Recombinant Idiomarina loihiensis Putative phosphotransferase IL1317 (IL1317)

What is Idiomarina loihiensis and where was it discovered?

Idiomarina loihiensis is a γ-proteobacterium isolated from hydrothermal vents on the Lōihi submarine volcano in Hawaii at a depth of approximately 1,300 meters . This organism inhabits partially oxygenated cold waters at the periphery of hydrothermal vents and demonstrates remarkable adaptability, surviving across a wide range of growth temperatures (4°C to 46°C) and salinities (0.5% to 20% NaCl) . The complete genome of I. loihiensis consists of a single circular chromosome of 2,839,318 base pairs with an average G+C content of 47% . Unlike many other microorganisms found in similar environments, I. loihiensis represents a distinct lineage among γ-proteobacteria, having branched from the main trunk of the γ-proteobacterial phylogenetic tree after the Pseudomonas lineage but before the Vibrio cluster, as determined by phylogenetic analysis of concatenated ribosomal protein sequences .

What is known about the IL1317 gene and its product?

The IL1317 gene in Idiomarina loihiensis L2TR is located at genomic position 1418014..1418823 on the positive strand and encodes a hypothetical protein of 269 amino acids in length . The gene has a G+C content of 43.33%, which is slightly lower than the genome average (47%), and is identified with protein ID 56460425 . Although annotated as a hypothetical protein in genomic databases, computational predictions suggest it may function as a phosphotransferase, though this function has not been experimentally verified. The gene is situated in a genomic region where several other hypothetical proteins are encoded (such as IL1318), suggesting it might be part of a functional module that has yet to be fully characterized . Structural predictions would likely place IL1317 among phosphotransferase enzyme families, potentially involved in phosphoryl group transfer reactions critical for cellular signaling or metabolic pathways.

How does IL1317 fit into the metabolic strategy of Idiomarina loihiensis?

Given that Idiomarina loihiensis relies primarily on amino acid catabolism rather than sugar fermentation for carbon and energy, IL1317 may play a role in this specialized metabolic strategy . The genome analysis of I. loihiensis reveals an abundance of amino acid transport and degradation enzymes coupled with a loss of sugar transport systems and certain enzymes of sugar metabolism . As a putative phosphotransferase, IL1317 might participate in phosphorylation events related to amino acid metabolism, peptide utilization, or signal transduction pathways that are crucial for the organism's survival in nutrient-limited deep-sea environments. The protein could potentially be involved in the organism's strategy of colonizing proteinaceous particles in hydrothermal vent waters, contributing to processes that allow I. loihiensis to digest environmental proteins and metabolize the resulting peptides and amino acids . Understanding IL1317's exact role would require experimental verification through techniques such as gene knockout studies and metabolic flux analysis.

What expression systems are suitable for producing recombinant IL1317?

For recombinant expression of IL1317, bacterial expression systems such as pSpeedET vectors have been successfully employed with I. loihiensis genes, as evidenced by the availability of expression constructs for other I. loihiensis proteins . E. coli-based expression systems would typically be the first choice due to their well-established protocols, high yield potential, and versatility. When expressing IL1317, researchers should consider optimizing codon usage for E. coli, as differences in codon bias between the deep-sea bacterium and the expression host could affect translation efficiency. Temperature optimization is particularly important, given that I. loihiensis naturally grows in a wide temperature range (4°C to 46°C), suggesting that low-temperature expression strategies might improve protein solubility and proper folding . Additionally, considering the relatively high G+C content variation in the I. loihiensis genome, careful design of expression constructs, potentially including solubility tags like MBP or SUMO, may improve recombinant protein yield and quality.

What structural and functional analyses are recommended to characterize the enzymatic activity of IL1317?

Comprehensive characterization of IL1317's enzymatic activity requires a multi-faceted approach combining structural and biochemical techniques. Initially, researchers should purify the recombinant protein to high homogeneity using affinity chromatography, followed by size exclusion and ion-exchange chromatography if necessary. Structural determination through X-ray crystallography or cryo-electron microscopy would provide critical insights into the active site architecture and potential substrate binding pockets. For functional analysis, researchers should conduct phosphotransferase activity assays using various potential substrates, including ATP, GTP, phosphoenolpyruvate, and different target proteins or metabolites relevant to amino acid metabolism . Enzymatic parameters (Km, Vmax, kcat) should be determined under various pH and salt conditions that mimic the hydrothermal vent environment. Additionally, site-directed mutagenesis of predicted catalytic residues would help confirm the reaction mechanism. Isothermal titration calorimetry and surface plasmon resonance techniques can further elucidate substrate binding affinity and kinetics, providing a comprehensive picture of IL1317's biochemical function in relation to I. loihiensis' unique metabolic adaptations to the deep-sea environment.

How can transcriptomic and proteomic approaches be applied to understand IL1317 expression patterns?

To elucidate the expression patterns and regulation of IL1317, researchers should implement integrated transcriptomic and proteomic approaches under various physiological conditions. RNA-Seq analysis of I. loihiensis cultures grown under different conditions (varying amino acid availability, salt concentrations, temperatures, and oxygen levels) would reveal transcriptional regulation patterns of IL1317 and potentially co-regulated genes. Quantitative PCR could validate expression changes observed in high-throughput data. At the protein level, targeted proteomics using selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) would enable precise quantification of IL1317 abundance across different conditions. Phosphoproteomics analysis would be particularly valuable to identify potential phosphorylation sites on IL1317 itself or its substrates, providing functional context. Ribosome profiling could further reveal translational regulation mechanisms. To identify protein-protein interactions, researchers should employ co-immunoprecipitation followed by mass spectrometry or bacterial two-hybrid systems, potentially revealing functional complexes involving IL1317. Integration of these multi-omics data using systems biology approaches would situate IL1317 within the broader adaptive response network of I. loihiensis to its extreme environment.

What are the best approaches for investigating the physiological role of IL1317 through gene manipulation?

Investigating the physiological role of IL1317 requires systematic genetic manipulation approaches adapted for the challenging characteristics of Idiomarina loihiensis. Researchers should first develop or optimize genetic transformation protocols specifically for I. loihiensis, potentially adapting methods from related γ-proteobacteria. For gene deletion experiments, allelic exchange methods using suicide vectors carrying homologous flanking regions could create a clean IL1317 knockout strain. Complementation studies, reintroducing the wild-type gene or mutated versions under native or inducible promoters, would confirm phenotype specificity. CRISPR-Cas9 systems could be adapted for I. loihiensis to facilitate precise genome editing. Phenotypic characterization of mutant strains should include growth curve analysis under various amino acid sources, metabolic profiling using LC-MS/MS, and stress response assays mimicking hydrothermal vent conditions. Particularly important would be examining the mutant's ability to utilize proteinaceous particles, given I. loihiensis' specialized ecological niche . Conditional knockdown approaches using inducible antisense RNA or CRISPRi might be valuable if IL1317 proves essential. Additionally, researchers should consider in situ expression studies using reporter fusions to understand the spatial and temporal dynamics of IL1317 expression in simulated natural environments.

How does IL1317 compare to homologous proteins in related species, and what can comparative genomics reveal about its evolution?

Comparative genomic analysis of IL1317 would provide valuable insights into its evolutionary history and functional significance across the Idiomarina genus and related γ-proteobacteria. Researchers should begin with comprehensive sequence similarity searches using position-specific iterative BLAST (PSI-BLAST) and hidden Markov models to identify remote homologs beyond conventional BLAST detection limits. Multiple sequence alignment of identified homologs would reveal conserved motifs potentially associated with catalytic activity or substrate binding. Phylogenetic reconstruction using maximum likelihood or Bayesian methods could trace the evolutionary trajectory of IL1317, potentially identifying events of horizontal gene transfer or gene duplication. Synteny analysis examining the conservation of gene neighborhood across species would help identify functionally linked genes. Positive selection analysis using dN/dS ratios could highlight residues under evolutionary pressure, potentially indicating functional importance. Recent comparative studies of novel Idiomarina species from hypersaline Miocene deposits have employed Average Nucleotide Identity (ANI) and Average Amino Acid Identity (AAI) analyses (with threshold values of 95% and 95-96%, respectively) to establish evolutionary relationships . Similar approaches could position IL1317 within the broader context of Idiomarina adaptation to extreme environments. Function-based comparison tools, such as those provided by the RAST server, would help assess the presence of similar functional roles across different Idiomarina genomes .

The following table summarizes key features of IL1317 and methodological approaches for its study:

What strategies can overcome challenges in expressing soluble recombinant IL1317?

When encountering difficulties in obtaining soluble recombinant IL1317, researchers should implement a systematic troubleshooting approach addressing multiple aspects of the expression system. First, consider expression temperature optimization—lowering to 16-20°C can significantly improve folding of proteins from psychrophilic organisms like I. loihiensis, which naturally inhabits environments with varied temperatures . The addition of solubility-enhancing fusion partners such as MBP, SUMO, or thioredoxin often dramatically improves protein solubility. Researchers should experiment with different E. coli expression strains, particularly those designed for difficult proteins (Rosetta for rare codon issues, Origami for disulfide bond formation, or ArcticExpress for low-temperature expression). Codon optimization of the IL1317 sequence for E. coli is crucial given the different G+C content (43.33% for IL1317 versus ~50% for E. coli) . Altering buffer conditions during lysis and purification by screening various salt concentrations, pH values, and stabilizing additives (glycerol, reducing agents, specific ions) may also improve solubility. If inclusion bodies form despite these measures, researchers should develop refolding protocols using gradual dialysis or on-column refolding techniques. Finally, high-throughput screening of expression conditions using parallel small-scale cultures with varying induction parameters can efficiently identify optimal soluble expression conditions.

How can researchers address the challenges of determining substrate specificity for a putative phosphotransferase like IL1317?

Determining the substrate specificity of IL1317 presents significant challenges that require a methodical experimental approach. Researchers should begin with untargeted screening using phosphotransferase activity assays against a diverse panel of potential substrates, including different nucleotides (ATP, GTP, CTP), sugar phosphates, and phosphorylated compounds relevant to amino acid metabolism (given I. loihiensis' metabolic preferences) . Radiometric assays using 32P-labeled substrates offer high sensitivity for initial activity detection. Subsequently, researchers should employ protein microarrays or peptide libraries to identify potential protein substrates, focusing on proteins involved in amino acid utilization pathways. Metabolomic approaches using LC-MS/MS can identify changes in cellular phosphometabolite profiles when IL1317 is overexpressed or deleted. Structural studies through co-crystallization with substrate analogs or inhibitors can provide direct evidence of substrate binding modes. Computational approaches including molecular docking and molecular dynamics simulations may predict binding affinities for putative substrates. Researchers should also consider environmental context—examining potential substrates that would be abundant in deep-sea hydrothermal environments where I. loihiensis naturally occurs. Cross-linking mass spectrometry (XL-MS) could capture transient enzyme-substrate interactions, providing additional evidence for physiologically relevant substrates in complex mixtures.

What approaches help resolve contradictory results when analyzing IL1317 function in different experimental systems?

When faced with contradictory results regarding IL1317 function across different experimental systems, researchers should implement a strategic approach to resolve these discrepancies. First, standardize experimental conditions across all platforms, ensuring comparable protein concentrations, buffer compositions, and assay parameters. Verify protein quality through multiple methods (circular dichroism, size exclusion chromatography, dynamic light scattering) to confirm proper folding and oligomeric state in each experimental system. Consider the influence of different tags or fusion partners on protein function by creating multiple constructs with varied tag positions (N-terminal, C-terminal) or cleavable tags. When contradictions arise between in vitro and in vivo results, develop defined in vitro systems that more closely mimic the physiological environment of deep-sea bacteria, including appropriate salt concentrations and pressure conditions. Implement orthogonal functional assays that measure the same activity through different detection methods to rule out assay-specific artifacts. Collaborate with structural biologists to determine if conformational changes under different experimental conditions might explain functional variations. Consider performing evolutionary coupling analysis to identify co-evolving residues that might indicate functional interactions not evident in single-protein studies. Finally, develop a consensus model that accounts for seemingly contradictory data by considering context-dependent functionality, which may be particularly relevant for proteins from extremophiles like I. loihiensis that must function under varying environmental conditions .

What bioinformatic approaches can predict the three-dimensional structure and functional sites of IL1317?

Advanced bioinformatic approaches for predicting IL1317's structure require integration of multiple computational methods to achieve maximum accuracy. Researchers should begin with state-of-the-art deep learning-based structure prediction tools such as AlphaFold2 or RoseTTAFold, which have revolutionized protein structure prediction by accurately modeling proteins with limited sequence homology to known structures. These predictions should be refined through molecular dynamics simulations in explicit solvent conditions mimicking the high-salt environment of I. loihiensis' natural habitat . For functional site prediction, conservation analysis using the ConSurf server can identify evolutionarily conserved residues likely to be functionally important. Machine learning approaches like COACH-D and COFACTOR can predict ligand-binding sites and potential catalytic residues based on structural comparisons. Researchers should employ electrostatic surface analysis to identify potential phosphate-binding regions characteristic of phosphotransferases. Computational solvent mapping techniques can identify hotspots where small molecules tend to bind, potentially revealing substrate interaction sites. Graph-theoretical approaches analyzing residue interaction networks might identify allosteric sites that regulate enzyme activity. For validation, researchers should compare predictions across multiple methods and assess their statistical significance. Finally, integrating predicted structures with genomic context data—analyzing the functions of neighboring genes and their conservation across species—can provide additional functional insights within the broader metabolic context of I. loihiensis' adaptation to deep-sea hydrothermal environments.

How can systems biology approaches integrate IL1317 into the metabolic network of Idiomarina loihiensis?

Integrating IL1317 into the metabolic network of Idiomarina loihiensis requires sophisticated systems biology approaches that place this putative phosphotransferase within the context of the organism's unique metabolic adaptations. Researchers should begin by constructing a genome-scale metabolic model (GEM) of I. loihiensis, leveraging existing annotation data and filling gaps through computational predictions and manual curation. This model should specifically emphasize amino acid catabolism pathways, given I. loihiensis' specialization in protein utilization rather than sugar metabolism . Flux balance analysis (FBA) can then simulate metabolic flows under different environmental conditions, with particular attention to how variations in amino acid availability affect metabolic flux distributions. To position IL1317 within this network, researchers should implement enzyme constraint-based metabolic modeling (ECMM) incorporating kinetic parameters of IL1317 once experimentally determined. Network analysis tools can identify metabolic choke points and essential reactions, potentially highlighting critical roles for IL1317. Integration of transcriptomic, proteomic, and metabolomic data through multi-omics data integration platforms would enable contextualization of IL1317 expression patterns with metabolic shifts under different growth conditions. Researchers should apply machine learning approaches to predict regulatory interactions, potentially identifying transcription factors that control IL1317 expression. Finally, comparative systems biology approaches examining metabolic network differences between I. loihiensis and related species could reveal how IL1317 contributes to the unique ecological adaptations that allow this organism to thrive in deep-sea hydrothermal environments.

How might IL1317 contribute to Idiomarina loihiensis' adaptation to deep-sea hydrothermal environments?

The putative phosphotransferase IL1317 likely plays a specialized role in Idiomarina loihiensis' remarkable adaptation to the challenging deep-sea hydrothermal vent environment. Within these ecosystems, nutrient availability fluctuates dramatically as hydrothermal fluids mix with cold seawater, creating steep chemical and temperature gradients . As a potential phosphotransferase, IL1317 may facilitate rapid phosphorylation-based signaling responses to these environmental changes, enabling swift metabolic adjustments. The protein could be integral to I. loihiensis' distinctive strategy of colonizing proteinaceous particles in hydrothermal waters, potentially phosphorylating key proteins involved in exopolysaccharide production, which the organism uses for adhesion to these nutrient-rich particles . Given that I. loihiensis relies primarily on amino acid catabolism rather than sugar fermentation for carbon and energy, IL1317 might catalyze essential phosphorylation reactions in amino acid utilization pathways . The protein could also contribute to stress response mechanisms necessary for surviving the wide temperature range (4°C to 46°C) and salinity variations (0.5% to 20% NaCl) that I. loihiensis encounters in its natural habitat . Comparative genomic analyses with other hydrothermal vent microorganisms could reveal whether IL1317 represents a unique adaptation specific to Idiomarina or a more widespread strategy for surviving in these extreme environments.

What insights can be gained from studying IL1317 expression under simulated deep-sea conditions?

Studying IL1317 expression under simulated deep-sea conditions would provide crucial insights into its physiological role and regulation in Idiomarina loihiensis' natural habitat. Researchers should design specialized bioreactors capable of mimicking the hydrothermal vent environment, including high pressure (approximately 130 atmospheres at 1,300 meters depth), variable temperature gradients (4°C to 46°C), and chemical compositions characteristic of vent fluids . Time-course transcriptomic and proteomic analyses during transitions between different environmental conditions would reveal dynamic expression patterns of IL1317 and co-regulated genes. Particular attention should be paid to expression changes in response to various protein and amino acid sources, given I. loihiensis' specialization in amino acid catabolism . Researchers should implement reporter systems (such as transcriptional fusions to fluorescent proteins) to visualize IL1317 expression in real-time within the simulated environment. Comparison of expression patterns between laboratory and simulated deep-sea conditions might reveal environment-specific regulatory mechanisms not observable under standard laboratory cultivation. Single-cell approaches could identify potential heterogeneity in IL1317 expression within I. loihiensis populations, potentially indicating bet-hedging strategies for survival in fluctuating deep-sea environments. Additionally, researchers should examine IL1317 expression in the context of multispecies communities that mimic the microbial consortia found in hydrothermal vent ecosystems, potentially revealing interspecies interactions that influence its regulation.