Methylation at position 32 of tRNA catalyzed by TrmJ alters oxidative stress response in Pseudomonas aeruginosa

Bacteria respond to environmental stresses using a variety of signaling and gene expression pathways, with translational mechanisms being the least well understood. Here, we identified a tRNA methyltransferase in Pseudomonas aeruginosa PA14, trmJ, which confers resistance to oxidative stress. Analysis of tRNA from a trmJ mutant revealed that TrmJ catalyzes formation of Cm, Um, and, unexpectedly, Am. Defined in vitro analyses revealed that tRNAMet(CAU) and tRNATrp(CCA) are substrates for Cm formation, tRNAGln(UUG), tRNAPro(UGG), tRNAPro(CGG) and tRNAHis(GUG) for Um, and tRNAPro(GGG) for Am. tRNASer(UGA), previously observed as a TrmJ substrate in Escherichia coli, was not modified by PA14 TrmJ. Position 32 was confirmed as the TrmJ target for Am in tRNAPro(GGG) and Um in tRNAGln(UUG) by mass spectrometric analysis. Crystal structures of the free catalytic N-terminal domain of TrmJ show a 2-fold symmetrical dimer with an active site located at the interface between the monomers and a flexible basic loop positioned to bind tRNA, with conformational changes upon binding of the SAM-analog sinefungin. The loss of TrmJ rendered PA14 sensitive to H2O2 exposure, with reduced expression of oxyR-recG, katB-ankB, and katE. These results reveal that TrmJ is a tRNA:Cm32/Um32/Am32 methyltransferase involved in translational fidelity and the oxidative stress response.

[1]  S. Mongkolsuk,et al.  Analyses of the Regulatory Mechanism and Physiological Roles of Pseudomonas aeruginosa OhrR, a Transcription Regulator and a Sensor of Organic Hydroperoxides , 2010, Journal of bacteriology.

[2]  Angela T. Nguyen,et al.  The prrF-Encoded Small Regulatory RNAs Are Required for Iron Homeostasis and Virulence of Pseudomonas aeruginosa , 2014, Infection and Immunity.

[3]  Clemens Vonrhein,et al.  Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER , 2012, Acta crystallographica. Section D, Biological crystallography.

[4]  Guillermo Oliver,et al.  Elucidating the molecular characteristics of organogenesis in human embryos , 2010, Genome Biology.

[5]  Xavier Robert,et al.  Deciphering key features in protein structures with the new ENDscript server , 2014, Nucleic Acids Res..

[6]  H. Mori,et al.  Bmc Molecular Biology the Yfhq Gene of Escherichia Coli Encodes a Trna:cm32/um32 Methyltransferase , 2022 .

[7]  Ashish Patil,et al.  Translational infidelity-induced protein stress results from a deficiency in Trm9-catalyzed tRNA modifications , 2012, RNA biology.

[8]  G. Björk,et al.  The Conserved Cys-X1-X2-Cys Motif Present in the TtcA Protein Is Required for the Thiolation of Cytidine in Position 32 of tRNA from Salmonella enterica serovar Typhimurium , 2004, Journal of bacteriology.

[9]  Thomas J. Begley,et al.  Trm9-catalyzed tRNA modifications link translation to the DNA damage response. , 2007, Molecular cell.

[10]  P. Sergiev,et al.  The yfiC gene of E. coli encodes an adenine-N6 methyltransferase that specifically modifies A37 of tRNA1Val(cmo5UAC). , 2009, RNA.

[11]  Clement T Y Chan,et al.  Quantitative analysis of ribonucleoside modifications in tRNA by HPLC-coupled mass spectrometry , 2014, Nature Protocols.

[12]  Wolfgang Kabsch,et al.  Integration, scaling, space-group assignment and post-refinement , 2010, Acta crystallographica. Section D, Biological crystallography.

[13]  F. Murphy,et al.  Modifications Modulate Anticodon Loop Dynamics and Codon Recognition in E. coli tRNAArg1,2 , 2012, Journal of molecular biology.

[14]  M. Sekine,et al.  Conformational rigidity of specific pyrimidine residues in tRNA arises from posttranscriptional modifications that enhance steric interaction between the base and the 2'-hydroxyl group. , 1992, Biochemistry.

[15]  A. Drake,et al.  Optical studies of the base-stacking properties of 2'-O-methylated dinucleoside monophosphates. , 1974, Journal of molecular biology.

[16]  S. Mongkolsuk,et al.  Expression analysis and characterization of the mutant of a growth-phase- and starvation-regulated monofunctional catalase gene from Xanthomonas campestris pv. phaseoli. , 2000, Gene.

[17]  A. Hopper,et al.  Yeast Trm7 interacts with distinct proteins for critical modifications of the tRNAPhe anticodon loop. , 2012, RNA.

[18]  Clement T Y Chan,et al.  A Quantitative Systems Approach Reveals Dynamic Control of tRNA Modifications during Cellular Stress , 2010, PLoS genetics.

[19]  S. Worgall,et al.  Transcriptome Analysis of the Response of Pseudomonas aeruginosa to Hydrogen Peroxide , 2004, Journal of bacteriology.

[20]  A. Anderson,et al.  Cloning and mutational analysis of the gene for the stationary-phase inducible catalase (catC) from Pseudomonas putida , 1997, Journal of bacteriology.

[21]  Allan Matte,et al.  The structure of the RlmB 23S rRNA methyltransferase reveals a new methyltransferase fold with a unique knot. , 2002, Structure.

[22]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[23]  D. Hassett,et al.  The Major Catalase Gene (katA) of Pseudomonas aeruginosa PA14 Is under both Positive and Negative Control of the Global Transactivator OxyR in Response to Hydrogen Peroxide , 2009, Journal of bacteriology.

[24]  Janusz M Bujnicki,et al.  Trm7p catalyses the formation of two 2′‐O‐methylriboses in yeast tRNA anticodon loop , 2002, The EMBO journal.

[25]  B. Wanner,et al.  Conditionally replicative and conjugative plasmids carrying lacZ alpha for cloning, mutagenesis, and allele replacement in bacteria. , 1996, Plasmid.

[26]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[27]  G. Björk,et al.  Improvement of reading frame maintenance is a common function for several tRNA modifications , 2001, The EMBO journal.

[28]  G. Glinsky,et al.  Role of rpoS regulon in resistance to oxidative stress and near-UV radiation in delta oxyR suppressor mutants of Escherichia coli. , 1997, Free radical biology & medicine.

[29]  S. Solomon,et al.  Antibiotic resistance threats in the United States: stepping back from the brink. , 2014, American family physician.

[30]  Marcin Feder,et al.  Characterization of the cofactor-binding site in the SPOUT-fold methyltransferases by computational docking of S-adenosylmethionine to three crystal structures , 2003, BMC Bioinformatics.

[31]  S. Yokoyama,et al.  Structural basis for methyl-donor–dependent and sequence-specific binding to tRNA substrates by knotted methyltransferase TrmD , 2015, Proceedings of the National Academy of Sciences.

[32]  Clement T Y Chan,et al.  Highly Predictive Reprogramming of tRNA Modifications Is Linked to Selective Expression of Codon-Biased Genes , 2015, Chemical research in toxicology.

[33]  E. Wang,et al.  tRNA recognition by a bacterial tRNA Xm32 modification enzyme from the SPOUT methyltransferase superfamily , 2015, Nucleic acids research.

[34]  V. de Crécy-Lagard,et al.  Biosynthesis and function of posttranscriptional modifications of transfer RNAs. , 2012, Annual review of genetics.

[35]  D. Hanahan,et al.  Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[36]  D. Hassett,et al.  Differential roles of OxyR-controlled antioxidant enzymes alkyl hydroperoxide reductase (AhpCF) and catalase (KatB) in the protection of Pseudomonas aeruginosa against hydrogen peroxide in biofilm vs. planktonic culture. , 2009, FEMS microbiology letters.

[37]  Li Li,et al.  Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial , 2006, Genome Biology.

[38]  Clement T Y Chan,et al.  Identification of N6,N6-Dimethyladenosine in Transfer RNA from Mycobacterium bovis Bacille Calmette-Guérin , 2011, Molecules.

[39]  J. Steyaert,et al.  Characterization of two homologous 2′-O-methyltransferases showing different specificities for their tRNA substrates , 2014, RNA.

[40]  Paul F Agris,et al.  Bringing order to translation: the contributions of transfer RNA anticodon‐domain modifications , 2008, EMBO reports.

[41]  Clement T Y Chan,et al.  Reprogramming of tRNA modifications controls the oxidative stress response by codon-biased translation of proteins , 2012, Nature Communications.

[42]  Jef Rozenski,et al.  SOS: A simple interactive program for ab initio oligonucleotide sequencing by mass spectrometry , 2002, Journal of the American Society for Mass Spectrometry.

[43]  Robert E W Hancock,et al.  Pseudomonas aeruginosa: new insights into pathogenesis and host defenses. , 2013, Pathogens and disease.

[44]  D. Roop,et al.  Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. , 1995, Gene.

[45]  Y. Yamamoto,et al.  NMR analyses on the molecular mechanism of the conformational rigidity of 2‐thioribothymidine, a modified nucleoside in extreme thermophile tRNAs , 1983, FEBS letters.

[46]  D. Hassett,et al.  Role of the Pseudomonas aeruginosa oxyR-recG Operon in Oxidative Stress Defense and DNA Repair: OxyR-Dependent Regulation of katB-ankB, ahpB, andahpC-ahpF , 2000, Journal of bacteriology.

[47]  A. Byström,et al.  Prevention of translational frameshifting by the modified nucleoside 1-methylguanosine. , 1989, Science.

[48]  M. Niederman,et al.  Community-acquired pneumonia due to gram-negative bacteria and pseudomonas aeruginosa: incidence, risk, and prognosis. , 2002, Archives of internal medicine.

[49]  S. Douthwaite,et al.  YibK is the 2'-O-methyltransferase TrmL that modifies the wobble nucleotide in Escherichia coli tRNA(Leu) isoacceptors. , 2010, RNA.

[50]  M. Alexeyev.  The pKNOCK series of broad-host-range mobilizable suicide vectors for gene knockout and targeted DNA insertion into the chromosome of gram-negative bacteria. , 1999, BioTechniques.

[51]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[52]  T. Murray,et al.  Pseudomonas aeruginosa OspR is an oxidative stress sensing regulator that affects pigment production, antibiotic resistance and dissemination during infection , 2010, Molecular microbiology.

[53]  F. Hildebrand,et al.  Global regulation of gene expression by OxyR in an important human opportunistic pathogen , 2012, Nucleic acids research.