Biochemical characterization of bacteriophage IPhane7 repressor proteins

Bacteriophages are viruses that infect bacteria and can utilize two different pathways after infection; the lytic or lysogenic cycles. During the lytic cycle, the bacteriophage hijacks the bacterial machinery to replicate itself and destroy the bacterial cell. During the lysogenic cycle, the viral genome integrates into the bacterial genome, and the bacterium reproduces normally. The lysogenic cycle continues until a stimulus causes the virus to reenter the lytic replication cycle. Temperate bacteriophages use both the lytic and lysogenic cycles. Bacteriophage diversity is immense, and they are currently loosely classified into different genetic clusters based on sequence and gene content similarity. Cluster M bacteriophages are temperate and can be divided into 3 subclusters M1, M2, and M3. IPhane7 is a cluster M1 bacteriophage that was discovered at WCU in 2016. Temperate bacteriophages typically encode a repressor gene. The repressor protein binds to the viral genome during lysogeny and prevents expression of genes vital to lytic replication. The purpose of this project is to begin characterization of a novel cluster M repressor system. While cluster M bacteriophages have been shown to be temperate, bioinformatic analysis failed to provide a repressor gene candidate. A repressor gene candidate (gene 1) was identified using an overexpression screen by undergraduate student Erin Cafferty. Interestingly Erin also determined that gene 2 may also be playing a role in repression of select cluster M bacteriophages but is toxic during overexpression assays. Initial work using a single copy expression assay and a panel of cluster M viruses has established that gene 1 and gene 2 likely synergize during lysogeny to repress lytic gene replication. To begin biochemical characterization of IPhane7 gene 1, a solubility test has also been performed. The results of this test revealed that IPhane7 gene product 1 will likely be able to be expressed and purified and is soluble under conditions tested. Due to the Coronavirus pandemic, further biochemical characterization work will be a future direction of this project. Repressor proteins typically bind to specific DNA sequences located near promoters. In order to bind specific DNA sequences, repressor proteins typically contain a helix-turn-helix DNA binding motif. A detailed bioinformatic analysis was performed on cluster M genomes, as well as gene products 1 and 2. Cluster M genomes were searched for predicted promoters, terminators, and repetitive elements. This analysis helped reveal potential areas of the genome that are likely subject to repressor regulation and binding. Structural prediction programs were used to predict the 3-dimensional structure of gene products 1 and 2. Detailed amino acid analysis were then performed to determine the conserved areas of these gene products that are likely essential to their function. Gene 1 is unique to cluster M bacteriophages. However, preliminary BLAST analysis of gene 2 has revealed gene 2 is found in other bacteriophages and in a wide range of Actinobacterial species and may have been taken from one of these hosts