Recombinant Dehalococcoides sp. Methionyl-tRNA formyltransferase (fmt)

What is the role of methionyl-tRNA formyltransferase in bacterial translation?

Methionyl-tRNA formyltransferase (fmt or MTF) catalyzes the N-formylation of initiator methionyl-tRNA (Met-tRNA^Met), which is a critical step for translation initiation in bacteria, mitochondria, and chloroplasts. This formylation reaction irreversibly commits methionyl-tRNA^Met to initiation of translation in eubacteria, making it a key regulatory point in protein synthesis. The enzyme transfers a formyl group from a donor molecule (typically 10-formyltetrahydrofolate) to the amino group of the methionine esterified to the tRNA, resulting in formyl-methionyl-tRNA^Met (fMet-tRNA^Met) .

In metazoan mitochondria, the translation system uniquely uses a single methionine tRNA for both initiation and elongation, with only a portion being formylated for initiation. Disruptions in this process can significantly impact mitochondrial translation efficiency, which has been linked to oxidative phosphorylation deficiency and severe conditions like Leigh syndrome in humans with mutations in the mitochondrial MTF gene .

What is unique about Dehalococcoides species in environmental bioremediation?

Dehalococcoides species are remarkable microorganisms known for their ability to dechlorinate various chlorinated compounds, making them invaluable for bioremediation of contaminated environments. What makes these organisms particularly significant is their capacity to perform complete dechlorination of compounds like trichloroethene (TCE) to non-toxic ethene, a process critical for remediation of groundwater contaminants .

Dehalococcoides mccartyi strains exhibit specialized metabolic capabilities in environmental settings. For instance, D. mccartyi strain GEO12 demonstrates a natural tolerance to chloroform at concentrations up to 14 μM (1.6 mg·L^-1), while the same concentration effectively inhibits dechlorination in other Dehalococcoides strains like ANAS2, 11a, and BAV1. Through adaptation, strain GEO12 has been cultured to tolerate even higher chloroform concentrations (up to 83 μM or 10 mg·L^-1), demonstrating its metabolic flexibility and potential utility in sites with multiple contaminants .

How does methionyl-tRNA formyltransferase recognize its substrate?

Methionyl-tRNA formyltransferase exhibits high specificity toward its tRNA substrate, primarily through the recognition of structural features in the acceptor arm of the tRNA molecule. Unlike aminoacyl-tRNA synthetases that often recognize the anticodon loop, formyltransferase focuses on the acceptor arm's unique characteristics .

Crystal structure analysis of Escherichia coli methionyl-tRNA formyltransferase complexed with formyl-methionyl-tRNA^fMet at 2.8 Å resolution has revealed that the enzyme fits inside the L-shaped tRNA molecule on the D-stem side. The enzyme employs a distinctive recognition mechanism where a protein loop wedges into the major groove of the acceptor helix, causing the C1-A72 mismatch (characteristic of initiator tRNA) to split and the 3' arm to bend toward the active center. This recognition mechanism differs markedly from that used by elongation factor Tu, which binds elongator tRNAs on the T-stem side .

The main basis for specific formylation of eubacterial methionyl-tRNA^fMet is the lack of base pairing at the top of the acceptor helix (the C1-A72 mismatch in E. coli initiator tRNA). Additional specificity determinants include the A73 discriminator base and specific base pairs in the acceptor arm (G2-C71, C3-G70, and G4-C69) .

What substrates does methionyl-tRNA formyltransferase use as formyl donors?

Methionyl-tRNA formyltransferase typically uses 10-formyltetrahydrofolate (10-CHO-THF) as its primary formyl group donor substrate. This metabolite is produced through the conversion of 5,10-methylene tetrahydrofolate (5,10-CH₂-THF) to 10-formyl-THF by the bifunctional enzyme folate dehydrogenase-cyclohydrolase (FolD) .

Recent research has demonstrated that 10-formyldihydrofolate (10-CHO-DHF) may also serve as an alternative substrate for fmt, functioning as a formyl group donor to formylate Met-tRNA^fMet. This finding, supported by both in vivo and in vitro approaches, expands our understanding of the metabolic flexibility of the formylation process. The use of 10-CHO-DHF as an alternative substrate has been verified through LC-MS/MS analysis, which detected dihydrofolate (DHF) as a by-product in in vitro assays .

This substrate flexibility may have implications for the function of fmt under various metabolic conditions and could be particularly relevant in organisms like Dehalococcoides that may experience specific metabolic constraints in their specialized environmental niches.

How do corrinoids influence the growth and function of Dehalococcoides mccartyi?

Dehalococcoides mccartyi requires corrinoids (vitamin B12-like molecules) for its growth and dechlorination activities. These organisms engage in corrinoid-related interactions with supportive microorganisms in mixed communities. For example, in defined consortia where D. mccartyi strain 195 (Dhc195) was cultured alongside Desulfovibrio vulgaris Hildenborough (DvH) and Pelosinus fermentans R7 (PfR7), distinct patterns of interaction and dependency emerged .

In the triculture system, DvH provided hydrogen while PfR7 supplied corrinoids to Dhc195. The initiation of dechlorination activity and Dhc195 cell growth was highly dependent on the growth of PfR7. Detailed analysis showed that Dhc195 imported and remodeled the phenolic corrinoids produced by PfR7 into cobalamin (vitamin B12) in the presence of 5,6-dimethylbenzimidazole (DMB) .

Transcriptomic analyses revealed that during corrinoid salvaging and remodeling, Dhc195 induced genes in the CbiZ-dependent corrinoid-remodeling pathway and the BtuFCD corrinoid ABC transporter. Conversely, when cobalamin was exogenously provided, a different operon encoding a putative iron/cobalamin ABC transporter (DET1174-DET1176) was induced. This indicates sophisticated regulatory mechanisms for corrinoid acquisition and utilization in Dehalococcoides .

What are the molecular mechanisms of chloroform tolerance in Dehalococcoides mccartyi strain GEO12?

Dehalococcoides mccartyi strain GEO12 exhibits an unusual natural tolerance to chloroform, a compound that typically inhibits dechlorination in most Dehalococcoides strains. The molecular basis for this tolerance appears to involve alterations in the expression of reductive dehalogenase homologous (rdh) genes. Genomic analysis of strain GEO12 identified seven rdh genes, including tceA and vcrA, which are known to encode key enzymes in the dechlorination pathway .

Transcriptional analyses of the chloroform-adapted culture GEO12CF (tolerant to 83 μM or 10 mg·L^-1 chloroform) revealed that exposure to chloroform (45 μM; 5.3 mg·L^-1) significantly enhanced the transcription of tceA. This upregulation showed a median increase of 55.4 transcripts per 10^4 16S rRNA (CI 95% = [12.9, 125]). The effect on vcrA transcription was less conclusive, with a median increase of 109 transcripts per 10^4 16S rRNA but a confidence interval that spanned zero (CI 95% = [-13.6, 246]) .

These findings suggest that GEO12CF may overcome chloroform inhibition through upregulation of specific rdh genes, particularly tceA. This adaptation mechanism demonstrates the metabolic flexibility of Dehalococcoides and could have significant implications for bioremediation strategies at sites where chloroform and chloroethenes coexist as contaminants .

How does the structure of methionyl-tRNA formyltransferase relate to its function and substrate specificity?

The three-dimensional structure of methionyl-tRNA formyltransferase provides critical insights into its function and remarkable substrate specificity. Crystallographic studies of E. coli formyltransferase complexed with formyl-methionyl-tRNA^fMet at 2.8 Å resolution reveal a sophisticated recognition mechanism that explains the enzyme's high selectivity .

The enzyme comprises two domains connected by an elongated linker. The catalytic N-terminal domain (residues 1-189) contains a Rossmann fold and shares structural similarity with glycinamide ribonucleotide transformylase (GARF), which also uses 10-formyltetrahydrofolate as a formyl donor. Distinctive features of formyltransferase include an additional loop (loop 1, residues 34-49) within the catalytic domain and a β-barrel C-terminal domain (residues 209-314) .

In the enzyme-tRNA complex, both domains and the linker interact with the L-shaped tRNA molecule. The acceptor arm is clamped between the C-terminal domain and loop 1, while the β-barrel binds to both the D-arm and the minor groove of the acceptor stem. The 3' extremity of tRNA, carrying the formylated methionyl group, enters the N-terminal domain containing the catalytic site .

The specific recognition of initiator tRNA primarily depends on the C1-A72 mismatch at the top of the acceptor helix. When this mismatch is replaced with standard base pairs (C1-G72 or G1-C72), the catalytic efficiency drops dramatically to 0.04% and 0.01% of wild-type levels, respectively. The importance of loop 1 in this recognition was demonstrated by deletion and mutation experiments. Deletion of residues 38-47 of loop 1 (Δ38-47) or mutation of a single residue (R42A) substantially diminished the enzyme's ability to discriminate between initiator and elongator tRNAs, as shown in the following data :

Values represent catalytic efficiencies (K_cat/K_m) as percentages of those obtained with wild-type tRNA^fMet

These structural insights not only explain the enzyme's specificity but also provide a foundation for understanding how mutations in formyltransferase can lead to physiological consequences in various organisms .

What factors influence the interactions between Dehalococcoides and other microorganisms in mixed consortia?

The interactions between Dehalococcoides and other microorganisms in mixed consortia are influenced by several key factors, particularly those related to the exchange of essential metabolites. In defined consortia consisting of Dehalococcoides mccartyi strain 195 (Dhc195), Desulfovibrio vulgaris Hildenborough (DvH), and Pelosinus fermentans R7 (PfR7), specific metabolic dependencies have been observed .

Hydrogen supply is a critical factor, as Dehalococcoides requires hydrogen as an electron donor for dechlorination. In mixed consortia, DvH can provide this hydrogen through lactate fermentation, establishing a syntrophic relationship .

Corrinoid exchange represents another crucial interaction factor. Dhc195 requires corrinoids for its metabolic activities but cannot synthesize them de novo. In the absence of exogenous cobalamin (vitamin B12), Dhc195 becomes highly dependent on corrinoid-producing organisms like PfR7. In a triculture without added cobalamin (Dhc195/DvH/PfR7(-DMB)), the initiation of dechlorination and Dhc195 cell growth showed a 5-day lag period, tracking with the growth lag of PfR7. This lag was absent in cultures amended with cobalamin, indicating that Dhc195 relied on corrinoids produced by PfR7 only when exogenous cobalamin was unavailable .

The specific corrinoid type also matters. Dhc195 can import and remodel phenolic corrinoids produced by PfR7 into cobalamin in the presence of 5,6-dimethylbenzimidazole (DMB), suggesting a preference for specific corrinoid structures. This remodeling process involves the CbiZ-dependent pathway and requires the BtuFCD corrinoid ABC transporter system .

What alternative formyl donors can be utilized by methionyl-tRNA formyltransferase and what are the implications?

Recent research has expanded our understanding of the potential substrates that can serve as formyl donors for methionyl-tRNA formyltransferase. While 10-formyltetrahydrofolate (10-CHO-THF) is the canonical formyl donor, evidence now indicates that 10-formyldihydrofolate (10-CHO-DHF) can also function as an alternative substrate .

This flexibility in formyl donor utilization has several important implications:

• Metabolic resilience: The ability to use multiple formyl donors may provide bacteria with metabolic flexibility under different growth conditions or folate metabolism states. This could be particularly relevant for organisms in specialized niches, such as Dehalococcoides in contaminated environments.

• Antibiotic sensitivity: The connection between folate metabolism and fmt activity impacts antibiotic sensitivity. Research has shown that FolD-deficient mutants and fmt-overexpressing strains exhibit increased sensitivity to trimethoprim (TMP), an antibiotic that inhibits dihydrofolate reductase. This suggests that perturbing the balance of these pathways affects cellular resistance to folate-targeting antibiotics .

• Potential for targeted interventions: Understanding the alternative pathways for formylation could lead to new approaches for manipulating bacterial metabolism, either to enhance beneficial activities (such as bioremediation by Dehalococcoides) or to develop new antimicrobial strategies.

• Evolutionary implications: The ability to utilize multiple formyl donors might represent an evolutionary adaptation that allows bacteria to maintain translation initiation under varying metabolic conditions, potentially contributing to their survival in changing environments.

The verification of DHF as a by-product in the in vitro assay through LC-MS/MS analysis provides strong evidence for this alternative pathway and opens new avenues for research into folate metabolism and its connection to translation initiation .

How do mutations in methionyl-tRNA formyltransferase affect translation and cellular function?

Mutations in methionyl-tRNA formyltransferase can have profound effects on translation efficiency and cellular function, with implications ranging from bacterial growth inhibition to human disease. The severity of these effects depends on the specific mutation and the organism affected .

In humans, compound heterozygous mutations in the nuclear gene encoding mitochondrial MTF (mt-MTF) have been linked to combined oxidative phosphorylation deficiency and Leigh syndrome, a severe neurological disorder. Specific mutations identified in patients include a combination of a stop codon mutation in one MTF allele and an S209L mutation in the other, dramatically reducing mitochondrial translation efficiency .

The structural basis for how specific mutations affect enzyme function has been partially elucidated through crystallographic studies. For example, in E. coli formyltransferase, mutagenesis of key residues in loop 1 (such as the R42A mutation) significantly reduces the enzyme's ability to discriminate between initiator and elongator tRNAs, allowing inappropriate formylation of non-initiator tRNAs. This demonstrates how subtle changes in enzyme structure can substantially alter substrate specificity and function .

The biochemical characterization of pathogenic mutations provides insights into the structure-function relationships of formyltransferase and helps explain why certain mutations lead to disease. This knowledge is essential for understanding the molecular basis of translation-related disorders and may guide the development of potential therapeutic approaches .

What are the optimal conditions for expressing and purifying recombinant Dehalococcoides methionyl-tRNA formyltransferase?

When expressing and purifying recombinant Dehalococcoides methionyl-tRNA formyltransferase, researchers should consider multiple factors to maximize yield and activity. While specific protocols for Dehalococcoides fmt are not directly mentioned in the search results, we can extrapolate from related research on methionyl-tRNA formyltransferase from other organisms.

For expression, E. coli is typically the preferred host system due to its well-established genetic tools and rapid growth. The fmt gene should be cloned into an expression vector with an inducible promoter (such as T7) and potentially fused with affinity tags (like His-tag or GST) to facilitate purification. Expression conditions should be optimized through a systematic approach testing various parameters:

• Temperature: Lower temperatures (15-25°C) often enhance soluble protein expression compared to standard 37°C

• Induction timing: Mid-log phase (OD600 of 0.6-0.8) is typically optimal

• Inducer concentration: For IPTG, concentrations between 0.1-1 mM should be tested

• Expression duration: 4-16 hours depending on temperature and strain

For purification, a multi-step approach is recommended:

• Initial capture via affinity chromatography (Ni-NTA for His-tagged proteins)

• Ion exchange chromatography to remove contaminants

• Size exclusion chromatography for final polishing and buffer exchange

Throughout purification, maintain reducing conditions (with DTT or β-mercaptoethanol) to prevent oxidation of critical cysteine residues. The final buffer should contain glycerol (10-20%) for stability during storage at -80°C.

Activity of the purified enzyme can be assessed using a formylation assay with Met-tRNA^Met and 10-formyltetrahydrofolate as substrates, followed by thin-layer chromatography or HPLC analysis to detect the formylated product.

What methods are most effective for studying the interactions between Dehalococcoides and other microorganisms in defined consortia?

Studying interactions between Dehalococcoides and other microorganisms in defined consortia requires an integrated approach combining several methodologies. Based on the research with Dehalococcoides mccartyi strain 195 (Dhc195), Desulfovibrio vulgaris Hildenborough (DvH), and Pelosinus fermentans R7 (PfR7), the following methods have proven effective :

• Construction of defined consortia:

  • Begin with pure cultures of each organism

  • Establish co- and tricultures under controlled conditions

  • Test various combinations to identify key interactions

• Growth monitoring and dechlorination analysis:

  • Track cell numbers using quantitative PCR targeting specific genes (e.g., 16S rRNA)

  • Monitor dechlorination activity by measuring substrate depletion and product formation using gas chromatography

  • Compare growth and dechlorination patterns across different consortia compositions

• Metabolite exchange investigation:

  • Test growth with and without specific supplements (e.g., cobalamin, DMB)

  • Analyze corrinoid profiles using HPLC and mass spectrometry

  • Identify specific metabolites that mediate interactions

• Transcriptomic analysis:

  • Perform RNA-seq to identify genes differentially expressed in various consortia

  • Focus on genes involved in metabolite transport, utilization, and regulatory mechanisms

  • Use platforms like GEO (Gene Expression Omnibus) for data storage and sharing

• Time-course experiments:

  • Monitor growth, metabolite production, and gene expression over time

  • Identify temporal dependencies between organisms

  • Determine lag phases and growth kinetics under different conditions

In the study of Dhc195 interactions, these approaches revealed that DvH provided hydrogen while PfR7 supplied corrinoids, with Dhc195 growth and dechlorination activity highly dependent on PfR7 growth in the absence of exogenous cobalamin. Such comprehensive methodologies are essential for unraveling the complex interactions in microbial consortia .

How can researchers effectively measure the activity of methionyl-tRNA formyltransferase in experimental systems?

Measuring methionyl-tRNA formyltransferase activity requires specialized approaches that account for the enzyme's unique substrate requirements and reaction characteristics. Several complementary methods can be employed to effectively assess fmt activity:

• In vitro formylation assays:

  • Prepare methionyl-tRNA^Met substrate using purified methionyl-tRNA synthetase

  • Incubate with purified fmt and 10-formyltetrahydrofolate (or alternative formyl donors)

  • Detect formylated Met-tRNA^Met using:
    a) TLC separation of [³⁵S]-methionine-labeled tRNAs
    b) HPLC analysis with UV detection
    c) Mass spectrometry of the aminoacyl moiety after hydrolysis

• Detection of formylation by-products:

  • Monitor the formation of tetrahydrofolate or dihydrofolate (depending on the formyl donor)

  • Employ LC-MS/MS analysis to verify the identity of folate by-products, as demonstrated in studies of alternative formyl donors

  • Quantify formyl donor consumption through spectrophotometric methods

• Kinetic analysis:

  • Determine Km and kcat values for different substrates

  • Measure catalytic efficiencies (kcat/Km) to assess substrate preferences

  • Compare wild-type enzyme with mutants to identify critical residues

  • The data should be presented in table format similar to the enzyme specificity data shown for different tRNA variants

• In vivo assessment:

  • Use fmt-deficient strains complemented with recombinant fmt variants

  • Measure growth rates as an indirect indicator of translation efficiency

  • Assess antibiotic sensitivity (e.g., to trimethoprim) as fmt activity correlates with sensitivity to certain antibiotics

  • Quantify in vivo formylation rates using metabolic labeling

These methodologies provide complementary data on fmt activity and can be selected based on the specific research question being addressed. Combining multiple approaches provides the most comprehensive assessment of enzyme function.

What genomic approaches help identify and characterize rdh genes in Dehalococcoides strains with unique metabolic capabilities?

The identification and characterization of reductive dehalogenase homologous (rdh) genes in Dehalococcoides strains with unique metabolic capabilities, such as chloroform tolerance, require sophisticated genomic approaches. Based on the work with D. mccartyi strain GEO12, several effective methods can be employed :

• Whole genome sequencing and annotation:

  • Use next-generation sequencing platforms (Illumina, PacBio, or Oxford Nanopore)

  • Assemble the genome using specialized software for bacterial genomes

  • Identify rdh genes through similarity searches against known reductive dehalogenases

  • Annotate the genome to identify all potential rdh genes and their associated regulatory elements

• Comparative genomics:

  • Compare genomes of strains with different metabolic capabilities (e.g., chloroform-tolerant vs. sensitive)

  • Identify unique genetic elements that may contribute to specific capabilities

  • Analyze the genomic context of rdh genes to identify potential regulatory networks

• Transcriptomic profiling:

  • Perform RNA-seq under different conditions (e.g., presence vs. absence of chloroform)

  • Quantify transcription levels of rdh genes in response to specific conditions

  • Express results as transcripts per defined number of 16S rRNA (e.g., per 10^4 16S rRNA) with appropriate statistical analysis including confidence intervals

  • Compare transcriptional responses across multiple strains with different metabolic capabilities

• Adapting strains to selective pressures:

  • Perform successive transfers under increasing concentrations of selective agents (e.g., chloroform)

  • Monitor changes in rdh gene expression and function during adaptation

  • Sequence the genomes of adapted strains to identify potential mutations

• Functional characterization:

  • Clone and express identified rdh genes in heterologous hosts

  • Characterize substrate specificity and kinetic properties of the encoded enzymes

  • Correlate enzyme properties with strain-specific metabolic capabilities

These approaches, when integrated, provide a comprehensive understanding of the genetic basis for unique metabolic capabilities in Dehalococcoides strains. For instance, the analysis of D. mccartyi strain GEO12 identified seven rdh genes, including tceA and vcrA, with tceA showing significantly enhanced transcription in the presence of chloroform in the adapted strain GEO12CF .

How can researchers manipulate corrinoid pathways to enhance Dehalococcoides growth and dechlorination activity?

Manipulating corrinoid pathways to enhance Dehalococcoides growth and dechlorination activity represents a promising approach for improving bioremediation outcomes. Based on research into corrinoid-related interactions, several strategic approaches can be implemented :

• Supplement with exogenous corrinoids:

  • Add cobalamin (vitamin B12) directly to Dehalococcoides cultures

  • Test different concentrations to determine optimal supplementation levels

  • Consider the timing of supplementation relative to growth phase

• Co-culture with corrinoid-producing microorganisms:

  • Design defined consortia with efficient corrinoid producers like Pelosinus fermentans

  • Optimize the ratio of Dehalococcoides to corrinoid producers

  • Select corrinoid producers based on the specific phenolic corrinoids they generate

• Provide lower bases for corrinoid remodeling:

  • Add 5,6-dimethylbenzimidazole (DMB) to enable Dehalococcoides to remodel phenolic corrinoids into cobalamin

  • Optimize DMB concentration based on the specific strain and consortium composition

  • Consider timing of DMB addition relative to growth phases

• Enhance corrinoid transport and processing:

  • Target genetic modifications to upregulate corrinoid transporters (e.g., BtuFCD)

  • Enhance expression of the CbiZ-dependent corrinoid-remodeling pathway

  • Consider modifications to alternative transporters like the DET1174-DET1176 operon that responds to exogenous cobalamin

• Optimize electron donor supply:

  • Ensure sufficient hydrogen is available, either through direct addition or via syntrophic hydrogen producers like Desulfovibrio

  • Balance electron donor supply with corrinoid availability for maximum dechlorination efficiency

• Monitor key indicators of pathway effectiveness:

  • Track dechlorination products and ratios (e.g., ethene-to-vinyl chloride ratio)

  • Measure Dehalococcoides cell numbers using quantitative PCR

  • Perform transcriptomic analysis to confirm upregulation of desired pathways

By implementing these approaches, researchers can potentially overcome the corrinoid limitations that often constrain Dehalococcoides growth and activity in both laboratory cultures and field applications. The success of such manipulations should be evaluated through comprehensive monitoring of growth kinetics, dechlorination rates, and end-product formation .