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

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

Genome-Wide Identification of Host Genes Required for Toxicity of Bacterial Cytolethal Distending Toxin in a Yeast Model

BackgroundAggregatibacter actinomycetemcomitans, a periodontal pathogen, secretes a cytolethal distending toxin (AaCDT) that causes host cell cycle arrest and cell death. Although CDT could be an important virulence factor, it is unclear how it enters the nucleus to exert its cytotoxicity.ObjectiveTo investigate the mechanisms of AaCDT by genome-wide screening for host mutations that confer resistance to the catalytic subunit, AaCdtB, in a Saccharomyces cerevisiae model.MethodsWe transformed the yeast haploid deletion library, a collection of yeast mutants with single gene deletions of virtually all non-essential ORFs in the genome, with plasmids carrying galactose-inducible AaCdtB. Yeast mutants that showed resistance to AaCdtB were selected and rescreened by a spotting assay. AaCdtB expression was confirmed by western blot analysis; any mutants that showed no or weak expression of AaCdtB were omitted from the analysis. The lists of genes whose mutations confer resistance to AaCdtB were analyzed for Gene Ontology (GO) term enrichments. Localization of AaCdtB-EGFP was examined using fluorescent microscopy. Nuclear localization relative to EGFP control was calculated and compared to wild-type.ResultsOut of approximately 5,000 deletion mutants, we isolated 243 mutants that are resistant to AaCdtB. GO analyses indicated that genes associated with organic anion transport are significantly enriched (16 genes). Furthermore, several genes associated with the nucleus and endoplasmic reticulum (ER) were identified. Localization studies of AaCdtB, in mutants with the deletion of genes associated with the GO term organic anion transport, showed lower nuclear localization than wild-type. The results suggest that these genes may be required for AaCdtB translocation into the nucleus and its cytotoxicity.ConclusionThe genome-wide screen in the yeast deletion library allowed us to identify a large number of host genes required for AaCdtB cytotoxicity. Further investigation could lead to more insights into the mechanisms of CdtB intoxication

Methods for genetic manipulation of Burkholderia gladioli pathovar cocovenenans

<p>Abstract</p> <p>Background</p> <p><it>Burkholderia gladioli </it>pathovar <it>cocovenenans </it>(BGC) is responsible for sporadic food-poisoning outbreaks with high morbidity and mortality in Asian countries. Little is known about the regulation of virulence factor and toxin production in BGC, and studies in this bacterium have been hampered by lack of genetic tools.</p> <p>Findings</p> <p>Establishment of a comprehensive antibiotic susceptibility profile showed that BGC strain ATCC33664 is susceptible to a number of antibiotics including aminoglycosides, carbapenems, fluoroquinolones, tetracyclines and trimethoprim. In this study, we established that gentamicin, kanamycin and trimethoprim are good selection markers for use in BGC. Using a 10 min method for preparation of electrocompetent cells, the bacterium could be transformed by electroporation at high frequencies with replicative plasmids containing the pRO1600-derived origin of replication. These plasmids exhibited a copy number of > 100 in BGC. When co-conjugated with a transposase expressing helper plasmid, mini-Tn<it>7 </it>vectors inserted site- and orientation-specifically at a single <it>glmS</it>-associated insertion site in the BGC genome. Lastly, a <it>Himar1 </it>transposon was used for random transposon mutagenesis of BGC.</p> <p>Conclusions</p> <p>A series of genetic tools previously developed for other Gram-negative bacteria was adapted for use in BGC. These tools now facilitate genetic studies of this pathogen and allow establishment of toxin biosynthetic pathways and their genetic regulation.</p

Antifungal Potential of Extracellular Metabolites Produced by Streptomyces hygroscopicus against Phytopathogenic Fungi

Indigenous actinomycetes isolated from rhizosphere soils were assessed for in vitro antagonism against Colletotrichum gloeosporioides and Sclerotium rolfsii. A potent antagonist against both plant pathogenic fungi, designated SRA14, was selected and identified as Streptomyces hygroscopicus. The strain SRA14 highly produced extracellular chitinase and &#946;-1,3-glucanase during the exponential and late exponential phases, respectively. Culture filtrates collected from the exponential and stationary phases inhibited the growth of both the fungi tested, indicating that growth suppression was due to extracellular antifungal metabolites present in culture filtrates. The percentage of growth inhibition by the stationary culture filtrate was significantly higher than that of exponential culture filtrate. Morphological changes such as hyphal swelling and abnormal shapes were observed in fungi grown on potato dextrose agar that contained the culture filtrates. However, the antifungal activity of exponential culture filtrates against both the experimental fungi was significantly reduced after boiling or treatment with proteinase K. There was no significant decrease in the percentage of fungal growth inhibition by the stationary culture filtrate that was treated as above. These data indicated that the antifungal potential of the exponential culture filtrate was mainly due to the presence of extracellular chitinase enzyme, whereas the antifungal activity of the stationary culture filtrate involved the action of unknown thermostable antifungal compound(s).</p

Physiological and Expression Analyses of Agrobacterium tumefaciens trxA, Encoding Thioredoxin▿

Exposure of Agrobacterium tumefaciens to menadione, cumene hydroperoxide, and diamide strongly induced trxA expression. The trxA mutant showed a reduction in the aerobic growth rate and plating efficiency and was cytochrome c oxidase negative. Atypically, the mutant has decreased resistance to menadione but an increased H2O2 resistance phenotype