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. We have shown that when these genes are mutated, the ability of this bacterium to initiate virulence in its eukaryotic host is greatly reduced. Furthermore, homologues of the pvf gene cluster are widespread throughout bacterial genomes, affecting a variety of different eukaryotic hosts beyond D. melanogaster. 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. Our goal 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 PPSEEN0973-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 a more quantitative assay that measured the conversion of ONPG to o-nitrophenol, a reaction catalyzed by β-galactosidase. A total of 5,500 colonies were screened, and 49 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 PVF-initiated signaling pathway.

Characterizing a novel signaling pathway in Pseudomonas entomophila: Elucidation of the signal cascade initiated by pseudomonas virulence factor (PVF) through transposon mutagenesis

255th National Meeting (2018)

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