Recombinant Bdellovibrio bacteriovorus tRNA 2-thiocytidine biosynthesis protein TtcA (ttcA)

What is the biological role of TtcA in B. bacteriovorus?

TtcA (tRNA 2-thiocytidine biosynthesis protein) catalyzes the formation of 2-thiocytidine (s2C) in specific tRNAs. This modification occurs at position 32 of the anticodon loop and impacts tRNA structure and function. In B. bacteriovorus, this modification likely influences translational accuracy during its complex predatory life cycle. Based on studies in E. coli and S. enterica, TtcA proteins are characterized by a PP-loop and a conserved Cys-X1-X2-Cys motif that is essential for their function, with both cysteines required for s2C formation . As a predatory bacterium that undergoes significant metabolic shifts between attack phase and growth phase, B. bacteriovorus may rely on precise translational control that could be influenced by tRNA modifications.

How does the biphasic lifecycle of B. bacteriovorus influence TtcA function?

B. bacteriovorus has a unique biphasic lifecycle divided into an attack phase (AP) and an intraperiplasmic growth phase (GP), with a recently identified third transition phase . This complex lifecycle may require precise regulation of translation for phase-specific proteins. TtcA-mediated tRNA modifications could play a role in modulating translation efficiency during these transitions. During the attack phase, when the bacterium is searching for prey, versus the growth phase, when it is replicating within the bdelloplast, different sets of genes are expressed . The modification of specific tRNAs by TtcA may help regulate this differential gene expression by affecting the efficiency of translation of specific codons used in phase-specific proteins.

What expression systems are optimal for recombinant B. bacteriovorus TtcA production?

Based on approaches used for other B. bacteriovorus proteins and similar enzymes, the following expression systems are recommended:

For optimal expression, consider including:

• Reducing agents (DTT, β-mercaptoethanol) in buffers to maintain cysteine residues

• Codon optimization for the expression host

• IPTG concentration optimization (typically 0.1-0.5 mM)

• Induction at OD600 of 0.6-0.8 for balanced growth and expression

How can I assess the enzymatic activity of recombinant B. bacteriovorus TtcA?

Multiple complementary approaches can be used to evaluate TtcA activity:

• In vivo complementation assays:

  • Transform a ttcA deletion strain with a plasmid expressing B. bacteriovorus ttcA

  • Extract tRNA and analyze for restoration of s2C modification

  • This approach has been successfully used for S. enterica TtcA functional validation

• In vitro enzymatic assays:

  • Radiolabeling assay: Incubate purified TtcA with 35SO42- labeled substrate, followed by tRNA extraction and quantification of incorporated radioactivity

  • HPLC analysis: Digest tRNA to nucleosides and separate using a method such as that developed by Gehrke and Kuo on a Supelcosil LC 18 column

  • Mass spectrometry: Analyze modified nucleosides using an electrospray ionization source as described for S. enterica TtcA studies

• Functional readouts:

  • Codon-specific translation efficiency: Using reporter constructs with specific codons known to be read by s2C-modified tRNAs

  • Frameshift reporter assays: Systems like pTH34 and pTH35 used in S. enterica studies to measure effects on frameshifting

What are the critical parameters for purifying active recombinant B. bacteriovorus TtcA?

Purification of active TtcA requires careful attention to:

• Buffer composition:

  • Maintain reducing conditions with 1-5 mM DTT or β-mercaptoethanol

  • Include 5-10% glycerol for stability

  • Use physiological pH (7.2-7.8)

  • Consider adding potential cofactors like Mg2+ and ATP

• Purification strategy:

  • Affinity chromatography (His-tag, FLAG-tag) followed by size-exclusion chromatography

  • Avoid harsh elution conditions; use gradient elution

  • Keep protein concentration below 2 mg/ml to prevent aggregation

  • Perform purification at 4°C to maintain stability

• Activity preservation:

  • Flash-freeze purified protein in small aliquots

  • Validate activity after each purification step

  • Consider including potential protein partners from B. bacteriovorus

• Quality control:

  • Verify protein folding using circular dichroism

  • Confirm oligomeric state using size-exclusion chromatography

  • Check for integrity of the Cys-X1-X2-Cys motif using mass spectrometry

How does B. bacteriovorus TtcA interact with the bacterial sulfur mobilization machinery?

TtcA requires sulfur for the thiolation of cytidine to form s2C. Based on studies of other thiolated nucleoside biosynthesis pathways, this process likely involves:

• Sulfur transfer pathway components:

  • IscS cysteine desulfurase is known to be involved in the synthesis of all five thiolated nucleosides present in tRNA of E. coli and S. enterica

  • IscS forms a persulfide intermediate (IscS-SSH) that can transfer sulfur to target proteins

  • TtcA likely receives sulfur from IscS or a similar sulfur carrier protein

• Proposed interaction model:

  • The conserved Cys-X1-X2-Cys motif in TtcA likely accepts the sulfur atom from IscS

  • ATP hydrolysis may drive the transfer of sulfur to the substrate cytidine

  • The PP-loop domain characteristic of TtcA proteins is typically involved in ATP binding

• Research approaches to investigate sulfur transfer:

  • Co-immunoprecipitation of TtcA with potential sulfur carrier proteins

  • In vivo crosslinking followed by mass spectrometry analysis

  • Radiolabeling with 35S to track sulfur transfer in reconstituted systems

  • Site-directed mutagenesis of the conserved cysteines in TtcA to confirm their role in sulfur transfer

What is the impact of ttcA gene knockout on B. bacteriovorus predatory efficiency?

Creating a ttcA knockout in B. bacteriovorus would require specialized techniques due to the unique biology of this predatory bacterium:

How does TtcA-mediated tRNA modification influence B. bacteriovorus stress responses?

The relationship between tRNA modifications and stress response is particularly relevant for B. bacteriovorus given its exposure to various stresses during predation:

• Potential roles in stress response:

  • Modified tRNAs can affect translation of stress-response genes

  • s2C32 may stabilize tRNA structure under stress conditions

  • Specific codon usage in stress genes may be optimized for s2C-modified tRNAs

• Experimental approaches:

  • Compare survival of wild-type and ΔttcA strains under various stresses (oxidative, pH, temperature)

  • Analyze expression of stress-response genes using RNA-seq and proteomics

  • Measure translation efficiency of stress proteins using ribosome profiling

  • Investigate tRNA fragmentation patterns, which are known stress responses in bacteria

• Connection to predatory lifecycle:

  • B. bacteriovorus faces various stresses when entering prey periplasm

  • The bdelloplast environment undergoes significant biochemical changes

  • Predation-specific stress response genes may rely on optimized translation via modified tRNAs

How conserved is TtcA structure and function across different bacterial species?

TtcA belongs to a protein family characterized by specific structural elements that can be compared across species:

• Structural conservation patterns:

  • The TtcA protein family can be divided into two distinct groups based on the presence and location of additional Cys-X1-X2-Cys motifs in terminal regions of the sequence

  • The central Cys-X1-X2-Cys motif is universally conserved and both cysteines are required for function

  • The PP-loop domain is another conserved feature across TtcA proteins

• Phylogenetic distribution:

  • TtcA is found in organisms from both Archaea and Bacteria domains

  • Interestingly, similar genes exist in eukaryotic organisms despite no s2C having been found in their tRNAs

  • B. bacteriovorus TtcA likely shares core functional domains with other bacterial TtcAs but may have unique adaptations related to its predatory lifestyle

• Functional conservation:

  • The mechanism of s2C formation appears conserved where studied

  • Substrate specificity (which tRNAs get modified) may vary between species

  • Regulation of ttcA expression and TtcA activity might differ between free-living and predatory bacteria

Can B. bacteriovorus TtcA complement ttcA mutations in other bacterial species?

Cross-species complementation provides insights into functional conservation:

What are common technical challenges when working with recombinant B. bacteriovorus TtcA?

Researchers frequently encounter several challenges:

• Expression issues:

  • Low solubility due to improper folding

  • Toxicity to expression host

  • Inefficient translation due to codon bias differences between B. bacteriovorus and expression hosts

• Activity problems:

  • Loss of activity during purification due to oxidation of critical cysteine residues

  • Missing cofactors or partner proteins needed for full activity

  • Substrate specificity differences between B. bacteriovorus and test systems

• Troubleshooting strategies:

  • Use solubility-enhancing fusion tags (SUMO, MBP, TrxA)

  • Optimize redox conditions by including reducing agents

  • Co-express with potential partner proteins from B. bacteriovorus

  • Try multiple expression hosts and conditions

  • Consider cell-free expression systems for toxic proteins

How can researchers reconcile contradictory results in TtcA activity studies?

When facing inconsistent experimental results:

• Sources of experimental variability:

  • Differences in protein preparation methods affecting conformation

  • Variation in assay conditions (pH, temperature, salt concentration)

  • Differences in substrate preparation and quality

  • Detection method sensitivity and specificity variations

• Standardization approaches:

  • Establish standard operating procedures for enzyme purification

  • Use multiple activity assay methods in parallel

  • Include appropriate positive and negative controls

  • Carefully document all experimental parameters

• Validation strategies:

  • Verify protein activity using complementary approaches

  • Perform structure-function studies to confirm active site integrity

  • Test activity under a range of conditions to identify optimal parameters

  • Consider the influence of post-translational modifications

What are the most promising avenues for advancing B. bacteriovorus TtcA research?

Several research directions show particular promise:

• Structural biology approaches:

  • Determine the crystal or cryo-EM structure of B. bacteriovorus TtcA

  • Perform comparative structural analysis with TtcA from non-predatory bacteria

  • Investigate structural changes during the catalytic cycle

• Integration with predation biology:

  • Analyze ttcA expression patterns during the predatory cycle

  • Investigate the role of TtcA in prey preference and host range

  • Explore potential connections between TtcA and B. bacteriovorus-specific metabolic adaptations

• Systems biology perspectives:

  • Map the complete tRNA modification landscape across the B. bacteriovorus lifecycle

  • Integrate transcriptomics, proteomics, and tRNA modification data

  • Develop predictive models for the role of tRNA modifications in predation efficiency

• Biotechnological applications:

  • Explore the use of B. bacteriovorus TtcA for in vitro RNA modification

  • Investigate potential applications in synthetic biology

  • Develop TtcA-based sensors for sulfur metabolism studies

How might genetic engineering tools improve our ability to study B. bacteriovorus TtcA?

Advanced genetic tools could accelerate research:

• Emerging genetic technologies for B. bacteriovorus:

  • The genetic toolbox for B. bacteriovorus is still limited compared to model organisms

  • Promising approaches include:

    • Suicide plasmids like pSSK10 containing counter-selectable markers

    • Transposon mutagenesis protocols established using facultative HI isolates

    • Expression of antisense RNA for downregulation of genes

    • Synthetic riboswitches for regulated expression

• Adaptation of CRISPR-based tools:

  • Development of CRISPRi systems for conditional knockdowns

  • Precise genome editing for tag insertion at native loci

  • Base editing to create specific mutations in ttcA

• Reporter systems for tracking TtcA activity:

  • Development of fluorescent or luminescent reporters sensitive to s2C modification

  • Live-cell imaging techniques to track TtcA localization during predation

  • High-throughput screening systems for TtcA modulators

What are the most reliable protocols for B. bacteriovorus genetic manipulation relevant to ttcA studies?

Key protocols and their applications include:

• Gene deletion techniques:

  • Suicide vector method using pK18mobsacB with sacB counterselection

  • Allelic exchange procedures optimized for B. bacteriovorus

  • Linear transformation approaches that have been successful in other bacteria

• Expression systems:

  • Heterologous expression in E. coli using pET vectors

  • Expression in B. bacteriovorus using IncQ-type plasmids

  • Conjugation protocols for transferring DNA from E. coli to B. bacteriovorus

• RNA and tRNA analysis:

  • Synchronized predatory cultures for stage-specific RNA extraction

  • tRNA isolation using LiCl extraction

  • Nucleoside analysis by HPLC and mass spectrometry

What specialized equipment and reagents are needed for comprehensive TtcA functional studies?

Essential resources include:

• Equipment requirements:

  • HPLC system with C18 column for nucleoside analysis

  • Mass spectrometer with electrospray ionization source

  • Flow scintillation analyzer for radiolabeling experiments

  • Advanced microscopy for predation studies

• Critical reagents:

  • Purified tRNA substrates from B. bacteriovorus

  • 35S-labeled substrates for sulfur transfer studies

  • Specific antisera against B. bacteriovorus TtcA

  • Fluorescent D-amino acids like HADA for prey cell wall labeling

• Specialized bacterial strains:

  • Reporter strains for measuring translation effects

  • B. bacteriovorus host-independent strains for specific genetic manipulations

  • E. coli strains optimized for heterologous expression of B. bacteriovorus proteins