Recombinant Nitrosomonas europaea Chaperone protein ClpB (clpB), partial

What is the molecular structure of Nitrosomonas europaea ClpB?

N. europaea ClpB belongs to the AAA+ protein family and likely shares the conserved structural organization seen in bacterial ClpB proteins. The protein forms functional hexamers of identical monomers. Each monomer comprises four domains: an N-terminal domain connected via a conserved linker, the first nucleotide binding domain (NBD-1) containing the flexible middle (M) domain, and a second NBD (NBD-2) . The M-domain is particularly important as it mediates interactions with the DnaK chaperone system and stabilizes the hexameric structure . Unlike human ClpB/Skd3, bacterial ClpB contains a characteristic coiled-coil domain and lacks the ankyrin-repeat domain found in metazoan homologs .

How does N. europaea ClpB differ from ClpB in other bacterial species?

While the search results don't specifically address differences between N. europaea ClpB and other bacterial ClpB proteins, it's important to note that ClpB is highly conserved across bacterial species . Given N. europaea's specialized metabolism as an ammonia oxidizer and its complex genome with numerous repetitive elements , its ClpB may have adaptations specific to the stresses encountered in its ecological niche. Researchers should perform sequence alignments and structural comparisons with well-characterized ClpB proteins (such as E. coli ClpB) to identify any unique features that might relate to N. europaea's specific lifestyle.

What is the primary function of ClpB in Nitrosomonas europaea?

Based on the conserved function of ClpB across bacteria, N. europaea ClpB likely serves as a molecular chaperone with disaggregase activity, critical for survival under various stress conditions, particularly heat shock . The protein would work cooperatively with the DnaK system to recover functional proteins from aggregates formed during stress . This function would be particularly important for N. europaea, which as an obligate chemolithoautotroph has specific metabolic constraints and requires efficient protein quality control systems to maintain cellular homeostasis under environmental stresses .

How does the ATP hydrolysis mechanism of N. europaea ClpB enable protein disaggregation?

The disaggregation mechanism likely follows the conserved pattern of bacterial ClpB proteins. The protein's two nucleotide binding domains (NBD-1 and NBD-2) couple their ATPase activity to drive the translocation of unfolded protein substrates through the axial channel of the hexameric structure . This process provides the mechanical force necessary to extract individual polypeptides from aggregates. The energy derived from ATP hydrolysis powers conformational changes in the hexamer that enable the threading of substrate proteins through the central channel, effectively dissolving protein aggregates formed during stress conditions.

What expression systems are most effective for producing recombinant N. europaea ClpB?

For recombinant expression of N. europaea ClpB, E. coli-based expression systems are likely most appropriate. When designing an expression strategy, researchers should consider:

• Selection of an appropriate E. coli strain (BL21(DE3), Rosetta, or Arctic Express for potentially challenging proteins)

• Optimization of codon usage for efficient expression

• Use of a vector with an inducible promoter (such as T7) for controlled expression

• Inclusion of affinity tags (His6, GST, or MBP) to facilitate purification

• Temperature and induction conditions optimization to ensure proper folding of the hexameric complex

Given the large size of ClpB and its oligomeric nature, lower induction temperatures (16-25°C) and longer induction times may improve soluble protein yields.

What purification strategy provides the highest yield of functional N. europaea ClpB?

A multi-step purification strategy is recommended for obtaining high-purity, functional recombinant ClpB:

• Initial capture using affinity chromatography (IMAC for His-tagged constructs)

• Ion exchange chromatography to separate ClpB from contaminants with different charge properties

• Size exclusion chromatography to isolate properly formed hexamers and remove aggregates

Throughout purification, include ATP or non-hydrolyzable ATP analogs in buffers to stabilize the oligomeric state. Ensure that the purification buffers maintain physiological pH (7.0-8.0) and include reducing agents to prevent oxidation of cysteine residues that might affect protein function.

How can the ATPase activity of purified recombinant N. europaea ClpB be assessed?

The ATPase activity of purified recombinant N. europaea ClpB can be assessed using several approaches:

• Malachite green assay: Measures inorganic phosphate released during ATP hydrolysis, providing a colorimetric readout

• Coupled enzymatic assay: Utilizes pyruvate kinase and lactate dehydrogenase to couple ATP hydrolysis to NADH oxidation, which can be monitored spectrophotometrically

• Isothermal titration calorimetry: Provides detailed thermodynamic parameters of ATP binding and hydrolysis

Activity should be measured both in the basal state and in the presence of substrate proteins or peptides known to stimulate ClpB ATPase activity. Comparison with well-characterized ClpB proteins (e.g., from E. coli) would provide valuable reference points.

How can recombinant N. europaea ClpB be used to study stress response in ammonia-oxidizing bacteria?

Recombinant N. europaea ClpB can serve as a valuable tool for investigating stress responses in ammonia-oxidizing bacteria through several approaches:

• In vitro disaggregation assays using aggregated N. europaea proteins to assess ClpB's substrate specificity

• Protein-protein interaction studies to identify the components of the N. europaea chaperone network

• Structure-function analyses to determine adaptations specific to ammonia oxidizers

• Comparative studies with ClpB from other bacteria to identify unique features related to N. europaea's specialized metabolism

• Development of ClpB inhibitors to probe its role in N. europaea stress tolerance

Such studies could illuminate how this specialized bacterium maintains cellular homeostasis despite its metabolic constraints as an obligate chemolithoautotroph .

What role might ClpB play in N. europaea's adaptation to environmental stressors encountered during wastewater treatment?

N. europaea plays important roles in wastewater treatment through nitrification . In these environments, the bacteria encounter stressors including temperature fluctuations, pH changes, and oxidative stress. ClpB likely contributes significantly to N. europaea's survival under these conditions by:

• Restoring functional proteins from stress-induced aggregates, particularly after heat shock

• Maintaining the integrity of key metabolic enzymes involved in ammonia oxidation

• Supporting recovery from oxidative stress generated during nitrification

• Facilitating adaptation to fluctuating conditions in treatment systems

Research utilizing recombinant ClpB could help optimize nitrification processes by identifying conditions that maintain optimal chaperone function and bacterial survival.

How does the genomic context of clpB in N. europaea inform its regulation and function?

The genomic context of the clpB gene in N. europaea may provide insights into its regulation and functional associations. N. europaea's genome has several interesting features, including complex repetitive elements constituting approximately 5% of the genome and 85 predicted insertion sequence elements in eight different families . Analysis of the genomic neighborhood of clpB could reveal:

• Co-regulated genes that might form a stress-responsive regulon

• Transcription factor binding sites that control clpB expression

• Potential operon structures that suggest functional relationships

• Evolutionary history through comparative genomics with other ammonia oxidizers

Could N. europaea ClpB be involved in Type VI secretion system function similar to what has been observed in Francisella?

An intriguing research question emerges from the observation that ClpB can regulate secretion of bacterial effector molecules related to Type VI secretion systems (T6SS) in some bacteria, notably Francisella tularensis . In F. tularensis, ClpB serves as a functional homolog of ClpV, providing energy through ATP hydrolysis for depolymerization of the T6SS sheath .

While N. europaea is not known to possess a T6SS, investigation of potential non-canonical roles of its ClpB in secretion systems or other cellular processes beyond protein disaggregation would represent an advanced research direction. This could involve:

• Protein interaction studies to identify non-chaperone binding partners

• Localization studies to determine if ClpB concentrates at specific cellular sites

• Functional assays examining potential roles in membrane-associated processes

• Comparative studies with ClpB proteins known to have dual functions

What structural adaptations might exist in N. europaea ClpB related to the bacterium's unusual metabolism?

N. europaea has a highly specialized metabolism as an obligate chemolithoautotroph, deriving energy solely from ammonia oxidation and fixing carbon dioxide for biomass production . This unusual lifestyle might be reflected in adaptations of its stress response proteins, including ClpB. Advanced structural studies could examine:

Such studies would require advanced structural biology techniques including X-ray crystallography, cryo-electron microscopy, and hydrogen-deuterium exchange mass spectrometry.