Recombinant Bartonella henselae tRNA dimethylallyltransferase (miaA)

What is the function of tRNA dimethylallyltransferase (MiaA) in bacterial systems?

MiaA functions as a critical tRNA modifying enzyme that catalyzes the prenylation of adenosine-37 (A-37) within tRNAs that decode UNN codons. Specifically, MiaA adds a prenyl group to the N⁶-nitrogen of A-37 to create i⁶A-37 tRNA . This modification is subsequently methylthiolated by another enzyme, MiaB, to create ms²i⁶A-37 . The bulky and hydrophobic nature of the ms²i⁶A-37 modification enhances tRNA interactions with UNN target codons, promoting reading frame maintenance and translational fidelity during protein synthesis . This function is highly conserved across both prokaryotic and eukaryotic organisms, highlighting its fundamental importance in cellular protein synthesis mechanisms .

How does the structure of Bartonella henselae MiaA compare to homologous enzymes in other bacterial species?

While the search results don't provide specific structural information about B. henselae MiaA, research on homologous tRNA modifying enzymes indicates that MiaA proteins are relatively well conserved across bacterial species . In prokaryotes, MiaA and MiaB homologues demonstrate consistent functional mechanisms across tested bacterial species . Based on conservation patterns observed in the 17-kDa antigen gene within various Bartonella species, we can infer that functional domains of MiaA likely maintain structural conservation while allowing for species-specific variations that may relate to host adaptation and pathogenicity . Methodologically, researchers can employ comparative protein modeling using known bacterial MiaA structures as templates to predict B. henselae MiaA structure-function relationships.

What expression systems are most effective for producing recombinant Bartonella henselae MiaA?

For effective recombinant expression of B. henselae MiaA, E. coli-based expression systems offer practical advantages, particularly using vectors like pRR48 or pUC19 that have been successfully employed for expressing other Bartonella proteins . When designing expression constructs, researchers should consider incorporating appropriate purification tags (such as Flag or 6xHis tags) at the C-terminus to minimize interference with enzymatic function, as demonstrated with other tRNA modifying enzymes . Expression optimization typically involves:

• Codon optimization for E. coli expression

• Temperature modulation (often 16-30°C) during induction to enhance solubility

• Testing various induction conditions (IPTG concentration and induction timing)

• Supplementation with precursors that may enhance folding

Research indicates that tags like Flag do not interfere with MiaA function in complementation assays, suggesting similar approaches would be viable for B. henselae MiaA .

What are appropriate controls when studying MiaA enzyme activity in vitro?

When studying B. henselae MiaA activity in vitro, researchers should implement the following controls:

Additionally, researchers should consider time-course analyses to determine optimal reaction kinetics and include controls for potential contaminating enzymes that might influence results .

How does stress affect the expression and activity of MiaA in Bartonella henselae, and what are the regulatory mechanisms involved?

Research in related bacterial systems has shown that MiaA levels can shift in response to stress via post-transcriptional mechanisms . For B. henselae, which must adapt to diverse host environments including arthropod vectors and mammalian hosts, stress adaptation is particularly critical. Researchers investigating stress responses should:

• Examine MiaA expression under various stress conditions (oxidative stress, pH changes, nutrient limitation, temperature fluctuation)

• Quantify both mRNA and protein levels to identify post-transcriptional regulation

• Investigate potential regulatory sRNAs or RNA-binding proteins that might influence MiaA expression

• Analyze promoter regions for stress-responsive regulatory elements

Studies in E. coli have demonstrated that the shift in MiaA levels during stress results in marked changes in fully modified MiaA substrates, suggesting a similar mechanism might exist in B. henselae as an adaptation strategy during host infection . Researchers should employ quantitative RT-PCR, western blotting, and tRNA modification analysis techniques to fully characterize these stress-responsive mechanisms.

What role does MiaA play in Bartonella henselae pathogenesis and host adaptation?

Based on research in extraintestinal pathogenic E. coli (ExPEC), MiaA is crucial for bacterial fitness and virulence . To investigate MiaA's role in B. henselae pathogenesis, researchers should:

• Generate MiaA deletion and catalytic mutants in B. henselae

• Assess colonization ability in relevant cell culture models

• Evaluate adherence, invasion, and intracellular persistence phenotypes

• Analyze proteome changes in wild-type versus mutant strains under host-mimicking conditions

Experimental models should include human endothelial cells and cat-derived cell lines, reflecting B. henselae's natural hosts. The research approach should include:

$\text{Invasion efficiency} = \frac{\text{Intracellular bacteria (CFU/ml)}}{\text{Initial inoculum (CFU/ml)}} \times 100\%$

Researchers should pay particular attention to the role of MiaA in regulating virulence factors through its effects on translational fidelity, as both deletion and overexpression of MiaA have been shown to stimulate translational frameshifting and profoundly alter bacterial proteomes .

How do site-directed mutations in the catalytic domain of B. henselae MiaA affect enzyme kinetics and substrate specificity?

To investigate structure-function relationships in B. henselae MiaA, researchers should generate a panel of site-directed mutants targeting conserved residues in the catalytic domain. This methodological approach should include:

• Identification of conserved residues through multiple sequence alignment with characterized MiaA enzymes

• Generation of point mutations using site-directed mutagenesis (similar to approaches used for E. coli MiaA)

• Purification of wild-type and mutant proteins

• In vitro enzyme kinetic analysis with various tRNA substrates

The experimental design should measure:

Analysis of these parameters will provide insights into how specific residues contribute to catalysis and how B. henselae MiaA might be adapted to function during host infection .

What is the effect of MiaA-catalyzed tRNA modifications on the Bartonella henselae translatome during infection?

Given that MiaA modifications enhance tRNA interactions with UNN target codons, variations in MiaA activity likely influence the bacterial translatome, particularly for UNN-enriched transcripts . To investigate this complex relationship, researchers should:

• Apply ribosome profiling (Ribo-seq) to wild-type and MiaA-deficient B. henselae under infection-relevant conditions

• Analyze translation efficiency across the transcriptome with focus on UNN codon usage

• Identify genes with altered translation rates and correlate with UNN content

• Validate findings using reporter constructs with modified UNN codon content

This approach can be complemented with mass spectrometry-based proteomics to directly assess protein level changes. Research in E. coli has shown that MiaA influences translational frameshifting and can markedly alter the spectrum of expressed proteins , suggesting B. henselae may employ similar mechanisms to regulate its proteome during different stages of infection.

What are the optimal conditions for measuring MiaA enzymatic activity in vitro?

For optimal measurement of B. henselae MiaA activity in vitro, researchers should establish conditions that maintain enzyme stability while promoting catalytic efficiency. Based on research with related enzymes, the following methodological approach is recommended:

Activity can be monitored via:

• Radiolabeled substrate incorporation

• HPLC analysis of modified tRNA

• Mass spectrometry detection of modified nucleosides

Researchers should validate assay conditions specifically for B. henselae MiaA, as optimal conditions may vary from those established for E. coli or other bacterial MiaA enzymes .

How can researchers distinguish between direct and indirect effects of MiaA deletion on the bacterial proteome?

Distinguishing direct from indirect effects of MiaA deletion presents a significant methodological challenge. A comprehensive approach would include:

• Codon usage analysis: Compare the UNN codon frequency in genes with altered expression to identify direct translational effects.

• Complementation studies: Use plasmid-based expression of wild-type MiaA and catalytically inactive MiaA to determine which phenotypes are directly dependent on enzymatic activity .

• Temporal proteomics: Analyze proteome changes at multiple timepoints after MiaA depletion to differentiate primary from secondary effects.

• Targeted reporter assays: Develop luciferase reporters with varying UNN content to directly measure translational effects .

• tRNA modification analysis: Quantify changes in tRNA modification profiles using mass spectrometry to correlate with protein expression alterations.

The experimental design should include careful controls to account for potential pleotropic effects, including analysis of tRNA abundance, ribosome association, and mRNA stability, as these could all contribute to observed phenotypes independent of direct tRNA modification effects .

How can recombinant B. henselae MiaA be utilized for developing diagnostic tools for Bartonella infections?

While MiaA itself has not been extensively explored as a diagnostic target for Bartonella infections, the approach used for the 17-kDa antigen of B. henselae provides a methodological framework . Researchers could:

• Express and purify recombinant B. henselae MiaA

• Evaluate its antigenicity using sera from confirmed Bartonella infection cases

• Determine species specificity by comparing cross-reactivity with MiaA from other bacterial species

• Develop ELISA or Western blot assays using the recombinant protein

Research on the 17-kDa antigen of B. henselae demonstrated that recombinant proteins can be effective serological reagents . If MiaA shows similar immunogenic properties with minimal cross-reactivity with human proteins, it could serve as a candidate diagnostic marker. Researchers should evaluate sensitivity and specificity with diverse patient samples, including those with confirmed Bartonella infections and appropriate control groups .

What potential exists for targeting MiaA in antimicrobial development against Bartonella henselae?

Given MiaA's essential role in bacterial fitness and virulence, as demonstrated in ExPEC , it represents a potential antimicrobial target. Researchers pursuing this direction should:

• Confirm essentiality of MiaA in B. henselae using conditional expression systems

• Develop high-throughput screening assays for MiaA inhibitors

• Evaluate selectivity by comparing activity against bacterial versus human homologs

• Assess inhibitor efficacy in cellular infection models

A methodological approach for inhibitor screening might include:

The development pathway should consider the potential for resistance development and examine combination approaches with existing antimicrobials, particularly since alterations in translational fidelity can influence susceptibility to various antibiotics .