Collections > UNC Chapel Hill Undergraduate Honors Theses Collection > Characterizing a novel signalling pathway in Pseudomonas entomophila: Elucidation of the signal cascade initiated by Pseudomonas virulence factor (PVF) through transposon mutagenesis
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Characterizing a novel signalling pathway in Pseudomonas entomophila: Elucidation of the signal cascade initiated by Pseudomonas virulence factor (PVF) through transposon mutagenesis

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  • Date Deposited: 2018-04-30
  • Date Created: 2018-04-01

Path:  Collections > UNC Chapel Hill Undergraduate Honors Theses Collection > Characterizing a novel signalling pathway in Pseudomonas entomophila: Elucidation of the signal cascade initiated by Pseudomonas virulence factor (PVF) through transposon mutagenesis

Pseudomonas virulence factor (PVF) is a signaling molecule synthesized by proteins encoded in the pvf biosynthetic gene cluster of the Drosophila melanogaster pathogen Pseudomonas entomophila. Previous research has shown that when these genes are mutated, the ability of this bacterium to persist in a eukaryotic host and have virulent effects is greatly reduced.1 Furthermore, the Li lab has identified homologues of the pvf gene cluster in over 300 strains of bacteria with a variety of different hosts that span the domain, Eukarya. While its ubiquity and importance to bacterial virulence has been established, little is actually known about the signaling pathway PVF initiates in neighboring P. entomophila cells. The goal of this project was to identify the genes involved in this novel signaling pathway, starting from the reception of the PVF signaling molecule at the cell membrane to the initiation of transcription. To this end, Tn5 mutagenesis was used to disrupt individual genes in a reporter strain of P. entomophila containing a PSEEN0973::lacZ insertion and a reporter strain of P. entomophila containing a PSEEN5493::lacZ insertion. Cells that took up the Tn5 plasmid were selected by antibiotic resistance and screened using a blue/white screen. Resultant white colonies exhibited disruptions in genes vital to the signaling pathway as they were not able to produce β-galactosidase, whereas blue colonies had fully functional pathways. These white colonies were further screened with quantitative assay that measured the conversion of ortho-nitrophenyl-β-galactosidase to orthonitrophenol, a reaction catalyzed by β-galactosidase. A total of 8,500 colonies were screened, and 86 white colonies were identified and quantitatively screened. Arbitrary PCR was used to investigate the locations of the Tn5 insertions in the genome. With the aid of bioinformatics, we have identified a putative histidine kinase and methylesterase that are likely involved in the PVFinitiated signaling pathway.

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