Recombinant Bdellovibrio bacteriovorus Lon protease 1 (lon1), partial

What is Bdellovibrio bacteriovorus Lon protease 1 and how does it differ from canonical Lon proteases?

Bdellovibrio bacteriovorus encodes two Lon protease homologs: Lon-1 and Lon-2. While Lon-2 functions similarly to the canonical ATP-dependent Lon protease found in most bacteria, Lon-1 appears to have evolved distinctive functions. Recent studies suggest that Lon-1 functions differently from prototypical Lon proteases despite sharing structural similarities in the ATPase and proteolytic domains .

Unlike canonical Lon proteases and B. bacteriovorus Lon-2, Lon-1:

• Does not complement E. coli Lon mutant phenotypes

• Shows different substrate specificity due to variations in its substrate-binding domain

• Does not degrade SsrA-tagged proteins (a typical substrate of canonical Lon)

• Is upregulated transcriptionally by exposure to blood in vitro

These differences suggest Lon-1 has evolved specialized functions potentially related to the predatory lifestyle of B. bacteriovorus.

What are the characterized functional domains of B. bacteriovorus Lon-1?

B. bacteriovorus Lon-1 maintains the essential domains characteristic of ATP-dependent proteases with some unique features:

The proteolytic domain contains the catalytic serine residue at position 714, which is essential for its function. Mutation of this residue to alanine (S714A) abolishes proteolytic activity and results in loss of bacterial infection capability, confirming the importance of this residue .

What role does Lon-1 play in B. bacteriovorus biology and pathogenesis?

Lon-1 plays critical roles in multiple aspects of B. bacteriovorus biology:

• Infection capability: Lon-1 is essential for the infection of host bacteria; lon-1 deletion mutants show severely attenuated infectivity in murine infection models

• Growth regulation: lon-1 mutants display growth defects in regular BSK-II medium

• Stress resistance: Lon-1 contributes to bacterial resistance to osmotic stress and oxidative stress (tert-butyl hydroperoxide)

• Virulence factor regulation: Production of several virulence factors (BosR, RpoS, OspC) is elevated in lon-1 mutants, suggesting Lon-1 may regulate these factors

• Host adaptation: Lon-1 is upregulated during blood exposure and may be important in the transition from arthropod vector to warm-blooded host

The proteolytic activity of Lon-1 appears essential for bacterial infection, as the lon-1(S714A) mutant failed to infect mice .

How is Lon-1 expression regulated in B. bacteriovorus?

The expression of lon-1 in B. bacteriovorus is regulated in response to environmental conditions:

• Blood exposure: lon-1 is upregulated transcriptionally by exposure to blood in vitro, while lon-2 is not affected by blood exposure

• Animal infection: lon-1 is highly expressed during animal infection, suggesting an important function in the infection process

• Developmental stage-specific expression: Expression patterns differ between the predatory cycle stages, with highest expression during infection phases

This regulation pattern differs from that of lon-2, which appears to be more consistently expressed across different growth conditions, supporting the hypothesis that Lon-1 has evolved specialized functions related to the predatory lifestyle and host adaptation.

What experimental approaches can be used to assess the enzymatic activity of recombinant Lon-1?

Several methodological approaches can be employed to characterize the enzymatic activity of recombinant Lon-1:

Proteolytic activity assays:

• ATP and Mg²⁺-dependent proteolytic activity can be measured using fluorogenic peptide substrates

• Degradation of model substrates can be monitored by SDS-PAGE and western blotting

• For the S714A mutant variant, comparative assays can confirm the importance of the catalytic serine

Chaperone activity assessment:

• Protein refolding assays using denatured model proteins

• Prevention of protein aggregation measured by light scattering

• ATP-dependent conformational changes monitored by fluorescence spectroscopy or circular dichroism

Substrate specificity determination:

• Proteomic approaches to identify natural substrates (e.g., pull-down assays followed by mass spectrometry)

• In vitro degradation assays with purified potential substrate proteins (e.g., BosR, RpoS, OspC)

• Comparison of degradation rates between different substrates to establish preference profiles

For optimal results, recombinant protein should be purified to >90% homogeneity using affinity chromatography (e.g., His-tag purification) .

What methodological approaches can be used to study Lon-1's role in B. bacteriovorus predatory behavior?

Investigating Lon-1's role in predation requires multifaceted approaches:

Genetic manipulation strategies:

• Generation of lon-1 deletion mutants via allelic exchange using suicide plasmids (e.g., pSSK10 or pK18 mobsacB)

• Construction of site-directed mutants (e.g., S714A) to target specific functions

• Complementation studies to confirm phenotype specificity

Predation assays:

• Host-dependent growth curves measuring prey killing efficiency

• Plaque formation assays on prey lawns (clear vs. turbid plaques)

• Time-lapse microscopy to directly observe predation dynamics

• Quantification of prey population reduction (log reduction)

Specific phenotypic analyses:

• Stress response assays (osmotic stress, oxidative stress)

• Growth rate determination in standard media

• Virulence factor expression analysis via western blotting

• Infection models (e.g., murine models)

Expression analysis during predation cycle:

• qRT-PCR to measure lon-1 expression at different stages of predation

• Reporter gene fusions to monitor expression patterns

• Proteomics to identify Lon-1 interaction partners and substrates during predation

The complementary use of these approaches can provide a comprehensive understanding of Lon-1's role in the predatory behavior of B. bacteriovorus.

How can researchers investigate the substrate specificity differences between Lon-1 and Lon-2?

The substrate specificity differences between Lon-1 and Lon-2 can be investigated through:

Comparative structural analysis:

• Generate homology models of both proteases focusing on substrate-binding domains

• Identify key amino acid differences that may influence substrate recognition

• Perform molecular docking simulations with potential substrates

Domain swapping experiments:

• Create chimeric proteins with swapped substrate-binding domains between Lon-1 and Lon-2

• Express and purify these chimeric proteins

• Assess changes in substrate preference and catalytic efficiency

Substrate profiling:

• Perform comparative degradation assays with identical potential substrates

• Use peptide libraries to identify preferred cleavage motifs

• Employ proteomic approaches (e.g., SILAC) to identify differentially degraded proteins in vivo

Complementation studies:

• Test whether Lon-1 can complement Lon-2 mutants and vice versa

• Assess whether chimeric proteins can restore wild-type phenotypes

• Compare complementation in different stress conditions

This integrated approach can reveal the molecular basis for the functional divergence between these two Lon proteases and potentially identify unique substrates for each.

What techniques can be used to investigate the ATP-dependence of Lon-1 protease activity?

The ATP-dependence of Lon-1 can be systematically characterized using:

Enzymatic activity assays with ATP manipulation:

• Compare proteolytic activity in the presence and absence of ATP

• Perform titration experiments with varying ATP concentrations to determine K₍ₘ₎

• Investigate the effects of non-hydrolyzable ATP analogs (e.g., ATPγS) on activity

• Assess the impact of ATPase inhibitors on proteolytic function

Site-directed mutagenesis of the ATPase domain:

• Identify and mutate key residues in the ATPase domain predicted to be involved in ATP binding/hydrolysis

• Express and purify these mutant proteins

• Compare their ATPase and proteolytic activities to wild-type Lon-1

ATP hydrolysis measurements:

• Directly measure ATP hydrolysis rates using colorimetric assays (e.g., malachite green)

• Compare ATP hydrolysis in the presence and absence of substrates

• Determine the coupling between ATP hydrolysis and proteolytic activity

Structural dynamics studies:

• Use fluorescence spectroscopy to monitor ATP-induced conformational changes

• Employ hydrogen-deuterium exchange mass spectrometry to identify regions affected by ATP binding

• Utilize small-angle X-ray scattering to characterize large-scale structural changes upon ATP binding

These approaches would provide a comprehensive understanding of how ATP regulates Lon-1 activity and whether this regulation differs from canonical Lon proteases.

How can one investigate the potential role of Lon-1 in B. bacteriovorus stress response?

To systematically investigate Lon-1's role in stress response:

Stress challenge experiments:

• Generate lon-1 deletion and point mutants

• Challenge with various stressors (oxidative, osmotic, temperature, pH)

• Quantify survival rates compared to wild-type strains

• Determine minimum inhibitory concentrations of stressors

Gene expression analysis under stress conditions:

• Perform RNA-seq or qRT-PCR under various stress conditions

• Compare expression profiles between wild-type and lon-1 mutants

• Identify stress response genes regulated directly or indirectly by Lon-1

Proteome stability assessment:

• Use pulse-chase experiments to measure protein turnover rates under stress

• Perform 2D gel electrophoresis to identify proteins differentially accumulated in lon-1 mutants

• Employ quantitative proteomics to identify stress-induced Lon-1 substrates

Biochemical characterization of stress-induced changes:

• Measure enzymatic activities of key metabolic enzymes in wild-type vs. mutant strains

• Assess changes in Lon-1 substrate specificity under stress conditions

• Determine whether stress conditions alter Lon-1 activity or localization

Research has already shown that lon-1 mutants display reduced resistance to osmotic stress and oxidative stress (tert-butyl hydroperoxide) , providing a foundation for more detailed investigations.

Reference Table: Key Findings on B. bacteriovorus Lon-1 Function