Recombinant Geobacter sulfurreducens Isoleucine--tRNA ligase (ileS), partial

What is Geobacter sulfurreducens and why is its Isoleucine--tRNA ligase of interest?

Geobacter sulfurreducens is a gram-negative metal- and sulfur-reducing proteobacterium that was first isolated in Norman, Oklahoma from materials found around a contaminated ditch . It is characterized as a rod-shaped, aerotolerant anaerobe with non-fermentative metabolism, featuring flagellum and type four pili . The organism was discovered by Derek R. Lovley in 1987 and has since become a model organism for studying electroactive bacteria due to its ability to transfer electrons to metallic minerals in natural environments, essentially "breathing" metals .

The Isoleucine--tRNA ligase (ileS) from G. sulfurreducens is of particular interest because aminoacyl-tRNA synthetases (aaRSs) are essential enzymes responsible for accurately charging tRNAs with their cognate amino acids, a crucial step in protein synthesis. Within the context of G. sulfurreducens' unique metabolism, which is heavily dependent on cytochromes and characterized by high lipid and iron content compared to other bacteria, understanding the function and regulation of ileS provides insights into how this organism maintains translational fidelity under its distinctive physiological conditions .

How does Isoleucine--tRNA ligase function in protein synthesis?

Isoleucyl-tRNA synthetase (IleRS) is responsible for decoding isoleucine codons in all three domains of life . As a class I aminoacyl-tRNA synthetase, IleRS catalyzes a two-step reaction: first, it activates isoleucine with ATP to form isoleucyl-AMP, and second, it transfers the activated isoleucine to the 3'-end of the appropriate tRNA molecule to form isoleucyl-tRNA^Ile .

The enzyme is built around a conserved N-terminal Rossmann fold catalytic domain that encloses the synthetic site where amino acid activation occurs . Additionally, IleRS contains a connective peptide 1 (CP1) inserted between the two halves of the catalytic domain, which folds into an independent domain hosting the post-transfer editing site . This structural arrangement is shared with closely related class I valyl- and leucyl-tRNA synthetases .

A critical aspect of IleRS function is its ability to distinguish between the cognate amino acid isoleucine and structurally similar non-cognate amino acids such as valine. The discrimination factor between isoleucine and valine can be as low as 200, necessitating editing mechanisms to prevent misincorporation of incorrect amino acids during protein synthesis .

What methods are commonly used to express and purify recombinant G. sulfurreducens Isoleucine--tRNA ligase?

The expression and purification of recombinant G. sulfurreducens IleRS typically follows standard protocols for recombinant protein production, adapted to the specific characteristics of this enzyme. Based on methodologies described for related aminoacyl-tRNA synthetases, the following approach is recommended:

• Cloning Strategy:

  • PCR amplification of the ileS gene from G. sulfurreducens genomic DNA

  • Insertion into an expression vector (commonly pET series) with an appropriate affinity tag (His6 or GST)

  • Transformation into an E. coli expression strain (BL21(DE3) or derivatives)

• Expression Conditions:

  • Culture in LB or rich media (e.g., TB) at 30°C until OD600 reaches 0.6-0.8

  • Induction with 0.1-0.5 mM IPTG

  • Post-induction growth at 18-25°C for 12-16 hours to minimize inclusion body formation

• Purification Protocol:

  • Cell lysis by sonication or French press in buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 5 mM β-mercaptoethanol, and 10% glycerol

  • Affinity chromatography using Ni-NTA or glutathione resin

  • Ion exchange chromatography (typically Q-Sepharose)

  • Size exclusion chromatography for final polishing

• Activity Assessment:

  • ATP-PPi exchange assay to measure amino acid activation

  • Aminoacylation assay using radioactive amino acids or other detection methods

  • Editing assay to assess hydrolysis of misacylated tRNA species

The purified enzyme should be stored in buffer containing 20 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM DTT, and 50% glycerol at -80°C for long-term stability.

What are the basic biochemical properties of G. sulfurreducens Isoleucine--tRNA ligase?

Based on studies of IleRS from related organisms and the unique characteristics of G. sulfurreducens, the following biochemical properties can be anticipated for G. sulfurreducens IleRS:

The enzyme is expected to have a relatively high thermal stability compared to IleRS from mesophilic organisms, consistent with G. sulfurreducens' adaptation to various environmental conditions. Additionally, given the high iron content of G. sulfurreducens cells, the enzyme might display specific adaptations related to function in an iron-rich cellular environment .

How does the editing mechanism of G. sulfurreducens Isoleucine--tRNA ligase compare to other bacterial species?

G. sulfurreducens IleRS likely employs both pre-transfer and post-transfer editing mechanisms similar to other bacterial IleRS enzymes, but with potential adaptations reflecting its unique metabolism. In bacterial IleRS enzymes like that from E. coli, tRNA-dependent pre-transfer editing contributes up to 30% of total editing activity . This mechanism involves hydrolysis of non-cognate aminoacyl-AMP intermediates before transfer to tRNA.

The post-transfer editing activity, which hydrolyzes misacylated tRNAs and is located in the CP1 domain, is more universally conserved. This domain in G. sulfurreducens IleRS would be expected to efficiently hydrolyze Val-tRNA^Ile with rates potentially similar to those observed in other bacterial enzymes (around 40 s^-1) . Given G. sulfurreducens' unique ecological niche and metabolic adaptations, its IleRS might have evolved specific features in its editing domain to accommodate its particular cellular environment.

The discrimination factors for non-cognate amino acids in G. sulfurreducens IleRS are likely similar to other bacterial IleRS enzymes: approximately 200-fold against valine and potentially 6000-7000-fold against α-aminobutyrate (Abu) . These discrimination factors would necessitate editing mechanisms to prevent misincorporation during protein synthesis, particularly under stress conditions where non-proteinogenic amino acids might accumulate.

What is the relationship between G. sulfurreducens' alternative isoleucine biosynthesis pathway and ileS function?

G. sulfurreducens possesses a unique isoleucine biosynthesis pathway that distinguishes it from model organisms like E. coli. While E. coli synthesizes the isoleucine precursor 2-oxobutanoate primarily from threonine via threonine ammonia-lyase, G. sulfurreducens utilizes both this pathway and an alternative citramalate pathway, with the latter serving as the main biosynthetic route .

This metabolic adaptation has several implications for ileS function:

• Substrate Availability: The citramalate pathway, which synthesizes 2-oxobutanoate from acetyl-coenzyme A and pyruvate, likely provides a more abundant supply of isoleucine under G. sulfurreducens' typical growth conditions. The ileS enzyme must function efficiently with potentially different steady-state concentrations of isoleucine compared to organisms using only the threonine pathway.

• Metabolic Integration: The genes encoding citramalate synthase (GSU1798) and threonine ammonia-lyase (GSU0486) have been identified in G. sulfurreducens, with knockout studies demonstrating that mutants lacking both enzymes are isoleucine auxotrophs, while single mutants can grow without isoleucine supplementation . This metabolic flexibility may influence the regulation of ileS expression and activity.

• Evolutionary Adaptation: The citramalate synthase of G. sulfurreducens represents the first characterized member of a phylogenetically distinct clade of citramalate synthases with representatives across diverse microorganisms . This suggests that the alternative isoleucine biosynthesis pathway and potentially the ileS enzyme have co-evolved specific features adapted to G. sulfurreducens' metabolic needs.

• Response to Environmental Conditions: The dual pathway for isoleucine biosynthesis may allow G. sulfurreducens to maintain adequate isoleucine production under varying environmental conditions, potentially affecting the substrate availability for ileS and thus influencing its catalytic efficiency and fidelity.

How does the cellular composition of G. sulfurreducens affect the function and regulation of Isoleucine--tRNA ligase?

G. sulfurreducens exhibits a unique cellular composition characterized by high lipid and iron content compared to other bacteria . This distinctive composition likely influences ileS function and regulation in several ways:

The cumulative effect of these factors suggests that G. sulfurreducens ileS may possess unique structural and functional adaptations not observed in ileS enzymes from model organisms with different cellular compositions and metabolic strategies.

What experimental approaches can be used to study the impact of metal reduction on ileS activity in G. sulfurreducens?

Given G. sulfurreducens' unique ability to reduce metals and its adaptation to metal-rich environments, several specialized experimental approaches can be employed to investigate the relationship between metal reduction and ileS activity:

• Bioelectrochemical Systems:

  • Grow G. sulfurreducens in three-electrode systems with controlled electrode potentials

  • Compare ileS expression, protein levels, and activity across different redox conditions

  • Correlate ileS function with current production as a measure of electron transfer activity

• Metal Reduction Assays:

  • Culture cells with various electron acceptors (Fe(III), Mn(IV), electrodes)

  • Extract ileS and measure activity to determine if electron acceptor type affects enzyme function

  • Compare amino acid misincorporation rates under different respiratory conditions

• Redox-Dependent Proteomics:

  • Apply redox proteomics techniques to identify potential redox-sensitive residues in ileS

  • Perform ICAT (isotope-coded affinity tag) labeling to quantify cysteine oxidation states

  • Use pulsed SILAC to measure ileS turnover under different redox conditions

• In vivo Translation Fidelity Assays:

  • Construct reporter strains containing engineered isoleucine codons at critical positions

  • Measure mistranslation rates under varying metal reduction conditions

  • Correlate with direct measurements of tRNA charging accuracy

• Integrated Multi-omics Approach:

  • Combine transcriptomics, proteomics, and metabolomics to create a systems-level view

  • Track isoleucine pathway intermediates, ileS expression, and protein error rates simultaneously

  • Develop computational models to predict ileS regulation in response to metal reduction

How can researchers accurately measure the editing activity of G. sulfurreducens Isoleucine--tRNA ligase?

The accurate measurement of editing activity in G. sulfurreducens IleRS requires several complementary approaches to distinguish between pre-transfer and post-transfer editing mechanisms and to quantify their relative contributions. The following methodologies are recommended:

• ATP/PPi Exchange Assay for Amino Acid Activation:

  • Reaction mixture: 100 mM HEPES (pH 7.5), 10 mM MgCl₂, 10 mM KCl, 2 mM ATP, 2 mM [32P]PPi (1-5 cpm/pmol), and varying concentrations of amino acids

  • Include purified IleRS at 10-50 nM concentration

  • Incubate at 37°C for time points from 30 seconds to 10 minutes

  • Quantify [32P]ATP formation by thin-layer chromatography or charcoal adsorption

  • Calculate activation rates for isoleucine vs. non-cognate amino acids like valine

• tRNA Aminoacylation Assay:

  • Reaction mixture: 20 mM HEPES (pH 7.5), 10 mM MgCl₂, 50 mM KCl, 4 mM ATP, 2 mM DTT, 0.1 μg/μl BSA, 0.004 units/μl inorganic pyrophosphatase, and 200 μM ATP

  • Include 10-15 μM tRNA^Ile and either 2 mM isoleucine or 20 mM valine

  • Use 15-50 nM IleRS depending on the amino acid used

  • Quantify aminoacylated tRNA by acid precipitation and filter binding or by using [14C/3H]-labeled amino acids

• Post-transfer Editing Assay:

  • Prepare misacylated Val-tRNA^Ile using a mutant IleRS with compromised editing

  • Reaction mixture: 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 50 mM KCl, 5 mM DTT

  • Add wild-type IleRS at 10-50 nM concentration

  • Monitor deacylation by acid precipitation and filter binding

  • Calculate deacylation rate constants (kdeacyl)

• Pre-transfer Editing Analysis:

  • Perform aminoacylation with and without tRNA to detect tRNA-dependent pre-transfer editing

  • Measure AMP formation using [α-32P]ATP and thin-layer chromatography

  • Compare AMP production rates with different amino acids and in the presence/absence of tRNA

  • Use kinetic modeling to separate contributions of different editing pathways

• Pulse-Chase Approach for Pathway Discrimination:

  • Pulse with radioactive amino acid and ATP to form enzyme-bound intermediates

  • Chase with non-radioactive amino acid and analyze products

  • The pattern of labeled products can distinguish between pre- and post-transfer editing

For mutations or modifications to the enzyme, site-directed mutagenesis of key residues in the editing domain (typically conserved threonine or histidine residues) can be used to create editing-deficient variants for comparative studies .

What genetic manipulation techniques are most effective for studying G. sulfurreducens ileS in vivo?

Genetic manipulation of G. sulfurreducens presents unique challenges due to its specialized metabolism and growth requirements. The following techniques have proven effective for in vivo studies of genes like ileS:

• Targeted Gene Knockout using Homologous Recombination:

  • Design primers to amplify regions flanking the ileS gene

  • Clone these regions into a suicide vector containing an antibiotic resistance marker

  • Transform into G. sulfurreducens using electroporation (typically 1.5-2.0 kV, 400 Ω, 25 μF)

  • Select for double recombinants on appropriate media containing antibiotics

  • Since ileS is essential, this approach requires complementation with a plasmid-borne copy of the gene

  • For complementation, use an inducible promoter system like the lac or tet promoters adapted for G. sulfurreducens

• Site-Directed Mutagenesis for Structure-Function Studies:

  • Create point mutations in ileS using overlap extension PCR

  • Introduce the mutated gene on a complementation plasmid

  • Exchange the chromosomal wild-type gene with a knockout cassette in the presence of the complementation plasmid

  • Use riboswitch or other regulatory elements to control expression levels

• Chromosomal Tagging for Localization and Protein Interaction Studies:

  • Design constructs to add epitope or fluorescent protein tags to the C-terminus of ileS

  • Use homologous recombination to integrate these constructs into the native locus

  • Verify functionality of the tagged protein by complementation tests

  • Perform immunofluorescence microscopy or pull-down assays to study localization and interactions

• Conditional Expression Systems:

  • Implement a tetracycline-responsive system adapted for G. sulfurreducens

  • Create strains where the chromosomal ileS is under control of an inducible promoter

  • Titrate expression levels by varying inducer concentration

  • Monitor growth, protein synthesis rates, and error frequencies under different expression conditions

• CRISPR-Cas9 System for G. sulfurreducens:

  • Adapt CRISPR-Cas9 for use in G. sulfurreducens using vectors containing origin of replication functional in this organism

  • Design sgRNAs targeting ileS or related genes

  • Use for precise genome editing, including introduction of point mutations

  • Employ CRISPRi for partial repression studies when complete knockout is lethal

These techniques should be combined with phenotypic assays measuring growth rates, metal reduction capability, and protein synthesis fidelity to fully characterize the impact of ileS modifications on G. sulfurreducens physiology.

How can researchers investigate the potential connection between ileS function and electron transport in G. sulfurreducens?

Investigating the connection between ileS function and electron transport in G. sulfurreducens requires specialized techniques that bridge protein synthesis and electrochemical activity. The following methodological approaches are recommended:

• Bioelectrochemical Cultivation with Controlled Gene Expression:

  • Construct strains with ileS under control of inducible promoters

  • Grow cells in three-electrode bioelectrochemical systems (working electrode set at specific potentials)

  • Monitor current production while manipulating ileS expression levels

  • Correlate current density with ileS activity using quantitative assays

• In situ Assessment of Translation Fidelity:

  • Develop reporter systems containing sequences sensitive to isoleucine misincorporation

  • Measure reporter activity during growth with different electron acceptors

  • Compare translation error rates during Fe(III) reduction versus electrode respiration

  • Correlate with direct measurements of misaminoacylated tRNA levels using acid gel electrophoresis

• Metabolic Flux Analysis with Isotope Labeling:

  • Culture G. sulfurreducens with 13C-labeled acetate under different electron-accepting conditions

  • Track isotope incorporation into isoleucine and related amino acids

  • Correlate flux through isoleucine biosynthesis pathways with electron transport rates

  • Develop computational models linking central metabolism to aminoacyl-tRNA synthesis

• Redox-State Dependent Proteomics:

  • Harvest cells at defined redox states (controlled electrode potential or Fe(II)/Fe(III) ratios)

  • Perform quantitative proteomics to measure ileS levels and post-translational modifications

  • Apply crosslinking mass spectrometry to identify potential interaction partners

  • Correlate findings with electrophysiological measurements

• Combined Transcriptomic-Proteomic-Electrochemical Analysis:

  • Subject G. sulfurreducens to controlled electrochemical cultivation with varied potentials

  • Extract RNA and protein at defined time points during adaptation to new redox conditions

  • Perform RNA-Seq and quantitative proteomics to track ileS expression and protein levels

  • Correlate with electrochemical parameters like current density and impedance spectra

A particularly powerful approach would be to combine these methods with genetic manipulations, such as introducing specific mutations in the editing domain of ileS and measuring the impact on electron transfer capabilities and growth under various electron-accepting conditions.

What computational approaches can predict the impact of ileS mutations on G. sulfurreducens metabolism?

Computational approaches offer powerful tools for predicting how mutations in ileS might affect the broader metabolism of G. sulfurreducens. The following methodologies can be applied:

Example workflow for predicting effects of editing domain mutations:

• Generate homology model of G. sulfurreducens IleRS based on crystal structures

• Introduce mutations in silico and perform molecular dynamics simulations

• Calculate binding affinities for Ile vs. Val in wild-type and mutant enzymes

• Determine kinetic parameters from computational predictions

• Feed these parameters into a kinetic model of aminoacylation and editing

• Predict mischarging rates and impact on translation fidelity

• Integrate with genome-scale metabolic model to predict growth phenotypes

• Validate predictions with experimental measurements

This multi-scale approach allows for predictions ranging from atomic-level interactions to organism-level phenotypes, providing a comprehensive view of how ileS mutations might affect G. sulfurreducens metabolism.

How can understanding G. sulfurreducens ileS contribute to bioelectrochemical applications?

Understanding the function of ileS in G. sulfurreducens offers several potential benefits for bioelectrochemical applications, given the organism's unique capability to transfer electrons to external acceptors including electrodes. The following perspectives highlight key research directions:

• Enhanced Biofilm Formation and Current Production:

  • Optimizing ileS function could improve protein synthesis fidelity under electroactive conditions

  • Engineered ileS variants with adjusted specificity could enhance stress tolerance during electrode respiration

  • Manipulation of isoleucine incorporation in key cytochromes might improve electron transfer efficiency

• Controlled Protein Expression for Bioelectronic Interfaces:

  • Understanding how ileS function responds to different redox environments could inform electrode design

  • Engineered ileS variants might allow for redox-responsive protein synthesis control

  • The connection between ileS activity and electron transfer could be exploited for bioelectronic sensing applications

• Metabolic Engineering for Improved Performance:

  • Coordinating isoleucine biosynthesis pathway and ileS function could optimize resource allocation during electrode respiration

  • Tailoring the balance between citramalate and threonine pathways might enhance electrochemical activity

  • Engineering ileS to function optimally under specific electrode potentials could improve biocatalytic current production

• Bioelectrochemical Sensing Applications:

  • Understanding how ileS responds to environmental changes could enable development of whole-cell biosensors

  • The connection between translation fidelity and electron transfer could be exploited for detecting metabolic stressors

  • Engineered ileS variants could serve as components of bioelectronic detection systems

These applications depend on fundamental research establishing clear relationships between ileS function, protein synthesis fidelity, and electron transfer mechanisms in G. sulfurreducens.

What are the evolutionary implications of G. sulfurreducens' unique isoleucine metabolism and ileS function?

The evolutionary aspects of G. sulfurreducens' isoleucine metabolism and ileS function offer insights into bacterial adaptation to specialized ecological niches. Several key evolutionary implications can be drawn:

• Dual Pathway Evolution:

  • G. sulfurreducens possesses both the threonine and citramalate pathways for isoleucine biosynthesis, with the latter being the dominant route

  • This metabolic flexibility likely represents an adaptation to environments where threonine might be limiting or where acetyl-CoA (a precursor for the citramalate pathway) is abundant

  • The presence of a phylogenetically distinct citramalate synthase suggests independent evolution or horizontal gene transfer events

• Adaptation to Redox-Active Environments:

  • G. sulfurreducens thrives in metal-rich, redox-active environments where iron cycling occurs

  • The ileS enzyme may have evolved features allowing it to function optimally under fluctuating redox conditions

  • Selection pressure for accurate protein synthesis during metal respiration could have driven specific adaptations in the editing domain

• Coevolution with Electron Transfer Systems:

  • The extensive cytochrome network of G. sulfurreducens represents a significant investment in iron-containing proteins

  • Accurate translation of these proteins is crucial for electron transfer efficiency

  • The ileS enzyme may have coevolved with these systems to ensure proper synthesis of electron transport components

• Metabolic Integration with Lipid Biosynthesis:

  • The high lipid content of G. sulfurreducens cells suggests a unique metabolic configuration

  • The connection between isoleucine metabolism (which shares precursors with lipid biosynthesis) and ileS function may reflect adaptation to this lipid-rich cellular environment

  • The balance between amino acid and lipid biosynthesis pathways may have shaped ileS evolution

• Broader Implications for Aminoacyl-tRNA Synthetase Evolution:

  • The presence or absence of tRNA-dependent pre-transfer editing in different IleRS enzymes suggests evolutionary plasticity in editing mechanisms

  • G. sulfurreducens ileS may represent an interesting case study in how aminoacyl-tRNA synthetases adapt to specialized metabolic contexts

  • Comparative analysis across Geobacter species could reveal evolutionary trajectories of ileS in electroactive bacteria

These evolutionary considerations provide a framework for understanding G. sulfurreducens ileS not just as an isolated enzyme but as part of an integrated system that has evolved to support the organism's unique lifestyle and metabolic capabilities.