Recombinant Bdellovibrio bacteriovorus Glutamyl-tRNA (Gln) amidotransferase subunit A (gatA)

What is the function of GatA in Bdellovibrio bacteriovorus?

GatA functions as a critical subunit of the heterotrimeric GatCAB enzyme complex in B. bacteriovorus. This amidotransferase plays a dual role in protein synthesis pathways:

• It catalyzes the conversion of misacylated Glu-tRNA^Gln^ to correctly charged Gln-tRNA^Gln^

• It facilitates the amidation of Asp-tRNA^Asn^ to Asn-tRNA^Asn^

This transamidation pathway provides B. bacteriovorus with a secondary route for asparagine synthesis, making it not strictly an asparagine auxotroph as previously predicted . The GatCAB complex represents an essential component of the indirect aminoacylation pathway that appears to be retained in B. bacteriovorus despite the presence of both AsnRS and GlnRS genes, which is unusual in bacterial systems.

How is the gatCAB operon organized in Bdellovibrio bacteriovorus?

The gatCAB operon in B. bacteriovorus follows the typical bacterial arrangement, with genes ordered as gatC, gatA, and gatB in a single transcriptional unit. Comparative analysis with other bacterial species shows this organization is conserved, similar to what has been observed in Bacillus subtilis and Bacillus stearothermophilus . The GatCAB subunits demonstrate significant sequence homology with those from other bacterial species:

Expression analysis indicates that the gatCAB operon is more highly expressed during the growth phase (GP) compared to the attack phase (AP), consistent with its role in protein synthesis during the predator's replicative stage .

What methods can be used to express recombinant B. bacteriovorus GatA?

Several expression methods have been developed for recombinant production of B. bacteriovorus proteins, which can be applied to GatA:

• Plasmid-based expression systems:

  • Use of pSUP404.2 vector which can be transferred to B. bacteriovorus via conjugation from E. coli S17-1

  • RSF1010-derived plasmids with broad-host range origins of replication that can autonomously replicate in B. bacteriovorus

• Promoter selection:

  • Strong native promoters such as P1753, P3184, PAPSRNA5, and PmerRNA, which are highly active during the attack phase

  • Inducible expression using theophylline-activated riboswitches for controlled expression

• Chromosomal integration:

  • Tn7-mediated chromosome insertion for monocopy gene expression

  • Marker-free deletion or allelic exchange using suicide plasmids containing counter-selectable markers like sacB

Methodological approach: Clone the gatA gene into a suitable expression vector under control of a strong promoter, transform into E. coli, and transfer to B. bacteriovorus via conjugation. Alternatively, gatA can be expressed in E. coli with a His-tag for purification, similar to protocols used for other GatCAB components .

How does B. bacteriovorus GatCAB contribute to the predatory lifestyle?

B. bacteriovorus GatCAB represents a unique adaptation that enables amino acid synthesis during predation, with several important implications:

• Metabolic flexibility: The dual route for Asn-tRNA^Asn^ formation (direct via AsnRS and indirect via ND-AspRS/GatCAB) provides metabolic redundancy that may be advantageous during predation .

• Predatory stage-specific function: Transcriptomic analysis reveals differential expression of the GatCAB components between attack phase (AP) and growth phase (GP), suggesting stage-specific roles:

  • Lower expression in AP when predator is searching for prey

  • Higher expression in GP when rapid protein synthesis is needed for replication within prey

• Evolutionary significance: Unlike Deinococcus radiodurans and Thermus thermophilus which have two AspRSs, B. bacteriovorus has retained only one non-discriminating AspRS (ND-AspRS) that can charge both tRNA^Asp^ and tRNA^Asn^ . This suggests evolutionary pressure to maintain the indirect pathway despite encoding AsnRS.

Experimental approach to study this question: Use genetic knockout of gatA combined with metabolic labeling to track changes in amino acid incorporation during predation cycles. Compare predation efficiency of ΔgatA mutants against various prey bacteria in both nutrient-rich and nutrient-limited conditions.

What is the role of GatA in the context of tRNA-dependent asparagine synthesis in B. bacteriovorus?

B. bacteriovorus presents an interesting case where both direct (AsnRS) and indirect (ND-AspRS/GatCAB) pathways for Asn-tRNA^Asn^ formation coexist . The role of GatA in this context includes:

• Catalytic function: GatA possesses the amidase domain responsible for generating the ammonia used in the transamidation reaction, converting Asp-tRNA^Asn^ to Asn-tRNA^Asn^.

• Interdomain communication: GatA works in concert with GatB (ATP-binding and acyl-tRNA binding) and GatC (structural role) to execute transamidation.

• Physiological relevance: Experimental evidence supports the functionality of the indirect pathway in B. bacteriovorus:

  • The ND-AspRS readily forms Asp-tRNA^Asn^

  • GatCAB successfully amidates this to Asn-tRNA^Asn^

  • Co-expression of B. bacteriovorus AspRS and GatCAB rescues an E. coli asparagine auxotroph (JF448)

Research methodology: To study GatA's specific contribution, employ site-directed mutagenesis targeting conserved residues in the amidase domain, followed by in vitro transamidation assays using radioactively labeled Asp-tRNA^Asn^ substrates. Monitor conversion to Asn-tRNA^Asn^ by thin-layer chromatography.

What structural and functional differences exist between B. bacteriovorus GatA and homologs from other bacteria?

Comparative analysis of GatA from B. bacteriovorus and other bacterial species reveals several important differences:

Functional implications of these differences:

• The retention of GatCAB despite encoding AsnRS and GlnRS suggests it provides an evolutionary advantage for B. bacteriovorus

• The dual-substrate capability may reflect the predatory lifestyle where adaptability is crucial

Methodological approach for structural studies: Express and purify recombinant GatA with a His-tag, perform X-ray crystallography or cryo-EM analysis, and compare with existing structures from other bacteria. Specific attention should be paid to the active site configuration and substrate-binding pockets.

How can genetic engineering of gatA be used to study or enhance B. bacteriovorus predation capabilities?

Genetic engineering of gatA offers several approaches to investigate and potentially enhance predation:

• Promoter replacement strategies:

  • Replace native promoter with characterized attack phase-specific promoters (P1753, P3184, PAPSRNA5) to study effects of altered expression timing

  • Implement theophylline-responsive riboswitches for externally controlled expression

• Domain engineering approaches:

  • Create chimeric proteins with GatA domains from organisms with different substrate specificities

  • Introduce mutations to enhance catalytic efficiency based on structural models

• Experimental designs to leverage GatA engineering:

  • Predation assays comparing wild-type and engineered strains against diverse prey bacteria

  • Competition experiments between engineered variants in mixed prey environments

  • Host-range expansion studies to assess potential for targeting new pathogens

Recent technological advances that facilitate this work:

• Development of genetic tools specifically for B. bacteriovorus, including marker-free deletion methods

• Tn7-mediated chromosomal insertion systems for stable gene expression

• Golden Gate-based destination vectors adapted from SEVA plasmids

What methods can be used to assay the enzymatic activity of recombinant B. bacteriovorus GatA?

Several complementary approaches can be used to assess GatA activity:

• In vitro biochemical assays:

  • Thin-layer chromatography (TLC): Monitor conversion of [³H]-Asp or [¹⁴C]-Glu from mischarged tRNAs

  • ATP consumption assay: Measure ATP hydrolysis during transamidation reaction

  • Coupled enzyme assays: Link ammonia production to NADH consumption via glutamate dehydrogenase

• Genetic complementation approaches:

  • Use of E. coli JF448 asparagine auxotroph rescue system

  • Complementation of heterologous GatCAB knockouts in model organisms

• Activity assessment protocol:

  a. Purify recombinant GatCAB complex containing the target GatA:

  • Co-express GatC, GatA, and GatB in E. coli

  • Purify using affinity chromatography with a His-tag on one subunit

  b. Prepare substrate:

  • Generate Asp-tRNA^Asn^ using purified ND-AspRS and in vitro transcribed tRNA^Asn^

  c. Reaction conditions:

  • Buffer: 50 mM HEPES-KOH pH 7.5, 30 mM KCl, 15-25 mM CaCl₂

  • Temperature: 30-35°C (optimal for B. bacteriovorus activity)

  • Include ATP, ammonia donor (glutamine), and Asp-tRNA^Asn^ or Glu-tRNA^Gln^

  d. Analysis methods:

  • Acid gel electrophoresis to resolve charged vs. uncharged tRNAs

  • Mass spectrometry to identify amino acid attached to tRNA

  • Radioactive amino acid incorporation assays

How can researchers study the interaction between GatA and other subunits of the GatCAB complex?

The interaction between GatA and other subunits can be studied through multiple approaches:

• Biochemical interaction assays:

  • Pull-down assays: Express tagged versions of GatA, GatB, and GatC individually and assess co-purification

  • Surface plasmon resonance (SPR): Determine binding kinetics and affinities between purified subunits

  • Isothermal titration calorimetry (ITC): Measure thermodynamic parameters of subunit association

• Structural approaches:

  • X-ray crystallography of the complete GatCAB complex (as performed for other species)

  • Cryo-EM analysis to visualize the assembled complex

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction surfaces

• Genetic approaches:

  • Bacterial two-hybrid system: Assess interactions in a heterologous host

  • Suppressor mutation analysis: Identify compensatory mutations that restore function in interface mutants

  • Co-expression studies: Test the effects of coordinated vs. separate expression of GatCAB subunits

• In vivo complex assembly studies:

  • Fluorescence-based approaches with differentially tagged subunits

  • Split fluorescent protein complementation to visualize interaction

Methodology for studying GatA-GatC interaction specifically:

• Generate truncation variants of GatA to identify minimal interaction domains

• Introduce site-specific mutations at predicted interface residues

• Perform co-purification assays to determine effects on complex stability

• Compare results with known structures from related GatCAB complexes

What approaches can be used to study the role of GatA in B. bacteriovorus under different predatory conditions?

Understanding GatA's role in predation requires examination across diverse conditions:

• Genetic manipulation strategies:

  • Generate conditional gatA knockdown using riboswitch-controlled expression

  • Create point mutations in catalytic residues to produce partially functional variants

  • Use inducible promoters to control expression levels during different predatory stages

• Predatory activity assays:

  • Plaque formation assays on different prey lawns

  • Liquid culture predation efficiency measurements (viable prey count reduction over time)

  • Microscopic observation of predatory cycle progression with fluorescently labeled components

• Experimental matrix for comprehensive analysis:

• Analytical approaches:

  • RNA-seq to monitor gene expression changes during predation under varied conditions

  • Proteomics to quantify GatA abundance relative to other cellular proteins

  • Metabolic labeling to track amino acid incorporation patterns

Suggested methodology: Use a combination of conditional gatA expression strains with fluorescent prey to track predation efficiency across conditions. Monitor both predator and prey populations simultaneously using flow cytometry and time-lapse microscopy.

How can the three-dimensional structure of B. bacteriovorus GatA be determined and what insights might it provide?

Determining the 3D structure of B. bacteriovorus GatA requires a multi-faceted approach:

• Expression and purification strategy:

  • Clone gatA gene with an N-terminal His-tag for affinity purification

  • Co-express with GatB and GatC for stable complex formation

  • Use size exclusion chromatography to ensure homogeneity of the sample

• Structural determination methods:

  • X-ray crystallography: Attempt crystallization of purified GatA alone and in complex with GatB and GatC

  • Cryo-electron microscopy: Particularly useful for the complete GatCAB complex

  • NMR spectroscopy: Applicable for individual domains if expression of isotope-labeled protein is possible

  • Homology modeling: Utilize existing structures of GatA from other organisms as templates

• Functional validation of the structure:

  • Site-directed mutagenesis of predicted catalytic residues

  • Activity assays to correlate structural features with catalytic function

  • Substrate docking simulations to understand binding interactions

• Potential structural insights:

  • Identification of catalytic residues specific to B. bacteriovorus GatA

  • Structural adaptations that might explain dual substrate specificity (both Asp-tRNA^Asn^ and Glu-tRNA^Gln^)

  • Interface regions that mediate interaction with GatB and GatC

  • Conformational changes associated with substrate binding and catalysis

Example pipeline for structural studies:

• Recombinant expression in E. coli BL21(DE3) using pET-based vectors

• Purification via Ni-NTA affinity chromatography followed by ion exchange and size exclusion

• Crystallization screening using commercial sparse matrix screens

• X-ray diffraction data collection at synchrotron facilities

• Structure determination using molecular replacement with homologous GatA structures

• Validation and refinement of the final structure

What are common challenges in expressing and purifying recombinant B. bacteriovorus GatA?

Researchers face several technical challenges when working with recombinant B. bacteriovorus GatA:

• Expression challenges:

  • Protein solubility issues: GatA may form inclusion bodies in heterologous hosts

  • Proper folding: As part of a multi-subunit complex, GatA may require co-expression with GatB and GatC

  • Expression toxicity: Overexpression may be toxic to the host cell

• Purification difficulties:

  • Complex stability: The GatA subunit may be unstable when purified separately from GatB and GatC

  • Enzymatic activity retention: Maintaining native conformation during purification

  • Contaminating host proteins: Especially problematic when purifying from B. bacteriovorus

• Troubleshooting strategies:

• Recommended expression systems:

  • E. coli BL21(DE3) with pET-based vectors for initial attempts

  • E. coli Arctic Express for difficult-to-fold proteins

  • Baculovirus-insect cell system for complex multi-subunit expression

  • Native expression in B. bacteriovorus with C-terminal tags for authentic folding environment

How can researchers troubleshoot issues with GatA activity in transamidation assays?

Transamidation assays can be challenging to optimize. Here are strategies to address common issues:

• Low or no detectable activity:

  • Cause: Inactive enzyme, improper assay conditions, or substrate issues

  • Solutions:

    • Verify enzyme integrity by SDS-PAGE and circular dichroism

    • Test activity across a range of pH (6.5-8.5) and temperature conditions (25-40°C)

    • Include positive controls using commercial GatCAB preparations

    • Check substrate quality using other aminoacyl-tRNA synthetases

• High background or non-specific activity:

  • Cause: Contaminating aminoacyl-tRNA hydrolases or contaminating ammonia sources

  • Solutions:

    • Include RNase inhibitors in reaction buffers

    • Purify enzyme preparations more stringently

    • Use DEPC-treated water for all solutions

    • Include negative controls lacking ATP or ammonia donor

• Inconsistent results between replicates:

  • Cause: Batch-to-batch variation in enzyme or substrate preparation

  • Solutions:

    • Standardize protein expression and purification protocols

    • Prepare large batches of substrates and store in single-use aliquots

    • Include internal calibration standards in each assay

• Optimized transamidation assay protocol:

  a. Reaction components:

  • 50 mM HEPES-KOH (pH 7.5)

  • 30 mM KCl

  • 15-25 mM CaCl₂ (optimal for B. bacteriovorus)

  • 10 mM ATP

  • 10 mM glutamine (ammonia donor)

  • 5 µM Asp-tRNA^Asn^ (prepared with ND-AspRS)

  • 0.5 µM purified GatCAB complex

  b. Controls to include:

  • No enzyme control

  • No ATP control

  • Heat-inactivated enzyme control

  • Positive control with well-characterized GatCAB (e.g., from B. subtilis)

  c. Detection methods:

  • Thin-layer chromatography of hydrolyzed aminoacyl-tRNAs

  • HPLC analysis of amino acids after deacylation

  • Mass spectrometry of intact charged tRNAs

What strategies can be employed to study GatA function in B. bacteriovorus when genetic manipulation is challenging?

Working with B. bacteriovorus presents unique challenges due to its predatory lifestyle. Alternative approaches to study GatA function include:

• Heterologous expression and complementation:

  • Express B. bacteriovorus GatA in model organisms lacking endogenous GatA

  • Test functionality through complementation of GatA-deficient strains

  • Create chimeric proteins with domains from well-characterized GatA proteins

• Inhibitor-based approaches:

  • Develop specific chemical inhibitors targeting GatA

  • Use competitive inhibitors of the amidation reaction

  • Apply in vitro inhibition results to predation studies

• Antisense RNA and CRISPR interference:

  • Design antisense RNA targeting gatA mRNA to reduce expression

  • Utilize CRISPR interference (CRISPRi) with catalytically inactive Cas9 (dCas9)

  • Express these elements under inducible control for temporal regulation

• Alternative genetic approaches:

  • Isolate temperature-sensitive gatA mutants through random mutagenesis

  • Use transposon mutagenesis to identify genetic interactions with gatA

  • Generate dominant negative gatA variants that interfere with wildtype function

• Biochemical approaches:

  • Use activity-based protein profiling to study GatA in cell lysates

  • Employ crosslinking mass spectrometry to map interaction networks

  • Develop gatA-specific antibodies for immunoprecipitation and localization studies

Methodological workflow for antisense RNA approach:

• Design antisense RNAs targeting different regions of gatA mRNA

• Clone under control of inducible promoters in suitable vectors for B. bacteriovorus

• Introduce via conjugation from E. coli S17-1

• Induce expression at different stages of the predatory cycle

• Monitor effects on predation efficiency, growth rate, and protein synthesis

How might engineered variants of GatA be utilized to expand B. bacteriovorus predation capabilities?

Engineering GatA to enhance B. bacteriovorus predation presents several promising research directions:

• Protein engineering approaches:

  • Directed evolution to select for GatA variants with enhanced catalytic efficiency

  • Structure-guided mutagenesis targeting substrate binding and catalytic sites

  • Domain swapping with GatA from thermophilic organisms to increase stability

• Potential applications of engineered GatA variants:

  • Expansion of temperature range for predation (cold or heat-adapted variants)

  • Enhanced predation under challenging environmental conditions (pH extremes, presence of inhibitors)

  • Altered substrate specificity to accommodate non-canonical amino acids

• Integration with synthetic biology tools:

  • Coupling GatA expression to prey-sensing mechanisms for targeted activation

  • Creating feedback loops that enhance gatA expression upon successful predation

  • Designing genetic circuits that coordinate GatA activity with other predation-related proteins

• Research methodology for GatA engineering:

  • Create a library of gatA variants using error-prone PCR or site-directed mutagenesis

  • Screen variants in E. coli JF448 complementation system for initial assessment

  • Introduce promising variants into B. bacteriovorus via optimized genetic tools

  • Assess predation efficiency across various conditions and prey types

  • Perform competition assays between engineered strains to identify optimal variants

• Potential impact on applications:

  • Enhanced biocontrol properties for targeting antibiotic-resistant pathogens

  • Improved performance in environmental bioremediation

  • More efficient reduction of recombinant bacterial populations in controlled environments

What is the relationship between GatA function and the two-phase lifecycle of B. bacteriovorus?

Understanding how GatA functions across the B. bacteriovorus lifecycle presents important research opportunities:

• Phase-specific expression patterns:

  • RNA-seq data indicates differential expression between AP and GP phases

  • The relationship between tRNA-dependent amino acid synthesis and the metabolic demands of each phase requires further investigation

• Key research questions:

  • Is GatA activity regulated post-translationally during the transition between phases?

  • Does the availability of host-derived amino acids affect GatA utilization during predation?

  • How does the balance between direct (AsnRS) and indirect (GatCAB) pathways shift across the lifecycle?

• Experimental approaches:

  • Temporal expression analysis: Use fluorescently tagged GatA to track abundance throughout predation

  • Metabolic labeling: Apply pulse-chase labeling with amino acid isotopes to track synthesis patterns

  • Conditional expression: Deploy riboswitches to modulate GatA levels at specific lifecycle stages

  • Single-cell analysis: Develop microfluidic systems to monitor individual predation events with reporter strains

• Mechanistic hypotheses to test:

  • GatA activity is upregulated during GP when rapid protein synthesis occurs within the bdelloplast

  • The dual pathway for Asn-tRNA^Asn^ formation provides metabolic flexibility during transitions between AP and GP

  • GatA function is coordinated with prey cell resource utilization via regulatory mechanisms

• Methodological design:

  • Create reporter strains with phase-specific promoters (AP vs. GP) controlling fluorescent protein expression

  • Synchronize B. bacteriovorus cultures by isolating AP cells via filtration

  • Monitor changes in GatA expression, localization, and activity throughout predation cycle

  • Compare wildtype with gatA mutants for progression through predatory cycle phases

What synthetic biology applications might emerge from understanding B. bacteriovorus GatA function?

Understanding GatA function could enable several synthetic biology applications:

• Engineered predatory modules:

  • Design minimal synthetic predators with optimized GatCAB pathways

  • Create specialized predators for targeting specific pathogens

  • Develop self-limiting predatory systems for controlled environmental release

• Expanded genetic code applications:

  • Leverage GatCAB's natural ability to modify aminoacyl-tRNAs to incorporate non-canonical amino acids

  • Engineer GatA to facilitate synthesis of novel amino acids on tRNA

  • Develop orthogonal translation systems based on modified GatCAB pathways

• Biological containment systems:

  • Design B. bacteriovorus strains dependent on GatA function for containment

  • Create auxotrophic strains with engineered GatA that function only under specific conditions

  • Develop kill switches based on inducible toxic GatA variants

• Therapeutic protein production:

  • Use B. bacteriovorus with engineered GatA as "living factories" that produce therapeutic proteins during predation

  • Harness the indirect aminoacylation pathway to incorporate specialized amino acids into therapeutic proteins

  • Release bioactive compounds upon prey lysis through coordination with GatA activity

• Research methodology for synthetic applications:

  • Design modular genetic parts based on GatCAB components for standardized assembly

  • Develop computational models of GatA-related metabolic pathways to predict system behavior

  • Create high-throughput screening platforms for assessing engineered GatA variants

• Potential research directions:

  • Investigate the compatibility of B. bacteriovorus GatA with orthogonal tRNA/aaRS pairs

  • Explore the possibility of compartmentalizing GatA activity within synthetic cells

  • Develop GatA-based biosensors that detect specific metabolic states