Recombinant Sulfurovum sp. ATP-dependent zinc metalloprotease FtsH (ftsH)

What is the structural composition of Sulfurovum sp. ATP-dependent zinc metalloprotease FtsH?

Sulfurovum sp. FtsH shares the characteristic structural features of the FtsH protease family. It consists of an N-terminal transmembrane domain with one or two transmembrane helices, followed by a highly conserved AAA+ ATPase domain containing Walker A and B structural elements, and a downstream M41 peptidase domain with a zinc-binding proteolytic site . The AAA+ domain contains a second region of homology motif responsible for ATP binding and hydrolysis . Like other FtsH proteases, the Sulfurovum sp. variant likely forms hexameric complexes, with the soluble ATPase and protease domains interacting with neighboring protomers to create a hexagonal particle, as this oligomeric structure is essential for proteolytic activity .

How does the mechanism of substrate processing work in Sulfurovum sp. FtsH?

Sulfurovum sp. FtsH likely follows the conserved mechanism observed in other FtsH proteases:

• Substrate recognition: A phenylalanine residue positioned on the top surface near the central pore of the ATPase domain (in the FVG motif) is responsible for substrate binding

• Substrate unfolding: ATP hydrolysis powers the unfolding of the target protein

• Translocation: The unfolded substrate is moved through the central pore of the ATPase complex to the protease domain

• Degradation: The zinc-binding proteolytic site in the M41 domain digests the substrate into approximately 12 amino-acid long oligopeptides

The ~20 amino acid flexible linker between the transmembrane and ATPase domains creates space for substrates to access the protease .

What physiological roles does FtsH likely play in Sulfurovum species?

Based on FtsH functions in other bacteria and Sulfurovum's ecological niche, FtsH in Sulfurovum sp. likely has multiple important functions:

• Protein quality control: Degrading misfolded and damaged proteins

• Regulatory roles: Participating in stress response pathways

• Adaptation to environmental stressors: Managing protein homeostasis under conditions of extreme pH, temperature fluctuations, and high sulfide concentrations

• Maintenance of cellular processes: Supporting sulfur oxidation and hydrogen oxidation pathways that are central to Sulfurovum's metabolism

Sulfurovum species function as sulfur-oxidizing bacteria in hydrothermal vent systems, and FtsH may be particularly important for maintaining cellular integrity in these extreme environments .

How might the FtsH protease from Sulfurovum sp. differ from those in non-extremophilic bacteria?

Sulfurovum species inhabit hydrothermal vent systems with dynamic acidic (pH 5.5-8.1) and sulfidic (9-3000 μM) conditions . The FtsH protease in Sulfurovum likely exhibits several adaptations:

These adaptations would be critical for Sulfurovum's survival in hydrothermal vents where it has been identified as a dominant bacterial genus .

What role might Sulfurovum sp. FtsH play in symbiotic relationships with hydrothermal vent metazoans?

Sulfurovum species form symbiotic relationships with vent organisms such as the brachyuran vent crab, Xenograpsus testudinatus . In this context, FtsH could serve several functions:

Fluorescence in situ hybridization (FISH) studies have shown that Sulfurovum-related bacteria are widely distributed in the afferent vessels of vent crab gills and in epithelial principal cells and pilaster cells on the lamella , suggesting a close symbiotic relationship where FtsH could play important regulatory roles.

How does the ATP-dependency of Sulfurovum sp. FtsH affect its function in energy-limited environments?

Hydrothermal vent systems present unique energy constraints, potentially affecting how ATP-dependent enzymes like FtsH operate:

The optimization of ATP usage by FtsH in Sulfurovum may parallel adaptations seen in ATP synthases, where the number of c-subunits is adjusted to achieve a specific proton per ATP ratio for balanced performance .

What are the optimal conditions for expressing recombinant Sulfurovum sp. FtsH in E. coli?

Based on the commercial product information and general knowledge of recombinant protein expression:

• Expression system: pET expression system in E. coli BL21(DE3) or similar strains

• Induction conditions:

  • IPTG concentration: 0.2-0.5 mM

  • Induction temperature: 16-20°C (lower temperatures may improve solubility of membrane-associated proteins)

  • Induction time: 16-20 hours (overnight induction)

• Growth media supplements:

  • ZnSO₄ (1-10 μM) to ensure zinc incorporation into the metalloprotease domain

  • Glucose (0.5-1%) to suppress basal expression

• Buffer considerations:

  • Include mild detergents (0.05-0.1% DDM or CHAPS) to solubilize the transmembrane domain

  • Maintain pH 7.5-8.0 for optimal stability

  • Include 5-10% glycerol to enhance protein stability

Researchers should validate these conditions through small-scale expression trials before scaling up production.

What purification strategies are most effective for isolating active Sulfurovum sp. FtsH?

A multi-step purification protocol would likely include:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using the N-terminal His-tag

  • Buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5-10% glycerol, 0.05% detergent

  • Gradient elution with imidazole (20-300 mM)

• Secondary purification: Ion exchange chromatography

  • Anion exchange (Q Sepharose) at pH 8.0

  • Salt gradient: 50-500 mM NaCl

• Final polishing: Size exclusion chromatography

  • To isolate properly assembled hexameric complexes

  • Buffer: 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 0.03% detergent, 5 mM MgCl₂, 1 mM DTT

• Quality control assessments:

  • SDS-PAGE to confirm purity

  • Native PAGE to verify oligomeric state

  • ATPase activity assay to confirm functionality

  • Zinc content analysis to verify metalloprotease domain integrity

How can researchers accurately measure the proteolytic activity of recombinant Sulfurovum sp. FtsH?

Several complementary approaches can be used:

• Fluorogenic peptide substrates:

  • FRET-based peptides containing FtsH cleavage sites

  • Monitor fluorescence increase upon cleavage

  • Reaction conditions: 25 mM HEPES pH 7.5, 150 mM KCl, 5 mM MgCl₂, 2 mM ATP, 37°C

• Model protein substrates:

  • Known FtsH substrates (e.g., σ32, SsrA-tagged proteins)

  • Follow degradation via SDS-PAGE and western blotting

  • Reaction conditions: Include ATP regeneration system (10 mM phosphoenolpyruvate, 10 U/ml pyruvate kinase)

• ATPase activity coupling:

  • Measure ATP hydrolysis rate as a proxy for proteolytic activity

  • NADH-coupled assay with pyruvate kinase and lactate dehydrogenase

  • Monitor NADH oxidation at 340 nm

• Activity modulation tests:

  • Test activity at varying pH (5.5-8.0) to mimic hydrothermal vent conditions

  • Assess effects of sulfide compounds (9-3000 μM range)

  • Evaluate temperature dependence (20-60°C)

How should researchers design experiments to investigate the role of Sulfurovum sp. FtsH in extreme environment adaptation?

A comprehensive experimental approach should include:

• Comparative analysis with non-extremophile FtsH proteins:

  • Express and purify FtsH from mesophilic bacteria

  • Compare enzymatic parameters (Km, kcat, substrate specificity)

  • Assess stability under varying pH, temperature, and sulfide concentrations

• Substrate identification:

  • Perform proteomics analysis of Sulfurovum cells under normal and stress conditions

  • Identify proteins with altered abundance

  • Validate candidate substrates using in vitro degradation assays

• Structure-function analysis:

  • Generate site-directed mutants targeting unique residues in Sulfurovum FtsH

  • Assess effects on activity under standard and extreme conditions

  • Perform structural studies (X-ray crystallography or cryo-EM) to identify adaptations

• In vivo studies:

  • Develop genetic manipulation systems for Sulfurovum sp.

  • Create FtsH mutants or modulate FtsH expression

  • Monitor effects on growth and survival under varying conditions

• Symbiosis investigation:

  • Co-culture experiments with vent crab cells or tissues

  • Assess effects of FtsH inhibition on symbiotic interactions

  • Analyze spatial distribution of FtsH expression in symbiotic contexts using techniques like FISH

What approaches can be used to study the interaction between Sulfurovum sp. FtsH and other proteins in hydrothermal vent environments?

Researchers can employ several complementary approaches:

• Proteomic identification of interaction partners:

  • Co-immunoprecipitation using anti-FtsH antibodies

  • Crosslinking mass spectrometry to capture transient interactions

  • Bacterial two-hybrid screening to identify protein-protein interactions

• Functional characterization of regulators:

  • Screen for homologs of known FtsH regulators (e.g., SPFH family proteins)

  • Express and purify candidate regulators

  • Assess effects on FtsH activity in vitro

• Membrane protein complex analysis:

  • Blue native PAGE to preserve native complexes

  • Density gradient ultracentrifugation to isolate intact complexes

  • Cryo-electron microscopy of membrane fractions

• In situ visualization:

  • Develop specific antibodies against Sulfurovum FtsH

  • Perform immunofluorescence microscopy in host tissues

  • Correlate FtsH localization with other symbiosis markers

These approaches would help reveal how FtsH functions within the complex cellular network of Sulfurovum and its potential role in symbiotic relationships with hydrothermal vent organisms.