Recombinant Rhodopirellula baltica Arginine--tRNA ligase (argS), partial

What is Rhodopirellula baltica and why is it significant as a model organism?

Rhodopirellula baltica (R. baltica) is a marine representative of the globally distributed bacterial phylum Planctomycetes, first isolated from the water column in the Kiel Fjord (Baltic Sea). This organism is significant as a model system due to several unique characteristics that distinguish it within the bacterial domain.

R. baltica possesses several remarkable features that make it valuable for research: peptidoglycan-free proteinaceous cell walls, intracellular compartmentalization, and a distinctive reproduction mode via budding that resembles that of Caulobacter crescentus . The organism exhibits a complex life cycle with morphological transitions between motile swarmer cells and sessile cells that produce holdfast substances .

The complete genome sequence revealed R. baltica has one of the largest circular bacterial genomes sequenced, comprising 7.145 megabases . This extensive genetic repertoire includes unexpected metabolic capabilities for an aerobic heterotroph, including genes for heterolactic acid fermentation, C1 compound interconversion, and an impressive array of 110 sulfatases .

What is the function of Arginine--tRNA ligase (argS) in bacterial systems?

Arginine--tRNA ligase, encoded by the argS gene, catalyzes the attachment of arginine to its cognate tRNA molecule, producing arginyl-tRNA that is essential for protein synthesis. This enzyme belongs to the aminoacyl-tRNA synthetase family and plays a critical role in translation by ensuring accurate incorporation of arginine residues into nascent polypeptides.

In R. baltica specifically, the argS gene is part of the complex regulatory network involved in amino acid metabolism. During growth phase transitions, R. baltica modifies expression of genes involved in amino acid biosynthesis pathways, including arginine metabolism . The expression profile of argS may fluctuate in response to changing nutrient conditions, as evidenced by transcriptional profiling that showed differential regulation of amino acid metabolism genes across growth phases .

How does the structure of Arginine--tRNA ligase from R. baltica compare to homologous enzymes from other bacterial species?

While the specific structural details of R. baltica Arginine--tRNA ligase are not fully elucidated in the provided literature, comparative genomic analyses indicate that aminoacyl-tRNA synthetases generally maintain highly conserved catalytic domains while exhibiting species-specific variations in auxiliary domains.

The enzyme likely maintains the canonical class I aminoacyl-tRNA synthetase architecture featuring a Rossmann fold for ATP binding. Phylogenetic analysis clearly affiliates Planctomycetes to the bacterial domain as a distinct phylum , suggesting that while core enzymatic functions are conserved, R. baltica's argS may incorporate structural adaptations reflecting its marine lifestyle and unique cellular organization.

Researchers investigating structural comparisons should employ multiple sequence alignment tools followed by homology modeling using resolved crystal structures from related organisms as templates. Computational approaches such as molecular dynamics simulations can further illuminate potential structural adaptations that influence substrate recognition and catalytic efficiency in the marine environment.

What are the optimal conditions for expressing recombinant R. baltica Arginine--tRNA ligase in heterologous systems?

Successful expression of recombinant R. baltica Arginine--tRNA ligase requires careful optimization of several parameters:

Codon Optimization: R. baltica exhibits distinctive codon usage patterns that may impede efficient translation in standard expression hosts. Codon optimization of the argS gene sequence for the selected expression host is recommended to improve protein yields.

Culture Conditions Protocol:

• Initiate cultures at 37°C until mid-log phase (OD600 ~0.6-0.8)

• Reduce temperature to 16-18°C prior to induction

• Induce with low IPTG concentrations (0.1-0.5 mM) when using T7-based expression systems

• Extend expression time to 16-24 hours at the reduced temperature

• Supplement media with 5-10% NaCl to mimic the marine environment, as R. baltica demonstrates halo-tolerance

Purification Strategy: A two-step chromatography approach is recommended:

• Initial capture using immobilized metal affinity chromatography (IMAC) if a histidine tag is incorporated

• Subsequent polishing step using ion exchange chromatography

• Maintain buffers at pH 7.5-8.0 with 100-250 mM NaCl throughout purification

How should researchers design experiments to assess the catalytic activity of R. baltica Arginine--tRNA ligase?

When designing experiments to evaluate the catalytic activity of R. baltica Arginine--tRNA ligase, researchers should implement a comprehensive assay system:

ATP-PPi Exchange Assay:

• Prepare reaction mixture containing purified enzyme (10-100 nM), arginine (1-5 mM), ATP (2-5 mM), [32P]PPi, and appropriate buffer (typically HEPES or Tris at pH 7.5-8.0)

• Incubate at 25-30°C (optimal for R. baltica enzymes based on growth temperature preferences)

• At timed intervals, remove aliquots and quench with activated charcoal in 2% TCA

• Wash charcoal and measure radioactivity via liquid scintillation counting

• Calculate initial velocities and determine kinetic parameters (kcat, Km)

tRNA Aminoacylation Assay:

• Generate substrate tRNAArg through in vitro transcription or isolation from appropriate source

• Combine purified enzyme (10-100 nM) with tRNAArg (1-10 μM), arginine (including trace [³H]- or [¹⁴C]-arginine), ATP (2-5 mM), and buffer containing Mg²⁺

• Monitor aminoacylation by acid-precipitable counts or by using filter-binding techniques

• For real-time analysis, consider using fluorescently labeled tRNA substrates and monitoring changes in anisotropy

Data Analysis Protocol:

• Determine initial reaction velocities under varying substrate concentrations

• Plot data using appropriate enzyme kinetics models (Michaelis-Menten, Lineweaver-Burk)

• Calculate and compare kinetic parameters with those from related organisms

• Evaluate salt dependence given R. baltica's marine habitat

What approaches can be used to investigate the interaction between R. baltica Arginine--tRNA ligase and its cognate tRNA?

To elucidate the molecular basis of specific recognition between R. baltica Arginine--tRNA ligase and its cognate tRNA, researchers can employ several complementary techniques:

Binding Affinity Measurements:

• Surface Plasmon Resonance (SPR): Immobilize either the enzyme or tRNA on a sensor chip and measure binding kinetics by flowing the partner molecule at varying concentrations

• Microscale Thermophoresis (MST): Label either interaction partner fluorescently and detect binding-induced changes in thermophoretic mobility

• Isothermal Titration Calorimetry (ITC): Directly measure thermodynamic parameters of binding without requiring modification of either component

Mutational Analysis Workflow:

• Identify conserved residues across argS homologs through multiple sequence alignment

• Generate point mutations in predicted recognition elements using site-directed mutagenesis

• Express and purify mutant enzymes following optimized protocols

• Assess impact on tRNA binding and aminoacylation efficiency

• Create a comprehensive structure-function relationship map based on mutational data

Structural Studies Approach:

• Prepare stable complexes of enzyme and cognate tRNA

• Employ X-ray crystallography or cryo-electron microscopy to determine complex structure

• Validate key interactions through hydrogen-deuterium exchange mass spectrometry

• Compare recognition elements with those identified in related aminoacyl-tRNA synthetases

How can the study of R. baltica Arginine--tRNA ligase contribute to understanding marine bacterial adaptation?

R. baltica Arginine--tRNA ligase serves as an excellent model for investigating adaptations of marine microorganisms to their specific environmental conditions. Its study provides valuable insights in several key areas:

Osmoadaptation Mechanisms: Marine bacteria must maintain enzymatic function under fluctuating salinity conditions. Analysis of R. baltica Arginine--tRNA ligase activity across salt concentration gradients provides insights into osmoadaptive strategies. Growth studies demonstrate that R. baltica exhibits remarkable salt tolerance, maintaining similar growth patterns between 1.15% and 2.3% NaCl, with only slight growth delays at higher concentrations (4.6% NaCl) . This halo-tolerance suggests the enzyme has evolved structural adaptations to maintain stability and function across varying ionic strengths.

Temperature Adaptation: Examination of temperature-dependent enzyme kinetics reveals adaptations to the marine thermocline. Researchers should:

• Measure enzyme activity across 4-37°C temperature range

• Compare thermal stability parameters with terrestrial homologs

• Identify structural elements contributing to cold adaptation

• Correlate findings with transcriptomic data from different growth conditions

Nutrient Response Integration: The expression of argS in R. baltica is likely coordinated with broader metabolic adaptations to nutrient availability in marine environments. Transcriptomic profiling has revealed that R. baltica modifies expression of genes involved in amino acid metabolism during growth phase transitions . The table below summarizes gene regulation patterns observed during R. baltica's growth cycle:

Table 1: Differential gene expression during R. baltica growth phases

What role might Arginine--tRNA ligase play in the unusual cell cycle and morphological differentiation of R. baltica?

R. baltica exhibits a distinctive cell cycle featuring morphological differentiation between motile swarmer cells and sessile cells that form aggregates called rosettes . The potential involvement of Arginine--tRNA ligase in this process merits detailed investigation:

Cell Cycle-Dependent Expression Analysis:
Microscopic examination has shown that R. baltica cultures transition from predominantly swarmer and budding cells in early exponential phase to predominantly rosette formations in stationary phase . Researchers should:

• Synchronize R. baltica cultures using established techniques

• Sample cells at defined cell cycle stages for transcriptomic and proteomic analysis

• Quantify argS expression levels across morphological transitions

• Correlate argS expression with stage-specific protein synthesis requirements

Protein Localization Studies:
The compartmentalized nature of Planctomycetes cells raises questions about the subcellular distribution of translation machinery. To investigate:

• Generate fluorescently tagged Arginine--tRNA ligase constructs

• Perform live-cell imaging across the cell cycle

• Co-localize with markers for different cellular compartments

• Assess if protein localization changes during morphological transitions

Functional Inhibition Experiments:
To directly test the role of argS in morphological differentiation:

• Design antisense RNA constructs targeting argS

• Develop CRISPR interference systems for conditional knockdown

• Administer sub-inhibitory concentrations of relevant aminoacyl-tRNA synthetase inhibitors

• Monitor effects on cell cycle progression and morphological development

• Analyze impact on holdfast substance production and rosette formation

How does the genome context of argS in R. baltica inform our understanding of its evolutionary history and functional relationships?

The genomic neighborhood of argS provides critical insights into its evolutionary trajectory and functional connections within R. baltica's metabolic network.

Comparative Genomic Analysis:
R. baltica possesses one of the largest bacterial genomes sequenced (7.145 megabases) , suggesting extensive gene acquisition and metabolic versatility. Analysis of the argS locus and surrounding genes reveals:

• Conservation patterns across Planctomycetes and related phyla

• Evidence of horizontal gene transfer events

• Co-evolution with tRNA genes and other translation machinery components

• Potential operonic structures or regulatory units

Regulatory Element Identification:
Understanding the transcriptional control of argS requires:

• In silico prediction of promoters and regulatory motifs

• Experimental validation through reporter gene assays

• Chromatin immunoprecipitation to identify transcription factor binding

• Integration with global transcriptomic data from different growth conditions

Metabolic Network Integration:
The argS gene functions within a broader network connecting translation to arginine metabolism. R. baltica demonstrates upregulation of glutamate dehydrogenase (RB6930) during transition to stationary phase, which is involved in the biosynthesis pathway for arginine, glutamate, and proline . This suggests coordinated regulation between aminoacyl-tRNA synthetases and amino acid biosynthetic pathways.

What are the common challenges in purifying active recombinant R. baltica Arginine--tRNA ligase and how can they be addressed?

Researchers frequently encounter several challenges when purifying active R. baltica Arginine--tRNA ligase:

Solubility Issues:
R. baltica proteins often exhibit solubility challenges in standard expression systems due to their adaptation to marine environments.

Solution Protocol:

• Express protein at reduced temperatures (16-18°C)

• Incorporate solubility-enhancing fusion tags (MBP, SUMO, or Thioredoxin)

• Supplement lysis buffer with 5-10% glycerol and 1-5% non-ionic detergents

• Add low concentrations of arginine (50-200 mM) to the buffer to enhance solubility

• Consider on-column refolding procedures if inclusion bodies form

Enzyme Instability:
Loss of activity during purification is a common issue with aminoacyl-tRNA synthetases.

Stabilization Strategy:

• Maintain strict temperature control (4°C) throughout purification

• Add stabilizing agents to all buffers (glycerol 10%, DTT 1-5 mM)

• Include substrate analogs (ATP analogs or arginine) at low concentrations

• Minimize purification duration with streamlined protocols

• Add protease inhibitor cocktails to prevent degradation

Contaminant RNA Binding:
Aminoacyl-tRNA synthetases naturally bind RNA, leading to co-purification of cellular RNAs.

RNA Removal Procedure:

• Incorporate high-salt washes (500 mM - 1 M NaCl) during initial purification steps

• Add RNase treatment steps where appropriate

• Implement additional purification steps such as size exclusion chromatography

• Include polyethyleneimine precipitation steps to remove nucleic acids

How can researchers effectively design and interpret kinetic studies of R. baltica Arginine--tRNA ligase under various environmental conditions?

Designing rigorous kinetic studies for R. baltica Arginine--tRNA ligase requires careful consideration of experimental parameters and appropriate data analysis:

Environmental Parameter Matrix:
Create a comprehensive testing matrix incorporating:

• Temperature range (4-37°C in 5°C increments)

• Salt concentration series (0-1M NaCl)

• pH gradients (pH 6.0-9.0)

• Divalent metal ion variations (Mg²⁺, Mn²⁺, Ca²⁺)

Experimental Design Guidelines:

• Include sufficient replicates (n=3 minimum) for statistical validity

• Incorporate appropriate controls for spontaneous ATP hydrolysis

• Ensure initial rate conditions are maintained (<10% substrate consumption)

• Use multiple substrate concentration ranges to detect cooperative effects

Data Analysis Framework:

• Apply appropriate kinetic models:

  • Michaelis-Menten for standard kinetics

  • Hill equation if cooperativity is observed

  • Competitive, non-competitive, or mixed inhibition models when relevant

• Calculate key parameters (Km, kcat, kcat/Km) under each condition

• Create Arrhenius plots to determine activation energies

• Generate comprehensive surface response plots for multi-parameter analyses

Interpretation Strategy:

• Compare kinetic parameters across environmental conditions

• Correlate enzyme performance with R. baltica's known ecological parameters

• Identify conditions that significantly impact catalytic efficiency

• Relate findings to adaptation strategies in the marine environment

What sequencing and bioinformatic approaches should be used to analyze variants of the argS gene in R. baltica environmental isolates?

Effective analysis of argS variants requires an integrated approach combining modern sequencing technologies with sophisticated bioinformatic analysis:

Sequencing Strategy:

• Employ targeted approaches:

  • PCR amplification and Sanger sequencing for specific regions

  • Targeted enrichment followed by next-generation sequencing for higher throughput

• Implement whole-genome sequencing:

  • Short-read technologies (Illumina) for high accuracy

  • Long-read technologies (PacBio, Oxford Nanopore) to resolve complex genomic regions

• Consider metagenomic approaches for environmental samples:

  • Shotgun metagenomics for community-wide analysis

  • Targeted metatranscriptomics to assess expression levels

Assembly and Annotation Pipeline:
Modern assembly tools apply De Bruijn Graph (DBG) approaches that effectively handle high-volume sequencing data . The recommended workflow includes:

• Quality control and preprocessing:

  • Remove low-quality reads and adapters

  • Error correction to eliminate sequencing artifacts

• Assembly using appropriate tools:

  • SPAdes or Velvet for isolate genomes

  • Specialized metagenomic assemblers for environmental samples

• Gene prediction and annotation:

  • Identify protein-coding regions on contigs

  • Compare against reference databases using BLAST, USEARCH, or DIAMOND

• Comparative analysis:

  • Multiple sequence alignment of argS variants

  • Phylogenetic reconstruction to establish evolutionary relationships

Variant Analysis Approach:

• Implement ARIBA or GROOT tools to accurately map reads to reference sequences while accounting for sequence variation

• Generate variation graphs that represent sequence variation within populations

• Identify non-synonymous substitutions and assess their potential impact on protein function

• Map variants to protein structural models to evaluate functional significance

How might R. baltica Arginine--tRNA ligase be applied in synthetic biology and biotechnology applications?

R. baltica Arginine--tRNA ligase presents several promising applications in synthetic biology and biotechnology, leveraging its unique properties as an enzyme from a marine organism with distinctive cellular features:

Expanded Genetic Code Systems:
Aminoacyl-tRNA synthetases are critical components for incorporating non-canonical amino acids into proteins. R. baltica Arginine--tRNA ligase could be engineered to:

• Recognize and charge tRNAs with non-canonical arginine analogs

• Function as a starting scaffold for directed evolution of synthetases with novel specificities

• Enable salt-tolerant cell-free protein synthesis systems incorporating non-standard amino acids

Biosensor Development:
The specific substrate recognition properties of the enzyme can be harnessed for biosensing applications:

• Engineer conformational changes coupled to fluorescent reporters for arginine detection

• Develop ATP consumption assays based on the enzyme's activity

• Create whole-cell biosensors that link arginine availability to reporter gene expression

Halo-Tolerant Biocatalysis:
The salt tolerance of R. baltica proteins makes them attractive for industrial biocatalysis:

• Develop enzyme immobilization strategies preserving salt tolerance

• Create enzyme cascade systems functional in high-salt environments

• Engineer the enzyme for compatibility with organic solvents, leveraging its natural stability

What insights can comparative analysis of R. baltica Arginine--tRNA ligase provide for understanding translation system evolution?

The unique phylogenetic position and cellular features of R. baltica make its translation machinery, including Arginine--tRNA ligase, particularly valuable for evolutionary studies:

Ancient Translation System Components:
Planctomycetes represent a deep-branching bacterial phylum with distinctive features. Phylogenetic analysis clearly affiliates the Planctomycetes to the domain Bacteria as a distinct phylum, although the deepest branching hypothesis is not supported by comprehensive analyses . Comparative studies of R. baltica Arginine--tRNA ligase can:

• Identify conserved motifs representing the core catalytic machinery preserved throughout evolution

• Detect lineage-specific adaptations that reflect environmental specialization

• Evaluate the hypothesis that Planctomycetes translation systems retain ancestral features

Domain Architecture Analysis:
Aminoacyl-tRNA synthetases often acquire additional domains with novel functions. Analysis of R. baltica Arginine--tRNA ligase domain architecture can:

• Identify unique domains not present in model organism homologs

• Map the evolutionary acquisition of auxiliary domains

• Determine if domain shuffling has occurred between different synthetase classes

• Correlate domain architecture with the compartmentalized cellular organization of Planctomycetes

Horizontal Gene Transfer Assessment:
The large genome of R. baltica (7.145 megabases) suggests extensive gene acquisition events . Investigation of argS can:

• Identify potential horizontal gene transfer events through phylogenetic incongruence

• Evaluate codon usage patterns for evidence of recent acquisition

• Compare genomic context across related species to track evolutionary rearrangements

How does the study of R. baltica Arginine--tRNA ligase contribute to our understanding of translation regulation in environmentally stressed bacteria?

R. baltica thrives in dynamic marine environments requiring sophisticated regulatory mechanisms to maintain cellular functions under stress. The study of its Arginine--tRNA ligase provides a window into these adaptations:

Stress-Responsive Gene Expression:
Transcriptomic profiling reveals that R. baltica undergoes extensive gene expression changes during transition to stationary phase, with approximately 12% of genes showing differential regulation . This includes upregulation of genes associated with amino acid biosynthesis, signal transduction, transcriptional regulation, stress response, and protein folding . Understanding argS regulation in this context provides insights into:

• How translation components respond to nutrient limitation

• Coordination between amino acid metabolism and tRNA charging

• Mechanisms maintaining translation fidelity under stress

Post-Translational Regulation:
Aminoacyl-tRNA synthetases are often subject to post-translational modifications that regulate their activity. Research should investigate:

• Phosphorylation, acetylation, or other modifications of R. baltica Arginine--tRNA ligase

• How these modifications change under different environmental conditions

• The role of modifications in rapid adaptation to environmental fluctuations

Integration with Global Stress Responses:
R. baltica has been shown to modulate expression of genes involved in transcriptional regulation and stress response during stationary phase . The upregulation of genes for phenylalanine, tyrosine, and tryptophan biosynthesis is consistent with proteome data but its physiological significance remains unknown . Investigation of argS regulation in this context can elucidate:

• How aminoacyl-tRNA synthetase activity is coordinated with stress response pathways

• The role of translation regulation in facilitating morphological transitions

• Mechanisms for prioritizing protein synthesis under stress conditions