Evolutionary repair reveals an unexpected role of the tRNA modification m1G37 in aminoacylation

The tRNA modification m1G37, which is introduced by the tRNA methyltransferase TrmD, is thought to be essential for growth in bacteria due to its role in suppressing translational frameshift errors at proline codons. However, because bacteria can tolerate high levels of mistranslation, it is unclear why loss of m1G37 is not tolerated. Here, we addressed this question by performing experimental evolution of trmD mutant strains of E. coli. Surprisingly, trmD mutant strains were viable even if the m1G37 modification was completely abolished, and showed rapid recovery of growth rate, mainly via tandem duplication or coding mutations in the proline-tRNA ligase gene proS. Growth assays and in vitro aminoacylation assays showed that G37-unmodified tRNAPro is aminoacylated less efficiently than m1G37-modified tRNAPro, and that growth of trmD mutant strains can be largely restored by single mutations in proS that restore aminoacylation of G37-unmodified tRNAPro. These results show that inefficient aminoacylation of tRNAPro is the main reason for growth defects observed in trmD mutant strains and that the ProRS enzyme may act as a gatekeeper of translational accuracy, preventing the use of error-prone unmodified tRNAPro in protein translation. Our work shows the utility of experimental evolution for uncovering the hidden functions of essential genes and has implications for the development of antibiotics targeting TrmD.

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Fragment-based discovery of a new class of inhibitors targeting mycobacterial tRNA modification

Nucleic Acids Research ◽

10.1093/nar/gkaa539 ◽

2020 ◽

Vol 48 (14) ◽

pp. 8099-8112 ◽

Cited By ~ 1

Author(s):

Sherine E Thomas ◽

Andrew J Whitehouse ◽

Karen Brown ◽

Sophie Burbaud ◽

Juan M Belardinelli ◽

...

Keyword(s):

Rapid Development ◽

Protein Translation ◽

Mycobacterium Abscessus ◽

Attractive Potential ◽

Trna Modification ◽

Reading Frame ◽

New Class ◽

Translational Frameshift ◽

New Antibiotics

Abstract Translational frameshift errors are often deleterious to the synthesis of functional proteins and could therefore be promoted therapeutically to kill bacteria. TrmD (tRNA-(N(1)G37) methyltransferase) is an essential tRNA modification enzyme in bacteria that prevents +1 errors in the reading frame during protein translation and represents an attractive potential target for the development of new antibiotics. Here, we describe the application of a structure-guided fragment-based drug discovery approach to the design of a new class of inhibitors against TrmD in Mycobacterium abscessus. Fragment library screening, followed by structure-guided chemical elaboration of hits, led to the rapid development of drug-like molecules with potent in vitro TrmD inhibitory activity. Several of these compounds exhibit activity against planktonic M. abscessus and M. tuberculosis as well as against intracellular M. abscessus and M. leprae, indicating their potential as the basis for a novel class of broad-spectrum mycobacterial drugs.

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Fragment-based discovery of a new class of inhibitors targeting mycobacterial tRNA modification

10.1101/564013 ◽

2019 ◽

Author(s):

Sherine E. Thomas ◽

Andrew J. Whitehouse ◽

Karen Brown ◽

Juan M. Belardinelli ◽

Ramanuj Lahiri ◽

...

Keyword(s):

Bacterial Infections ◽

Protein Translation ◽

Mycobacterium Abscessus ◽

Trna Modification ◽

Macrophage Infection ◽

Reading Frame ◽

New Class ◽

Translational Frameshift ◽

Fragment Library

AbstractTranslational frameshift errors are often deleterious to the synthesis of functional proteins as they lead to the production of truncated or inactive proteins. TrmD (tRNA-(N(1)G37) methyltransferase) is an essential tRNA modification enzyme in bacteria that prevents +1 errors in the reading frame during protein translation and has been identified as a therapeutic target for several bacterial infections. Here we validate TrmD as a target inMycobacterium abscessusand describe the application of a structure-guided fragment-based drug discovery approach for the design of a new class of inhibitors against this enzyme. A fragment library screening followed by structure-guided chemical elaboration of hits led to the development of compounds with potentin vitroTrmD inhibitory activity. Several of these compounds exhibit activity against planktonicM. abscessus and Mycobacterium tuberculosis.The compounds were further active in macrophage infection models againstMycobacterium lepraeandM. abscessussuggesting the potential for novel broad-spectrum mycobacterial drugs.

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Bacterial tRNA 2′-O-methylation is dynamically regulated under stress conditions and modulates innate immune response

Nucleic Acids Research ◽

10.1093/nar/gkaa1123 ◽

2020 ◽

Vol 48 (22) ◽

pp. 12833-12844

Author(s):

Adeline Galvanin ◽

Lea-Marie Vogt ◽

Antonia Grober ◽

Isabel Freund ◽

Lilia Ayadi ◽

...

Keyword(s):

Gene Expression Regulation ◽

Current Knowledge ◽

Protein Translation ◽

Stress Conditions ◽

Transcriptional Level ◽

Trna Modification ◽

Rna Modifications ◽

Swarming Motility ◽

E Coli ◽

Response To Stress

Abstract RNA modifications are a well-recognized way of gene expression regulation at the post-transcriptional level. Despite the importance of this level of regulation, current knowledge on modulation of tRNA modification status in response to stress conditions is far from being complete. While it is widely accepted that tRNA modifications are rather dynamic, such variations are mostly assessed in terms of total tRNA, with only a few instances where changes could be traced to single isoacceptor species. Using Escherichia coli as a model system, we explored stress-induced modulation of 2′-O-methylations in tRNAs by RiboMethSeq. This analysis and orthogonal analytical measurements by LC-MS show substantial, but not uniform, increase of the Gm18 level in selected tRNAs under mild bacteriostatic antibiotic stress, while other Nm modifications remain relatively constant. The absence of Gm18 modification in tRNAs leads to moderate alterations in E. coli mRNA transcriptome, but does not affect polysomal association of mRNAs. Interestingly, the subset of motility/chemiotaxis genes is significantly overexpressed in ΔTrmH mutant, this corroborates with increased swarming motility of the mutant strain. The stress-induced increase of tRNA Gm18 level, in turn, reduced immunostimulation properties of bacterial tRNAs, which is concordant with the previous observation that Gm18 is a suppressor of Toll-like receptor 7 (TLR7)-mediated interferon release. This documents an effect of stress induced modulation of tRNA modification that acts outside protein translation.

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Molecular basis for t6A modification in human mitochondria

Nucleic Acids Research ◽

10.1093/nar/gkaa093 ◽

2020 ◽

Vol 48 (6) ◽

pp. 3181-3194 ◽

Cited By ~ 2

Author(s):

Jing-Bo Zhou ◽

Yong Wang ◽

Qi-Yu Zeng ◽

Shi-Xin Meng ◽

En-Duo Wang ◽

...

Keyword(s):

Base Pair ◽

Molecular Basis ◽

Protein Modification ◽

Trna Modification ◽

Anticodon Loop ◽

Recognition Mechanism ◽

Trna Recognition ◽

Translational Accuracy

Abstract N 6-Threonylcarbamoyladenosine (t6A) is a universal tRNA modification essential for translational accuracy and fidelity. In human mitochondria, YrdC synthesises an l-threonylcarbamoyl adenylate (TC-AMP) intermediate, and OSGEPL1 transfers the TC-moiety to five tRNAs, including human mitochondrial tRNAThr (hmtRNAThr). Mutation of hmtRNAs, YrdC and OSGEPL1, affecting efficient t6A modification, has been implicated in various human diseases. However, little is known about the tRNA recognition mechanism in t6A formation in human mitochondria. Herein, we showed that OSGEPL1 is a monomer and is unique in utilising C34 as an anti-determinant by studying the contributions of individual bases in the anticodon loop of hmtRNAThr to t6A modification. OSGEPL1 activity was greatly enhanced by introducing G38A in hmtRNAIle or the A28:U42 base pair in a chimeric tRNA containing the anticodon stem of hmtRNASer(AGY), suggesting that sequences of specific hmtRNAs are fine-tuned for different modification levels. Moreover, using purified OSGEPL1, we identified multiple acetylation sites, and OSGEPL1 activity was readily affected by acetylation via multiple mechanisms in vitro and in vivo. Collectively, we systematically elucidated the nucleotide requirement in the anticodon loop of hmtRNAs, and revealed mechanisms involving tRNA sequence optimisation and post-translational protein modification that determine t6A modification levels.

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Advantage of the F2:A1:B- IncF Pandemic Plasmid over IncC Plasmids in In Vitro Acquisition and Evolution of blaCTX-M Gene-Bearing Plasmids in Escherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01130-19 ◽

2019 ◽

Vol 63 (10) ◽

Cited By ~ 5

Author(s):

Anne-Claire Mahérault ◽

Harry Kemble ◽

Mélanie Magnan ◽

Benoit Gachet ◽

David Roche ◽

...

Keyword(s):

Escherichia Coli ◽

Experimental Evolution ◽

Negative Impact ◽

Fitness Cost ◽

M Gene ◽

Content Type ◽

E Coli ◽

Conjugative Plasmids ◽

K 12

ABSTRACT Despite a fitness cost imposed on bacterial hosts, large conjugative plasmids play a key role in the diffusion of resistance determinants, such as CTX-M extended-spectrum β-lactamases. Among the large conjugative plasmids, IncF plasmids are the most predominant group, and an F2:A1:B- IncF-type plasmid encoding a CTX-M-15 variant was recently described as being strongly associated with the emerging worldwide Escherichia coli sequence type 131 (ST131)-O25b:H4 H30Rx/C2 sublineage. In this context, we investigated the fitness cost of narrow-range F-type plasmids, including the F2:A1:B- IncF-type CTX-M-15 plasmid, and of broad-range C-type plasmids in the K-12-like J53-2 E. coli strain. Although all plasmids imposed a significant fitness cost to the bacterial host immediately after conjugation, we show, using an experimental-evolution approach, that a negative impact on the fitness of the host strain was maintained throughout 1,120 generations with the IncC-IncR plasmid, regardless of the presence or absence of cefotaxime, in contrast to the F2:A1:B- IncF plasmid, whose cost was alleviated. Many chromosomal and plasmid rearrangements were detected after conjugation in transconjugants carrying the IncC plasmids but not in transconjugants carrying the F2:A1:B- IncF plasmid, except for insertion sequence (IS) mobilization from the fliM gene leading to the restoration of motility of the recipient strains. Only a few mutations occurred on the chromosome of each transconjugant throughout the experimental-evolution assay. Our findings indicate that the F2:A1:B- IncF CTX-M-15 plasmid is well adapted to the E. coli strain studied, contrary to the IncC-IncR CTX-M-15 plasmid, and that such plasmid-host adaptation could participate in the evolutionary success of the CTX-M-15-producing pandemic E. coli ST131-O25b:H4 lineage.

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Antimicrobial Drug Resistance Genes Do Not Convey a Secondary Fitness Advantage to Calf-Adapted Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.72.1.443-448.2006 ◽

2006 ◽

Vol 72 (1) ◽

pp. 443-448 ◽

Cited By ~ 25

Author(s):

Artashes R. Khachatryan ◽

Dale D. Hancock ◽

Thomas E. Besser ◽

Douglas R. Call

Keyword(s):

Antimicrobial Resistance ◽

Resistance Genes ◽

Null Mutant ◽

Antimicrobial Drug Resistance ◽

E Coli ◽

Fitness Advantage ◽

Antimicrobial Resistance Genes ◽

Mutant Strains

ABSTRACT Maintenance of antimicrobial drug resistance in bacteria can be influenced by factors unrelated to direct selection pressure such as close linkage to other selectively advantageous genes and secondary advantage conveyed by antimicrobial resistance genes in the absence of drug selection. Our previous trials at a dairy showed that the maintenance of the antimicrobial resistance genes is not influenced by specific antimicrobial selection and that the most prevalent antimicrobial resistance phenotype of Escherichia coli is specifically selected for in young calves. In this paper we examine the role of secondary advantages conveyed by antimicrobial resistance genes. We tested antimicrobial-susceptible null mutant strains for their ability to compete with their progenitor strains in vitro and in vivo. The null mutant strains were generated by selection for spontaneous loss of resistance genes in broth supplemented with fusaric acid or nickel chloride. On average, the null mutant strains were as competitive as the progenitor strains in vitro and in newborn calves (in vivo). Inoculation of newborn calves at the dairy with antimicrobial-susceptible strains of E. coli did not impact the prevalence of antimicrobial-resistant E. coli. Our results demonstrate that the antimicrobial resistance genes are not responsible for the greater fitness advantage of antimicrobial-resistant E. coli in calves, but the farm environment and the diet clearly exert critical selective pressures responsible for the maintenance of antimicrobial resistance genes. Our current hypothesis is that the antimicrobial resistance genes are linked to other genes responsible for differential fitness in dairy calves.

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The Ribosome-Bound Chaperones RAC and Ssb1/2p Are Required for Accurate Translation in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.24.20.9186-9197.2004 ◽

2004 ◽

Vol 24 (20) ◽

pp. 9186-9197 ◽

Cited By ~ 68

Author(s):

Magdalena Rakwalska ◽

Sabine Rospert

Keyword(s):

Translation Termination ◽

Ribosomal Subunit ◽

Reporter System ◽

Translational Fidelity ◽

Growth Defects ◽

Mutant Strains ◽

Translation Termination Factor ◽

Combined Data

ABSTRACT The chaperone homologs RAC (ribosome-associated complex) and Ssb1/2p are anchored to ribosomes; Ssb1/2p directly interacts with nascent polypeptides. The absence of RAC or Ssb1/2p results in a similar set of phenotypes, including hypersensitivity against the aminoglycoside paromomycin, which binds to the small ribosomal subunit and compromises the fidelity of translation. In order to understand this phenomenon we measured the frequency of translation termination and misincorporation in vivo and in vitro with a novel reporter system. Translational fidelity was impaired in the absence of functional RAC or Ssb1/2p, and the effect was further enhanced by paromomycin. The mutant strains suffered primarily from a defect in translation termination, while misincorporation was compromised to a lesser extent. Consistently, a low level of soluble translation termination factor Sup35p enhanced growth defects in the mutant strains. Based on the combined data we conclude that RAC and Ssb1/2p are crucial in maintaining translational fidelity beyond their postulated role as chaperones for nascent polypeptides.

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Host Mutations (miaA and rpsL) Reduce Tetracycline Resistance Mediated by Tet(O) and Tet(M)

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.1.59 ◽

1998 ◽

Vol 42 (1) ◽

pp. 59-64 ◽

Cited By ~ 13

Author(s):

Diane E. Taylor ◽

Catharine A. Trieber ◽

Gudrun Trescher ◽

Michelle Bekkering

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Tetracycline Resistance ◽

Acceptor Site ◽

Amino Acid Substitutions ◽

Trna Modification ◽

E Coli ◽

Translational Accuracy ◽

Host Genes ◽

Ribosomal Protection

ABSTRACT The effects of mutations in host genes on tetracycline resistance mediated by the Tet(O) and Tet(M) ribosomal protection proteins, which originated in Campylobacter spp. andStreptococcus spp., respectively, were investigated by using mutants of Salmonella typhimuriumand Escherichia coli. The miaA,miaB, and miaAB double mutants of S. typhimurium specify enzymes for tRNA modification at the adenosine at position 37, adjacent to the anticodon in tRNA. InS. typhimurium, this involves biosynthesis ofN 6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A). The miaA mutation reduced the level of tetracycline resistance mediated by both Tet(O) and Tet(M), but the latter showed a greater effect, which was ascribed to the isopentenyl (i6) group or to a combination of the methylthioadenosine (ms2) and i6 groups but not to the ms2 group alone (specified by miaB). In addition, mutations in E. coli rpsL genes, generating both streptomycin-resistant and streptomycin-dependent strains, were also shown to reduce the level of tetracycline resistance mediated by Tet(O) and Tet(M). The single-site amino acid substitutions present in the rpsL mutations were pleiotropic in their effects on tetracycline MICs. These mutants affect translational accuracy and kinetics and suggest that Tet(O) and Tet(M) binding to the ribosome may be reduced or slowed in theE. coli rpsL mutants in which the S12 protein is altered. Data from both the miaA and rpsL mutant studies indicate a possible link between stability of the aminoacyl-tRNA in the ribosomal acceptor site and tetracycline resistance mediated by the ribosomal protection proteins.

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Candida glabrata MSH2 deletion increases antifungal tolerance in vitro and during intra-abdominal candidiasis (IAC), it does not impact pathogenesis of peritonitis or intra-abdominal abscesses

Access Microbiology ◽

10.1099/acmi.cc2021.po0088 ◽

2021 ◽

Vol 3 (12) ◽

Author(s):

Shaoji Cheng ◽

Guojun Liu ◽

Cornelius Joseph Clancy ◽

Minh Hong Thi Nguyen

Keyword(s):

Common Type ◽

Dna Mismatch ◽

Growth Defects ◽

Abdominal Abscesses ◽

Solid Media ◽

Damage Response ◽

Mutant Strains ◽

Fluconazole Resistant ◽

Resistant Mutants

Background: IAC is the second most common type of invasive Candidiasis, but its pathogenesis is poorly understood. We have shown that Candida albicans DNA damage response genes are strongly induced within intra-abdominal abscesses. Deletion of C. glabrata MSH2, A DNA mismatch repair (MMR) gene, results in a mutator phenotype that facilitates multidrug resistance in vitro and in mouse gastrointestinal tracts. Our goal was to determine if CGMSH2 Contributed to pathogenesis or resistance to the new antifungal rezafungin during IAC. Methods: We createdΔMSH2 in BG2 using SAT-Flipper, and tested virulence and rezafungin responses in a mouse model of IAC. Results: ΔMSH2 displayed no growth defects at 30°C in liquid (YPD, Ypglycerol) or solid media (YPD+0.02% MMS, 1MM H2O2, 1M NACL, 20 UG/ML CW, 250 UG/ML OR 0.02% SDS). ΔMSH2 longevity in YPD was comparable to BG2. Caspofungin-, Rezafungin- and Fluconazole-resistant mutants arose 24-, 16- and 3-fold more often, respectively, for ΔMSH2 than BG2 (108-106 CFU overnight in YPD, selected on 8XMIC-Containing plates). However, respective minimum inhibitory concentrations (MICS) were not different, nor were rezafungin time-kills.ΔMSH2 was comparable to BG2 in peritonitis and abscess burdens in mouse IAC.ΔMSH2 demonstrated significantly greater caspofungin- and fluconazole-tolerance than BG2 in abscesses. Rezafungin reduced peritonitis and abscess burdens ofΔMSH2,BG2 ANDFKS mutant strains to similar extents. Conclusions: CgMSH2 deletionincreased the frequency of spontaneously-arising echinocandin- and fluconazole-resistant colonies in vitro and tolerance in intra-abdominal abscesses, but it did not attenuate virulence or rezafungin responses during IAC.

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Characterization of the RcsC Sensor Kinase from Erwinia amylovora and Other Enterobacteria

Phytopathology ◽

10.1094/phyto-09-10-0258 ◽

2011 ◽

Vol 101 (6) ◽

pp. 710-717 ◽

Cited By ~ 17

Author(s):

Dongping Wang ◽

Schuyler S. Korban ◽

P. Lawrence Pusey ◽

Youfu Zhao

Keyword(s):

Histidine Kinase ◽

Erwinia Amylovora ◽

Kinase Domain ◽

Sensor Kinase ◽

Pear Fruit ◽

E Coli ◽

Pantoea Stewartii ◽

Mutant Strains

RcsC is a hybrid sensor kinase which contains a sensor domain, a histidine kinase domain, and a receiver domain. We have previously demonstrated that, although the Erwinia amylovora rcsC mutant produces more amylovoran than the wild-type (WT) strain in vitro, the mutant remains nonpathogenic on both immature pear fruit and apple plants. In this study, we have comparatively characterized the Erwinia RcsC and its homologs from various enterobacteria. Results demonstrate that expression of the Erwinia rcsC gene suppresses amylovoran production in various amylovoran overproducing WT and mutant strains, thus suggesting the presence of a net phosphatase activity of Erwinia RcsC. Findings have also demonstrated that rcsC homologs from other enterobacteria could not rescue amylovoran production of the Erwinia rcsC mutant in vitro. However, virulence of the Erwinia rcsC mutant is partially restored by rcsC homologs from Pantoea stewartii, Yersinia pestis, and Salmonella enterica but not from Escherichia coli on apple shoots. Domain-swapping experiments have indicated that replacement of the E. coli RcsC sensor domain by those of Erwinia and Yersinia spp. partially restores virulence of the Erwinia rcsC mutant, whereas chimeric constructs containing the sensor domain of E. coli RcsC could not rescue virulence of the Erwinia rcsC mutant on apple. Interestingly, only chimeric constructs containing the histidine kinase and receiver domains of Erwinia RcsC are fully capable of rescuing amylovoran production. These results suggest that the sensor domain of RcsC may be important in regulating bacterial virulence, whereas the activity of the histidine kinase and receiver domains of Erwinia RcsC may be essential for amylovoran production in vitro.

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