Recombinant Marinobacter aquaeolei ATP-dependent zinc metalloprotease FtsH (ftsH)

What is ATP-dependent zinc metalloprotease FtsH in Marinobacter aquaeolei?

ATP-dependent zinc metalloprotease FtsH in Marinobacter aquaeolei is a membrane-bound AAA+ protease (ATPases Associated with diverse cellular Activities) that plays critical roles in protein quality control and regulatory mechanisms. While specific research on M. aquaeolei FtsH is limited, similar FtsH proteins across bacterial species participate in degrading misfolded membrane proteins, certain regulatory proteins, and proteins involved in stress responses. M. aquaeolei, recognized as a ubiquitous marine bacterium with versatile metabolic capabilities, likely relies on FtsH for membrane protein homeostasis in its adaptable lifestyle across various marine environments, including hydrocarbon-rich habitats and the deep sea .

How does M. aquaeolei FtsH relate to the genomic adaptability of this organism?

M. aquaeolei's genomic adaptability is characterized by multiple metabolic pathways that enable its survival in diverse environments. The organism possesses four variations of the TCA cycle, complete pathways for glycolysis, and mechanisms for degrading complex hydrocarbons . In this context, FtsH likely plays a crucial regulatory role in protein quality control under changing environmental conditions. Its ATP-dependent proteolytic function would help maintain cellular homeostasis during transitions between aerobic and anaerobic metabolism, as M. aquaeolei is known to be a facultative anaerobe with remarkable metabolic versatility . The protease may also be involved in stress responses when the organism encounters hydrocarbon-rich or nutrient-limited environments.

What is the difference between basic FtsH characterization and advanced functional studies?

Basic FtsH characterization:

• Identification of protein sequence and primary structure

• Expression and purification of the recombinant protein

• Assessment of basic enzymatic activity (ATP hydrolysis, proteolytic function)

• Determination of essential cofactors (zinc, ATP requirements)

Advanced functional studies:

• Structural analysis through crystallography or cryo-EM

• Identification of specific substrate recognition mechanisms

• Elucidation of regulatory networks controlled by FtsH activity

• Investigation of FtsH roles in stress response pathways

• Analysis of FtsH contribution to M. aquaeolei's adaptation to extreme environments

• Integrative studies connecting FtsH function to the organism's unique metabolic capabilities, including its hydrocarbon degradation and extremophilic lifestyles

What are the optimal conditions for heterologous expression of recombinant M. aquaeolei FtsH?

For successful heterologous expression of recombinant M. aquaeolei FtsH, an E. coli-based expression system is typically recommended based on similar successes with other M. aquaeolei proteins . The following methodological approach is advised:

• Vector selection: pET-based expression vectors with N-terminal His-tag fusion for purification

• Host strain: E. coli BL21(DE3) or C43(DE3) for membrane proteins

• Expression conditions:

  • Induction at OD600 of 0.6-0.8

  • IPTG concentration: 0.1-0.5 mM

  • Post-induction temperature: 16-18°C for 16-18 hours (to minimize inclusion body formation)

  • Supplementation with 0.1 mM ZnSO4 in the growth medium to ensure proper zinc incorporation

Similar approaches have been successfully employed for other M. aquaeolei enzymes, as documented in studies on fatty aldehyde dehydrogenases where multiple enzymes were heterologously expressed in E. coli . For membrane proteins like FtsH, the addition of mild detergents such as n-dodecyl-β-D-maltoside (DDM) at 0.03-0.05% during cell lysis can facilitate solubilization while maintaining protein activity.

What purification strategy yields the highest activity for recombinant FtsH?

Based on methodologies used with similar metalloproteases, a multi-step purification strategy is recommended:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Binding buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 0.03% DDM, 10% glycerol, 10 mM imidazole

  • Wash buffer: Same as binding buffer with 20-30 mM imidazole

  • Elution buffer: Same as binding buffer with 250-300 mM imidazole

• Secondary purification: Size exclusion chromatography (SEC)

  • Buffer: 25 mM HEPES pH 7.5, 150 mM NaCl, 0.015% DDM, 5% glycerol, 5 mM MgCl2, 1 mM DTT

  • Column: Superdex 200

• Activity preservation:

  • Addition of 10% glycerol to final preparation

  • Storage at -80°C in small aliquots

  • Avoid repeated freeze-thaw cycles

Activity assays should be performed after each purification step to monitor specific activity, with typical yields of 2-5 mg of purified protein per liter of bacterial culture. Similar purification approaches have successfully maintained activity for other M. aquaeolei enzymes .

How can I assess the purity and activity of the purified FtsH protein?

Purity assessment:

• SDS-PAGE analysis (>90% purity is typically achievable)

• Western blot using anti-His tag antibodies

• Mass spectrometry for identity confirmation

Activity assessment methodologies:

• ATP hydrolysis assay:

  • Measure inorganic phosphate release using malachite green assay

  • Typical reaction conditions: 50 mM Tris-HCl pH 8.0, 150 mM KCl, 5 mM MgCl2, 1 mM DTT, 2 mM ATP, 0.5-2 μM FtsH

  • Incubation at 37°C with time points at 0, 5, 10, 15, 30 minutes

• Proteolytic activity assay:

  • Using fluorescent model substrates (FITC-casein)

  • ATP-dependent degradation monitored by fluorescence increase

  • Control reactions without ATP to confirm ATP dependence

• Zinc dependence verification:

  • Activity assays in presence of metal chelators (EDTA)

  • Restoration of activity by zinc supplementation

What domains are typically present in bacterial FtsH proteins and how might they be organized in M. aquaeolei FtsH?

Based on comparative analysis with other bacterial FtsH proteins, M. aquaeolei FtsH likely contains four key domains:

• N-terminal transmembrane domain: Typically comprising 1-2 transmembrane helices that anchor the protein to the cytoplasmic membrane

• AAA+ ATPase domain: Contains Walker A and B motifs for ATP binding and hydrolysis

• Zinc-binding metalloprotease domain: Characterized by an HEXXH motif that coordinates the zinc ion essential for proteolytic activity

• C-terminal region: Involved in substrate recognition and oligomerization

These domains would likely work in concert, with the AAA+ domain utilizing ATP hydrolysis to unfold substrate proteins and translocate them to the proteolytic chamber where the zinc-binding domain performs the actual proteolysis. The structural organization would presumably involve hexameric assembly, which is characteristic of FtsH proteins, forming a barrel-like structure with a central pore for substrate entry. This arrangement allows for coupling of ATP hydrolysis to protein unfolding and degradation in a controlled, processive manner.

How does the substrate specificity of FtsH in M. aquaeolei likely compare to other bacterial FtsH proteases?

While specific substrate profiles for M. aquaeolei FtsH have not been extensively characterized, the following comparison can be inferred based on known FtsH functions in other bacteria and M. aquaeolei's ecological niche:

M. aquaeolei's unique metabolic flexibility, including its ability to thrive in both aerobic and anaerobic conditions and degrade complex hydrocarbons , suggests that its FtsH protease may have evolved specificity for substrates involved in these metabolic transitions. The enzyme might recognize specific degradation signals that allow for rapid adaptation to changing environmental conditions in marine ecosystems.

What role might FtsH play in the extremophilic adaptations of M. aquaeolei?

M. aquaeolei demonstrates remarkable adaptability to extreme conditions, including psychrophily (cold adaptation), oligotrophy (low-nutrient environments), and halotolerance (salt tolerance) . FtsH likely contributes to these adaptations through several mechanisms:

• Cold adaptation:

  • Maintains membrane fluidity by removing misfolded proteins that could disrupt membrane integrity at low temperatures

  • Potentially degrades cold-sensitive proteins that might inhibit growth at lower temperatures

• Nutrient limitation response:

  • Participates in protein recycling to conserve amino acids during oligotrophic conditions

  • Regulates the abundance of nutrient transporters to optimize uptake efficiency

  • May degrade regulatory factors that repress alternative metabolic pathways

• Halotolerance mechanisms:

  • Removes salt-damaged proteins that could accumulate in high-salt environments

  • May regulate membrane protein composition to maintain proper osmotic balance

  • Potentially controls expression of salt stress response proteins

• Hydrocarbon metabolism:

  • May regulate the expression or activity of hydrocarbon degradation enzymes

  • Could remove oxidatively damaged proteins resulting from hydrocarbon metabolism

The versatility of M. aquaeolei's metabolic network, including four variations of the TCA cycle and complete pathways for glycolysis and hydrocarbon degradation , suggests that FtsH may play a critical role in the rapid protein turnover required for switching between different metabolic modes in response to environmental changes.

What are the most effective approaches for studying substrate specificity of M. aquaeolei FtsH?

Determining substrate specificity requires a systematic approach combining biochemical, proteomic, and genetic techniques:

• In vitro degradation assays:

  • Purified FtsH incubated with candidate substrate proteins

  • Time-course SDS-PAGE analysis to monitor degradation

  • Confirmation of ATP dependence by parallel reactions without ATP

  • Controls with catalytically inactive FtsH (mutation in the HEXXH motif)

• Proteomic approaches:

  • Comparative proteomics of wild-type vs. FtsH-depleted M. aquaeolei

  • SILAC (Stable Isotope Labeling with Amino acids in Cell culture) to quantify protein turnover rates

  • Pulse-chase experiments combined with immunoprecipitation of FtsH to identify interacting substrates

• Degradation signal mapping:

  • Creation of fusion proteins with potential degradation signals

  • Systematic mutagenesis to identify critical residues for recognition

  • Competition assays with synthetic peptides containing potential recognition motifs

• Structural studies:

  • Cryo-EM analysis of FtsH-substrate complexes

  • Crosslinking mass spectrometry to identify substrate contact points

  • Molecular dynamics simulations to model substrate interactions

Researchers have successfully employed similar approaches with fatty aldehyde dehydrogenases from M. aquaeolei, where substrate specificity was characterized by testing multiple enzymatic reactions with different aldehydes . These studies revealed that M. aquaeolei enzymes can often recognize a broader range of substrates than initially anticipated, which may also apply to its FtsH protease.

How can I develop a reliable in vitro assay system for M. aquaeolei FtsH activity?

A robust in vitro assay system for FtsH activity should address both ATPase and proteolytic functions:

ATPase activity assay:

• Reaction setup:

  • Buffer: 50 mM Tris-HCl pH 8.0, 150 mM KCl, 5 mM MgCl2, 1 mM DTT

  • Purified FtsH protein: 0.5-2 μM

  • ATP: 1-5 mM

  • Total volume: 50-100 μL

  • Incubation at 37°C (or temperature gradient for kinetic studies)

• Measurement methods:

  • Malachite green assay for inorganic phosphate quantification

  • Coupled enzyme assay (pyruvate kinase/lactate dehydrogenase) monitoring NADH oxidation

  • Radioactive [γ-32P]ATP hydrolysis assay for highest sensitivity

Proteolytic activity assay:

• Fluorescent substrate approach:

  • FITC-labeled casein (general protease substrate)

  • Custom fluorescent peptides based on known FtsH substrates

  • Reaction conditions: Same buffer as ATPase assay plus 2 mM ATP and fluorescent substrate

  • Monitoring fluorescence increase as degradation proceeds

• Model substrate approach:

  • Using known FtsH substrates from other bacteria (e.g., σ32, λ CI repressor)

  • SDS-PAGE visualization of substrate degradation over time

  • Western blot quantification for more sensitive detection

• Controls and validations:

  • ATP-omission control

  • Zinc chelation control (EDTA treatment)

  • Temperature and pH optimization

  • Catalytically inactive mutant as negative control

What are the critical controls needed when studying FtsH function in vivo in M. aquaeolei?

When investigating FtsH function within M. aquaeolei cells, several critical controls must be implemented:

• Genetic manipulation controls:

  • Complementation study: Restoration of wild-type phenotype when FtsH is reintroduced to knockout/knockdown strains

  • Catalytic mutants: HEXXH→AEXXA mutation to eliminate proteolytic activity while maintaining ATPase function

  • Walker A/B mutants: K→A mutation in Walker A or E→Q in Walker B to eliminate ATP hydrolysis

  • Expression level verification: qRT-PCR and Western blotting to confirm appropriate expression levels

• Physiological response controls:

  • Multiple stress conditions: Test responses to temperature, osmotic stress, nutrient limitation, and oxidative stress

  • Growth phase considerations: Separate analysis of exponential vs. stationary phase cells

  • Adaptation period: Allow sufficient time for cells to adapt to FtsH depletion (avoid acute effects)

• Omics approach controls:

  • Time-course analysis: Capture dynamic changes rather than single time points

  • Multiple biological replicates: Minimum of three independent experiments

  • Technical controls for specificity: Compare FtsH depletion effects to depletion of unrelated proteases

  • Spatial resolution: Separate analysis of membrane vs. cytosolic fractions

• Substrate validation controls:

  • Direct vs. indirect effects: Distinguish primary FtsH substrates from secondary effects

  • Pulse-chase experiments: Determine actual protein half-lives

  • In vitro confirmation: Validate candidate substrates with purified components

These controls are particularly important given M. aquaeolei's metabolic versatility and ability to adapt to various environmental conditions , which could confound interpretation of FtsH function if not properly controlled.

How might FtsH contribute to the hydrocarbon degradation capabilities of M. aquaeolei?

M. aquaeolei is known for its ability to degrade complex hydrocarbons, including octane oxidation and cyclohexanol degradation . FtsH may contribute to these capabilities through several mechanisms:

• Regulatory control:

  • FtsH likely regulates the abundance of transcription factors that control hydrocarbon degradation pathways

  • It may degrade repressors of hydrocarbon metabolism genes under inducing conditions

  • Could control the half-life of key enzymes to fine-tune metabolic flux

• Quality control during hydrocarbon stress:

  • Hydrocarbons can damage membrane proteins through direct solubilization effects

  • FtsH could remove damaged membrane proteins to maintain membrane integrity

  • May be essential for tolerance to toxic intermediates of hydrocarbon metabolism

• Metabolic adaptation mechanisms:

  • FtsH may facilitate rapid switching between different carbon sources

  • Could regulate the abundance of electron transport chain components during transitions between aerobic and anaerobic metabolism

  • May control membrane composition to adjust permeability for different hydrocarbons

• Integration with other systems:

  • Potential coordination with fatty aldehyde dehydrogenases (FAldDHs) that are known to be important in M. aquaeolei lipid metabolism

  • Possible regulation of the four variations of the TCA cycle present in M. aquaeolei

  • May interact with stress response pathways activated during hydrocarbon metabolism

Research approaches to investigate these connections could include comparative proteomics of wild-type vs. FtsH-depleted strains grown on different hydrocarbon substrates, combined with metabolic flux analysis to identify pathway alterations.

What insights could structural studies provide about M. aquaeolei FtsH that aren't obtainable through biochemical methods alone?

Structural studies would offer unique insights into M. aquaeolei FtsH that complement biochemical approaches:

• Substrate binding pocket architecture:

  • High-resolution structures could reveal adaptations specific to M. aquaeolei's ecological niche

  • Comparison with structures like the fatty aldehyde binding pocket in related enzymes would highlight functional specializations

  • Visualization of potential binding sites for hydrocarbon-related substrates

• Conformational dynamics during ATP hydrolysis:

  • Cryo-EM structures in different nucleotide-bound states could capture the conformational changes driving substrate processing

  • Single-molecule FRET studies could reveal the coordination between ATP hydrolysis and proteolytic activities

  • Molecular dynamics simulations could predict how conformational changes propagate through the protein

• Hexameric assembly specializations:

  • Structural details of the oligomeric interfaces might reveal adaptations for stability under extreme conditions

  • Potential identification of marine-specific features not present in mesophilic FtsH homologs

  • Visualization of how membrane association influences hexamer formation and stability

• Substrate translocation pathway:

  • Structures with substrate analogs could map the precise path of substrate proteins from recognition to proteolysis

  • Identification of key residues forming the central pore and their role in substrate discrimination

  • Visualization of how ATP hydrolysis couples to mechanical force generation for substrate unfolding

Similar structural studies of fatty aldehyde dehydrogenase enzymes from M. aquaeolei have provided valuable insights into substrate binding and catalysis mechanisms , suggesting that structural analysis of FtsH would be equally informative.

How might systems biology approaches enhance our understanding of FtsH's role in M. aquaeolei's ecological adaptations?

• Multi-omics integration:

  • Combined analysis of transcriptomics, proteomics, and metabolomics data from wild-type and FtsH-depleted strains

  • Identification of regulatory networks centered on FtsH activity

  • Mapping of metabolic consequences of FtsH dysfunction across central carbon metabolism

• Ecological context modeling:

  • Simulation of how FtsH activity changes across different environmental conditions relevant to M. aquaeolei's marine habitats

  • Prediction of how FtsH contributes to the "opportunotroph" lifestyle described for M. aquaeolei

  • Modeling of FtsH's role in transitions between different metabolic states

• Comparative genomics and evolution:

  • Analysis of FtsH sequence conservation across Marinobacter species from different marine environments

  • Identification of specific adaptations in M. aquaeolei FtsH compared to terrestrial bacteria

  • Investigation of potential horizontal gene transfer events that might have shaped FtsH function

• Protein interaction networks:

  • Identification of the complete FtsH interactome under different growth conditions

  • Mapping of how FtsH interacts with other quality control systems

  • Network analysis to identify critical nodes that connect FtsH activity to stress responses

Such systems-level analysis would provide a comprehensive understanding of how FtsH contributes to M. aquaeolei's remarkable ability to thrive in diverse marine environments, from the water column to the deep sea, and in association with hydrothermal plume particles and marine snow .

What are common pitfalls in recombinant FtsH expression and how can they be addressed?

Researchers frequently encounter several challenges when expressing recombinant FtsH:

When expressing M. aquaeolei proteins in E. coli, researchers have found that supplementing with 6% trehalose in storage buffers can significantly improve stability, as demonstrated with other recombinant proteins from this organism . Additionally, specialized E. coli strains like Rosetta(DE3) that supply rare tRNAs may improve expression of M. aquaeolei proteins, as they have a different codon usage pattern compared to E. coli.

How can I distinguish between direct and indirect effects of FtsH depletion in functional studies?

Distinguishing direct from indirect effects requires a multi-faceted experimental approach:

• Temporal resolution strategies:

  • Acute vs. chronic depletion: Use inducible depletion systems to observe immediate effects

  • Time-course analysis: Direct substrates should accumulate earlier than indirect effects

  • Pulse-chase experiments: Directly measure protein stability changes upon FtsH depletion

• Substrate validation approaches:

  • In vitro degradation: Confirm direct substrate processing with purified components

  • Substrate mutations: Modify potential recognition motifs to prevent degradation

  • Co-immunoprecipitation: Detect physical interaction between FtsH and substrates

  • Crosslinking: Capture transient interactions during substrate processing

• Catalytic mutant comparisons:

  • Proteolytically inactive FtsH: Maintains structure and ATP hydrolysis but lacks proteolysis

  • ATPase-deficient FtsH: Maintains structure and substrate binding but lacks unfolding activity

  • Comparison of phenotypes helps separate scaffolding from enzymatic functions

• Systems-level controls:

  • Parallel analysis of other AAA+ protease depletion (e.g., Lon, ClpXP) to identify protease-specific effects

  • Metabolic flux analysis to trace physiological consequences through biochemical pathways

  • Mathematical modeling to predict network effects of protein stabilization

This comprehensive approach is particularly important for M. aquaeolei given its complex metabolic network with four variations of the TCA cycle and multiple hydrocarbon degradation pathways , where perturbation of one regulatory node could have far-reaching consequences.

What analytical techniques are most informative for studying FtsH-substrate interactions in the context of M. aquaeolei's unique physiology?

Several specialized analytical techniques can provide unique insights into FtsH-substrate interactions within M. aquaeolei's physiological context:

• Advanced biophysical methods:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map conformational changes upon substrate binding

  • Surface plasmon resonance (SPR) to determine binding kinetics for different substrates

  • Microscale thermophoresis (MST) to measure affinity constants under varying conditions

  • Single-molecule FRET to observe real-time conformational dynamics during processing

• Specialized proteomics approaches:

  • N-terminomics to identify specific cleavage sites in FtsH substrates

  • Ubiquitin-like protein identification (TULIP) to tag and identify degradation intermediates

  • Thermal proteome profiling (TPP) to detect proteins stabilized by FtsH interactions

  • Crosslink-MS to map interacting regions between FtsH and substrates

• In situ visualization techniques:

  • FRAP (Fluorescence Recovery After Photobleaching) to assess FtsH mobility in membranes

  • Super-resolution microscopy to localize FtsH activity centers within cells

  • Split fluorescent protein complementation to visualize substrate interactions in live cells

  • Proximity labeling (BioID, APEX) to identify neighboring proteins in native conditions

• Environmental simulation approaches:

  • Microfluidic devices to mimic changing marine conditions

  • Recreating oil-water interfaces to study FtsH function during hydrocarbon degradation

  • High-pressure chambers to simulate deep-sea conditions where Marinobacter species are found

  • Time-resolved omics during environmental transitions to capture dynamic FtsH contributions

These techniques should be applied with consideration of M. aquaeolei's natural environmental conditions, including its adaptations to hydrocarbon-rich environments, variable oxygen availability, and temperature fluctuations . Such environmentally relevant analyses will provide the most meaningful insights into FtsH's role in this organism's remarkable ecological adaptability.