Recombinant Legionella pneumophila tRNA uridine 5-carboxymethylaminomethyl modification enzyme MnmG (mnmG), partial

What is the MnmG enzyme and what role does it play in bacterial physiology?

The MnmG enzyme (also known as tRNA uridine 5-carboxymethylaminomethyl modification enzyme) is responsible for tRNA modification that reduces frameshift errors in translation. It functions as a 5-carboxymethylaminomethyluridine-tRNA synthase that helps maintain protein synthesis quality, particularly under stress conditions . The enzyme contains a main catalytic domain with nucleotide binding sites (typically before amino acid position 370) and a flexibly linked independent domain (approximately spanning amino acids 479-629) .

MnmG's primary function is to consolidate protein synthesis quality by ensuring proper tRNA modifications, which facilitates bacterial stress responses. This becomes particularly important when bacteria face environmental challenges such as antibiotic exposure, as these modifications help maintain translational fidelity despite cellular stress .

How does MnmG contribute to antibiotic resistance in bacteria?

MnmG contributes to antibiotic resistance through a multi-stage model of bacterial adaptation:

• Early stage response: MnmG-mediated tRNA modifications help maintain translation quality during initial antibiotic exposure, providing prompt protection before other resistance mechanisms develop .

• Oxidative stress management: Many antibiotics generate reactive oxygen species (ROS), and MnmG's tRNA modifications appear to counteract ROS-induced damage, allowing bacteria to survive the initial oxidative stress .

• Selective applicability: This resistance mechanism applies specifically to ROS-generating antibiotics. For example, studies have shown that polymyxin B, which does not generate ROS at sub-MIC concentration, cannot be resisted through this mechanism .

This positions MnmG as part of a general defense mechanism rather than a specific resistance factor, explaining why targeting bacterial DNA recombination systems and tRNA could potentially retard spontaneous drug resistance .

What are the optimal methods for expressing and purifying recombinant MnmG from Legionella pneumophila?

When working with recombinant Legionella pneumophila MnmG, researchers should consider the following methodological approaches:

Expression Systems:

• Bacterial expression systems (particularly E. coli) with temperature-inducible promoters are commonly used

• Expression vectors containing His-tags facilitate subsequent purification steps

• Codon optimization may be necessary due to differences between Legionella and expression host codon usage

Purification Strategy:

• Affinity chromatography using nickel or cobalt resins for His-tagged proteins

• Size exclusion chromatography to remove aggregates and achieve higher purity

• Ion exchange chromatography as a final polishing step

Activity Preservation:

• Addition of reducing agents (DTT or β-mercaptoethanol) to preserve enzymatic activity

• Inclusion of nucleotide cofactors during purification may enhance stability

• Storage in glycerol-containing buffers at -80°C to maintain long-term activity

How can researchers assess MnmG activity in experimental settings?

Researchers can assess MnmG activity through several complementary approaches:

In vitro Enzymatic Assays:

• Substrate conversion assays using purified tRNAs and monitoring modified nucleoside formation

• Coupled enzyme assays measuring co-substrate consumption or product formation

• Spectrophotometric measurement of enzymatic reactions using appropriate chromogenic substrates

Structural and Functional Analysis:

• Mutation analysis of key residues (particularly in the nucleotide binding sites before 370 aa in the main domain)

• Assessment of truncation effects (such as the 497-629 aa region in the flexibly linked domain)

• Complementation studies in MnmG-deficient bacterial strains

Table: Comparative Methods for MnmG Activity Assessment

How do structural variations in the genome influence MnmG expression and function?

Structural variations (SVs) in the bacterial genome significantly impact MnmG expression and function, particularly under antibiotic stress conditions:

• Preferential SV occurrence near tRNA genes: Research has demonstrated that SVs occur significantly more frequently near tRNA genes than would be expected by random distribution. In one study examining E. coli under ciprofloxacin stress, 63 out of 341 SVs detected in the first 7 generations were located near tRNA genes (±3,000 nt), representing a highly significant enrichment (P = 4.74 × 10^-35, Fisher Exact Test) .

• Validation by multiple sequencing technologies: This preferential distribution was confirmed using both short-read sequencing (MGISEQ-2000) and long-read Nanopore sequencing, which identified 57 SV events near tRNA genes out of 1408 total events (P = 8.97 × 10^-6, Fisher exact test) .

• Direct impact on MnmG: A specific deletion between positions 3,916,925 and 3,917,502 was detected after 21 generations of antibiotic exposure, resulting in truncation of the mnmG gene. Remarkably, this truncation led to elevated expression of the gene rather than loss of function .

• Functional consequences of MnmG truncation: The observed truncation removed amino acids 497-629, affecting the flexibly linked independent domain while preserving the main catalytic domain (containing nucleotide binding sites before amino acid 370). This likely maintained catalytic function while potentially altering regulation .

These findings suggest that genomic structural variations, particularly those occurring under stress conditions, can strategically modify tRNA processing systems to enhance bacterial survival.

What role does homologous recombination play in MnmG evolution in Legionella pneumophila?

Homologous recombination serves as a major driver of genetic diversity and adaptation in Legionella pneumophila, with profound implications for genes like mnmG:

• Extensive contribution to genetic diversity: Recombination accounts for an extraordinarily high proportion (>96%) of diversity within several major disease-associated sequence types (STs) of L. pneumophila . This suggests it represents a potentially important force shaping adaptation and virulence.

• Source of recombined DNA: Genomic analysis shows that L. pneumophila isolates most frequently import DNA from isolates belonging to their own clade, but also occasionally from other major clades of the same subspecies . This provides opportunities for horizontal exchange of adaptations between different lineages.

• Subspecies recombination barriers: Acquisition of recombined regions from another subspecies (L. pneumophila subsp. fraseri) is rarely observed, suggesting the existence of a recombination barrier and/or the possibility of ongoing speciation between subspecies .

• Multi-fragment recombination mechanism: Evidence suggests that L. pneumophila may undergo multi-fragment recombination, whereby multiple non-contiguous segments originating from the same donor DNA molecule are imported into a recipient genome during a single recombination event . This could allow coordinated acquisition of functionally related genes or regulatory elements.

These recombination dynamics provide a mechanism for the rapid adaptation of L. pneumophila to new environments and stresses, potentially including modifications to tRNA processing enzymes like MnmG.

How might understanding MnmG function contribute to new diagnostic approaches for Legionella pneumonia?

While MnmG itself is not currently a primary diagnostic target, understanding its role in Legionella pneumophila might inform novel diagnostic approaches:

• Potential as a molecular marker: As a conserved enzyme with specific sequence variations between Legionella strains, mnmG could potentially serve as an additional target in molecular diagnostic panels, particularly those using nucleic acid amplification techniques with sensitivity ranging from 17-100% .

• Context within current diagnostic landscape: Current Legionella pneumonia diagnosis relies on multiple methods, each with specific advantages and limitations:

(Adapted from information in source )

• Integration with metagenomic approaches: Next-generation sequencing approaches (mNGS) could potentially incorporate analysis of tRNA modification genes like mnmG as part of comprehensive pathogen identification and characterization, offering simultaneous identification of co-infecting pathogens within 48 hours .

How does MnmG contribute to Legionella pneumophila pathogenesis?

The relationship between MnmG and Legionella pneumophila pathogenesis involves several interconnected mechanisms:

• Translation quality control: By ensuring accurate translation through proper tRNA modifications, MnmG helps L. pneumophila maintain the protein synthesis fidelity necessary for virulence factor expression, particularly under stress conditions encountered during infection .

• Stress adaptation: During infection, L. pneumophila encounters various stresses, including oxidative stress from host immune responses. MnmG-mediated tRNA modifications likely contribute to bacterial adaptation to these stresses, similar to observations in other bacteria exposed to antibiotics .

• Evolutionary context: Given that recombination represents a significant force shaping adaptation and virulence in L. pneumophila (accounting for >96% of diversity within major disease-associated sequence types) , variations in MnmG function resulting from recombination events may contribute to differences in virulence between strains.

• Potential role in persistence: The multi-stage adaptation model observed in antibiotic resistance (early tRNA-mediated responses followed by more specific adaptations) may have parallels in host adaptation, with MnmG potentially contributing to the early persistence of Legionella during establishment of infection.

What experimental approaches would best elucidate the relationship between MnmG activity and bacterial stress responses?

To thoroughly investigate the relationship between MnmG activity and bacterial stress responses, researchers should consider these advanced experimental approaches:

• Comparative transcriptomics/proteomics under stress conditions:

  • RNA-seq analysis of wild-type vs. mnmG-deficient strains under various stresses

  • Quantitative proteomics to identify proteins differentially expressed

  • Ribosome profiling to examine translation efficiency and accuracy changes

• tRNA modification profiling:

  • Liquid chromatography-mass spectrometry (LC-MS) to quantify changes in tRNA modifications

  • High-throughput sequencing of tRNA populations (tRNA-seq)

  • Correlation of modification levels with stress intensity and duration

• In vivo infection models:

  • Comparison of wild-type and mnmG-mutant strains in appropriate infection models

  • Assessment of bacterial survival, replication, and virulence

  • Host response characterization (immunological and transcriptional)

• Structural biology approaches:

  • Cryo-EM or X-ray crystallography of MnmG under different conditions

  • Molecular dynamics simulations to understand conformational changes

  • Structure-guided mutagenesis to identify functionally critical residues

• Time-resolved studies of adaptation:

  • Longitudinal sampling during stress exposure to track the temporal sequence of:

    • Early stage: Recombination → tRNA up-regulation → translational regulation

    • Medium stage: Up-regulation of efflux pumps

    • Late stage: Resistant mutations

How might targeting MnmG function serve as a strategy for developing novel antimicrobials?

The potential of MnmG as an antimicrobial target offers several strategic advantages and considerations:

• Disruption of early resistance mechanisms:

  • Evidence suggests tRNA up-regulation provides prompt protection at the early stage of antibiotic exposure, before upregulating efflux pumps and evolving resistance mutations

  • By targeting MnmG, it may be possible to block this early protective response, potentially making bacteria more vulnerable to existing antibiotics

• Synergistic approaches:

  • Combining MnmG inhibitors with conventional antibiotics might prevent the development of resistance

  • Targeting both DNA recombination systems and tRNA modification could more effectively retard spontaneous drug resistance

• Specificity considerations:

  • MnmG inhibitors would likely be most effective against infections involving ROS-generating antibiotics

  • Limited effectiveness expected against non-ROS-generating antimicrobials like polymyxin B

• Potential structural vulnerabilities:

  • The distinct domains of MnmG (main catalytic domain with nucleotide binding sites and flexibly linked independent domain) offer multiple potential binding sites for inhibitor development

  • Structure-based drug design could target the nucleotide binding region critical for catalytic function

• Comparative screening approaches:

  • High-throughput screening of compound libraries against MnmG from multiple bacterial pathogens

  • Identification of broad-spectrum vs. species-specific inhibitors

  • Assessment of resistance development frequency compared to conventional antibiotics

This approach represents a departure from traditional antibiotic targets, potentially addressing the urgent need for novel strategies to combat antimicrobial resistance in clinically important pathogens like Legionella pneumophila.