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Description
Project 1: Our lab has screened synthetic peptide libraries to find peptides that trap Holliday junction intermediates. Out of those, the peptide (wrwycr)₂ binds to Holliday junction intermediates and inhibits resolution. It is also a potent antimicrobial. It was further tested in more detail for its activities in Gram negative bacteria. Herein, we explored its effects on the cell membrane and observed that the peptide leads to disruption of osmoregulation in E. coli. Cytoplasmic ATP, NAD⁺, and NADH levels were quantified and the levels dropped after treatment with the peptide. While investigating the mechanism of membrane depolarization, we discovered a drop in cytoplasmic pH. Because acidification of the cytoplasm may lead to depurination and depyrimidation, we tested and found that E. coli mutants defective in the repair of abasic sites are hypersensitive to the peptide. These findings show the peptide’s activity is multipronged. Project 2: Bacteriophages (Phages) are highly abundant in the marine environment and play a critical role in contributing to dissolved organic matter in the ocean. Understanding the role of phages in their environment is stymied, however, by the large fraction of virus-encoded genes with unknown function. We have synthesized, cloned and expressed unknown predicted non-structural phage genes in E. coli. We have developed a stress screen assay using multi-phenotypic assay plates (MAPs) to determine whether cells expressing single phage genes have a growth advantage or disadvantage when exposed to an environmental stress. (from the E. coli ASKA and GOS study¹). Physiological changes were compared using metabolomics. Putative unknown phage genes that showed similar growth characteristics to reference genes were investigated further. We have observed that the phage gene expressed in strain EDT-5953, encoded by a phage present in the benthic water from Starbuck in the Southern Line Islands showed some growth properties similar to those of the transcription dual regulator fur of E. coli. Thus, we have described a protocol encompassing the use of stress screens in combination with metabolomics and MAPs to characterize the putative function of unknown phage genes.
