Recombinant Nitrosomonas europaea Chaperone surA (surA)

What is the genomic context of the surA gene in Nitrosomonas europaea?

The surA gene in Nitrosomonas europaea is located within its single circular chromosome of 2,812,094 bp . Based on comparative genomic analyses, the surA gene likely resides in a cluster related to outer membrane protein assembly and maintenance, consistent with its function in other gram-negative bacteria. The complete genome sequencing of N. europaea has enabled precise identification of the surA gene locus and its genetic neighborhood, facilitating targeted genetic manipulation for recombinant expression.

How does SurA function in Nitrosomonas europaea compared to other chaperones?

SurA functions as a periplasmic chaperone in N. europaea, assisting in the folding and assembly of outer membrane proteins. Unlike the heat shock proteins GroEL (NE0028) and DnaK (NE1949) that are upregulated under stress conditions , SurA specifically facilitates the biogenesis of β-barrel outer membrane proteins. The specialized function of SurA becomes particularly important in N. europaea as it maintains membrane integrity during ammonia oxidation, which can generate reactive intermediates that potentially damage membrane structures.

What is the relationship between SurA and stress response in Nitrosomonas europaea?

While not directly identified in the stress response proteomic studies, SurA likely plays a significant role in N. europaea's adaptation to various stressors. Under salt stress conditions, N. europaea exhibits production of osmolytes, regulation of cell permeability, and oxidative stress responses . As a chaperone involved in maintaining outer membrane integrity, SurA presumably contributes to cell permeability regulation and protection against environmental stressors by ensuring proper assembly of protective outer membrane proteins. Its function would complement the documented upregulation of stress proteins like GroEL and DnaK observed during chloroform exposure .

How do different expression systems compare for recombinant production of N. europaea SurA?

The recombinant production of N. europaea SurA can be approached through several expression systems, each with distinct advantages:

For most laboratory applications, the E. coli pET system provides sufficient yields, though careful optimization of induction conditions is necessary to minimize inclusion body formation. The synthetic arabinose-inducible promoter system described for S. islandicus could be adapted for N. europaea proteins, potentially providing better folding conditions for this specialized chaperone.

What structural and functional domains characterize N. europaea SurA?

N. europaea SurA likely shares the canonical domain organization of other bacterial SurA proteins:

• N-terminal domain: Critical for chaperone activity and substrate binding

• Two peptidyl-prolyl isomerase (PPIase) domains: Facilitate proper protein folding

• C-terminal domain: Essential for periplasmic targeting and function

Structural analyses would reveal specific adaptations of N. europaea SurA to its unique ecological niche as an ammonia oxidizer. These adaptations may include modified substrate specificity for outer membrane proteins involved in ammonia transport or detoxification of nitrification intermediates.

How does salt stress affect SurA expression and function in N. europaea?

N. europaea demonstrates distinct responses to increasing salt concentrations, with significant metabolic modifications observed at conductivities from 5 to 30 mS cm⁻¹ . While direct evidence for SurA's role is not available in the search results, its function in outer membrane protein assembly suggests it would be critically involved in the cell permeability regulation observed during salt stress adaptation. Proteomic analyses of N. europaea exposed to salinity revealed regulation of cell permeability mechanisms , which would likely involve SurA-dependent pathways for outer membrane protein biogenesis and maintenance.

What purification strategies yield optimal purity and activity of recombinant N. europaea SurA?

A systematic purification protocol for recombinant N. europaea SurA would typically include:

• Affinity chromatography: Utilizing a hexahistidine (6×His) tag system as implemented in the pSeSD and pEXA vectors

• Ion exchange chromatography: To separate charged contaminants

• Size exclusion chromatography: For final polishing and buffer exchange

Critical considerations include:

• Inclusion of protease inhibitors throughout purification to prevent degradation

• Maintaining reducing conditions (typically 1-5 mM DTT) to preserve native disulfide bonding

• Buffer optimization to mimic the periplasmic environment (pH 7.0-7.5)

• Incorporation of removal tags for the hexahistidine sequence using engineered protease sites

This approach typically yields >95% pure protein with preserved chaperone activity.

How can the chaperone activity of recombinant N. europaea SurA be assayed in vitro?

The functional assessment of recombinant N. europaea SurA can be performed through multiple complementary assays:

• Protein aggregation prevention assay: Measuring the ability of SurA to prevent aggregation of model substrates (such as citrate synthase or rhodanese) under denaturing conditions using light scattering techniques

• β-barrel protein folding assay: Monitoring SurA-assisted folding of outer membrane proteins like OmpA or OmpF using intrinsic tryptophan fluorescence or circular dichroism

• Thermal stability assessment: Determining the protective effect of SurA on substrate proteins during thermal denaturation using differential scanning fluorimetry

These functional assays should be coupled with structural verification through circular dichroism spectroscopy to confirm proper folding of the recombinant SurA itself.

What experimental approaches can determine SurA's role in N. europaea biofilm formation?

Given that N. europaea demonstrates enhanced biofilm formation when co-cultured with Pseudomonas aeruginosa , investigating SurA's role in this process would be valuable. Experimental approaches should include:

• Generation of surA knockout or conditional expression mutants in N. europaea

• Flow cell biofilm formation assays comparing wild-type and surA-modified strains

• Confocal microscopy analysis of biofilm architecture using fluorescently labeled strains

• Complementation studies using recombinant SurA

The dual-species biofilm experimental protocol described for N. europaea and P. aeruginosa provides an excellent framework for such studies. This would involve pre-establishing P. aeruginosa biofilms for 3 days before introducing wild-type or surA-modified N. europaea, followed by confocal imaging on days 3 and 5 after inoculation.

How can recombinant N. europaea SurA contribute to synthetic biology applications?

Recombinant N. europaea SurA offers unique opportunities for synthetic biology applications, particularly in creating engineered microorganisms for environmental applications. The chaperone could be employed to facilitate proper folding and assembly of designer outer membrane proteins in synthetic microbial consortia designed for enhanced nitrification processes. Such applications could utilize the synthetic arabinose-inducible promoter systems described for hyperthermophilic archaea , adapted for controlled expression in environmental bioprocesses.

What is the potential role of SurA in N. europaea's adaptation to environmental pollutants?

N. europaea demonstrates distinct transcriptional responses to environmental pollutants such as chloroform, with upregulation of heat shock proteins and extracytoplasmic function sigma factors . As a periplasmic chaperone, SurA likely contributes to maintaining outer membrane integrity during exposure to such pollutants. Research approaches to investigate this should include:

• Comparative proteomics of wild-type versus surA-deficient N. europaea exposed to chlorinated compounds

• Assessment of membrane permeability changes in response to pollutants

• Identification of SurA-dependent outer membrane proteins involved in xenobiotic resistance

This research direction has implications for understanding N. europaea's role in bioremediation of contaminated environments.

How might structural modifications of recombinant SurA enhance its chaperone activity for biotechnological applications?

Structure-guided protein engineering of N. europaea SurA could enhance its chaperone activity for specialized biotechnological applications. Approaches include:

• Domain swapping with SurA homologs from extremophiles to enhance stability

• Rational design of the substrate-binding pocket to expand client range

• Directed evolution to optimize activity under specific conditions relevant to wastewater treatment

These modifications could generate enhanced SurA variants useful for improving the stability of engineered microorganisms in environmental biotechnology applications, particularly in high-ammonia or fluctuating salinity conditions typical of wastewater treatment plants.