Project description:we performed ChIP-seq assays to identify in vivo targets of GltR. The plasmid mini-gltR-flag-lacz was constructed and the resultant plasmid was fused to P. aeruginosa strain PAO1, yielding PAO1/mini-gltR-flag-lacz. We investigated GltR-binding to the chromosome of PAO1 during growth with glucose by ChIP-Seq. Sequence reads obtained from three independent ChIP-Seq experiments using anti-flag antibody and mapped to the P. aeruginosa PAO1 genome.Using the MACS software,we identified 55 enriched loci (q-value < 0.05) harboring GltR-binding peaks, that were enriched > 3-fold, but were absent in control sample conducted without anti-flag antibody.
Project description:SbrI and SbrR are an extracytoplasmic function sigma factor and its cognate anti-sigma factor, respectively. To identify the SbrIR regulon, we measured gene expression in wild type PAO1 , PAO1 ∆sbrR, and PAO1 ∆sbrIR mutants using microarrays.
Project description:P. aeruginosa PAO1 grown as lawns on Nematode Growth Medium prepared without supplementation (NGM Pi<0.1 mM) has high killing ability against C. elegans, however, no mortality in worms has been observed during 48 hrs when feeding on PAO1 lawns grown on phosphate supplemented full NGM Pi 25 mM, pH 6.0 medium. We used a microarray to define the virulence-related genes in P. aeruginosa grown as lawns in NGM Pi<0.1 mM vs NGM Pi25 mM pH 6.0
Project description:The Pseudomonas aeruginosa response regulator AlgR is critical for the organism's virulence and controls up to 155 different genes. In order to determine which genes are controlled by phosphorylated and unphosphorylated AlgR, phosphomimetic and phosphoablative alleles were recombined onto the chromosome of PAO1. The algR gene was mutated at aspartate 54 to asparagine (D54N) for the phosphoablative allele and mutated at aspartete 54 to glutamate (D54E) for the phosphomimetic allele. These alleles were recombined into the PAO1 chromosome.
Project description:SbrI and SbrR are an extracytoplasmic function sigma factor and its cognate anti-sigma factor, respectively. To identify the SbrIR regulon, we measured gene expression in wild type PAO1 , PAO1 âsbrR, and PAO1 âsbrIR mutants using microarrays. WT PAO1 pPSV38 (empty vector), PAO1 âsbrR pPSV38, PAO1 âsbrR pSbrR, and PAO1 âsbrIR pPSV38 were grown to mid-log. RNA was extracted and reverse transcribed into cDNA. The cDNA was biotinylated and hybridized to Pseudomonas aeruginosa Affymetrix microarrays.
Project description:Although Pseudomonas aeruginosa is an opportunistic pathogen that does not often naturally infect alternate hosts such as plants, the plant-P. aeruginosa model has become a widely recognized system for identifying new virulence determinants and studying pathogenesis of this organism. Here we examine how both host factors and P. aeruginosa PAO1 gene expression are affected in planta after infiltration into incompatible and compatible cultivars of tobacco (Nicotiana tabacum L.) Nicotiana tabacum has a resistance gene (N) against tobacco mosaic virus; and although resistance to PAO1 infection correlated to the presence of a dominant N-gene, our data suggests that it is not a factor in resistance against Pseudomonas. We did observe that the resistant tobacco cultivar had higher basal levels of salicylic acid, and a stronger salicylic acid response upon infiltration of PAO1. Salicylic acid acts as a signal to activate defense responses in plants, limiting the spread of the pathogen and preventng access to nutrients. It has also been shown to have direct virulence modulating effects on P. aeruginosa. We also examined host effects on the pathogen by analyzing global gene expression profiles of bacteria removed from the intracellular fluid of the two plant hosts. We discovered that the availability of micronutrients, particularly sulfate and Pi, are important factors in in planta pathogenesis, and that the amounts of these nutrients made available to the bacteria may in turn have an effect on virulence gene expression. Indeed, there are several reports suggesting that P. aeruginosa virulence is influenced in mammalian hosts by the availability of iron and by levels of O2. Keywords: comparative genomic hybridization, disease state analysis