Recombinant Photobacterium profundum Chaperone protein hscA homolog (hscA), partial

What is the HscA chaperone protein in Photobacterium profundum and what is its primary function?

The HscA protein in Photobacterium profundum is a specialized member of the Hsp70 family of molecular chaperones that plays a critical role in bacterial stress responses. Similar to other bacterial chaperones like DnaK (Hsp70), GroEL (Hsp60), and ClpB (Hsp100), HscA functions within complex molecular networks to maintain cellular protein homeostasis under stressful conditions such as high pressure, temperature fluctuations, or oxidative stress .

In P. profundum, which thrives in deep-sea environments, HscA is particularly important for adaptation to high hydrostatic pressure environments. Its specialized function likely includes facilitating proper protein folding and preventing aggregation under pressure stress, similar to how other chaperones in this piezophilic bacterium have adapted for high-pressure functionality .

Unlike general chaperones, HscA appears to have more specialized functions, potentially involved in iron-sulfur cluster assembly and the maturation of iron-sulfur proteins, which are essential for numerous cellular processes including respiration and DNA repair.

How does the HscA homolog in P. profundum differ from those found in mesophilic bacteria?

The HscA homolog in P. profundum exhibits several adaptations that distinguish it from mesophilic counterparts, reflecting specialization for life under high hydrostatic pressure. While specific sequence comparisons for HscA aren't provided in the search results, we can infer potential differences based on other pressure-adapted proteins in P. profundum:

• Structural modifications: Like other pressure-adapted proteins in P. profundum, HscA likely contains amino acid substitutions that maintain conformational flexibility under pressure. This may include reduced hydrophobic core packing and increased surface hydrophilicity.

• Functional adaptation: The HscA homolog in P. profundum likely exhibits pressure-optimized activity, similar to how its flagellar motility systems show enhanced functionality at elevated pressures (30 MPa) compared to atmospheric pressure .

• Co-chaperone interactions: The interactions between HscA and its co-chaperones may differ from mesophilic systems. Similar to how M. mazei DnaK shows functional similarities to E. coli DnaK in vitro but significant differences in vivo, HscA likely has unique interactional profiles adapted to deep-sea conditions .

What expression systems are most suitable for producing recombinant P. profundum HscA?

For successful expression of recombinant P. profundum HscA, several expression systems can be considered based on methodologies used for similar deep-sea bacterial proteins:

When expressing the recombinant protein, consider using approaches similar to those employed for other P. profundum proteins. For instance, cultures can be grown in 2216 medium at 16°C as described for P. profundum SS9 strains . For homologous expression, high-pressure growth can be performed anaerobically at 16°C in 2216 medium supplemented with 20 mM glucose and 100 mM HEPES buffer (pH 7.5) .

What approaches can be used to characterize the pressure-dependent activity of recombinant HscA from P. profundum?

Characterizing the pressure-dependent activity of recombinant HscA requires specialized techniques to maintain and monitor protein function under high hydrostatic pressure:

• High-pressure spectroscopic analysis: Utilize high-pressure optical cells compatible with UV-visible, fluorescence, or CD spectroscopy to monitor conformational changes and activity at increasing pressure points (0.1-150 MPa).

• ATPase activity assays under pressure: Measure ATPase activity using a high-pressure stopped-flow apparatus to determine how pressure affects the ATP hydrolysis cycle. Similar to experiments with M. mazei DnaK, measure how co-chaperones affect this activity .

• Protein refolding assays: Assess chaperone activity using protein substrates like luciferase under varying pressure conditions. This approach was successfully used for archaeal DnaK characterization and can be adapted for HscA.

• High-pressure microscopic chamber: Similar to the approach used for studying P. profundum flagellar motility, develop assays using a high-pressure cell fixed to an inverted microscope with temperature and pressure monitoring via transducers .

For these methodologies, pressure vessels similar to those described for P. profundum growth (stainless-steel pressure vessels with sealed polyethylene transfer pipettes) could be adapted for biochemical assays .

How can we investigate the interaction between HscA and its co-chaperones under high-pressure conditions?

Investigating HscA-co-chaperone interactions under high pressure requires specialized techniques:

• High-pressure pull-down assays: Perform co-immunoprecipitation or pull-down assays in a pressure-stable buffer system within pressure vessels to identify pressure-dependent interactions.

• FRET-based interaction studies: Develop fluorescently labeled HscA and co-chaperones and measure Förster resonance energy transfer in a high-pressure optical cell to quantify interaction dynamics under pressure.

• Surface plasmon resonance under pressure: Modified SPR systems capable of operating under pressure can measure binding kinetics between HscA and its co-chaperones at various pressure points.

• Cross-linking coupled with mass spectrometry: Perform protein cross-linking at different pressures followed by digestion and mass spectrometry to map interaction interfaces.

Based on studies of other chaperones, it would be valuable to investigate whether interactions between HscA and its co-chaperones are enhanced at 30 MPa (the pressure optimum for P. profundum SS9) compared to atmospheric pressure, similar to how DnaK from M. mazei shows different co-chaperone preferences and functional outcomes .

What genetic approaches can be used to study the in vivo function of hscA in P. profundum?

To study the in vivo function of hscA in P. profundum, several genetic approaches can be employed based on successful methods used for other genes in this organism:

• Gene disruption mutants: Create hscA disruption mutants using suicide vector systems like pMUT100, similar to the approach used for recD gene disruption . Specific primers can be designed to amplify internal portions of the hscA gene, which can then be cloned into vectors like pCR2.1 and subcloned into pMUT100 for integration into the P. profundum genome.

• Complementation assays: Test functionality by complementing the disrupted gene with wild-type or mutated variants. This can be performed using conjugation methods as described for P. profundum, where exconjugants are selected on appropriate media incubated at 15°C for 3-5 days .

• Gene expression analysis: Monitor hscA expression under different pressure conditions using β-galactosidase reporter constructs, similar to how flagellin gene expression was studied in P. profundum .

• Conditional mutants: For essential genes, develop pressure-sensitive or temperature-sensitive conditional mutants to study function under controlled conditions.

Table of expected phenotypes for hscA mutants:

How does the domain structure of P. profundum HscA compare to other bacterial chaperones?

The domain structure of P. profundum HscA likely follows the conserved architecture of Hsp70 family chaperones but with specific adaptations for pressure tolerance:

• N-terminal nucleotide-binding domain (NBD): Contains the ATPase activity essential for chaperone function, likely with pressure-specific substitutions that maintain ATP binding and hydrolysis under high pressure.

• Substrate-binding domain (SBD): Responsible for binding unfolded protein substrates, potentially with adaptations that maintain substrate affinity under pressure.

• C-terminal lid domain: Regulates substrate access to the binding pocket in an ATP-dependent manner.

Interestingly, the pressure adaptation strategy may differ from that seen in ClpB, where the middle domain plays crucial roles in interactions with DnaK and in stabilizing the hexameric structure . In HscA, pressure adaptations may instead focus on maintaining domain flexibility and preventing pressure-induced dissociation of substrate complexes.

What experimental approaches can determine how HscA contributes to pressure adaptation in P. profundum?

To determine HscA's contribution to pressure adaptation, several experimental approaches can be employed:

• Comparative growth assays: Compare growth characteristics of wild-type and hscA mutant strains under varying pressure conditions, similar to motility phenotype characterization in flagellin mutants . This can be performed in pressure vessels at various pressures (0.1-50 MPa).

• Protein aggregation assays: Measure accumulation of protein aggregates in wild-type versus hscA mutant strains under pressure stress using biochemical fractionation or fluorescent reporters of protein aggregation.

• Transcriptome and proteome analysis: Compare global gene expression and protein levels between wild-type and hscA mutants under different pressure conditions to identify connected pathways.

• Synthetic lethality screens: Identify genes that become essential in an hscA-deficient background specifically under pressure, revealing functional relationships.

• In vitro reconstitution: Reconstitute protein folding systems with and without HscA and test functionality under pressure using model substrates, similar to luciferase renaturation assays used for archaeal DnaK .

These approaches can reveal whether HscA has specialized functions in pressure adaptation beyond the general chaperone activities typical of Hsp70 family proteins.

How can researchers distinguish between the roles of different chaperone systems in P. profundum pressure adaptation?

Distinguishing between the roles of different chaperone systems in P. profundum pressure adaptation requires methodical approaches:

Analysis should focus on identifying whether HscA functions in specialized pathways (like iron-sulfur cluster assembly) under pressure or has broader roles similar to other chaperones. The approach could be similar to how flagellar systems in P. profundum were characterized, where two distinct systems (polar and lateral) with different pressure adaptations were identified .

How can recombinant P. profundum HscA be utilized to improve protein expression of difficult targets?

Recombinant P. profundum HscA could improve expression of difficult protein targets through several applications:

• Co-expression systems: Develop expression vectors for co-expression of P. profundum HscA with difficult-to-express proteins, particularly those requiring iron-sulfur cluster assembly or pressure adaptation for proper folding.

• In vitro protein folding: Establish in vitro refolding protocols using purified HscA, potentially allowing recovery of proteins from inclusion bodies under conditions mimicking high pressure.

• Engineered expression hosts: Create specialized E. coli strains expressing P. profundum HscA and other pressure-adapted chaperones for expression of proteins that normally require high pressure for proper folding.

• Cell-free protein synthesis: Develop cell-free protein synthesis systems supplemented with P. profundum HscA and co-chaperones, potentially operating under moderate pressure to facilitate folding of challenging proteins.

These applications could be particularly valuable for expression of proteins from other deep-sea organisms, metalloproteins requiring specialized assembly factors, or proteins with complex folding requirements that current systems cannot adequately address.

What are the current technical challenges in studying pressure effects on HscA function?

Several technical challenges exist in studying pressure effects on HscA function:

• High-pressure equipment limitations: Most biochemical and structural analysis equipment is not designed to operate under high pressure, requiring specialized adaptations or custom-built apparatus like the high-pressure microscopic chamber described for motility studies .

• Protein stability during decompression: Analyzing proteins after pressure treatment requires decompression, which may alter protein states or interactions that existed under pressure.

• Time-resolved measurements: Capturing dynamic processes (like ATP hydrolysis cycles or substrate binding kinetics) under pressure requires sophisticated time-resolved techniques that can operate under pressure.

• Structural analysis under pressure: Obtaining high-resolution structural information (through crystallography or cryo-EM) under pressure remains extremely challenging.

• Genetic manipulation at high pressure: Performing genetic manipulations and selections under constant high pressure requires specialized equipment not readily available in most laboratories.

Addressing these challenges requires interdisciplinary approaches combining expertise in high-pressure biophysics, protein biochemistry, and bacterial genetics, potentially using systems like the high-pressure direct visualization system described for motility studies as a starting point .

How might the study of P. profundum HscA inform our understanding of protein folding in extreme environments?

The study of P. profundum HscA provides valuable insights into protein folding in extreme environments:

• Pressure adaptation principles: By comparing pressure-adapted HscA with mesophilic homologs, researchers can identify specific structural features that enable protein function under high hydrostatic pressure, potentially revealing general principles of pressure adaptation.

• Evolutionary adaptations: Understanding how chaperone systems have evolved in deep-sea bacteria reveals evolutionary strategies for adaptation to extreme environments and may uncover novel mechanisms of protein quality control.

• Crossover with other extremes: P. profundum grows at low temperatures (16°C) and high pressures, allowing investigation of how chaperone systems manage the combined challenges of cold and pressure—environments that both restrict protein dynamics but in different ways.

• Molecular ecology insights: Studies of specialized chaperones like HscA can inform how protein homeostasis mechanisms contribute to niche adaptation and ecological distribution of microorganisms, similar to how flagellar motility adaptations were shown to be critical for P. profundum's deep-sea lifestyle .

These insights have broad implications beyond deep-sea biology, potentially informing fields from astrobiology (studying potential life in high-pressure extraterrestrial environments) to biotechnology (designing pressure-stable enzymes for industrial applications).

Are there conflicting reports about the role of HscA in iron-sulfur cluster assembly under high pressure?

While the search results don't specifically address conflicting reports about HscA in iron-sulfur cluster assembly under high pressure, this question addresses potential research controversies:

Currently, there appear to be knowledge gaps rather than direct conflicts in our understanding of HscA function under pressure. The specialized role of HscA in iron-sulfur cluster assembly is well-established in model organisms like E. coli, but its specific adaptations and potential modified functions in piezophiles like P. profundum remain underexplored.

A critical research question is whether HscA maintains its specialized role under high pressure or adopts broader chaperone functions. This parallels observations in other systems where specialized functions can be altered under extreme conditions. For example, in P. profundum, the ClpB protein appears to have adopted functions similar to ClpV in the Type VI Secretion System, demonstrating functional adaptability of chaperones in specialized environments .

Researchers should investigate whether HscA's substrate specificity changes under pressure and whether it can compensate for other chaperone systems under stress conditions, similar to the functional overlap observed between different flagellar systems in P. profundum .

How does horizontal gene transfer influence the evolution of chaperone systems in deep-sea bacteria?

Horizontal gene transfer (HGT) likely plays a significant role in the evolution of chaperone systems in deep-sea bacteria like P. profundum:

• Evidence of high HGT rates: Deep-sea bacterial communities, including vibrios like P. profundum, show evidence of high rates of horizontal gene transfer among strains co-existing in the water column . This suggests chaperone genes could be transferred between different deep-sea species.

• Mosaic chaperone systems: The genomic and metabolic diversity observed in vibrios may extend to their chaperone systems, potentially resulting in mosaic arrangements of chaperone genes with different evolutionary origins and pressure adaptations.

• Selective pressure on transferred genes: Chaperone genes acquired through HGT would face strong selective pressure to maintain compatibility with existing cellular machinery while providing adaptive advantages for high-pressure environments.

• Core versus flexible genome dynamics: Chaperone genes may exist in both the core genome (highly conserved) and the flexible genome (more variable between strains) of P. profundum, with different rates of turnover and evolution . Understanding which category HscA falls into could reveal its evolutionary importance.

This understanding has implications for how we interpret the evolution of pressure adaptation in deep-sea bacteria and could inform bioprospecting efforts for novel chaperones with unique properties.

What is the relationship between HscA and the Type VI Secretion System in P. profundum?

While the search results don't directly address a relationship between HscA and the Type VI Secretion System (T6SS) in P. profundum, this question explores potential connections between different stress-response and virulence systems:

In several bacteria, the Type VI Secretion System requires specialized chaperones for assembly and function. For example, in Francisella tularensis, ClpB serves as a functional homolog of ClpV, providing energy for depolymerization of the IglA-IglB sheath . Similar functional relationships might exist in P. profundum.

P. profundum is closely related to Vibrio species, and as noted in search result , vibrios display great genetic and metabolic diversity. Given that some Vibrio species like V. cholerae possess T6SS that can function partially without ClpV , it's possible that P. profundum might utilize alternative chaperones like HscA to support T6SS function under high pressure.

Investigation could focus on:

• Determining if HscA expression correlates with T6SS activity under different pressure conditions

• Testing if HscA interacts with T6SS components using co-immunoprecipitation or bacterial two-hybrid assays

• Examining whether hscA mutation affects T6SS sheath dynamics or secretion activity

This research direction could reveal novel functional relationships between specialized chaperones and secretion systems in deep-sea bacteria.