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

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

Haplogroup Distribution of 309 Thais from Admixed Populations across the Country by HVI and HVII Sanger-Type Sequencing

The mitochondrial DNA (mtDNA) control region sequences for the hypervariable regions I (HVI) and II (HVII) of 309 Thai citizens were investigated using Sanger-type sequencing to generate an mtDNA reference dataset for forensic casework, and the haplogroup distribution within geographically proximal Asian populations was analyzed. The population sample set contained 264 distinct haplotypes and showed high haplotype diversity, low matching probability, and high powers of discrimination, at 0.9985, 0.4744%, and 0.9953, respectively, compared with previous reports. Subhaplogroup F1a showed the highest frequency in the Thai population, similar to Southeast Asian populations. The haplotype frequencies in the northern, northeastern, and southern populations of Thailand illustrate the relevance of social, religious, and historical factors in the biogeographical origin of the admixed Thai population as a whole. The HVI and HVII reference datasets will be useful for forensic casework applications, with improved genetic information content and discriminatory power compared to currently available techniques

Integrated Continuous-Flow Production of Wax Esters Combining Whole-Cell and <i>In Vitro</i> Biocatalysis

A first-of-its-kind, fully continuous synthesis of wax esters from biobased precursors (glucose, fatty acids) was developed using metabolically engineered cells and in vitro enzyme catalysis. The cells, overexpressing fatty acyl-CoA reductase and xylose reductase, could be immobilized onto polyesters and packed in a continuous reactor. The immobilized cells were employed in the bioconversion, incorporating in situ extraction using dodecane as the solvent. Such extractive bioconversion was capable of producing fatty alcohols continuously at a productivity of 8.2 mg/(L·h). The immiscible aqueous-dodecane flow stream from the extractive bioconversion was then separated using an in-line membrane-based separator. The dodecane-rich phase was directed into an enzymatic reactor containing Novozyme 435 for the esterification of fatty alchols and fatty acids into the wax esters. A continuous production of wax esters (6.38–23.35 mg/(L·h)) was achieved as a result of the successful streamlining of the cascade biocatalytic process

Regulation of Organic Hydroperoxide Stress Response by Two OhrR Homologs in <i>Pseudomonas aeruginosa</i>

<div><p><i>Pseudomonas aeruginosa ohrR</i> and <i>ospR</i> are gene homologs encoding oxidant sensing transcription regulators. OspR is known to regulate <i>gpx</i>, encoding a glutathione peroxidase, while OhrR regulates the expression of <i>ohr</i> that encodes an organic peroxide specific peroxiredoxin. Here, we show that <i>ospR</i> mediated <i>gpx</i> expression, like <i>ohrR</i> and <i>ohr</i>, specifically responds to organic hydroperoxides as compared to hydrogen peroxide and superoxide anion. Furthermore, the regulation of these two systems is interconnected. OspR is able to functionally complement an <i>ohrR</i> mutant, i.e. it regulates <i>ohr</i> in an oxidant dependent manner. In an <i>ohrR</i> mutant, in which <i>ohr</i> is derepressed, the induction of <i>gpx</i> expression by organic hydroperoxide is reduced. Likewise, in an <i>ospR</i> mutant, where <i>gpx</i> expression is constitutively high, oxidant dependent induction of <i>ohr</i> expression is reduced. Moreover, <i>in vitro</i> binding assays show that OspR binds the <i>ohr</i> promoter, while OhrR binds the <i>gpx</i> promoter, albeit with lower affinity. The binding of OhrR to the <i>gpx</i> promoter may not be physiologically relevant; however, OspR is shown to mediate oxidant-inducible expression at both promoters. Interestingly, the mechanism of OspR-mediated, oxidant-dependent induction at the two promoters appears to be distinct. OspR required two conserved cysteines (C24 and C134) for oxidant-dependent induction of the <i>gpx</i> promoter, while only C24 is essential at the <i>ohr</i> promoter. Overall, this study illustrates possible connection between two regulatory switches in response to oxidative stress.</p></div

Long-term storage limits PCR-based analyses of malaria parasites in archival dried blood spots.

International audienceABSTRACT: BACKGROUND: Blood samples collected in epidemiological and clinical investigations and then stored, often at room temperature, as blood spots dried on a filter paper has become one of the most popular source of material for further molecular analyses of malaria parasites. The dried blood spots are often archived so that they can be used for further retrospective investigations of parasite prevalence, or as new genetic markers come to the fore. However, the suitability of the template obtained from dried blood spots that have been stored for long periods for DNA amplification is not known. METHODS: DNA from 267 archived blood spots collected over a period of 12 years from persons with microscopically confirmed Plasmodium falciparum infection was purified by one of two methods, Chelex and Qiagen columns. These templates were subjected to highly sensitive nested PCR amplification targeting three parasite loci that differ in length and/or copy number. RESULTS: When a 1.6 kb fragment of the parasites' small subunit ribosomal RNA was targeted (primary amplification), the efficiency of P. falciparum detection decreased in samples archived for more than six years, reaching very low levels for those stored for more than 10 years. Positive amplification was generally obtained more often with Qiagen-extracted templates. P. falciparum could be detected in 32 of the 40 negative Qiagen-extracted templates when a microsatellite of about 180 bp was targeted. The remaining eight samples gave a positive amplification when a small region of 238 bp of the higher copy number (20 to 200) mitochondrial genome was targeted. CONCLUSIONS: The average length of DNA fragments that can be recovered from dried blood spots decrease with storage time. Recovery of the DNA is somewhat improved, especially in older samples, by the use of a commercial DNA purification column, but targets larger than 1.5 kb are unlikely to be present 10 years after the initial blood collection, when the average length of the DNA fragments present is likely to be around a few hundred bp. In conclusion, the utility of archived dried blood spots for molecular analyses decreases with storage time

Mapping of the OspR binding sites on the <i>P</i>. <i>aeruginosa gpx</i> (A) and <i>ohr</i> (B) promoter fragments by DNase I footprinting.

<p>PCR-generated probe fragments were labeled on one strand by end-labeling one of the primers with ³²P prior to amplification. The sequencing ladder (G, A, T, C) used to localize the binding sites on the promoters were generated using the promoter fragment itself as a template and the same labeled oligonucleotide as was used to generate the probe as a primer. Numbers above each lane indicate amounts of the OpsR and OhrR protein (μM) used in each reaction. The regions protected by OspR or OhrR are indicated by vertical lines. Hypersensitive site is indicated by asterisk.</p

Expression analysis of <i>gpx</i> and <i>ospR</i> in the presence of oxidants.

<p>Quantitative real-time PCR was performed to measure relative expression levels (2^-ΔΔCt) of <i>gpx</i> (A) and <i>ospR</i> (B) in wild-type <i>Pseudomonas aeruginosa</i> with various oxidant treatments for 15 min. The uninduced sample was set to 1. UN, uninduced; CHP500, 500 μM CHP; tBOOH500, 500 μM tBOOH; LOOH100, 100 μM linoleic hydroperoxide; H₂O₂500, 500 μM H₂O₂; H₂O₂1000, 1000 μM H₂O₂; H₂O₂4000, 4000 μM H₂O₂; MD50, 50 μM menadione; MD500, 500 μM menadione; PQ50, 50 μM paraquat; PQ100, 100 μM paraquat; PQ500, 500 μM paraquat. Significant differences (<i>P</i> < 0.05) between the treated samples and uninduced sample are denoted with asterisks.</p