Recombinant Bordetella bronchiseptica CCA-adding enzyme (cca)

How does B. bronchiseptica CCA-adding enzyme relate to virulence and pathogenesis?

While no direct evidence in the search results links CCA-adding enzyme to virulence, we can infer potential relationships based on general bacterial physiology. Proper tRNA processing is essential for efficient protein synthesis, which directly impacts the production of virulence factors. B. bronchiseptica relies on several virulence factors including adenylate cyclase-hemolysin, filamentous hemagglutinin, and pertactin for colonization and infection . Disruption of tRNA maturation through CCA-adding enzyme dysfunction would likely impair the translation of these virulence factors, potentially attenuating bacterial pathogenicity. Additionally, since B. bronchiseptica responds to environmental signals through the BvgAS regulatory system to modulate virulence gene expression , proper tRNA function is necessary for this adaptation process.

What are the optimal cloning strategies for B. bronchiseptica CCA-adding enzyme gene?

The optimal cloning strategy for B. bronchiseptica CCA-adding enzyme would follow similar approaches to those used for other bacterial tRNA nucleotidyltransferases. Based on methodologies described for related enzymes, researchers should:

• Identify and amplify the complete open reading frame encoding the CCA-adding enzyme using PCR with high-fidelity polymerase.

• Design primers with appropriate restriction sites for directional cloning into expression vectors.

• Consider using vectors like pET series (such as pET19B or pET11A) that have been successfully used for other nucleotidyltransferases .

• Include affinity tags (such as His-tag) to facilitate purification, preferably at the N-terminus as demonstrated successful for other similar enzymes .

The approach used for Bacillus halodurans provides a useful template: PCR products corresponding to the open reading frames were prepared and cloned into expression vectors like pET19B, allowing for the expression of N-terminal His-tagged derivatives purifiable by immobilized metal ion affinity chromatography .

What expression systems are most effective for producing active recombinant B. bronchiseptica CCA-adding enzyme?

Based on successful expression of related enzymes, E. coli-based expression systems are likely most effective for producing recombinant B. bronchiseptica CCA-adding enzyme. Specifically:

• BL21(DE3) or similar E. coli strains carrying the T7 RNA polymerase system are recommended for controllable high-level expression.

• Expression vectors containing T7 promoters (pET series) have proven effective for other bacterial nucleotidyltransferases .

• Induction conditions should be optimized, typically using IPTG at concentrations between 0.1-1 mM.

• Lower induction temperatures (16-25°C) may improve solubility and proper folding of the enzyme.

• Consider co-expression with chaperone proteins if initial attempts yield insoluble protein.

The effectiveness of the expression system can be verified by SDS-PAGE analysis of purified protein, with expected molecular weight estimation based on amino acid sequence . Activity assays using tRNA substrates should follow to confirm that the recombinant enzyme retains its catalytic function.

What purification protocols yield the highest purity and activity for recombinant B. bronchiseptica CCA-adding enzyme?

Optimal purification of recombinant B. bronchiseptica CCA-adding enzyme would likely involve a multi-step process similar to those used for other bacterial nucleotidyltransferases:

• Affinity chromatography: If expressing His-tagged protein, immobilized metal ion affinity chromatography (IMAC) using Ni-NTA or similar matrices offers an efficient first purification step .

• Ion exchange chromatography: For proteins that don't bind well to affinity columns or as a secondary purification step, ion exchange chromatography can be effective, as demonstrated with B. halodurans NTSFII .

• Hydrophobic interaction chromatography: This can serve as an additional purification step, particularly effective after ion exchange chromatography .

• Size exclusion chromatography: As a final polishing step to remove aggregates and achieve highest purity.

• Buffer optimization: The enzyme activity is pH-dependent, so final buffers should be optimized based on activity assays. Different pH optima may exist for different nucleotide incorporation activities, as seen with B. halodurans enzymes (pH 9.5 for CTP incorporation, pH 8 for ATP incorporation) .

Purification success should be monitored by SDS-PAGE and activity assays at each step to ensure retention of enzymatic function. Protein purity of >95% as assessed by SDS-PAGE is typically desired for detailed enzymological studies.

How can site-directed mutagenesis be used to identify critical residues in B. bronchiseptica CCA-adding enzyme catalysis?

Site-directed mutagenesis represents a powerful approach to investigate structure-function relationships in the B. bronchiseptica CCA-adding enzyme. Based on successful approaches with related enzymes:

The approach that successfully transformed CCA-adding enzymes into (U,G)-adding enzymes through targeted mutations provides an excellent model . These experiments demonstrated that alterations to the nucleotide recognition domain can fundamentally change substrate specificity. Similar strategies could identify residues in B. bronchiseptica CCA-adding enzyme responsible for nucleotide selection and the mechanism of template-independent sequential addition.

What experimental approaches can determine if B. bronchiseptica possesses a single CCA-adding enzyme or separate CC- and A-adding enzymes?

Determining whether B. bronchiseptica uses a single CCA-adding enzyme or separate CC- and A-adding enzymes requires systematic biochemical and genetic approaches:

• Genomic analysis: Search the B. bronchiseptica genome for nucleotidyltransferase superfamily members and analyze their phylogenetic relationships with known CC-, A-, or CCA-adding enzymes from other species .

• Gene expression analysis: Use RT-PCR with gene-specific primers to verify expression of candidate genes, as was done for B. halodurans NTSF genes .

• Biochemical characterization:

  • Clone and express each candidate gene

  • Perform enzyme assays with varying pH conditions and nucleotide substrates

  • Analyze reaction products by gel electrophoresis to determine if enzymes add C, CC, A, or complete CCA

• pH profiling: Test enzyme activity across pH range 7-10 with different nucleotide substrates. Distinct pH optima for CTP versus ATP incorporation (as seen with B. halodurans enzymes) would suggest separate enzymes .

• Product analysis: Use 3'-end labeled tRNA substrates and analyze reaction products at single-nucleotide resolution to determine exactly which nucleotides are added by each enzyme.

The methodological approach employed for B. halodurans provides an excellent template, where distinct pH optima and substrate preferences clearly distinguished CC-adding from A-adding activities .

How does the nucleotide specificity mechanism of B. bronchiseptica CCA-adding enzyme compare with the engineered (U,G)-adding enzyme variants?

Understanding the nucleotide specificity mechanism of B. bronchiseptica CCA-adding enzyme in comparison to engineered variants requires detailed structural and biochemical analysis:

• Comparative sequence analysis: Align B. bronchiseptica CCA-adding enzyme with native and engineered variants to identify conserved and divergent regions, particularly focusing on nucleotide recognition domains.

• Specificity testing: Assay the enzyme with all four nucleotides (ATP, CTP, GTP, UTP) under various conditions to determine its natural specificity range.

• Structural modeling: Generate homology models based on solved structures of related enzymes to predict nucleotide binding pocket architecture.

• Targeted mutations: Introduce mutations corresponding to those that transformed CCA-adding enzymes into (U,G)-adding enzymes to determine if similar changes alter the specificity of the B. bronchiseptica enzyme.

The research on reengineering CCA-adding enzymes demonstrated that specific mutations could transform these enzymes to incorporate UTP and GTP instead of CTP and ATP . The transformability of B. bronchiseptica's enzyme would provide insights into evolutionary conservation of nucleotide selection mechanisms across bacterial species and potentially identify unique features of this particular enzyme.

What are the optimal conditions for assaying B. bronchiseptica CCA-adding enzyme activity in vitro?

Based on methodologies established for related tRNA nucleotidyltransferases, the optimal conditions for assaying B. bronchiseptica CCA-adding enzyme activity would include:

• Buffer composition:

  • Tris-HCl or HEPES buffer (50-100 mM)

  • Divalent cation (typically 5-10 mM MgCl₂)

  • DTT or β-mercaptoethanol (1-5 mM) as reducing agent

  • Potential addition of spermidine (0.5-2 mM)

• pH optimization:

  • Test range from pH 7.0-9.5

  • Potentially different optima for different nucleotides (as seen with B. halodurans enzymes where CTP incorporation was optimal at pH 9.5, while ATP incorporation was optimal at pH 8.0)

• Temperature:

  • Likely 30-37°C based on B. bronchiseptica's growth temperature

• Reaction components:

  • Purified tRNA or tRNA-like substrates lacking CCA terminus

  • NTPs (ATP and CTP, 50-500 μM each)

  • Radiolabeled NTPs for detection ([α-³²P]ATP or [α-³²P]CTP)

  • Purified enzyme (concentration determined empirically)

• Time course:

  • Typically 5-30 minutes to establish linear range of activity

Analysis of reaction products should be performed by gel electrophoresis (typically denaturing PAGE) followed by autoradiography or phosphorimaging to visualize and quantify nucleotide addition .

What techniques can distinguish between CCA-adding activity and other nucleotidyltransferase activities in B. bronchiseptica cellular extracts?

Distinguishing CCA-adding activity from other nucleotidyltransferase activities in B. bronchiseptica cellular extracts requires selective experimental approaches:

• Substrate specificity:

  • Use defined tRNA substrates lacking specific 3' nucleotides (tRNA-N, tRNA-NC, tRNA-NCC)

  • Monitor addition of specific nucleotides to determine if separate or combined activities exist

• Differential inhibition:

  • Test sensitivity to inhibitors that differentially affect tRNA nucleotidyltransferases versus poly(A) polymerases (e.g., cordycepin)

• Product analysis:

  • Analyze reaction products by high-resolution gel electrophoresis or sequencing

  • CCA-adding enzymes produce discrete bands of defined length

  • Poly(A) polymerases produce heterogeneous products of variable length

• Fractionation approaches:

  • Use ion exchange or size exclusion chromatography to separate different nucleotidyltransferase activities

  • Assay fractions for specific activities with different substrates and nucleotides

• pH profiling:

  • Test activity across pH range (7-10) with different nucleotide substrates

  • CCA-adding enzymes often show different pH optima for CTP versus ATP incorporation

The methods used for B. halodurans provide an excellent template, where gel electrophoresis clearly distinguished the discrete products of tRNA nucleotidyltransferases from the heterogeneous high-molecular-weight products of poly(A) polymerase .

What expression and purification methods yield the most enzymatically active recombinant B. bronchiseptica CCA-adding enzyme?

To obtain maximally active recombinant B. bronchiseptica CCA-adding enzyme, researchers should consider:

• Expression vector optimization:

  • Test multiple vectors (pET19B, pET11A) as different tag positions and fusion partners can affect activity

  • Compare N-terminal versus C-terminal affinity tags (His-tag, MBP, GST)

  • Consider tag removal options via protease sites

• Expression conditions:

  • Optimize induction parameters (IPTG concentration: 0.1-1.0 mM)

  • Test lower temperatures (16-25°C) to enhance proper folding

  • Evaluate different growth media (LB, TB, auto-induction)

  • Consider shorter induction times to minimize inclusion body formation

• Cell lysis and initial extraction:

  • Use gentle lysis methods (lysozyme treatment followed by mild sonication)

  • Include protease inhibitors to prevent degradation

  • Maintain reducing environment with DTT or β-mercaptoethanol

• Purification strategy:

  • For His-tagged constructs, optimize imidazole concentrations in binding and elution buffers

  • Consider a combination of purification techniques as used for B. halodurans nucleotidyltransferases :

    • Immobilized metal affinity chromatography

    • Ion exchange chromatography

    • Hydrophobic interaction chromatography

• Activity preservation:

  • Test stabilizing additives (glycerol 10-20%, BSA 0.1 mg/ml)

  • Optimize storage conditions (-80°C with flash freezing versus 4°C)

  • Evaluate buffer components that maintain activity during storage

The specific activity of the enzyme should be monitored throughout the purification process to ensure that activity is preserved, as some purification steps might yield higher purity but lower specific activity.

How can researchers design experiments to determine the substrate specificity of B. bronchiseptica CCA-adding enzyme?

To comprehensively characterize the substrate specificity of B. bronchiseptica CCA-adding enzyme, researchers should design experiments that examine both nucleotide and tRNA substrate preferences:

• Nucleotide specificity:

  • Test all four nucleotides (ATP, CTP, GTP, UTP) individually and in combinations

  • Determine incorporation rates and patterns for each nucleotide

  • Use gel electrophoresis to analyze products qualitatively

  • Employ kinetic assays to determine Km and kcat values for each nucleotide

• tRNA substrate recognition:

  • Prepare tRNA substrates with different 3'-end status (tRNA-N, tRNA-NC, tRNA-CC)

  • Use tRNAs from different species to test cross-species activity

  • Create truncated or mutated tRNAs to identify essential recognition elements

  • Compare activity on tRNA versus synthetic minihelix substrates

• Methodological approach:

  • Establish pH profiles for each nucleotide incorporation (as done for B. halodurans enzymes)

  • Perform competition assays between nucleotides

  • Use template challenge experiments (presenting enzyme with choice of different tRNA substrates)

• Data analysis and visualization:

The approach used to characterize B. halodurans tRNA nucleotidyltransferases provides an excellent model, where pH profiling and product analysis clearly distinguished the activities and specificities of different enzymes .

How can recombinant B. bronchiseptica CCA-adding enzyme be engineered for altered nucleotide specificity?

Engineering B. bronchiseptica CCA-adding enzyme for altered nucleotide specificity would build upon successful approaches used with other bacterial tRNA nucleotidyltransferases:

• Structure-guided mutation design:

  • Target residues in the nucleotide recognition pocket based on homology modeling

  • Focus on amino acids that interact with the base, ribose, and triphosphate moieties

  • Consider mutations that have successfully altered specificity in other CCA-adding enzymes

• Experimental approach:

  • Create a library of point mutations at key residues

  • Screen mutants for altered nucleotide incorporation patterns

  • Use iterative approaches, combining beneficial mutations

  • Test activity with non-standard nucleotides (e.g., dNTPs, modified nucleotides)

• Characterization of engineered variants:

  • Determine kinetic parameters for different substrates

  • Analyze product profiles by gel electrophoresis and sequencing

  • Assess thermostability and pH profiles of variants

The successful transformation of related CCA-adding enzymes into (U,G)-adding enzymes demonstrates the feasibility of this approach . Additionally, the conversion of the B. stearothermophilus CCA-adding enzyme into a poly(C,A) polymerase through mutations in helix J suggests multiple strategies for altering specificity .

What role might the B. bronchiseptica CCA-adding enzyme play in bacterial adaptation to environmental stresses?

The potential role of B. bronchiseptica CCA-adding enzyme in stress adaptation involves several mechanistic possibilities:

• tRNA maturation regulation:

  • Under stress conditions, modulation of CCA-adding enzyme activity could alter the pool of functional tRNAs

  • This would impact translation efficiency of specific proteins, potentially including stress-response factors

• Connection to virulence regulation:

  • B. bronchiseptica uses the BvgAS two-component system to regulate virulence factor expression in response to environmental cues

  • Changes in tRNA availability could influence the translation of regulators and virulence factors

• Potential experimental approaches:

  • Monitor CCA-adding enzyme expression and activity under various stress conditions

  • Create conditional mutants to assess the impact of reduced CCA-adding activity on stress survival

  • Examine global translation patterns when CCA-adding enzyme function is compromised

• Comparison with related species:

  • B. bronchiseptica is closely related to B. pertussis, the causative agent of whooping cough

  • Comparative analysis of CCA-adding enzymes across Bordetella species could reveal adaptation mechanisms

Understanding these relationships would provide insights into bacterial physiology and potentially identify new targets for therapeutic intervention in Bordetella infections.

How does the evolution of B. bronchiseptica CCA-adding enzyme compare with that of other bacterial species?

Evolutionary analysis of B. bronchiseptica CCA-adding enzyme would reveal important insights about bacterial tRNA processing systems:

• Phylogenetic analysis:

  • Compare B. bronchiseptica CCA-adding enzyme sequence with those from diverse bacterial species

  • Determine if it clusters with single CCA-adding enzymes or with separate CC- and A-adding enzymes

  • Examine evolutionary relationships with enzymes from other Bordetella species

• Functional conservation assessment:

  • Compare substrate specificities across evolutionary diverse enzymes

  • Identify conserved versus variable regions in the protein sequence

  • Correlate sequence conservation with functional domains

• Evolutionary trajectories:

  • Investigate whether B. bronchiseptica has a single CCA-adding enzyme or separate CC- and A-adding enzymes

  • Compare with deep-branching bacterial species that have separate enzymes

  • Examine potential gene duplication or fusion events in the evolutionary history

• Potential data visualization:

This evolutionary analysis would provide context for understanding the diversity of tRNA maturation systems across bacteria and potential adaptations specific to the Bordetella genus.