Recombinant Idiomarina loihiensis N utilization substance protein B homolog (nusB)

What is Idiomarina loihiensis and where was it discovered?

Idiomarina loihiensis is a halophilic γ-proteobacterium isolated from a hydrothermal vent at 1,300-m depth on the Lōihi submarine volcano, Hawaii. It represents a distinct lineage among γ-proteobacteria that branched after the Pseudomonas lineage but before the Vibrio cluster . Interestingly, this organism shares 99.9% 16S rRNA gene sequence similarity over 1415 nucleotides with an uncultured eubacterium from sediment at 11,000 m depth in the Mariana Trench, though its nearest cultivated neighbor is Idiomarina abyssalis KMM 227T (98.9% 16S rRNA similarity) .
Methodology for identification: Modern identification involves both 16S rRNA gene sequence analysis and whole genome-based approaches including core genome analysis, digital DNA-DNA hybridization (dDDH), and Orthologous Average Nucleotide Identity (OrthoANI) analysis . For novel Idiomarina species, genome-based approaches are strongly recommended due to heterogeneity in 16S rRNA gene sequences within this genus .

What are the basic characteristics of Idiomarina loihiensis genome?

The I. loihiensis genome consists of a single circular chromosome of 2,839,318 base pairs with the following characteristics:

What is the NusB protein, and what is its general function?

The N utilization substance B (NusB) protein modulates the efficiency of transcription termination at nut (N utilization) sites of various bacterial genes. In model organisms like Escherichia coli, NusB participates in transcription antitermination mechanisms that allow RNA polymerase to read through termination signals .
Methodological investigation: NusB function can be studied through genetic approaches (deletion mutants and complementation studies) and biochemical methods (in vitro transcription assays). The protein can be structurally characterized using NMR spectroscopy with samples labeled with 15N, 13C, and 2H in various combinations .

How can I clone and express the nusB gene from Idiomarina loihiensis?

To clone and express the I. loihiensis nusB gene, researchers should consider the following methodological approach:

• Gene identification and primer design:

  • Identify the nusB homolog in the I. loihiensis genome using BLAST searches against characterized NusB sequences

  • Design primers with appropriate restriction sites for directional cloning

• Expression system selection:

  • For high-yield expression, E. coli BL21(DE3) strains with pET-based vectors are recommended

  • Consider using a system capable of producing approximately 10% of total cell protein, as demonstrated with E. coli NusB

• Optimization protocol:

  • Test multiple induction conditions (IPTG concentration, temperature, induction time)

  • For halophilic proteins, expression at lower temperatures (16-20°C) often improves solubility

  • Include appropriate affinity tags (His6 or GST) for purification

• NIH Guidelines compliance:

  • Ensure research complies with NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules

  • Obtain Institutional Biosafety Committee (IBC) approval before initiation of experiments

What purification strategies are most effective for recombinant I. loihiensis NusB?

Based on successful purification of E. coli NusB, the following methodological approach is recommended:

• Initial extraction:

  • Lyse cells using sonication or French press in a buffer containing salt concentrations appropriate for halophilic proteins

  • Consider including low concentrations of non-ionic detergents to improve solubility

• Chromatography sequence:

  • Primary purification using affinity chromatography (Ni-NTA for His-tagged constructs)

  • Secondary purification using anion-exchange chromatography

  • Final polishing using gel-permeation chromatography

• Quality assessment:

  • Determine purity using SDS-PAGE

  • Verify oligomeric state using analytical ultracentrifugation (expected to be monomeric based on E. coli NusB)

  • Confirm identity using mass spectrometry

  • Assess activity through functional assays

How can I determine if the I. loihiensis NusB protein forms a complex with RNA?

To investigate RNA binding properties of I. loihiensis NusB:

• Electrophoretic mobility shift assays (EMSA):

  • Incubate purified NusB with labeled RNA fragments containing potential binding sites

  • Analyze complex formation by native gel electrophoresis

• Surface plasmon resonance (SPR):

  • Immobilize either NusB or RNA on a sensor chip

  • Measure binding kinetics and affinity constants

• Fluorescence anisotropy:

  • Use fluorescently labeled RNA to measure changes in anisotropy upon NusB binding

  • Determine binding constants and stoichiometry

• UV crosslinking:

  • Identify specific RNA binding sites by crosslinking followed by mass spectrometry

How does the structure of I. loihiensis NusB compare to other bacterial NusB proteins?

While the specific structure of I. loihiensis NusB has not been directly reported in the provided references, a comparative structural analysis can be approached methodologically as follows:

• Structure prediction and modeling:

  • Generate homology models based on the known E. coli NusB structure

  • E. coli NusB contains seven α-helices in a primarily α-helical structure

  • Validate models using energy minimization and Ramachandran plot analysis

• Experimental structure determination:

  • Express isotopically labeled protein (15N, 13C, 2H) for NMR studies

  • Use heteronuclear three-dimensional triple-resonance NMR experiments combined with semi-automatic assignment procedures

  • Analyze secondary structure using scalar couplings, chemical shift values, amide-exchange data, and NOE data

• Comparative analysis:

  • Identify conserved structural features across NusB homologs

  • Map sequence conservation onto structural models

  • Correlate structural differences with adaptation to extreme environments

How might the extreme environmental adaptations of I. loihiensis affect its NusB function?

I. loihiensis has several adaptations to its extreme deep-sea hydrothermal vent environment that could potentially influence NusB function:

• Halophilic adaptations:

  • I. loihiensis can grow in medium containing up to 20% (w/v) NaCl

  • Methodology: Compare salt tolerance of I. loihiensis NusB activity to mesophilic homologs using transcription assays across salt gradients

• Temperature adaptations:

  • I. loihiensis grows across a wide temperature range (4-46°C)

  • Methodology: Assess thermostability using differential scanning calorimetry and circular dichroism across temperature ranges

• Pressure adaptations:

  • Being isolated from 1,300m depth, potential adaptations to high pressure

  • Methodology: Investigate protein activity and stability under varying pressure conditions using specialized high-pressure spectroscopic equipment

• Amino acid metabolism connection:

  • I. loihiensis relies primarily on amino acid catabolism rather than sugar fermentation

  • Methodology: Investigate potential regulatory connections between NusB function and amino acid metabolism using transcriptomics and metabolomics approaches

What is the molecular basis for recognition of specific RNA elements by I. loihiensis NusB?

To elucidate the molecular basis of RNA recognition:

• Identification of RNA binding motifs:

  • Perform RNA-seq and CLIP-seq experiments to identify in vivo binding sites

  • Use in vitro selection methods (SELEX) to determine preferred binding sequences

• Mutagenesis studies:

  • Generate alanine scanning mutants of conserved residues

  • Assess impact on RNA binding using quantitative binding assays

  • Correlate with functional effects in transcription antitermination assays

• Structural characterization of complexes:

  • Solve structures of NusB-RNA complexes using X-ray crystallography or Cryo-EM

  • Identify key residues involved in RNA recognition

  • Compare with known NusB-RNA complexes from model organisms

How can I determine if I. loihiensis NusB interacts with other transcription factors?

To characterize protein-protein interactions:

• Pull-down assays and co-immunoprecipitation:

  • Use tagged NusB to pull down interacting partners

  • Identify binding partners through mass spectrometry

• Yeast two-hybrid or bacterial two-hybrid screening:

  • Screen for interactions with known transcription factors

  • Perform genome-wide screens to identify novel interactions

• Surface plasmon resonance:

  • Quantify binding kinetics and affinities with purified potential partners

  • Determine effects of mutations on complex formation

• Structural studies of complexes:

  • Use NMR, X-ray crystallography, or Cryo-EM to determine structures of protein complexes

  • Map interaction interfaces through hydrogen-deuterium exchange mass spectrometry

How do I analyze potential functional differences between I. loihiensis NusB and E. coli NusB?

To compare functional properties between homologs:

• Complementation studies:

  • Express I. loihiensis NusB in E. coli nusB deletion strains

  • Assess ability to restore antitermination function

  • Measure growth under various conditions

• In vitro transcription assays:

  • Reconstitute transcription systems with purified components

  • Compare ability to promote readthrough at termination sites

  • Measure kinetics of antitermination

• Domain swap experiments:

  • Create chimeric proteins containing domains from both homologs

  • Identify domains responsible for functional differences

• Differential expression analysis:

  • Perform RNA-seq to identify differentially regulated genes when expressing either homolog

What approaches should I use to study the role of NusB in I. loihiensis gene regulation?

To investigate the regulatory role of NusB in I. loihiensis:

• Genetic manipulation:

  • Develop genetic tools for I. loihiensis (currently limited)

  • Create conditional knockdown or deletion mutants

  • Perform complementation with wild-type and mutant variants

• Transcriptome analysis:

  • Compare transcriptomes of wild-type and NusB-depleted strains

  • Identify genes affected by NusB deficiency

  • Focus on changes in amino acid metabolism pathways, which are central to I. loihiensis metabolism

• Chromatin immunoprecipitation (ChIP-seq):

  • Map genomic binding sites of NusB

  • Correlate with transcriptional changes

  • Identify consensus binding motifs

• In vitro reconstitution:

  • Reconstitute transcription complexes with I. loihiensis components

  • Study effects of NusB on transcription termination/antitermination

How does the study of I. loihiensis NusB contribute to our understanding of deep-sea microbial adaptations?

Studying I. loihiensis NusB provides insights into deep-sea adaptations through:

• Transcriptional regulation in extreme environments:

  • I. loihiensis inhabits hydrothermal vents with fluctuating conditions

  • Methodology: Compare transcription regulation mechanisms across bacteria from different extreme environments

• Metabolic specialization:

  • I. loihiensis relies primarily on amino acid catabolism rather than sugar fermentation

  • Methodology: Investigate whether NusB regulates genes involved in protein/peptide utilization

• Biofilm formation connection:

  • I. loihiensis produces exopolysaccharide and has a 32-gene cluster for its synthesis

  • Methodology: Examine if NusB influences expression of biofilm-related genes

• Evolutionary considerations:

  • I. loihiensis represents a distinct lineage among γ-proteobacteria

  • Methodology: Perform phylogenetic analyses of NusB across bacterial lineages to trace evolutionary adaptations

What role might NusB play in the ecological success of Idiomarina species in deep-sea environments?

NusB may contribute to ecological fitness through:

• Regulation of stress responses:

  • Methodology: Compare expression profiles under various stressors (temperature, pressure, salt) between wild-type and NusB-depleted strains

  • Focus on genes involved in protein quality control and stress response

• Coordination of metabolic adaptation:

  • I. loihiensis genome reveals an integrated mechanism of metabolic adaptation to constantly changing deep-sea hydrothermal ecosystems

  • Methodology: Investigate if NusB regulates metabolic switching in response to nutrient availability

• Influence on community interactions:

  • Idiomarina species develop biofilms by secreting exopolysaccharides

  • Methodology: Examine if NusB affects expression of genes involved in cell-cell communication and biofilm formation

• Adaptation to nutrient limitation:

  • I. loihiensis has incomplete biosynthetic pathways for several amino acids (Leu, Ile, Val, Thr, and Met)

  • Methodology: Test whether NusB regulates expression of transporters for these essential amino acids