Recombinant Desulfovibrio vulgaris Aspartyl/glutamyl-tRNA (Asn/Gln) amidotransferase subunit C (gatC)

What is the function of gatC in Desulfovibrio vulgaris?

Aspartyl/glutamyl-tRNA (Asn/Gln) amidotransferase subunit C (gatC) is one component of the heterotrimeric GatCAB complex that catalyzes the transamidation of misacylated Asp-tRNA^Asn and Glu-tRNA^Gln to correctly charged Asn-tRNA^Asn and Gln-tRNA^Gln, respectively. In D. vulgaris, this indirect aminoacylation pathway is essential for accurate protein synthesis, as this organism (like many bacteria) lacks separate aminoacyl-tRNA synthetases for asparagine and glutamine. The gatC subunit works in concert with gatA and gatB to enable this critical transamidation process, making it an integral part of the translation machinery in D. vulgaris.

Why study gatC in Desulfovibrio vulgaris rather than in model organisms?

D. vulgaris presents a unique experimental system due to its anaerobic lifestyle, role in environmental sulfur cycling, and increasing recognition as a human gut pathobiont. The gatC subunit in D. vulgaris may possess distinct characteristics adapted to the organism's lifestyle. Studying gatC in D. vulgaris can provide insights into protein synthesis adaptations in anaerobic environments, potential therapeutic targets (as D. vulgaris has been implicated in inflammatory bowel disease and other conditions), and evolutionary biology of indirect aminoacylation pathways. Recent research has highlighted D. vulgaris as significantly increased in ulcerative colitis patients, correlating with disease severity , making its cellular components potential targets for therapeutic intervention.

What are the challenges in expressing recombinant D. vulgaris proteins?

Expressing recombinant D. vulgaris proteins presents several challenges: (1) D. vulgaris is an anaerobic organism with different codon usage and protein folding environments than common expression hosts; (2) The organism has specialized metabolic requirements, necessitating careful optimization of expression systems; (3) Traditional molecular tools have historically been limited for D. vulgaris, though recent advances have improved genetic manipulation capabilities . When expressing gatC specifically, additional challenges may include maintaining proper interaction with gatA and gatB subunits if the complete functional complex is desired. Researchers have successfully used E. coli as a heterologous host for D. vulgaris proteins, as demonstrated by the conjugative transfer of vectors carrying D. vulgaris genes .

What expression systems are most effective for recombinant D. vulgaris gatC?

For recombinant expression of D. vulgaris gatC, several systems have proven effective with careful optimization. E. coli-based expression systems (particularly BL21(DE3) or specialized derivatives) remain the most commonly used platform due to their versatility and high yield potential. When using E. coli, codon optimization of the gatC sequence may improve expression by addressing codon bias differences between the organisms. For more native-like production, homologous expression within modified D. vulgaris strains or closely related Desulfovibrio species can be achieved using the genetic manipulation methods established for these organisms . For example, plasmid vectors can be transferred by conjugation from E. coli to Desulfovibrio species, as demonstrated with other D. vulgaris genes . Expression should be verified through multiple methods, including Western blotting, activity assays, and mass spectrometry.

How should I design primers for amplifying the gatC gene from D. vulgaris genomic DNA?

Primer design for amplifying D. vulgaris gatC requires careful consideration of several factors. First, obtain the complete sequence of the gatC gene and its flanking regions from reliable genomic databases (such as NCBI GenBank). Design primers with the following characteristics:

• Length: 20-30 nucleotides with complementarity to the template sequence

• GC content: 40-60% for stable annealing

• Melting temperature (Tm): 55-65°C with forward and reverse primers within 5°C of each other

• Add appropriate restriction enzyme sites (with 3-6 nucleotide overhangs) for subsequent cloning

• Consider adding sequences for affinity tags if desired

Given the established protocols for D. vulgaris genetic manipulation, you may also consider designing primers compatible with Gateway cloning or SLIC (sequence- and ligation-independent cloning) techniques that have been successfully applied to D. vulgaris . When amplifying from D. vulgaris genomic DNA, ensure complete cell lysis under anaerobic conditions and verify PCR product specificity by sequencing before proceeding to cloning.

What purification strategy yields the highest purity and activity of recombinant gatC?

Purification of recombinant D. vulgaris gatC typically employs a multi-step chromatography approach to achieve high purity while maintaining protein activity. The optimal strategy includes:

Throughout purification, maintain reducing conditions (typically 1-5 mM DTT or 2-mercaptoethanol) to prevent oxidation of cysteine residues, which may be particularly important given D. vulgaris' anaerobic nature. Verify purity by SDS-PAGE and protein identity by mass spectrometry or Western blotting. For functional studies, consider co-purification with gatA and gatB subunits to maintain the native complex structure and activity.

How can I establish a functional assay for recombinant D. vulgaris gatC activity?

A comprehensive functional assay for D. vulgaris gatC should measure its activity as part of the GatCAB complex, as the individual subunit alone has limited catalytic capability. The assay should quantify the transamidation of mis-acylated tRNAs to their correctly charged forms. A typical workflow includes:

• Substrate preparation: Generate mis-acylated Asp-tRNA^Asn and Glu-tRNA^Gln using purified aminoacyl-tRNA synthetases (ND-AspRS and ND-GluRS).

• Reaction setup:

  • Combine purified GatCAB complex (or reconstituted complex using individually purified subunits)

  • Add mis-acylated tRNA substrate

  • Include amide donor (usually glutamine)

  • Buffer components: 50 mM HEPES pH 7.5, 10 mM MgCl₂, 5 mM ATP, 2 mM DTT

• Activity measurement: Monitor reaction progress through:

  • Thin-layer chromatography to separate amino acids after deacylation

  • HPLC analysis of amino acid content

  • Mass spectrometry to precisely determine tRNA charging states

  • Radiometric assays using ¹⁴C or ³H-labeled amino acids

When establishing this assay, prepare appropriate controls including reactions without enzyme, without amide donor, and with known GatCAB complexes from other organisms. Consider the anaerobic nature of D. vulgaris when optimizing reaction conditions, potentially conducting assays in an anaerobic chamber to maintain native-like environments.

What structural studies can reveal gatC's role in the GatCAB complex of D. vulgaris?

Structural characterization of D. vulgaris gatC can provide critical insights into its specific role within the GatCAB complex and potential unique adaptations in this anaerobic bacterium. A comprehensive structural investigation would include:

Compare the resulting structures with GatCAB complexes from other organisms to identify unique features in D. vulgaris that might relate to its anaerobic lifestyle or potential role in pathogenesis. These structural insights could guide the development of specific inhibitors that might have therapeutic potential for conditions associated with D. vulgaris overgrowth .

How can gatC be used to study translational fidelity in D. vulgaris?

Using gatC to study translational fidelity in D. vulgaris provides valuable insights into how this anaerobic bacterium maintains protein synthesis accuracy, particularly under stress conditions. A comprehensive approach includes:

• Conditional gatC expression systems: Create strains with regulated gatC expression using the genetic tools developed for D. vulgaris . This allows modulation of gatC levels and observation of effects on proteome accuracy.

• Reporter systems: Develop dual-luciferase reporters containing programmed errors (near-cognate codons) to quantify mistranslation rates under varying gatC expression levels.

• Proteomics approach: Use mass spectrometry-based proteomics to directly measure amino acid misincorporation rates at Asn and Gln codons when gatC function is compromised.

• Stress response studies: Examine how environmental stressors (oxidative stress, nutrient limitation, pH changes) affect gatC expression and function, and consequently, translational fidelity.

• Comparative analysis: Compare translational error rates between wild-type D. vulgaris and strains with modified gatC under conditions mimicking gut inflammation, as D. vulgaris has been implicated in inflammatory conditions .

This research provides fundamental insights into bacterial adaptation mechanisms and may identify conditions where translational fidelity becomes compromised, potentially contributing to stress tolerance or pathogenicity in this organism.

What strategies can overcome low expression yields of recombinant D. vulgaris gatC?

Low expression yields of recombinant D. vulgaris gatC can be addressed through systematic optimization of several parameters:

• Codon optimization: Analyze the gatC sequence for rare codons in the expression host and synthesize a codon-optimized version tailored to the host's preference.

• Expression vector selection: Test multiple promoter strengths (T7, tac, araBAD) and vector backbones to identify optimal expression control.

• Host strain selection: Compare expression in different E. coli strains (BL21(DE3), Rosetta, ArcticExpress) or consider Desulfovibrio host systems using established conjugation methods .

• Culture conditions optimization:

• Fusion partners: Test expression with solubility-enhancing fusion partners (MBP, SUMO, TrxA) if protein solubility is an issue.

• Scale-up considerations: Once optimal conditions are identified at small scale, carefully translate these to larger volumes maintaining key parameters like oxygen transfer rates and nutrient availability.

If expression in E. coli remains problematic, consider the genetic manipulation tools developed specifically for D. vulgaris to express the protein in its native host or closely related Desulfovibrio species.

How can I troubleshoot protein aggregation issues with recombinant gatC?

Protein aggregation is a common challenge when working with recombinant proteins, including D. vulgaris gatC. Implement these troubleshooting strategies to overcome aggregation:

• Expression temperature reduction: Lower cultivation temperature to 15-20°C to slow protein synthesis and improve folding kinetics.

• Buffer optimization:

  • Screen various pH conditions (pH 6.0-9.0)

  • Test different salt concentrations (50-500 mM NaCl)

  • Include stabilizing additives (5-10% glycerol, 0.5-1 M arginine, 1-5 mM TCEP as reducing agent)

• Co-expression with chaperones: Introduce plasmids encoding folding chaperones (GroEL/ES, DnaK/J/GrpE) to assist proper folding during expression.

• Purification under denaturing conditions: If native purification fails, extract protein under denaturing conditions (6-8 M urea or 4-6 M guanidinium HCl) followed by controlled refolding using dialysis or rapid dilution.

• Co-expression with binding partners: Consider co-expressing gatC with its natural partners gatA and gatB, as the complete complex may exhibit better solubility than individual subunits.

• Limited proteolysis approach: If domains of gatC aggregate less than the full-length protein, identify stable domains through limited proteolysis and express these independently.

Monitor aggregation status using dynamic light scattering, size-exclusion chromatography, and thermal shift assays to quantitatively assess improvements in protein homogeneity and stability through these interventions.

How should I address cross-reactivity or specificity issues in immunological detection of D. vulgaris gatC?

Addressing cross-reactivity and specificity challenges in immunological detection of D. vulgaris gatC requires a systematic approach:

• Antibody selection/production strategies:

  • Identify unique epitopes in D. vulgaris gatC not conserved in related bacteria

  • Use synthetic peptides representing these regions for immunization rather than whole protein

  • Consider recombinant antibody approaches (phage display) with stringent selection parameters

• Validation protocols:

  • Test antibodies against recombinant gatC proteins from multiple species

  • Verify specificity using wild-type and gatC knockout/mutant D. vulgaris strains

  • Include heterologous expression systems with and without gatC as controls

• Improved detection methods:

  • Implement sandwich ELISA with capture and detection antibodies targeting different epitopes

  • Use competitive binding assays with purified gatC to confirm specificity

  • Consider aptamer-based detection as an alternative to antibodies

• Sample preparation optimization:

  • Test different protein extraction methods to maximize target availability

  • Include blocking agents specific to the bacterial species being studied

  • Incorporate pre-absorption steps with related bacterial lysates to remove cross-reactive antibodies

Verification of specificity is particularly important when studying D. vulgaris in complex microbial communities such as gut microbiome samples, where multiple related species may be present simultaneously . Document antibody specificity comprehensively before applying to experimental samples.

How might gatC function be exploited for antimicrobial development against D. vulgaris?

GatC's essential role in protein synthesis through the indirect aminoacylation pathway makes it a potential target for selective antimicrobial development against D. vulgaris, which has been implicated in inflammatory conditions . A comprehensive antimicrobial development strategy would include:

• Target validation:

  • Confirm essentiality of gatC in D. vulgaris through conditional knockdown or CRISPR interference

  • Assess phenotypic consequences of partial inhibition on growth, metabolism, and virulence

  • Quantify effects on protein synthesis accuracy using global proteomics

• High-throughput screening approaches:

  • Develop biochemical assays measuring the complete GatCAB complex activity

  • Structure-based virtual screening targeting gatC-specific interfaces

  • Fragment-based screening to identify starting chemical matter

• Structural biology support:

  • Obtain high-resolution structures of D. vulgaris GatCAB in various functional states

  • Identify binding pockets unique to D. vulgaris gatC compared to human homologs or beneficial microbes

  • Use molecular dynamics simulations to identify allosteric sites for inhibitor binding

• Selectivity engineering:

  • Compare gatC sequences across pathogenic and beneficial gut bacteria

  • Target regions unique to Desulfovibrio to minimize disruption of beneficial microbes

  • Test compound specificity against panels of gut bacteria

• Delivery considerations:

  • Develop strategies for localized delivery to the colon where D. vulgaris resides

  • Explore prodrug approaches activated by sulfate-reducing bacterial metabolism

  • Consider phage-based delivery systems specific to Desulfovibrio

This approach may be particularly relevant for inflammatory bowel diseases where D. vulgaris overgrowth correlates with disease severity , potentially offering a targeted approach to microbiome modulation without broad-spectrum antibiotics.

How can systems biology approaches integrate gatC function into metabolic models of D. vulgaris?

Integrating gatC function into metabolic models of D. vulgaris requires sophisticated systems biology approaches that connect translation fidelity with broader cellular physiology:

• Genome-scale metabolic model expansion:

  • Incorporate aminoacyl-tRNA synthesis and transamidation reactions into existing D. vulgaris metabolic reconstructions

  • Define stoichiometric coefficients for ATP, glutamine, and other cofactors in the transamidation process

  • Link gatC activity to amino acid biosynthesis and nitrogen metabolism pathways

• Multi-omics data integration:

  • Correlate gatC expression levels (transcriptomics) with proteome composition (proteomics)

  • Map metabolic shifts (metabolomics) associated with varying gatC expression

  • Identify regulatory networks connecting gatC expression with stress responses

• Flux balance analysis applications:

  • Simulate growth phenotypes under varying levels of gatC activity

  • Predict metabolic adaptations to compromised transamidation capacity

  • Model energetic costs of translation with varying fidelity levels

• Host-microbe interaction modeling:

  • Extend models to include interactions between D. vulgaris and other gut microbes

  • Incorporate host factors that might influence gatC expression or activity

  • Simulate community-level effects of targeting gatC function

• In silico mutation analysis:

  • Predict functional consequences of naturally occurring gatC variants

  • Model evolutionary trajectories of the indirect aminoacylation pathway

  • Simulate adaptation to different environmental conditions

The genetic manipulation techniques established for D. vulgaris provide valuable tools for validating these computational models through targeted genetic modifications. This systems-level understanding could reveal emergent properties not obvious from studying gatC in isolation.

What role might D. vulgaris gatC play in horizontal gene transfer and antibiotic resistance?

Investigating the potential role of D. vulgaris gatC in horizontal gene transfer (HGT) and antibiotic resistance presents an intriguing research direction with implications for both evolutionary biology and clinical microbiology:

• Sequence analysis for HGT signatures:

  • Compare gatC sequences across Desulfovibrio species and other sulfate-reducing bacteria

  • Identify potential recombination events or mobile genetic element insertions

  • Conduct phylogenetic analyses to detect incongruence suggesting HGT events

• Experimental evolution approaches:

  • Subject D. vulgaris to conditions favoring genetic exchange (biofilms, stress conditions)

  • Monitor gatC sequence stability and potential transfer to other community members

  • Use the genetic manipulation tools developed for D. vulgaris to mark and track gatC transfer

• Connection to antibiotic resistance mechanisms:

  • Investigate whether altered gatC function affects translation of resistance determinants

  • Test if gatC mutations provide protection against antibiotics targeting protein synthesis

  • Examine if gatC overexpression affects minimum inhibitory concentrations of various antibiotics

• Functional implications in mixed communities:

  • Study whether D. vulgaris gatC function influences gene transfer in gut microbial communities

  • Determine if gatC activity affects biofilm formation, which often facilitates HGT

  • Explore if the presence of D. vulgaris influences resistance profiles of other community members

This research direction is particularly relevant given D. vulgaris' presence in the gut microbiome and its association with inflammatory conditions , where altered microbial community dynamics and antibiotic treatments are common.