Identification of a novel transcriptional repressor gene in Kumao, a genetically unique bacteriophage

WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
Lori Marie Neri (Creator)
Western Carolina University (WCU )
Web Site:
Maria Gainey

Abstract: Kumao is a genetically unique bacteriophage that infects Mycobacterium smegmatis (M. smegmatis) and was selected for further study because of its noncanonical genome arrangement. As an example, integrase genes are typically located at the center of Mycobacteriophage genomes. However, Kumao and cluster M bacteriophages contain an integrase gene located on the right side of the genome. Kumao and cluster M bacteriophages also contain a leftward transcribed operon containing genes of unknown function directly upstream of their structural genes. Kumao lysogens were successfully generated, proving that Kumao is indeed a temperate bacteriophage, but bioinformatics has failed to reveal an obvious repressor gene candidate. Repressor genes are typically located at a transcriptional directional change and are the first gene of a leftward transcribed operon. Repressor genes are also usually located near the integrase gene and contain an easily predicted helix-turn-helix DNA binding motif. Current work is focused on identifying the location of Kumao’s repressor gene using two different functional screens. The first involves overexpressing individual Kumao genes in M. smegmatis and performing a viral challenge experiment. If the gene is the repressor then productive viral replication should be inhibited. This approach was recently used to identify the repressor gene of cluster M bacteriophages, gene 1. Gene 1 is located at the end of the leftward transcribed operon upstream of the structural genes. Twenty-one Kumao genes were selected for testing, based on their proximity to the integrase gene, nearness to a transcriptional direction change, or potential promoter region. Excitingly, the results reveal that genes 5 and 44 have been found to be promising repressor candidates. The second approach involves CRISPRi of Kumao’s operons during lysogeny. If the operon containing the repressor is silenced, Kumao should reenter the lytic replication cycle. CRISPRi vector constructs that will target Kumao’s operonsand a positive control targeting IPhane7’s repressor operon have been successfully generated. The CRISPRi system constructs were validated using genes 1 and 2 from IPhane7. However, further testing is needed to optimize the CRISPRi system.

Additional Information

Language: English
Date: 2020
Bacteriophages -- Genetics
Bacteriophages -- Genetics -- Testing
Repressors, Genetic -- Analysis
Viral genetics
Molecular genetics
CRISPR (Genetics)

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