Recombinant Nitrobacter winogradskyi UPF0060 membrane protein Nwi_1459 (Nwi_1459)

What is Nwi_1459 and what organism does it come from?

Nwi_1459 is a UPF0060 membrane protein derived from Nitrobacter winogradskyi strain Nb-255 (ATCC 25391). Nitrobacter winogradskyi is a gram-negative facultative chemolithoautotroph belonging to the alphaproteobacteria class. This organism is capable of extracting energy from the oxidation of nitrite to nitrate and plays a significant role in the nitrogen cycle. The complete genome of Nitrobacter winogradskyi consists of a single circular chromosome of 3,402,093 bp encoding 3,143 predicted proteins, with Nwi_1459 being one of these proteins . The organism shows extensive genomic similarities to other alphaproteobacteria, particularly Bradyrhizobium japonicum USDA110 and Rhodopseudomonas palustris CGA009, which share 1,300 and 815 similar genes respectively with N. winogradskyi .

What expression systems are used for recombinant Nwi_1459 production?

Recombinant Nwi_1459 is typically produced in E. coli expression systems for research purposes. The protein is commonly expressed with a histidine tag to facilitate purification through affinity chromatography . The expression covers the full length of the protein (amino acids 1-108) and results in a functional recombinant protein suitable for various biochemical and structural studies . When selecting an expression system, researchers should consider that membrane proteins often require specialized approaches to maintain proper folding and functionality.

What are the optimal storage and handling conditions for recombinant Nwi_1459?

For optimal preservation of recombinant Nwi_1459, the following storage conditions are recommended:

• Store the protein at -20°C for regular use

• For extended storage periods, maintain at either -20°C or -80°C

• Avoid repeated freezing and thawing cycles which can denature the protein

• Working aliquots may be stored at 4°C for up to one week

The protein is typically supplied in a Tris-based buffer containing 50% glycerol, which has been optimized specifically for this protein . When handling the protein, minimize exposure to room temperature and use sterile techniques to prevent contamination. For experimental work, it's advisable to create small working aliquots to prevent degradation of the entire stock.

How can researchers validate the expression and purification of recombinant Nwi_1459?

Validation of recombinant Nwi_1459 expression and purification can be accomplished through multiple complementary techniques:

• SDS-PAGE analysis to confirm protein size (expected ~12 kDa plus the size of any tags)

• Western blotting using anti-His antibodies (if His-tagged)

• Mass spectrometry for precise molecular weight determination and sequence confirmation

• Circular dichroism (CD) spectroscopy to assess secondary structure, particularly important for membrane proteins

• Limited proteolysis followed by mass spectrometry to verify protein folding and accessibility of cleavage sites

When analyzing membrane proteins like Nwi_1459, special consideration should be given to sample preparation to avoid aggregation. Using appropriate detergents or lipid nanodiscs may help maintain the protein's native structure during analysis.

What approaches are suitable for studying Nwi_1459's membrane localization?

To investigate the membrane localization of Nwi_1459, researchers can employ several experimental approaches:

• Fluorescence microscopy using GFP-fusion constructs

• Subcellular fractionation followed by Western blotting

• Protease accessibility assays to determine membrane topology

• Immunogold electron microscopy for high-resolution localization

• Membrane protein extraction using differential detergent solubilization

When designing experiments to study Nwi_1459's membrane localization, it's important to consider that Nitrobacter winogradskyi undergoes cell division by polar swelling, resulting in asymmetric cells . This morphological characteristic may impact the distribution of membrane proteins and should be accounted for in localization studies.

What is the potential functional role of Nwi_1459 in Nitrobacter winogradskyi?

While the specific function of Nwi_1459 has not been fully characterized, its classification as a UPF0060 membrane protein and genomic context can provide insights into its potential roles:

• Given Nitrobacter winogradskyi's role in nitrification, Nwi_1459 might be involved in nitrite oxidation or related processes

• The presence of duplicated gene regions in the N. winogradskyi genome, including multiple copies of nitrite oxidoreductase and cytochrome c oxidase , suggests Nwi_1459 might function in electron transport or energy generation

• As a membrane protein, it could participate in substrate transport, signaling, or membrane integrity maintenance

Research approaches to elucidate Nwi_1459's function could include:

• Co-expression studies with known components of nitrite oxidation pathways

• Protein-protein interaction studies to identify binding partners

• Functional complementation in heterologous systems

• Transcriptomic analysis to determine expression patterns under different growth conditions

How can comparative genomics approaches help understand Nwi_1459?

Comparative genomics offers powerful tools for investigating Nwi_1459's evolutionary significance and potential function:

• Identify homologs across different nitrifying bacteria and related alphaproteobacteria

• Analyze conservation patterns of the UPF0060 protein family across bacterial lineages

• Examine syntenic relationships (gene neighborhood conservation) that might indicate functional associations

• Conduct phylogenetic analyses to trace the evolutionary history of Nwi_1459

The N. winogradskyi genome shows extensive similarities to other alphaproteobacteria, with 1,300 genes similar to Bradyrhizobium japonicum USDA110 and 815 similar to Rhodopseudomonas palustris CGA009 . Additionally, 85 genes show similarities to the ammonia-oxidizing betaproteobacterium Nitrosomonas europaea . Examining Nwi_1459 homologs in these organisms may provide insights into its function through evolutionary conservation patterns.

What structural analysis techniques would be most informative for characterizing Nwi_1459?

For thorough structural characterization of Nwi_1459, researchers should consider a multi-technique approach:

• X-ray crystallography (challenging for membrane proteins but potentially highly informative)

• Cryo-electron microscopy (cryo-EM) for near-atomic resolution without crystallization

• Nuclear Magnetic Resonance (NMR) spectroscopy for dynamic structural information

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for protein-ligand interactions and conformational changes

• Molecular dynamics simulations to predict behavior in membrane environments

When working with membrane proteins like Nwi_1459, special considerations include:

• Selection of appropriate detergents or lipid nanodiscs to maintain native conformation

• Optimization of protein stability conditions for long-duration experiments

• Careful interpretation of structural data in the context of the membrane environment

How can gene knockout or mutation studies of Nwi_1459 be designed?

Designing gene knockout or mutation studies for Nwi_1459 requires specialized approaches due to the characteristics of Nitrobacter winogradskyi:

• Homologous recombination-based techniques, utilizing the natural recombination machinery

• CRISPR-Cas9 systems adapted for alphaproteobacteria

• Transposon mutagenesis approaches, noting that the N. winogradskyi genome contains multiple IS elements

• Creation of conditional knockouts if Nwi_1459 proves essential

The table below outlines key considerations for different genetic modification approaches:

When analyzing phenotypes, researchers should monitor nitrite oxidation rates, growth under various conditions, and membrane integrity to connect Nwi_1459 function to cellular physiology.

What approaches can be used to investigate potential protein-protein interactions of Nwi_1459?

To identify and characterize protein interactions involving Nwi_1459, researchers can implement multiple complementary techniques:

• Co-immunoprecipitation (Co-IP) using antibodies against Nwi_1459 or its tags

• Bacterial two-hybrid (B2H) assays adapted for membrane proteins

• Cross-linking mass spectrometry (XL-MS) to capture transient interactions

• Biolayer interferometry (BLI) or surface plasmon resonance (SPR) for interaction kinetics

• Proximity-dependent biotin identification (BioID) or APEX2 proximity labeling in heterologous systems

When designing these experiments, researchers should consider potential interactions with components of nitrite oxidation pathways, particularly the nitrite oxidoreductase complex. The N. winogradskyi genome contains two copies of both the nitrite oxidoreductase α subunit (nwi0774 and nwi2068, 94% identity) and β subunit (nwi0776 and nwi0965, 97% identity) , suggesting the importance of this function to the organism.

How can transcriptomic and proteomic approaches contribute to understanding Nwi_1459 function?

Multi-omics approaches offer powerful insights into Nwi_1459's function by revealing expression patterns and regulatory networks:

• RNA-Seq to determine co-expression patterns under different growth conditions (autotrophic vs. heterotrophic)

• Quantitative proteomics to measure Nwi_1459 abundance relative to other membrane proteins

• Ribosome profiling to assess translational regulation

• Phosphoproteomics to identify potential regulatory post-translational modifications

• Chromatin immunoprecipitation sequencing (ChIP-seq) focusing on transcription factors that might regulate Nwi_1459

N. winogradskyi has 322 genes (approximately 10% of its genome) devoted to regulation and signaling, including σ32-, σ70-, and σ54-like transcription factors . Understanding how these regulatory systems affect Nwi_1459 expression would provide context for its physiological role.

What are the considerations for functional reconstitution of Nwi_1459 in artificial membrane systems?

For functional studies of membrane proteins like Nwi_1459, reconstitution into artificial membrane systems offers advantages:

• Proteoliposomes provide a controlled lipid environment for transport or enzymatic assays

• Nanodiscs allow for stable, monodisperse samples suitable for structural studies

• Planar lipid bilayers enable electrophysiological measurements if channel activity is suspected

• Giant unilamellar vesicles (GUVs) permit visualization of protein distribution and membrane effects

Key considerations for successful reconstitution include:

• Lipid composition optimization to mimic the native N. winogradskyi membrane

• Protein:lipid ratio determination for optimal function

• Orientation control to ensure physiologically relevant topology

• Buffer composition to support protein stability and function

• Validation of reconstitution success through activity assays or structural analysis

What bioinformatic tools are most useful for predicting Nwi_1459's function?

A systematic bioinformatic analysis workflow for Nwi_1459 should include:

• Sequence-based analyses:

  • BLAST and PSI-BLAST for identifying distant homologs

  • Multiple sequence alignment to identify conserved residues

  • TMHMM, HMMTOP for transmembrane domain prediction

  • SignalP for signal peptide prediction

• Structure-based analyses:

  • AlphaFold or RoseTTAFold for structure prediction

  • ConSurf for mapping conservation onto structural models

  • CASTp for binding pocket prediction

  • Molecular docking simulations with potential substrates

• Genomic context analyses:

  • Gene neighborhood analysis in N. winogradskyi and related species

  • Protein-protein interaction network prediction

  • Co-expression data mining from public repositories

When interpreting results, researchers should consider the UPF0060 protein family characteristics and the specific metabolic context of Nitrobacter winogradskyi as a nitrite-oxidizing bacterium.

How should researchers address data inconsistencies in Nwi_1459 functional studies?

When encountering contradictory results in Nwi_1459 research, consider the following methodological approach:

• Evaluate experimental conditions systematically:

  • Buffer composition effects on protein stability

  • Detergent selection impact on membrane protein function

  • Expression system differences affecting post-translational modifications

• Consider biological variables:

  • Growth phase effects on Nwi_1459 expression and function

  • Nitrite concentration effects on nitrite-oxidizing bacteria physiology

  • Oxygen levels affecting respiratory chain components

• Technical validation strategies:

  • Independent replication with varied methodologies

  • Controls for tag interference with protein function

  • Complementary approaches to verify key findings

The complex physiology of N. winogradskyi, capable of chemolithoautotrophic growth on nitrite, chemoorganotrophic growth on organic compounds, and mixotrophic growth combining both metabolic modes , may contribute to variable results depending on growth conditions.