Recombinant Treponema denticola Methionine--tRNA ligase (metG), partial
Description
Introduction and Background
Methionyl-tRNA synthetase (MetRS), encoded by the metG gene, is an essential enzyme in Treponema denticola responsible for charging methionine to its cognate tRNA. This process is critical for translation initiation and elongation. The recombinant partial form of T. denticola MetRS (metG) has been studied for its structural, functional, and pathological roles in bacterial physiology and antibiotic resistance. Below is a detailed analysis of its molecular characteristics, functional roles, and research implications.
Aminoacylation and Proofreading
MetRS performs two critical functions:
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Aminoacylation: Charges methionine to tRNA<sup>Met</sup>, enabling translation initiation and elongation .
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Proofreading: Prevents misacylation by rejecting non-cognate substrates (e.g., homocysteine) .
Mutations in metG (e.g., substitutions at methionine- or tRNA-binding sites) reduce proofreading efficiency, leading to elevated homocysteine sensitivity and altered tRNA pools. These changes trigger stringent response pathways (via RelA) in T. denticola, increasing antibiotic tolerance .
Antibiotic Tolerance and Persistence
Mutations in metG (e.g., frameshift or substitutions) are linked to:
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Reduced Growth Rates: Slowed bacterial proliferation under stress .
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Increased Persistence: Enhanced survival during antibiotic challenge (e.g., meropenem) due to disrupted tRNA metabolism and stringent response activation .
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Cross-Tolerance: Tolerance extends to multiple antibiotics, complicating treatment .
Pathogenic Implications
In T. denticola, metG mutations may contribute to:
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Chronic Infection: By enabling survival under antibiotic pressure, thereby prolonging periodontal disease progression .
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Biofilm Formation: Altered tRNA pools may modulate virulence factor expression (e.g., Msp or CTLP proteins) .
Figure 1: Schematic of MetRS Domains
(Note: Full-length MetRS includes a C-terminal dimerization domain; the partial recombinant lacks this region.)
Drug Target Development
The metG enzyme is a potential therapeutic target. Mutations identified in T. denticola (e.g., binding-site alterations) could guide the design of inhibitors that block MetRS activity without inducing stringent responses .
Biomarker for Antibiotic Tolerance
metG mutations serve as markers for emerging antibiotic persistence in pathogens. For example, phage-plasmid-borne metG copies in Shigella sonnei correlate with cephalosporin resistance evolution .
Product Specs
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Target Background
KEGG: tde:TDE1063
STRING: 243275.TDE1063
Q&A
What is Treponema denticola and why is it significant in periodontal research?
Treponema denticola is a small anaerobic spirochete frequently isolated from periodontal lesions and strongly associated with periodontal diseases. It represents one of the keystone pathogens contributing to the dysregulation of tissue homeostatic processes that leads to the breakdown of tissue and bone supporting teeth . The bacterium possesses various virulence factors, including the Major Surface Protein (MSP), which stimulates the secretion of pro-inflammatory cytokines and chemokines, activating host-mediated destructive processes observed during periodontitis .
What is Methionine-tRNA ligase (metG) and what role does it play in T. denticola?
Methionine-tRNA ligase (metG) is an essential aminoacyl-tRNA synthetase responsible for attaching methionine to its cognate tRNA during protein synthesis. While the search results don't specifically address T. denticola metG, this enzyme would be critical for protein biosynthesis in this organism, especially considering the specialized metabolism of this anaerobic spirochete in periodontal environments.
How does recombinant expression of T. denticola proteins typically work?
Based on successful expression of other T. denticola proteins, such as oligopeptidase B (OpdB), recombinant expression typically involves cloning PCR products into expression vectors like pET30b and transforming them into E. coli expression strains such as BL21(DE3)/pLysS . This approach has been shown to produce enzymatically active proteins with characteristics similar to the native T. denticola proteins.
What are the optimal conditions for heterologous expression of T. denticola proteins?
For T. denticola proteins, E. coli-based expression systems with vectors containing strong inducible promoters have proven successful. Based on experimental data with OpdB peptidase, the pET30b vector system in E. coli BL21(DE3)/pLysS has demonstrated effectiveness in producing functionally active proteins . For anaerobic spirochete proteins, optimization of induction conditions including temperature, inducer concentration, and duration is typically necessary.
What challenges might researchers encounter when expressing recombinant T. denticola metG?
Expression of T. denticola proteins faces several challenges:
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Restriction-modification (R-M) barriers: T. denticola strains possess Type I and Type II R-M systems that can significantly reduce transformation efficiency .
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Codon usage differences: The spirochete's distinct codon usage pattern may not be optimal for expression in laboratory hosts like E. coli.
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Protein folding issues: Large, multi-domain proteins like aminoacyl-tRNA synthetases may experience folding challenges in heterologous hosts.
How can researchers overcome restriction-modification barriers in T. denticola?
Recent research has developed SyngenicDNA-based plasmids that lack restriction-modification target motifs, enabling enhanced transformation efficiency in T. denticola. This approach involves:
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Characterizing the methylome to identify R-M systems present in specific T. denticola strains
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Designing plasmids that avoid restricted motifs
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Accounting for phase-varying Type I systems found in T. denticola strains
This strategy has successfully improved both transformation efficiency and enabled the first high-efficiency transposon mutagenesis of T. denticola using an RM-silent, codon-optimized system .
What methods are recommended for validating the enzymatic activity of recombinant T. denticola metG?
While specific protocols for T. denticola metG are not mentioned in the search results, verification of recombinant enzyme activity typically includes:
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Aminoacylation assays measuring the attachment of methionine to tRNA
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ATP-PPi exchange assays to verify methionine activation
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Comparisons of kinetic parameters with other bacterial methionine-tRNA ligases
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Complementation assays in relevant genetic systems
The approach used for validating OpdB peptidase activity, where enzymatic activities in E. coli containing T. denticola opdB showed BANA-peptidase activity similar to that of T. denticola, provides a potential model .
How might researchers investigate structure-function relationships in T. denticola metG?
Structure-function studies would typically involve:
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Sequence alignments with characterized methionine-tRNA ligases to identify conserved catalytic residues
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Site-directed mutagenesis targeting key functional domains
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Truncation analyses to determine minimal functional units
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Co-crystallization with substrates or substrate analogs
The transposon mutagenesis system developed for T. denticola using RM-silent, codon-optimized, himarC9 transposase-based plasmids could be adapted for metG structural studies .
How can researchers effectively design mutant studies in T. denticola?
Based on recent advances, researchers can:
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Utilize SyngenicDNA approaches that optimize transformation efficiency
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Apply the newly reported high-efficiency transposon mutagenesis system using an RM-silent, codon-optimized, himarC9 transposase-based plasmid
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Design complementation studies using optimized shuttle plasmids, as demonstrated with the T. denticola ΔfhbB mutant strain
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Account for the specific R-M systems present in the target T. denticola strain
The table below summarizes major genetic tools now available for T. denticola manipulation:
What detection methods can be used to identify T. denticola in complex samples?
Recent methodological advances include:
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Electron microscopy (Hitachi TM4000) for rapid detection of Treponema in clinical specimens
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Passive and mechanical filtration for cultivation
This combined approach has successfully detected and cultured T. denticola from oral samples, with 15 out of 44 oral samples (34%) testing positive for Treponema using electron microscopy and culture methods .
How can metagenomic approaches contribute to T. denticola research?
Metagenomic analysis has successfully detected T. denticola in various tissue samples, including ancient human remains. For example, researchers mapped metagenomic sequences against the T. denticola genome and identified reads specific to this opportunistic pathogen in Iceman tissue biopsies . The DNA damage pattern of these mapped reads suggested an ancient origin, demonstrating the potential of metagenomics to detect disease-associated microorganisms in historical samples.
How does T. denticola interact with the host immune system?
T. denticola interactions with host immunity involve several mechanisms:
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The Major Surface Protein (MSP) stimulates the synthesis of pro-inflammatory mediators in primary human monocytes, including Tumor necrosis factor-alpha (TNF-α), Interleukin 1-β (IL-1β), Interleukin-6 (IL-6), and Matrix metallo-Proteinase-9 (MMP-9) in a dose- and time-dependent manner .
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The outer membrane of T. denticola (OMT) induces apoptosis and heat shock protein expression (HO-1 and Hsp70) in endothelial cells, suggesting it can damage endothelium integrity .
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Bacterial motility and cell shape affect interactions with phagocytes - mutant strains lacking motility (FlgE-deficient) or exhibiting filamentation (CfpA-deficient) show increased uptake by macrophages compared to wild-type strains .
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Opsonization with specific antibodies significantly improves treponeme uptake by phagocytes .
What role might metG play in T. denticola virulence or survival?
While metG is primarily a housekeeping enzyme essential for protein synthesis, its role in virulence might include:
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Supporting bacterial growth and adaptation in the periodontal environment
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Contributing to stress responses through regulation of protein synthesis during host interaction
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Potentially presenting unique features that could be targeted for antimicrobial development
The importance of proper protein synthesis for expression of virulence factors like the Major Surface Protein suggests metG function is indirectly critical for pathogenesis.
What emerging genetic tools might advance T. denticola metG research?
Recent developments in T. denticola genetic manipulation include:
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Methylome-based characterization of R-M systems, including phase-varying Type I systems
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SyngenicDNA-based R-M-silent plasmid systems that overcome transformation barriers
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High-efficiency transposon mutagenesis using RM-silent, codon-optimized systems
These tools will facilitate more sophisticated genetic analysis of T. denticola, potentially enabling conditional expression systems or gene knockdowns to study essential genes like metG.
How might structural studies of T. denticola metG inform antimicrobial development?
Structural characterization of T. denticola metG could reveal:
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Unique binding pocket features that differ from human methionine-tRNA synthetases
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Potential allosteric sites for selective inhibition
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Differences in substrate recognition that could be exploited for antimicrobial design
Such insights could contribute to developing narrow-spectrum antimicrobials that target periodontal pathogens while preserving beneficial oral microbiota.
