statistical analysis to identify optimal conditions

How can recombinant L. plantarum expressing modified HslV be utilized in vaccine development research?

Recombinant L. plantarum has significant potential as a vaccine delivery vehicle, and HslV-based systems could enhance this application:

L. plantarum offers several advantages as a vaccine delivery system:

• It can persist in the human gastrointestinal tract for more than 6 days in an active form

• It has a record of safety and probiotic properties

• It can be engineered to express heterologous antigens

For vaccine development using recombinant L. plantarum expressing modified HslV or HslV fusion proteins:

• Antigen delivery strategies:

  • HslV can be engineered as a fusion protein with vaccine antigens

  • The ATP-dependent nature of the system could potentially provide controlled antigen release

  • Surface display versus secretion approaches can be utilized depending on immune response goals

• Genetic engineering approaches:

  • Similar to other recombinant systems in Lactobacillus, signal peptides can direct protein localization

  • For example, the L. brevis S-layer 30-amino acid signal peptide has been used successfully for other recombinant proteins

  • Codon optimization based on L. plantarum preferences enhances expression efficiency

• Experimental evaluation framework:

  • In vitro expression and stability analysis

  • Assessment of bacterial persistence in animal models (recombinant Lactobacillus strains can persist for at least 72 hours in vivo)

  • Immune response measurement (humoral and cellular)

  • Challenge studies to evaluate protective efficacy

This approach parallels successful work with other recombinant Lactobacillus systems, such as L. plantarum expressing S. aureus antigens, which have shown promise as mucosal vaccines .

What protocols are most effective for purifying recombinant HslV from L. plantarum for biochemical studies?

Purification of recombinant HslV from L. plantarum requires specific methodological considerations:

• Cell lysis optimization:

  • Lactobacillus has a thick cell wall requiring robust lysis methods

  • Enzymatic treatment (lysozyme, mutanolysin) combined with mechanical disruption

  • Buffer composition should include protease inhibitors to prevent degradation

  • Maintaining cold temperatures throughout processing is critical

• Affinity purification strategies:

  • Tagging the recombinant HslV (His-tag, FLAG-tag) facilitates selective purification

  • For functional studies, tag placement should avoid interference with active sites or complex formation

  • On-column refolding may improve recovery of functional protein

  • Elution conditions must be optimized to maintain protein stability

• Complex isolation considerations:

  • For studies requiring the HslV-HslU complex, co-expression or reconstitution approaches can be used

  • Co-immunoprecipitation can isolate native complexes

  • Size exclusion chromatography can separate assembled complexes from individual components

• Activity verification:

  • Purified HslV should be tested for ATP-dependent peptidase activity

  • The fluorogenic peptide Z-Gly-Gly-Leu-AMC is the preferred substrate

  • Activity should increase substantially (up to 150-fold) in the presence of ATP

Table 3: Troubleshooting Common Issues in HslV Purification

How can CRISPR-Cas9 technology improve the genetic manipulation of hslV in L. plantarum?

CRISPR-Cas9 technology offers significant advantages for precise genetic manipulation of hslV in L. plantarum:

While traditional approaches using suicide vectors have been employed successfully for generating knockouts in L. plantarum , CRISPR-Cas9 systems provide enhanced precision and efficiency:

• Advantages for hslV manipulation:

  • Higher precision in generating specific mutations without affecting neighboring genes

  • Increased efficiency compared to homologous recombination alone

  • Ability to introduce multiple modifications simultaneously

  • Reduced time required for strain construction

• Implementation strategies:

  • Two-plasmid systems (one carrying Cas9, one with sgRNA and repair template)

  • Temperature-sensitive vectors for transient expression

  • Counter-selection markers to facilitate plasmid curing after editing

  • Inducible Cas9 expression to minimize toxic effects

• Specific modifications enabled:

  • Point mutations to study structure-function relationships

  • Domain swapping with homologous proteases

  • Precise promoter modifications to alter expression patterns

  • Tag insertion for protein localization studies

• Verification protocols:

  • PCR amplification and sequencing of modified regions

  • Restriction fragment length polymorphism analysis

  • Protein expression verification via Western blotting

  • Functional assays to confirm expected phenotypic changes

These approaches complement existing methods for L. plantarum genetic manipulation while offering enhanced precision, especially for subtle modifications that are difficult to achieve with traditional techniques.

What are the key differences in proteolytic activity between wild-type and recombinant HslV expressed in L. plantarum?

Comparative analysis of wild-type and recombinant HslV reveals important functional differences:

When expressing recombinant HslV in L. plantarum, several factors can influence its proteolytic activity compared to the native enzyme:

• Expression level effects:

  • Recombinant systems typically achieve higher expression levels than native expression

  • Studies of similar proteolytic systems have shown activity increases of 10-fold in constitutive expression systems and up to 100-fold with high-copy plasmids

  • Higher concentration can alter reaction kinetics and substrate preferences

• Structural modifications:

  • Addition of affinity tags can affect complex formation with HslU

  • Mutations introduced for study purposes may alter catalytic efficiency

  • Codon optimization might affect folding kinetics and final conformation

• Interaction with native components:

  • Recombinant HslV must interact with endogenous HslU for full activity

  • Competition between native and recombinant HslV for HslU binding may occur

  • The stoichiometry of HslV:HslU may be altered in recombinant systems

• Substrate specificity differences:

  • Wild-type HslV-HslU complexes rapidly hydrolyze specific fluorogenic peptides like Z-Gly-Gly-Leu-AMC

  • Modifications to recombinant HslV may alter this specificity profile

  • Engineered variants might show activity toward non-native substrates

Methodological approaches for comparative analysis:

• Side-by-side activity assays with standardized substrate concentrations

• ATP-dependency profiles to assess functional complex formation

• Thermal stability comparisons to evaluate structural integrity

• Kinetic parameter determination (Km, Vmax) for various substrates