Regulation of f237 filamentous prophage excision and replication
Date
2020
Authors
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Publisher
University of Delaware
Abstract
Vibrio parahaemolyticus is a seafood-borne enteric pathogen found in marine environments, associated with fish and shellfish. A genetic marker for V. parahaemolyticus pandemic strains is the presence of a filamentous prophage f237 (also known as VfO3K6). The f237 prophage integrates site specifically via recombination into the dif site (attB site) on chromosome 1. f237 reproduces through episomal replication after excision from the chromosome. In this thesis, we examined the role of quorum sensing (QS) regulators on the replication and excision of f237 prophage. Quorum sensing is a bacterial regulatory system that allows cell to cell signaling via small molecules known as autoinducers. The level of autoinducer correlates with changes in cell density that result in a signaling cascade that activates and deactivate LuxO, the QS response regulator. In a ΔluxO mutant, the high cell density master regulator OpaR is constitutively expressed. In this mutant, most f237 genes were significantly upregulated. We performed PCR assays to examine the presence of the attachment sites of both integrated (attB) and excised (attP) forms of f237 prophage in wild type, luxO and opaR mutants. This analysis showed that the levels of attB and attP of f237 increased under high cell density conditions when OpaR is present. Whereas, we observed a reduction is attB and attP levels in the opaR deletion mutant suggesting it is a positive regulator of f237 excision and replication. We examined the binding of OpaR to the regulatory regions of f237 and showed binding in the promoter region of ORF10 and ORF1. Lastly, we showed that ORF1, the homolog of RstA, a phage replication protein, is required for the replication of f237. This is the first instance of direct regulation of a filamentous phage by a host quorum-sensing regulator.
Description
Keywords
Filamentous phage, Phage replication, Quorum sensing, Vibrio parahaemolyticus