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 tRNA[superscript Met(CAU)] and tRNA[superscript Trp(CCA)] are substrates for Cm formation, tRNA[superscript Gln(UUG)], tRNA[superscript Pro(UGG)], tRNA[superscript Pro(CGG)] and tRNA[superscript His(GUG)] for Um, and tRNA[superscript Pro(GGG)] for Am. tRNA[superscript Ser(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 tRNA[superscriptPro(GGG)] and Um in tRNA[superscript Gln(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.National Science Foundation (U.S.) (CHE-1308839)Agilent TechnologiesSingapore-MIT Alliance for Research and Technology (SMART

Macrophage Replication Screen Identifies a Novel Francisella Hydroperoxide Resistance Protein Involved in Virulence

Francisella tularensis is a Gram-negative facultative intracellular pathogen and the causative agent of tularemia. Recently, genome-wide screens have identified Francisella genes required for virulence in mice. However, the mechanisms by which most of the corresponding proteins contribute to pathogenesis are still largely unknown. To further elucidate the roles of these virulence determinants in Francisella pathogenesis, we tested whether each gene was required for replication of the model pathogen F. novicida within macrophages, an important virulence trait. Fifty-three of the 224 genes tested were involved in intracellular replication, including many of those within the Francisella pathogenicity island (FPI), validating our results. Interestingly, over one third of the genes identified are annotated as hypothetical, indicating that F. novicida likely utilizes novel virulence factors for intracellular replication. To further characterize these virulence determinants, we selected two hypothetical genes to study in more detail. As predicted by our screen, deletion mutants of FTN_0096 and FTN_1133 were attenuated for replication in macrophages. The mutants displayed differing levels of attenuation in vivo, with the FTN_1133 mutant being the most attenuated. FTN_1133 has sequence similarity to the organic hydroperoxide resistance protein Ohr, an enzyme involved in the bacterial response to oxidative stress. We show that FTN_1133 is required for F. novicida resistance to, and degradation of, organic hydroperoxides as well as resistance to the action of the NADPH oxidase both in macrophages and mice. Furthermore, we demonstrate that F. holarctica LVS, a strain derived from a highly virulent human pathogenic species of Francisella, also requires this protein for organic hydroperoxide resistance as well as replication in macrophages and mice. This study expands our knowledge of Francisella's largely uncharacterized intracellular lifecycle and demonstrates that FTN_1133 is an important novel mediator of oxidative stress resistance

The OxyR-regulated <i>phnW</i> gene encoding 2-aminoethylphosphonate:pyruvate aminotransferase helps protect <i>Pseudomonas aeruginosa</i> from <i>tert</i>-butyl hydroperoxide

<div><p>The LysR member of bacterial transactivators, OxyR, governs transcription of genes involved in the response to H₂O₂ and organic (alkyl) hydroperoxides (AHP) in the Gram-negative pathogen, <i>Pseudomonas aeruginosa</i>. We have previously shown that organisms lacking OxyR are rapidly killed by <2 or 500 mM H₂O₂ in planktonic and biofilm bacteria, respectively. In this study, we first employed a bioinformatic approach to elucidate the potential regulatory breadth of OxyR by scanning the entire <i>P</i>. <i>aeruginosa</i> PAO1 genome for canonical OxyR promoter recognition sequences (ATAG-N₇-CTAT-N₇-ATAG-N₇-CTAT). Of >100 potential OxyR-controlled genes, 40 were strategically selected that were <u><b><i>not</i></b></u> predicted to be involved in the direct response to oxidative stress (e.g., catalase, peroxidase, etc.) and screened such genes by RT-PCR analysis for potentially positive or negative control by OxyR. Differences were found in 7 of 40 genes when comparing an <i>oxyR</i> mutant vs. PAO1 expression that was confirmed by ß-galactosidase reporter assays. Among these, <i>phnW</i>, encoding 2-aminoethylphosphonate:pyruvate aminotransferase, exhibited reduced expression in the <i>oxyR</i> mutant compared to wild-type bacteria. Electrophoretic mobility shift assays indicated binding of OxyR to the <i>phnW</i> promoter and DNase I footprinting analysis also revealed the sequences to which OxyR bound. Interestingly, a <i>phnW</i> mutant was more susceptible to <i>t</i>-butyl-hydroperoxide (<i>t</i>-BOOH) treatment than wild-type bacteria. Although we were unable to define the direct mechanism underlying this phenomenon, we believe that this may be due to a reduced efficiency for this strain to degrade <i>t</i>-BOOH relative to wild-type organisms because of modulation of AHP gene transcription in the <i>phnW</i> mutant.</p></div

Determination of <i>phnW</i> expression levels in wild-type and mutant strains when exposed to <i>t</i>-BOOH.

<p>All bacteria which contained a <i>phnW</i>-<i>lacZ</i> transcriptional fusion plasmid (pQF-<i>phnW</i>) were grown to exponential phase and exposed to 250 μM <i>t</i>-BOOH. ß-galactosidase activity assays were reported as the mean +/- standard error compared to untreated bacteria. The assays were performed using three independent experiments. The white bars represent untreated bacteria while the gray bars represent <i>t</i>-BOOH treated organisms, respectively.</p

Semi-quantitative expression of PA <i>phnW</i>.

<p>Total RNA was isolated from exponential phase <i>PA</i> PAO1 or its isogenic <i>oxyR</i> mutant. Then, 1 μl of cDNA was used to amplify the <i>phnW</i> promoter region with specific primers. The <i>omlA</i> gene was used as an internal constitutive control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189066#pone.0189066.ref014" target="_blank">14</a>] and <i>ahpC</i> was used as a positive gene under OxyR control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189066#pone.0189066.ref006" target="_blank">6</a>].</p

Time course of <i>t</i>-BOOH degradation by various <i>PA</i> strains.

<p>The rate of PA <i>t</i>-BOOH degradation, an organic hydroperoxide, was investigated using xylenol orange–iron reaction system as described in the materials and methods section. The exponential phase of PAO1 (white bar), <i>oxyR</i> (light gray bar), <i>oxyR</i>/p<i>oxyR</i> (dotted bar), <i>phnW</i>::Gm/p<i>phnW</i> (black bar) and <i>phnW</i>::Gm (dark gray bar) cells were used in this study in the present of 200 μM of <i>t</i>-BOOH and measured the rate of degradation of <i>t</i>-BOOH in each cell at difference time points. The percentage of <i>t</i>-BOOH remaining was reported after 12 min of incubation. The experiments were independently repeated at least three times and typical results are shown.</p

Sensitivity of bacteria to <i>t</i>-BOOH.

<p>Bacteria from the aerobic exponential phase (<u><b>A,B).</b></u> PAO1, <i>phnW</i>::Gm and <i>phnW</i>::Gm/p<i>phnW</i>) or (<u><b>E,F).</b></u> <i>ohr ahpC</i> and <i>ohr ahpC</i>/p<i>phnW</i>) or stationary phase (<u><b>C,D</b></u>). PAO1, <i>oxyR</i>/p<i>oxyR</i>, <i>phnW</i>::Gm, <i>oxyR phnW</i>::Gm, <i>oxyR</i> and <i>oxyR</i>/p<i>phnW</i>) were used to determine sensitivity to <i>t</i>-BOOH at 0.3 M (exponential) or 0.5 M (stationary), respectively. The experiments were independent and repeated at least three times. The values shown are means +/- standard error.</p

Localization of the OxyR binding domain of the <i>phnW</i> promoter by DNase I footprinting analysis.

<p><b>(</b><u><b>A).</b></u> The 199-bp upstream sequence of <i>phnW</i> containing a putative OxyR domain was used to treat with DNase I in the presence of OxyR at 0, 500 and 1000 nM. The digested DNA was analyzed on a 5% denaturing polyacrylamide gel, followed by autoradiography. The sequence to which OxyR binds on this fragment was identified. (<u><b>B).</b></u> The upstream sequences of <i>phnW</i> that are underlined are the primers used in this study. The bold letters indicate the OxyR binding domain within the <i>phnW</i> upstream sequence while the lower case bold letters indicate the putative OxyR binding site based upon a match to the consensus ATAG-N₇-CTAT-N₇-ATAG-N₇-CTAT sequence used to search for OxyR-dependent gene candidates. The underlined and bold ATG indicates the translational initiation codon of the <i>phnW</i> gene.</p