Crystal structure and catalytic mechanism of the essential m1G37 tRNA methyltransferase TrmD from Pseudomonas aeruginosa.

The tRNA (m1G37) methyltransferase TrmD catalyzes m1G formation at position 37 in many tRNA isoacceptors and is essential in most bacteria, which positions it as a target for antibiotic development. In spite of its crucial role, little is known about TrmD in Pseudomonas aeruginosa (PaTrmD), an important human pathogen. Here we present detailed structural, substrate, and kinetic properties of PaTrmD. The mass spectrometric analysis confirmed the G36G37-containing tRNAs Leu(GAG), Leu(CAG), Leu(UAG), Pro(GGG), Pro(UGG), Pro(CGG), and His(GUG) as PaTrmD substrates. Analysis of steady-state kinetics with S-adenosyl-l-methionine (SAM) and tRNALeu(GAG) showed that PaTrmD catalyzes the two-substrate reaction by way of a ternary complex, while isothermal titration calorimetry revealed that SAM and tRNALeu(GAG) bind to PaTrmD independently, each with a dissociation constant of 14 ± 3 µM. Inhibition by the SAM analog sinefungin was competitive with respect to SAM (Ki = 0.41 ± 0.07 µM) and uncompetitive for tRNA (Ki = 6.4 ± 0.8 µM). A set of crystal structures of the homodimeric PaTrmD protein bound to SAM and sinefungin provide the molecular basis for enzyme competitive inhibition and identify the location of the bound divalent ion. These results provide insights into PaTrmD as a potential target for the development of antibiotics.

TrmB, a tRNA m7G46 methyltransferase, plays a role in hydrogen peroxide resistance and positively modulates the translation of katA and katB mRNAs in Pseudomonas aeruginosa.

Cellular response to oxidative stress is a crucial mechanism that promotes the survival of Pseudomonas aeruginosa during infection. However, the translational regulation of oxidative stress response remains largely unknown. Here, we reveal a tRNA modification-mediated translational response to H2O2 in P. aeruginosa. We demonstrated that the P. aeruginosa trmB gene encodes a tRNA guanine (46)-N7-methyltransferase that catalyzes the formation of m7G46 in the tRNA variable loop. Twenty-three tRNA substrates of TrmB with a guanosine residue at position 46 were identified, including 11 novel tRNA substrates. We showed that loss of trmB had a strong negative effect on the translation of Phe- and Asp-enriched mRNAs. The trmB-mediated m7G modification modulated the expression of the catalase genes katA and katB, which are enriched with Phe/Asp codons at the translational level. In response to H2O2 exposure, the level of m7G modification increased, consistent with the increased translation efficiency of Phe- and Asp-enriched mRNAs. Inactivation of trmB led to decreased KatA and KatB protein abundance and decreased catalase activity, resulting in H2O2-sensitive phenotype. Taken together, our observations reveal a novel role of m7G46 tRNA modification in oxidative stress response through translational regulation of Phe- and Asp-enriched genes, such as katA and katB.

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.

Pseudomonas aeruginosa GidA modulates the expression of catalases at the posttranscriptional level and plays a role in virulence.

Pseudomonas aeruginosa gidA, which encodes a putative tRNA-modifying enzyme, is associated with a variety of virulence phenotypes. Here, we demonstrated that P. aeruginosa gidA is responsible for the modifications of uridine in tRNAs in vivo. Loss of gidA was found to have no impact on the mRNA levels of katA and katB, but it decreased KatA and KatB protein levels, resulting in decreased total catalase activity and a hydrogen peroxide-sensitive phenotype. Furthermore, gidA was found to affect flagella-mediated motility and biofilm formation; and it was required for the full virulence of P. aeruginosa in both Caenorhabditis elegans and macrophage models. Together, these observations reveal the posttranscriptional impact of gidA on the oxidative stress response, highlight the complexity of catalase gene expression regulation, and further support the involvement of gidA in the virulence of P. aeruginosa.