Analysis of a Putative Promoter in Mycobacteriophage JacoRen57

JacoRen57 is a cluster AB mycobacteriophage that infects Mycobacterium smegmatis mc²155. We recently reported on the characterization of a putative promoter in JacoRen57 using an mCherry reporter construct. This promoter is present in a gap upstream of a gene that is present in all AB phages. In all cases, these are forward genes immediately following a long series of reverse genes. The genes are most frequently identified as a RecA-like DNA recombinases but also as RepA by bioinformatics. To further analyze this putative promoter and gene product, NWC Molecular Genetics students cloned the RecA-like DNA recombinase into an E. coli expression vector with a TVMV removable N-terminal His-tag. They expressed and we purified the tagged protein and are using it to immunize Balb/c mice. We plan to use the antiserum to confirm RecA-like DNA recombinase expression patterns when JacoRen57 infects M. smegmatis

Exploring Gene Functions and Phage-Host Protein Interactions in Mycobacteriophage Island3

Island3 is an I1 mycobacteriophage that infects Mycobacterium smegmatis mc²155. It has a total of 76 protein coding genes, but only 17 of these genes have functions assigned by bioinformatics. To discover the functions of the additional genes, we cloned 72 of Island3’s genes and are assaying each gene product for two functions when expressed in the host M. smegmatis: the ability to reduce growth of the host (cytotoxicity) and the ability to protect the host from infection by Island3 or another phage (defense). So far, we have assayed more than 60 of Island3’s genes and found 14 genes that exhibited cytotoxicity but none that exhibited definitive defense against phage infection. We are currently analyzing the remaining genes for cytotoxicity and defense. In addition, we are moving forward with bacterial two-hybrid assays on two of the genes that exhibited cytotoxicity, seeking to identify host proteins that interact with the cytotoxic phage gene products in an attempt to understand the mechanism of cytotoxicity.

Stormbreaker8 and A3Wally Bacteriophage Genome Annotations

Stormbreaker8 and A3Wally are two novel bacteriophages isolated and purified on Microbacterium foliorum NRRL B-24224 by students in the Fall 2020 Discovery course. Stormbreaker8, an EA1 cluster lytic phage, was isolated from soil collected in Orange City, IA. Its circular permuted genome contains 41,751 base-pairs with 63.4% GC content. A3Wally, a GD cluster phage, was isolated from soil collected in Sioux Center, IA. Its genome is 60.1% GC, contains 194,724 base-pairs, and its ends are direct terminal repeats. Spring 2021 Genetics students annotated the genomes using bioinformatics software

Investigating the Putative RecA-Like Recombinase Gene

Our Biochemistry: Molecular Genetics class has partnered with the Immunology class to investigate the expression of JacoRen57’s gene 50. The bacteriophage JacoRen57 – found in Sioux Center, Iowa (accession: MK279840). JacoRen57’s genome has sequenced by Pittsburg SEA-PHAGES Institute and fully annotated by Northwestern College students in 2018. A region between gene 49 and 50 caught our attention as there is a large gap between these genes. Almail et al., investigated if this is a transcription regulatory region for genes 49 and/or 50 (2021). This work demonstrated the region has a regulatory function in the direction of gene 50. Based on comparison genomics, gene 50 is a putative RecA-like recombinase (Almail et al., 2019). This protein has several functions including guiding the recombination of DNA within a gene. RecA-like recombinase allows the virus to evolve into new variants which can improve infection and replication. This is crucial for creating diversity in the genome and DNA repair mechanisms (Galletto and Kowalczykowski, 2007). To continue examination of gene 50 expression, we are working towards developing antibodies for this protein. To do this, the first step is to create an expression construct (Figure 1), express the protein in bacteria, purify the protein, and then use the purified protein to inoculate mice. This poster describes the construction of the expression vector. This work will provide valuable insight into the expression of gene 50, the RecA-like recombinase