Recombinant Nitrosomonas europaea LPS-assembly protein lptD (lptD), partial

What is Nitrosomonas europaea LPS-assembly protein lptD and what is its function?

The LPS-assembly protein lptD in Nitrosomonas europaea is a crucial outer membrane protein that functions in the lipopolysaccharide (LPS) assembly pathway. Similar to its well-characterized counterpart in Escherichia coli, the N. europaea lptD likely forms a complex with LptE to facilitate the final stage of LPS transport and assembly at the cell surface . This protein contains a soluble N-terminal domain that is predicted to be periplasmic and a C-terminal transmembrane domain that likely forms a β-barrel structure in the outer membrane . The primary function of this protein complex is to insert newly synthesized LPS molecules into the outer leaflet of the outer membrane, which is essential for maintaining outer membrane integrity and establishing bilayer asymmetry in this gram-negative chemolithotroph .

How does the structure of lptD in N. europaea compare to that in other bacteria?

The lptD protein in N. europaea shares structural similarities with its homologs in other gram-negative bacteria, particularly E. coli, though with species-specific variations. Like other lptD proteins, the N. europaea variant contains an N-terminal periplasmic domain and a C-terminal β-barrel transmembrane domain . The C-terminal domain interacts strongly with LptE to form the functional β-barrel fold. While the core structure is conserved across species due to its essential function in LPS assembly, sequence variations exist that may reflect adaptations to N. europaea's unique chemolithoautotrophic lifestyle and environmental niche . These structural variations may influence the specificity of LPS recognition and transport, potentially affecting the composition of the outer membrane and consequently the cell's response to environmental stressors such as pH changes, temperature fluctuations, and varying ammonia and nitrite concentrations .

What expression systems are available for recombinant N. europaea lptD production?

Based on available research, recombinant N. europaea lptD can be produced in various expression systems, each with distinct advantages for different research applications. The most commonly used systems include:

For functional studies of recombinant N. europaea lptD, the selection of an appropriate expression system is critical. E. coli-based expression (such as with the CSB-EP identifier products) is often preferred for initial characterization studies due to its relative simplicity and higher yields , though careful optimization of expression conditions is necessary to ensure proper folding of this membrane protein.

What are the optimal conditions for expressing recombinant N. europaea lptD in E. coli?

For optimal expression of recombinant N. europaea lptD in E. coli, researchers should implement the following methodology:

• Vector selection: Use pET expression vectors (such as pET24a) with codon-optimized sequences for E. coli expression .

• Strain selection: BL21(λDE3) strains are recommended for their reduced protease activity and tight control of T7 promoter expression .

• Culture conditions:

  • Growth medium: LB medium supplemented with appropriate antibiotics

  • Temperature: Initial growth at 37°C until OD600 reaches 0.6-0.8, followed by induction at lower temperatures (16-20°C)

  • Induction: Use lower IPTG concentrations (0.1-0.5 mM) for membrane proteins

  • Post-induction time: Extended expression (12-18 hours) at lower temperatures

• Cell lysis considerations:

  • Use mild detergents for membrane protein extraction

  • Include protease inhibitors to prevent degradation

  • Consider inclusion of low concentrations of glycerol (5-10%) to stabilize the protein

• Purification strategy:

  • Initial IMAC (immobilized metal affinity chromatography) using His-tagged constructs

  • Secondary purification via size exclusion chromatography

  • Detergent exchange during purification if necessary for downstream applications

It's worth noting that co-expression with its binding partner LptE may significantly improve the stability and proper folding of LptD, as these proteins form a functional complex in vivo .

How can researchers verify the proper folding and functionality of recombinant lptD?

Verification of proper folding and functionality of recombinant N. europaea lptD requires multiple complementary approaches:

• Biochemical characterization:

  • Size-exclusion chromatography to assess oligomeric state

  • Circular dichroism spectroscopy to evaluate secondary structure content (especially β-sheet content expected in the C-terminal domain)

  • Thermal stability assays to assess protein folding integrity

• Functional assays:

  • LPS binding assays to verify substrate recognition, similar to those performed for E. coli LptD/E complex

  • Complex formation with LptE, which can be assessed via co-immunoprecipitation or pull-down assays

  • Reconstitution into liposomes or nanodiscs for membrane insertion studies

• Structural validation:

  • Limited proteolysis to assess domain organization and stability

  • Surface accessibility studies using chemical modification reagents

  • Where feasible, structural studies via X-ray crystallography or cryo-EM

• In vivo complementation:

  • Functional complementation assays in lptD-depleted strains

  • Assessment of outer membrane integrity in complemented strains

The specific LPS binding capacity can be evaluated using methods similar to those described for E. coli LptD/E, where the purified complex demonstrated specific interaction with LPS molecules .

How does the function of lptD in N. europaea relate to the bacterium's stress responses?

The function of lptD in N. europaea is intricately connected to the bacterium's stress response mechanisms, particularly in relation to its unique chemolithoautotrophic lifestyle:

• Environmental stress adaptation: N. europaea is known to be susceptible to numerous environmental stressors including temperature fluctuations, pH changes, varying nitrite and ammonia concentrations, and the presence of heavy metals and organic/inorganic compounds . As a key component in outer membrane biogenesis, lptD likely plays a crucial role in maintaining membrane integrity under these stressful conditions.

• Integration with toxin-antitoxin systems: N. europaea possesses an unusually high number of toxin-antitoxin (TA) systems (more than 50 type II TA pairs), which are implicated in stress adaptation . The regulation of outer membrane composition via lptD-mediated LPS assembly may be coordinated with these TA systems to modulate cellular activities during stress.

• Oxygen limitation response: N. europaea has specific mechanisms to cope with growth under low dissolved oxygen concentrations . The LPS layer composition, which is directly influenced by lptD function, may be modified under oxygen limitation to optimize cellular metabolism and survival.

• Nitrite toxicity management: As an ammonia-oxidizing bacterium, N. europaea produces nitrite, which can reach toxic levels. Research has shown that N. europaea adapts to high nitrite concentrations by altering gene expression patterns . The outer membrane, maintained by proper lptD function, serves as a crucial barrier controlling nitrite influx and efflux.

The cross-talk between these stress response systems and lptD function represents an important area for further research to understand how N. europaea maintains cellular homeostasis in changing environments.

What are the implications of lptD research for understanding N. europaea's role in environmental nitrogen cycling?

Research on N. europaea lptD has significant implications for understanding this bacterium's crucial role in environmental nitrogen cycling:

• Biofilm formation and persistence: LPS composition, determined by proper lptD function, affects cell surface properties that influence biofilm formation. This is particularly relevant in wastewater treatment systems where N. europaea forms biofilms that oxidize ammonia to nitrite as part of the nitrification process .

• Resilience in engineered systems: Understanding lptD's role in membrane integrity can help explain N. europaea's resilience in wastewater treatment plants where conditions fluctuate. This knowledge could inform strategies to optimize nitrification performance in these systems .

• Environmental adaptability: The LPS layer is the primary interface between N. europaea and its environment. Research on lptD helps explain how this bacterium adapts to varied environments including wastewater treatment plants, sediments, and soils with different nitrogen loads .

• Interaction with environmental pollutants: N. europaea's ability to oxidize not only ammonia but also certain organic compounds (like chloroform) involves interactions at the cell surface . LptD's role in maintaining outer membrane composition may influence these secondary metabolic capabilities.

• Ecological niche determination: The specific LPS composition facilitated by lptD may contribute to N. europaea's ability to occupy specific ecological niches in the nitrogen cycle, particularly environments with high ammonia concentrations.

These implications highlight why lptD research extends beyond basic molecular biology to areas of environmental microbiology and biotechnology.

What techniques can be used to study the interaction between lptD and LptE in N. europaea?

Studying the interaction between lptD and LptE in N. europaea requires sophisticated molecular and biochemical approaches:

• Co-expression and co-purification:

  • Dual expression systems with differentially tagged proteins

  • Sequential affinity purification to isolate intact complexes

  • Native PAGE analysis to assess complex formation

• Biophysical interaction analysis:

  • Surface plasmon resonance (SPR) to determine binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Microscale thermophoresis (MST) for interaction studies in solution

• Structural studies:

  • X-ray crystallography of the co-purified complex

  • Cryo-electron microscopy for visualization of the intact complex

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

• In vivo interaction analysis:

  • Bacterial two-hybrid systems

  • Förster resonance energy transfer (FRET) with fluorescently tagged proteins

  • Split GFP complementation assays

• Computational modeling:

  • Molecular dynamics simulations to predict interaction modes

  • Homology modeling based on E. coli LptD/E complex structures

  • Interface prediction algorithms

The E. coli LptD/E complex can serve as a valuable model, as it has been demonstrated that LptE interacts strongly with the C-terminal domain of LptD to form the β-barrel fold and both are required for LPS assembly at the cell surface .

What are common challenges in purifying recombinant N. europaea lptD and how can they be addressed?

Purification of recombinant N. europaea lptD presents several challenges due to its nature as a membrane protein:

Co-expression with LptE significantly improves stability and purification outcomes as observed with the E. coli homolog . The stable 1:1 complex formation between LptD and LptE can enhance folding and reduce aggregation during expression and purification.

How can researchers overcome expression barriers when working with N. europaea proteins in heterologous systems?

Researchers can overcome expression barriers for N. europaea proteins, including lptD, in heterologous systems through these methodological approaches:

• Codon optimization strategies:

  • Analyze codon usage bias between N. europaea and the expression host

  • Optimize rare codons while maintaining key regulatory elements

  • Consider synonymous codon changes that affect translation rate and protein folding

• Host strain selection:

  • For E. coli expression, use specialized strains like Rosetta (DE3) for rare codon supplementation

  • C41/C43 strains derived from BL21(DE3) are particularly effective for membrane proteins

  • Consider alternative hosts like Pseudomonas for proteins that remain challenging in E. coli

• Vector design considerations:

  • Include solubility-enhancing fusion partners (MBP, SUMO, TrxA)

  • Incorporate cleavable tags for native protein recovery

  • Design constructs with varying N- and C-terminal boundaries to identify optimal expression domains

• Expression condition optimization:

  • Screen multiple induction parameters (temperature, inducer concentration, time)

  • Test various media formulations, including auto-induction media

  • Implement fed-batch cultivation to reduce metabolic burden

• Co-expression strategies:

  • Introduce molecular chaperones (GroEL/GroES, DnaK/DnaJ)

  • Co-express binding partners (like LptE for lptD)

  • Include key enzymes for post-translational modifications if required

This systematic approach has been successfully applied to express proteins from N. europaea, including those used for studying responses to oxidative stress and other environmental conditions .

How might studying lptD contribute to understanding N. europaea's unique metabolic adaptations?

Studying lptD provides valuable insights into N. europaea's unique metabolic adaptations through several research avenues:

• Membrane architecture and metabolic compartmentalization: The outer membrane, maintained by lptD-mediated LPS assembly, creates a compartment that houses specialized metabolic machinery. This architecture is crucial for N. europaea's chemolithoautotrophic lifestyle, where energy is derived from ammonia oxidation and carbon is fixed from CO2 .

• Stress response integration: N. europaea's metabolism must adapt to fluctuating environmental conditions. The lptD protein may play a role in modifying membrane permeability and composition during stress, potentially interfacing with the abundant toxin-antitoxin systems that regulate cellular activities under stress conditions .

• Electron transport chain functionality: The ammonia oxidation pathway that provides energy for N. europaea involves membrane-associated electron transport components. Proper LPS assembly via lptD is essential for maintaining the membrane environment where these components function .

• Substrate acquisition and toxic product export: The LPS layer influences the diffusion of ammonia (substrate) into the cell and nitrite (potentially toxic product) out of the cell. Understanding lptD's role in LPS assembly provides insight into how N. europaea optimizes this balance .

• Carbon fixation and energy conservation: N. europaea obtains energy from ammonia oxidation and carbon from CO2 fixation. The MazF endoribonuclease has been shown to potentially modulate translation of key enzymes in both pathways, including hydroxylamine dehydrogenase (hao) and ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcL) . The relationship between membrane integrity (maintained by lptD) and these regulatory mechanisms represents an important area for investigation.

Research in this area would contribute to understanding how N. europaea maintains its specialized metabolism in various environmental niches.

What are promising directions for developing genetic tools to study lptD function in N. europaea?

Several promising directions exist for developing genetic tools to study lptD function in N. europaea:

• Regulated expression systems:

  • Development of inducible promoter systems specifically calibrated for N. europaea

  • Creation of titratable knockdown systems using antisense RNA or CRISPR interference

  • Establishment of conditional mutants where lptD expression can be precisely controlled

• Reporter fusion strategies:

  • Fusion of lptD with fluorescent proteins to track localization, similar to GFP fusions that have been successfully employed in N. europaea

  • Development of split reporter systems to monitor protein-protein interactions in vivo

  • Creation of transcriptional and translational fusions to study regulation of lptD expression

• Genome editing approaches:

  • Adaptation of CRISPR-Cas9 systems for precise genome editing in N. europaea

  • Optimization of homologous recombination frequencies for targeted mutagenesis

  • Development of markerless deletion systems for clean genetic manipulation

• Heterologous complementation:

  • Engineering of chimeric lptD proteins combining domains from different bacterial species

  • Cross-species complementation assays to identify functional conservation and specialization

  • Development of shuttle vectors optimized for both E. coli and N. europaea

• High-throughput phenotyping platforms:

  • Microfluidic systems for single-cell analysis of N. europaea with modified lptD

  • Biosensor development to monitor membrane integrity and LPS composition in real-time

  • Transcriptomic and proteomic profiling platforms to assess global effects of lptD manipulation

These approaches build upon existing work with N. europaea, such as the successful construction of recombinant strains expressing GFP under control of specific promoters , and would significantly advance our ability to study lptD function in this environmentally important bacterium.

How does the lptD protein and its function in N. europaea compare to homologs in other ammonia-oxidizing bacteria?

The lptD protein in N. europaea exhibits both conserved and distinct features compared to homologs in other ammonia-oxidizing bacteria:

• Structural conservation: The basic domain architecture with an N-terminal periplasmic domain and C-terminal transmembrane β-barrel appears conserved across ammonia oxidizers, reflecting the fundamental importance of LPS transport for outer membrane integrity .

• Sequence divergence: Sequence analysis suggests species-specific adaptations that may reflect differences in LPS composition and environmental niches. These variations likely affect the specificity of LPS recognition and transport.

• Functional partners: While the lptD-LptE complex formation is likely conserved, the specific interaction interfaces and binding affinities may differ among ammonia oxidizers, potentially influencing the efficiency of LPS transport and assembly.

• Regulatory mechanisms: The expression patterns and regulatory controls of lptD likely vary among ammonia oxidizers that inhabit different environments, from wastewater treatment plants to soil and freshwater ecosystems.

• Environmental adaptations: N. europaea's lptD may have specific adaptations related to its unusual abundance of toxin-antitoxin systems (over 50 type II TA pairs) , which is not typical of all ammonia oxidizers.

A comprehensive comparative analysis of lptD across diverse ammonia oxidizers would provide valuable insights into how this essential protein has evolved to support specialized metabolic lifestyles in different environmental contexts.

What insights can be gained from studying lptD across bacterial species that inhabit different ecological niches?

Comparative analysis of lptD across bacterial species from diverse ecological niches offers several valuable insights:

• Evolutionary adaptation signatures: Patterns of sequence conservation and divergence can reveal which domains have undergone selective pressure in different environments, highlighting functionally critical regions versus adaptable ones.

• LPS structural diversity correlation: Variations in lptD structure likely correlate with differences in LPS composition across species. This relationship can provide insights into how membrane architecture has evolved to meet specific environmental challenges.

• Niche-specific functional modifications:

  • Extremophiles (temperature, pH, salinity) may show adaptations in lptD that maintain LPS transport under challenging conditions

  • Pathogens may have evolved specialized mechanisms to modulate LPS presentation for immune evasion

  • Symbionts may show cooperative adaptations in LPS assembly related to host interaction

• Horizontal gene transfer assessment: Analysis of lptD sequences can reveal potential horizontal gene transfer events that may have contributed to bacterial adaptation to new niches.

• Conservation of interaction networks: Comparing lptD-interacting partners across species can reveal conserved protein-protein interaction networks versus species-specific innovations.

This comparative approach is particularly valuable because LPS assembly is essential yet must adapt to diverse environmental conditions, making lptD an excellent lens through which to study bacterial adaptation strategies.

How does lptD expression correlate with other genes in N. europaea under different environmental conditions?

The expression patterns of lptD in N. europaea show complex correlations with other genes across varying environmental conditions:

• Co-regulation with stress response genes: Under conditions of environmental stress (temperature, pH, toxicants), lptD expression likely correlates with the numerous toxin-antitoxin systems that N. europaea employs for stress adaptation . The precise temporal coordination between membrane integrity maintenance and cellular stress responses represents an important area for investigation.

• Correlation with metabolic pathway genes: Under oxygen limitation, N. europaea shows increased expression of ammonia oxidation genes (amoA) and hydroxylamine oxidation genes (hao) . The relationship between these metabolic adaptations and outer membrane composition (influenced by lptD) would provide insight into integrated cellular responses.

• Nitrite stress response coordination: High nitrite conditions induce expression of nitrite reduction (nirK) and nitric oxide reduction (norB) genes in N. europaea . The coordination between these detoxification mechanisms and potential changes in membrane permeability via lptD-mediated LPS assembly represents an important regulatory network.

• Growth phase-dependent correlations: Expression patterns differ significantly between exponential and stationary phases in N. europaea , suggesting that lptD expression and its correlations with other genes are likely to show growth phase-dependent patterns.

Comprehensive transcriptomic analysis under various conditions would allow construction of gene co-expression networks to identify the key regulatory connections between lptD and other cellular systems in N. europaea.

What systems biology approaches can best integrate lptD function into models of N. europaea metabolism?

Several systems biology approaches can effectively integrate lptD function into comprehensive models of N. europaea metabolism:

• Multi-omics data integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Correlate lptD expression/activity with global cellular responses

  • Identify key regulatory nodes that connect membrane assembly with metabolic pathways

• Genome-scale metabolic modeling:

  • Incorporate membrane assembly costs into flux balance analysis models

  • Develop compartmentalized models that account for periplasmic vs. cytoplasmic processes

  • Simulate the metabolic impacts of altered LPS composition

• Protein-protein interaction networks:

  • Map the interaction partners of lptD beyond LptE

  • Identify connections to regulatory networks for stress response

  • Discover potential feedback mechanisms between membrane integrity and metabolic regulation

• Regulatory network reconstruction:

  • Identify transcription factors that regulate lptD expression

  • Map signaling pathways that respond to membrane stress

  • Model the temporal dynamics of membrane adaptation to environmental changes

• Agent-based modeling for population-level effects:

  • Model how cell-to-cell variations in lptD function affect population resilience

  • Simulate biofilm formation dynamics with varying LPS compositions

  • Predict ecological interactions based on membrane properties

These approaches would help develop a more comprehensive understanding of how N. europaea's specialized metabolism is integrated with membrane biogenesis and integrity, providing insights into this bacterium's environmental adaptations and potential biotechnological applications.

What are emerging techniques that could advance our understanding of lptD structure-function relationships?

Several cutting-edge techniques show promise for advancing our understanding of lptD structure-function relationships in N. europaea:

• Cryo-electron tomography:

  • Visualize lptD in its native membrane environment

  • Capture different conformational states during LPS transport

  • Map the spatial organization of lptD relative to other membrane proteins

• Single-molecule tracking:

  • Monitor the dynamics of individual lptD proteins in living cells

  • Measure diffusion rates and interaction kinetics in real-time

  • Correlate mobility with functional states

• Mass spectrometry-based footprinting:

  • Map LPS binding sites on lptD with high precision

  • Identify conformational changes upon substrate binding

  • Determine post-translational modifications that regulate function

• Directed evolution coupled with deep mutational scanning:

  • Generate comprehensive mutation libraries of lptD

  • Select for variants with altered function or stability

  • Map sequence-function relationships at single-residue resolution

• In-cell NMR spectroscopy:

  • Monitor structural changes in specific lptD domains in living cells

  • Probe ligand binding under physiological conditions

  • Identify dynamic regions important for function

• Artificial intelligence approaches:

  • Apply deep learning to predict structure-function relationships

  • Use machine learning to identify patterns in experimental data

  • Develop predictive models for lptD function based on sequence

These advanced techniques would complement traditional approaches and provide unprecedented insights into how lptD functions at the molecular level in N. europaea's outer membrane.

How might research on N. europaea lptD contribute to biotechnological applications in environmental remediation?

Research on N. europaea lptD has several potential applications in environmental biotechnology and remediation:

• Enhanced nitrification processes:

  • Engineering N. europaea with optimized lptD variants for increased resistance to environmental stressors in wastewater treatment

  • Developing strains with modified outer membrane properties for improved biofilm formation in bioreactors

  • Creating variants that maintain activity under wider ranges of temperature and pH conditions

• Bioremediation of nitrogen-rich environments:

  • Designing N. europaea strains with altered membrane permeability for more efficient ammonia uptake from contaminated sites

  • Developing variants with increased resistance to toxic compounds often found alongside ammonia pollution

  • Engineering strains with improved survival in field conditions

• Biosensor development:

  • Creating N. europaea-based biosensors with modified lptD to detect ammonia in environmental samples

  • Developing whole-cell bioreporters that respond to specific contaminants through altered membrane properties

  • Engineering systems that provide visual outputs based on ammonia oxidation activity

• Biocatalyst immobilization strategies:

  • Designing N. europaea with engineered cell surface properties for improved attachment to support materials

  • Developing methods to stabilize the outer membrane for prolonged catalyst lifetime

  • Creating systems for controlled release of immobilized cells based on environmental triggers

• Synthetic biology applications:

  • Using lptD as a component in engineered microbes designed for specialized environmental applications

  • Developing chimeric outer membrane proteins with novel functionalities

  • Creating synthetic consortia with optimized membrane properties for cooperative metabolic activities

These applications leverage N. europaea's natural capacity for ammonia oxidation while enhancing its resilience and functionality through targeted modifications of outer membrane composition and integrity.