Recombinant Nitrosomonas europaea Ribosome-binding factor A (rbfA)
Description
Definition of Recombinant Nitrosomonas europaea Ribosome-Binding Factor A (rbfA)
Ribosome-binding factor A (RbfA) is a bacterial cold-shock response protein essential for the efficient processing of the 5′ end of the 16S ribosomal RNA (rRNA) during the assembly of the small (30S) ribosomal subunit . In Escherichia coli, RbfA is encoded in an operon along with the cold-shock protein NusA and is constitutively expressed under normal growth conditions . Upon cold shock, the expression level of RbfA rapidly increases due to an up-regulation of the transcription of the rbfA mRNA, resulting in a several-fold increase in the amount of 30S-bound RbfA . The elevated levels of RbfA under cold-shock conditions are necessary to overcome the translational block at the reduced temperature, presumably by facilitating rapid maturation of the 30S subunits .
RbfA and RsgA Interaction
RsgA can promote the release of RbfA from the 30S subunit during a late stage of maturation of the 30S subunit when 17S pre-rRNA is processed into 16S rRNA . Cellular disorders upon depletion of RsgA result from prolonged retention of RbfA on the ribosome, and the interplay of RbfA and RsgA on the 30S subunit represents a novel process involving RbfA, RsgA, and possibly other assembly factors, including RimM and Era . Mutations in rbfA can suppress defects in growth and maturation of the 30S subunit of an rsgA− strain, allowing RbfA to release itself from the 30S subunit without the help of RsgA, leading to the suppression of impaired maturation of the ribosome due to the absence of RsgA . RsgA-GTP enhances the release of RbfA from the 30S subunit in vitro, and RsgA preferentially targets the 30S subunit in complex with RbfA rather than the free 30S subunit . RbfA binds to both immature 30S subunits containing 17S RNA and mature 30S subunits containing 16S rRNA with similar equilibrium affinity . RsgA prefers mature 30S subunits to immature 30S subunits in terms of both enhancement of GTP hydrolytic activity and equilibrium affinity, suggesting that RbfA binds to a pre-30S subunit and dissociates from nearly or completely maturated 30S with the assistance of RsgA . RbfA stays on immature 30S subunits with higher stability than on mature 30S subunits in the absence of RsgA, and RsgA has little or no effect on the stability of the complex of RbfA and immature 30S subunits, while it accelerates the release of RbfA from mature 30S subunits .
Rnf Complex in Fusobacterium nucleatum
The Rhodobacter nitrogen-fixation (Rnf) complex, encoded by the rnfCDGEAB gene cluster, is crucial for fusobacterial metabolic adaptation and virulence . Genetic disruption of the Rnf complex via non-polar, in-frame deletion of rnfC (Δ rnfC) abrogates polymicrobial interaction (or coaggregation) associated with adhesin RadD and biofilm formation . This defect is due to an increased level of extracellular lysine, which binds RadD and inhibits coaggregation, mirroring the phenotype of a mutant (Δ kamA) that fails to metabolize extracellular lysine . The Rnf complex is a highly conserved and evolutionarily ancient membrane-bound ferredoxin:NAD+ oxidoreductase that couples reversible electron transfer from reduced ferredoxin to NAD+ with the establishment of an ion-motive force, enabling substrate import and/or ATP biosynthesis . It acts as a versatile metabolic exchange center for nitrogen fixation, carbon dioxide fixation, metabolism of low-energy substrates, and gut colonization in mice . In F. nucleatum, disruption of the Rnf complex causes severe defects in many virulence traits, including coaggregation, biofilm formation, H2S production, and ATP production, in addition to altering cell morphology and growth . The defect in coaggregation is mechanistically linked to a failure of lysine catabolism, leading to an increased level of extracellular lysine that inhibits RadD-mediated coaggregation, while defects in other traits are attributed to a global reduction of amino acid metabolism . The rnfC mutant is severely impaired in inducing preterm birth in mice, establishing the Rnf complex as a central component in F. nucleatum .
Product Specs
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Target Background
KEGG: neu:NE0762
STRING: 228410.NE0762
Q&A
What is Ribosome-binding Factor A (RbfA), and what role does it play in Nitrosomonas europaea?
Ribosome-binding Factor A (RbfA) is a protein essential for the maturation of 16S rRNA in bacteria, facilitating the assembly of functional ribosomes. In Nitrosomonas europaea, RbfA contributes to the bacterium's ability to efficiently process ammonia into nitrite as part of its chemolithoautotrophic metabolism . This process is critical for nitrification, a key step in the biogeochemical nitrogen cycle. RbfA's structure and function have been extensively studied in model organisms like Escherichia coli, where it exhibits cold-shock adaptation properties and plays a role in ribosomal assembly under stress conditions .
The recombinant form of RbfA from Nitrosomonas europaea allows researchers to study its biochemical properties and structural dynamics using advanced techniques such as solution NMR spectroscopy . Understanding these mechanisms is crucial for elucidating how N. europaea adapts to environmental changes and maintains its metabolic efficiency.
How can researchers experimentally determine the structure of recombinant RbfA?
The structure of recombinant RbfA can be determined using solution Nuclear Magnetic Resonance (NMR) spectroscopy, which provides high-resolution insights into protein folding and dynamics. For example, studies on the truncated construct RbfAD25 from E. coli utilized heteronuclear single quantum coherence (HSQC) spectra combined with automated NOESY analysis to identify stable secondary structures and dynamic regions . Researchers can apply similar methodologies to recombinant RbfA from Nitrosomonas europaea, focusing on:
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Sample Preparation: Expressing recombinant RbfA in suitable host systems and purifying it under conditions that prevent aggregation.
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Spectral Analysis: Acquiring 3D 15N-edited NOESY and 13C-edited NOESY spectra for distance constraints.
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Structural Modeling: Using computational tools like DYANA for iterative refinement based on spectral data.
These approaches help identify key features such as KH-domain folds, hydrogen bonding patterns, and dynamic residues that influence protein function .
What experimental challenges might arise when studying recombinant RbfA?
Studying recombinant RbfA involves several challenges:
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Protein Aggregation: Full-length RbfA tends to aggregate at room temperature, complicating structural analysis. Truncated constructs like RbfAD25 may provide a solution by removing aggregation-prone regions .
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Spectral Line Broadening: NMR studies often encounter weak or unobservable resonances due to internal dynamics or exchange broadening effects .
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Environmental Adaptation: As a cold-shock protein, RbfA's activity may vary under different temperature conditions, requiring careful control during experiments.
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Functional Validation: Ensuring that recombinant constructs retain biological activity comparable to native proteins is critical for interpreting results.
Addressing these challenges requires optimizing experimental conditions, employing advanced analytical techniques, and validating findings through complementary methods such as X-ray crystallography or cryo-electron microscopy.
How does RbfA contribute to the ammonia oxidation process in Nitrosomonas europaea?
RbfA indirectly supports ammonia oxidation by ensuring efficient ribosomal function, which is vital for synthesizing enzymes involved in nitrification. Nitrosomonas europaea oxidizes ammonia (NH₃) to nitrite (NO₂⁻), a process mediated by enzymes like ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) . These enzymatic activities depend on robust protein synthesis machinery facilitated by mature ribosomes.
Furthermore, the genome of N. europaea reveals adaptations such as iron acquisition systems and nitrate reductase clusters that enhance its metabolic capabilities under aerobic and anaerobic conditions . The interplay between ribosomal efficiency and metabolic pathways underscores the importance of RbfA in maintaining cellular homeostasis.
What methodologies are used to assess the activity of recombinant RbfA?
To evaluate the activity of recombinant RbfA, researchers employ biochemical assays combined with structural analyses:
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RNA Binding Studies: Assessing interactions with 16S rRNA using electrophoretic mobility shift assays or fluorescence-based methods.
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Enzymatic Activity Assays: Measuring ribosomal assembly efficiency in vitro by monitoring translation rates or protein synthesis.
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Cold-shock Adaptation Tests: Evaluating functional responses under temperature stress conditions.
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Structural Validation: Confirming active conformations through NMR or crystallographic studies.
These methodologies provide insights into how recombinant RbfA mimics native protein functions and its potential applications in synthetic biology.
How does the genetic organization of Nitrosomonas europaea influence RbfA expression?
The genome of Nitrosomonas europaea consists of a single circular chromosome with genes evenly distributed across two replichores . This arrangement facilitates coordinated expression of essential genes, including those encoding ribosomal proteins and factors like RbfA. Regulatory elements such as promoters and operons ensure timely transcription under varying environmental conditions.
Studies have identified insertion sequence elements within the genome that may influence gene expression by promoting genetic rearrangements or horizontal gene transfer . Understanding these regulatory mechanisms provides clues about how N. europaea adapts its ribosomal machinery during nitrification.
What are the implications of studying recombinant RbfA for environmental research?
Research on recombinant RbfA has significant implications for environmental science:
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Nitrification Efficiency: Insights into ribosomal function can improve our understanding of ammonia oxidation rates in wastewater treatment systems .
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Greenhouse Gas Mitigation: By elucidating pathways involved in nitrous oxide production, researchers can develop strategies to reduce emissions during nitrification .
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Bioremediation Applications: Enhancing the metabolic capabilities of nitrifying bacteria through genetic engineering may aid in cleaning contaminated sites .
These applications highlight the broader impact of studying proteins like RbfA on ecological sustainability.
