Recombinant Campylobacter fetus subsp. fetus tRNA dimethylallyltransferase (miaA)

What is the biological function of MiaA in Campylobacter fetus?

MiaA (tRNA dimethylallyltransferase) catalyzes the first step of a two-step tRNA modification process in Campylobacter fetus. Specifically, MiaA, along with MiaB, facilitates the addition of the 2-methylthio-N6-(Δ2-isopentenyl), or ms2i6A, modification to adenine 37 of tRNAs that recognize codons beginning with uridine . This modification is crucial for translational fidelity and selective protein expression during bacterial responses to stress .

In C. fetus, MiaA plays a vital role in regulating gene expression through its effects on translation. Research has demonstrated that MiaA is necessary for the full expression of the stationary phase/general stress response sigma factor RpoS (σS). When miaA is mutated, RpoS expression decreases 2-3 fold, particularly upon entry into stationary phase (OD600 = 1.5 to 2.5) .

How is the miaA gene organized in C. fetus genomes?

The miaA gene in C. fetus is located in a complex operon upstream of the gene for the RNA chaperone Hfq . Based on genomic analysis, the miaA gene is highly conserved across C. fetus subspecies. In C. fetus subsp. venerealis 97/608, the miaA gene (designated as CFV97608_0219) is annotated as tRNA(i6A37) synthase in the KEGG Orthology database (K00791) .

Complete genome sequencing has revealed that the miaA gene is present in both C. fetus subspecies: C. fetus subsp. fetus and C. fetus subsp. venerealis. Comparative genomic analysis shows that despite the distinct host and niche preferences of these two subspecies, they share a high level of genetic similarity, including conservation of the miaA gene .

What phenotypic changes occur in C. fetus when miaA is mutated?

Mutations in the miaA gene result in pleiotropic phenotypes in C. fetus, affecting multiple cellular processes . Key phenotypic changes include:

• Decreased expression of RpoS (2-3 fold reduction), as demonstrated by both β-galactosidase assays and Western blot analysis

• Altered translational fidelity, with significant increases in frameshifting in both +1 and -1 directions

• Reduced virulence and fitness in host environments

• Impaired adaptation to various stress conditions

Research has shown that the miaA mutation was the only tRNA modification mutation among those tested that resulted in a Lac- phenotype in an rpoS750-lacZ translational fusion strain, suggesting a specific role for MiaA in RpoS expression regulation .

What are the most effective methods for cloning and expressing recombinant C. fetus MiaA?

Based on successful genetic tool development for C. fetus, the following methodology is recommended for cloning and expressing recombinant MiaA:

• Vector Selection: E. coli-Campylobacter shuttle vectors based on the C. coli plasmid pIP1455 replicon have been successfully used for C. fetus . The pRY series vectors (e.g., pRY111) carrying a chloramphenicol acetyltransferase (cat) gene are suitable starting points.

• Promoter Selection: For efficient expression in C. fetus, the C. fetus sapA promoter is critical. A 260-bp fragment homologous to the sapA promoter can be amplified by PCR and incorporated into the expression vector .

• Cloning Strategy:

  • Amplify the miaA gene via PCR using C. fetus genomic DNA

  • Digest both the PCR product and vector with appropriate restriction enzymes

  • Ligate the digested products

  • Transform into E. coli DH5α for verification before transferring to C. fetus

• Gene Transfer: Conjugative transfer from E. coli S17-1 λpir to C. fetus is more effective than electroporation, with typical transfer frequencies of ~10^-4 transconjugants per donor .

• Expression Verification: Western blotting or enzymatic activity assays can confirm successful expression.

Construction of a pBAD-miaA plasmid has been documented in the literature, where the miaA gene was amplified via PCR using primers targeting the gene, followed by restriction enzyme digestion, and ligation into a digested pBAD24 vector .

How can researchers effectively generate and characterize miaA mutants in C. fetus?

Creating and characterizing miaA mutants in C. fetus requires specific approaches due to the unique characteristics of this organism:

• Mutant Generation:

  • Insertional inactivation using a kanamycin resistance cassette (miaA::kan) has been successfully employed

  • Target the miaA gene through homologous recombination by constructing a vector with flanking sequences of the miaA gene surrounding a selectable marker

• Verification Techniques:

  • PCR confirmation of the insertion

  • RNA extraction and reverse transcription qPCR to confirm lack of miaA expression

  • Complementation tests with plasmid-borne wild-type miaA to confirm phenotype specificity

• Phenotypic Characterization:

  • β-galactosidase assays using rpoS-lacZ translational fusions

  • Western blot analysis of proteins affected by MiaA

  • Serum resistance assays

  • Invasion assays using epithelial cell lines

  • Animal models to assess virulence

• Control Experiments:

  • Ensure the mutant phenotype is not due to polarity on downstream genes (e.g., hfq) by performing complementation studies with both miaA and downstream genes

The effects of miaA mutation can be measured using rpoS-lacZ translational fusion on MacConkey-lactose plates, where miaA::kan mutation results in a Lac- phenotype .

What cell-based assays are appropriate for evaluating the impact of MiaA on C. fetus virulence?

Several cell-based assays have proven effective for assessing MiaA's role in C. fetus virulence:

• Gentamicin Protection (Invasion) Assays:

  • Methodology: Culture approximately 200,000 endometrial epithelial cells in a 25 cm² flask. Add bacteria (MOI 100:1 for C. fetus) diluted in DMEM and incubate for 2 hours at 37°C. Wash the monolayers three times with PBS and incubate with gentamicin to kill extracellular bacteria. Lyse cells and plate for viable counts or extract RNA for qPCR analysis .

  • This assay can be used to compare invasion efficiency between wild-type and miaA mutant strains.

• Cytokine Expression Analysis:

  • Following infection of epithelial cells, extract RNA and synthesize cDNA using commercial kits.

  • Perform qPCR to measure expression levels of proinflammatory cytokines such as IL-1β, IL-8, and IFN-γ.

  • Compare cytokine induction between wild-type and miaA mutant strains .

• Serum Resistance Assays:

  • Incubate bacterial strains with normal human serum and determine survival rates.

  • C. fetus wild-type strains typically show variable serum resistance, which may be altered in miaA mutants .

• Cytoskeleton Inhibition Assays:

  • Treat epithelial cells with cytochalasin D to inhibit cytoskeletal rearrangements before infection.

  • Analyze if MiaA influences bacterial invasion through cytoskeleton-dependent pathways .

Research has shown that C. fetus induces proinflammatory responses in bovine endometrial epithelial cells, with significant upregulation of IL-1β (4.62-fold increase) and IL-8 (3.62-fold increase) at 4 hours post-infection . Similar assays can be used to evaluate differences between wild-type and miaA mutant strains.

How does MiaA contribute to C. fetus stress adaptation and translational control?

MiaA plays a crucial role in C. fetus stress adaptation through its effects on translational control:

• Translational Fidelity Mechanism:

  • MiaA catalyzes the addition of the i6A modification to tRNAs, which affects codon-anticodon interactions.

  • Research in related bacteria shows that MiaA is critical for maintaining translational fidelity, particularly during stress responses .

  • MiaA-deficient strains show increased frameshifting in both +1 and -1 directions, affecting the accurate translation of stress-response proteins .

• Regulatory Impact:

  • MiaA modulates the expression of RpoS, a master regulator of stress response genes.

  • In miaA mutants, RpoS expression decreases 2-3 fold, compromising the cell's ability to respond to various stressors .

  • This regulation appears to be at the translational level rather than through transcriptional control.

• Dynamic Regulation:

  • Evidence suggests that bacteria can modulate MiaA levels in response to stress, providing a post-transcriptional mechanism to facilitate beneficial changes in their proteomes .

  • This represents a programmable mechanism that distressed cells can use to optimize their response to environmental challenges.

• Experimental Evidence:

  • Studies have shown that varying levels of MiaA can markedly alter the spectrum of expressed proteins, indicating its role in selective translation during stress .

  • MiaA's importance increases in challenging environments, such as during infection or colonization of hosts .

The regulatory role of MiaA extends beyond RpoS to potentially affect multiple stress response pathways, making it a key factor in C. fetus adaptation to the diverse environments it encounters during infection and transmission.

What is the relationship between MiaA activity and C. fetus host specificity?

The relationship between MiaA activity and C. fetus host specificity represents an intriguing research area with several key aspects:

• Subspecies-Specific Adaptation:

  • C. fetus comprises two main subspecies with distinct host preferences: C. fetus subsp. fetus (Cff) has a wider host range including humans and animals, while C. fetus subsp. venerealis (Cfv) is primarily adapted to the bovine genital tract .

  • Both subspecies possess the miaA gene, but its regulation and downstream effects may differ in ways that contribute to their distinct host tropisms.

• Translational Control of Host-Specific Factors:

  • MiaA's role in translational control may influence the expression of host-specific virulence factors.

  • Different codon usage in host-specific genes could make their translation differentially dependent on MiaA-mediated tRNA modifications.

• Interaction with Other Virulence Determinants:

  • MiaA likely works in concert with other host specificity determinants, such as:

    • Surface layer proteins (SLPs), which are critical for immune evasion and show variation between subspecies

    • Lipopolysaccharide (LPS) composition, which differs between sero-types A and B and affects host range

    • Type IV secretion systems (T4SS), which are differentially distributed among C. fetus strains and subspecies

• Research Approaches:

  • Comparative analysis of miaA expression levels between subspecies during infection of different host cells

  • Identification of differentially translated proteins in miaA mutants using proteomic approaches

  • Cross-complementation studies between subspecies to determine if MiaA from one subspecies can fully restore function in a miaA mutant of the other subspecies

The table below summarizes key differences between C. fetus subspecies that may interact with MiaA function:

While direct evidence linking MiaA to host specificity in C. fetus is still emerging, its fundamental role in translational control suggests it likely contributes to the expression patterns that determine host adaptation.

How do genomic islands and horizontal gene transfer events interact with MiaA function in C. fetus?

The interaction between genomic islands (GIs), horizontal gene transfer (HGT) events, and MiaA function in C. fetus represents a complex relationship that affects bacterial adaptation and virulence:

The T4SS distribution in C. fetus strains, which may interact with MiaA function, is summarized in the following points based on research findings:

• Three phylogenetically-different T4SS-encoding regions were identified in C. fetus genomes

• Some T4SSs are located in both chromosomes and plasmids, while others are exclusively chromosomal or plasmid-based

• C. fetus strains can contain multiple T4SSs simultaneously

• The genomic islands containing T4SSs differ mainly by the presence of fic genes, insertion sequence elements, and phage-related or hypothetical proteins

• T4SSs inserted in the same genomic locations were conserved across different C. fetus strains

Understanding how MiaA influences the expression and function of genes within these mobile genetic elements could provide insights into C. fetus evolution, adaptation, and pathogenesis.

What research approaches can elucidate the structural features of C. fetus MiaA and their relationship to function?

To investigate the structural features of C. fetus MiaA and their relationship to function, researchers can employ several cutting-edge approaches:

• Protein Structure Determination:

  • X-ray crystallography of purified recombinant MiaA to determine its three-dimensional structure

  • Cryo-electron microscopy (cryo-EM) as an alternative approach if crystallization proves challenging

  • Nuclear Magnetic Resonance (NMR) spectroscopy for analyzing dynamics and ligand interactions

• Computational Structure Analysis:

  • Homology modeling based on known structures of MiaA from other bacteria

  • Molecular dynamics simulations to study substrate binding and catalytic mechanisms

  • Protein-substrate docking to identify key interaction residues

• Structure-Function Analysis:

  • Site-directed mutagenesis of conserved residues identified through structural studies

  • Enzymatic assays to measure the effects of mutations on MiaA activity

  • Complementation studies in miaA knockout strains to correlate structural changes with in vivo function

• Comparative Analysis:

  • Alignment of MiaA sequences from different Campylobacter species to identify conserved and variable regions

  • Comparison with MiaA enzymes from other bacterial genera to identify unique features of C. fetus MiaA

  • Analysis of coevolution between MiaA and its tRNA substrates

• Experimental Protocol for Activity Assays:

  • Express and purify recombinant C. fetus MiaA with a His-tag

  • Prepare tRNA substrates either through in vitro transcription or extraction from cells

  • Conduct in vitro prenylation assays using dimethylallyl pyrophosphate (DMAPP) as the prenyl donor

  • Analyze reaction products using methods such as HPLC, mass spectrometry, or gel electrophoresis

• Domain Analysis Protocol:

  • Identify functional domains through limited proteolysis and mass spectrometry

  • Create truncated variants to map minimal functional units

  • Perform domain swapping experiments with MiaA from other species to identify subspecies-specific functional elements

While specific structural studies on C. fetus MiaA have not been reported in the provided search results, the enzyme belongs to the tRNA dimethylallyltransferase family (EC:2.5.1.75) , for which structural information from related organisms could serve as a starting point for comparative analysis.

How might understanding MiaA function contribute to improved diagnostic approaches for C. fetus infections?

Understanding MiaA function in C. fetus could enhance diagnostic approaches in several ways:

• Molecular Diagnostics:

  • While the miaA gene itself is conserved across C. fetus subspecies, its regulation and downstream effects may serve as subspecies-specific markers.

  • Diagnostic assays could target miaA expression patterns or MiaA-dependent translational products that differ between subspecies.

  • This could improve upon current diagnostic methods, as some established targets like the parA gene have been shown to be unreliable (detected in only 3 of 13 C. fetus subsp. venerealis isolates in one study) .

• Biomarker Development:

  • Proteins whose translation is specifically regulated by MiaA could serve as biomarkers for C. fetus infection.

  • Proteomic analysis comparing wild-type and miaA mutant strains could identify such biomarkers.

  • These could be particularly valuable for diagnosing infections in immunocompromised patients, where C. fetus causes more severe disease .

• Subspecies Differentiation:

  • C. fetus subspecies identification is clinically important as they cause different diseases:

    • C. fetus subsp. fetus causes intestinal illness and systemic infections in humans

    • C. fetus subsp. venerealis primarily causes bovine genital campylobacteriosis

  • MiaA-dependent translational profiles could potentially distinguish between these subspecies more accurately than current methods.

• Clinical Relevance:

  • C. fetus infections should be suspected in patients with nonspecific febrile illness who are immunocompromised or occupationally exposed to ruminants .

  • Understanding MiaA's role in virulence could help identify high-risk patient populations.

  • In pregnant women, C. fetus infections can lead to abortion or neonatal sepsis, making accurate diagnosis critical .

• Research Priorities:

  • Identifying MiaA-dependent proteins that are expressed during human infection

  • Developing antibody-based or nucleic acid tests targeting these proteins

  • Validating these approaches in clinical samples from both intestinal and systemic C. fetus infections

While MiaA itself may not become a direct diagnostic target due to its conservation across bacterial species, its downstream effects on protein expression patterns could yield valuable diagnostic markers for C. fetus detection and subspecies differentiation.

What is the relationship between MiaA and antimicrobial resistance mechanisms in C. fetus?

The relationship between MiaA and antimicrobial resistance in C. fetus represents an emerging area of research with several important dimensions:

• Translational Control of Resistance Genes:

  • MiaA's role in tRNA modification affects translational fidelity, which could influence the expression of antimicrobial resistance genes.

  • Codon bias in resistance genes might make their translation particularly dependent on MiaA-modified tRNAs.

  • Changes in MiaA activity under antibiotic stress could alter the translation efficiency of resistance proteins.

• Genomic Evidence and Resistance Genes:

  • Whole genome sequencing of C. fetus strains has identified antimicrobial resistance-related genes, including those encoding multidrug efflux pumps CmeABC and YkkCD .

  • Interestingly, research indicates that the presence of these genes alone is not sufficient to confer in vitro antimicrobial resistance .

  • This suggests that regulatory factors like MiaA might play a role in determining whether resistance genes are effectively translated into functional proteins.

• Stress Response Connection:

  • MiaA is necessary for robust expression of the stress response sigma factor RpoS .

  • Stress responses often overlap with antimicrobial resistance mechanisms.

  • Decreased MiaA function could potentially reduce resistance to antibiotics that induce stress responses.

• Experimental Observations:

  • In C. fetus subsp. venerealis isolates, no in vitro antimicrobial resistance was detected against tetracycline, penicillin, enrofloxacin, and streptomycin, despite the presence of resistance genes .

  • This discrepancy between genomic potential and phenotype could involve translational regulation mechanisms.

• Research Approaches:

  • Compare the antimicrobial susceptibility profiles of wild-type and miaA mutant C. fetus strains

  • Analyze the translation efficiency of specific resistance genes in the presence and absence of functional MiaA

  • Investigate whether antibiotic exposure alters MiaA expression or activity

  • Examine whether complementation with miaA can restore resistance in certain contexts

While direct experimental evidence linking MiaA to antimicrobial resistance in C. fetus is limited in the current literature, the fundamental role of MiaA in translational control suggests it could be an important factor in the expression of resistance determinants, particularly under stress conditions that bacteria encounter during antibiotic exposure.

How has the miaA gene evolved across Campylobacter species and what does this reveal about its functional importance?

The evolution of the miaA gene across Campylobacter species provides valuable insights into its functional importance and adaptation:

• Conservation and Essentiality:

  • The miaA gene is part of the core genome of Campylobacter species, suggesting its fundamental importance for bacterial viability and fitness.

  • The high conservation of miaA across different Campylobacter species, including C. fetus, C. jejuni, and C. coli, indicates strong selective pressure to maintain its function .

  • This conservation extends to the enzymatic function of MiaA as a tRNA dimethylallyltransferase (EC:2.5.1.75) .

• Subspecies Comparisons:

  • Comparative genomic analyses have shown that despite the distinct host preferences and pathogenicity of C. fetus subspecies, the miaA gene is conserved between them .

  • In whole genome analyses, C. fetus subspecies show approximately 92% sequence identity, with most conserved genes (including miaA) maintaining their syntenic relationships .

  • This conservation across subspecies that diverged to occupy different ecological niches further emphasizes the essential nature of MiaA function.

• Differential Regulation:

  • While the miaA gene itself is conserved, its regulation may differ between species and subspecies.

  • These regulatory differences could contribute to species-specific adaptations without requiring changes to the core enzymatic function.

  • Research suggests that bacteria can modulate MiaA levels in response to environmental conditions, providing a mechanism for adaptation without sequence changes .

• Research Approaches:

  • Phylogenetic analysis of miaA sequences across Campylobacter species and related genera

  • Functional complementation studies to test whether miaA from one species can restore function in another species

  • Analysis of selection pressures on different domains of the MiaA protein

  • Investigation of species-specific regulatory elements controlling miaA expression

• Broader Evolutionary Context:

  • The tRNA modification system involving MiaA is ancient and widespread across bacteria, reflecting its fundamental role in translational control.

  • Comparative analysis with more distantly related bacteria could reveal how C. fetus MiaA has adapted to specific niche requirements.

The conservation of miaA across Campylobacter species with diverse ecological niches and pathogenic potential suggests that its core function in tRNA modification is essential for bacterial fitness across different environments, while potentially subtle variations in sequence or regulation may contribute to species-specific adaptations.

What functional differences exist between MiaA enzymes from C. fetus subspecies fetus versus venerealis?

Investigating functional differences between MiaA enzymes from C. fetus subspecies fetus and venerealis requires careful comparative analysis:

While the search results do not provide direct experimental comparisons of MiaA function between the two subspecies, the fundamental importance of this enzyme combined with the distinct ecological niches of the subspecies suggests there may be subtle but important functional differences that contribute to their host adaptation and pathogenicity.