Using a Genetic Screen to Discover Gene Functions in Mycobacteriophages Sbash and Island3

Sbash is a temperate bacteriophage that infects Mycobacterium smegmatis. It was assigned to cluster I2 based on gene-content similarity of 35% or higher to sequenced bacteriophages present in the Actinobacteriophage database, phagesDB. Its genome was annotated in 2014 and found to include 89 protein-coding genes, only 22 of which were assigned functions based on bioinformatic analysis. We are using a genetic screen to identify functions of phage genes for which no function is currently known. We cloned 40 of the genes in Sbash’s genome with sizes ranging from 90 bp to 3,666 bp. We screened each gene for cytotoxicity and identified six genes that reduced growth of the host cells when expressed. We also screened for defense, the ability of each gene product to protect the host cell from infection by another phage. We identified eight Sbash gene products that defend host cells from infection by other mycobacteriophages. We have also analyzed genes in Mycobacteriophage Island3, a cluster I1 phage, for cytotoxicity and defense to complete the screen of this phage started by students in previous research groups

Mousing Around with Bacteriophages: ​Using Antibodies Raised in BALB/c Mice to Analyze Phages​

Bacteriophages are viruses that infect bacteria and can be used in phage therapy to combat bacterial infections resistant to traditional antibiotics. One current limitation of phage therapy is the lack of understanding about phage genetics and biochemistry. We can learn more about bacteriophages by identifying patterns in their protein structure. In our experiments, we immunized BALB/c mice using whole phage or ~71\u3ekDa proteins extracted from Sbash and Island3. We tail-bled the mice to collect antibodies raised against these phages, then used ELISA assays to detect anti-phage protein antibodies in mouse serum. We are using our antibodies and Western blot analysis to understand biochemical relationships between Island3, Sbash, and several other bacteriophages that infect Mycobacterium smegmatis

Understanding the Antiproliferative Activity of Plant Extracts

Many plants possess medicinal properties. Some, such as the Pacific yew, have yielded chemotherapeutic drugs (taxanes). Scientists report that other extracts such as the leaves of Calendula officinalis (marigold), Vinca rosea (periwinkle), Viscum cruciatum (mistletoe), and Rosmarinus officinalis (rosemary) have anti-tumor activity. In most cases, the chemical components responsible for antiproliferative activity have not been identified and it is unclear if any individual components are as effective in isolation as they are in the context of the whole extract. Furthermore, in most cases, there are no data indicating whether these extracts have synergistic effects or cause negative reactions when used with other drugs. We are using HeLa (adenocarcinoma), RAW 264.7 (leukemia), HepG2 (hepatoma), MDA-MB-231 (adenocarcinoma), and human foreskin fibroblasts (HFF, non-tumorigenic) to test the antiproliferative activity of several plant extracts. We identified five extracts, grapeseed, guava, yew, juniper berry, and Vinca, that slow the growth of all five cell lines in a dose-dependent manner. We are using a variety of methods to understand the mechanism by which these extracts are blocking cell growth

Genomic Annotation of Bacteriophages Clayda5, GShelby23, Santhid, and Wrigley

We annotated the genomes of four recently discovered Actinobacteriophages. Clayda5 and GShelby23 were isolated on Microbacterium foliorum NRRL B-24224. Clayda5 is a lytic, cluster EB phage, one of only 47 discovered to date. It has 10 base pair 3’ sticky overhanging ends and a GC content is 67.2%. It has 70 protein-coding genes and two tRNA genes in its 39,894 bp genome. Clayda5 was purified from soil collected in Hull, IA. GShelby23 was isolated from soil collected in Storm Lake, IA. It is a cluster EM phage, one of only six discovered to date. Its genome is circularly permuted and 53,603 bp long. Its GC content is 64.8%. Santhid and Wrigley are phages that infect Gordonia terrae 3612. Santhid is a cluster DY phage, one of only five discovered to date. It was isolated from soil collected in Orange City, IA. Its genome is 39,295 bp long and includes 60 protein-coding genes. Its GC content is 67.7% and has 10 base pair 3’ sticky overhanging ends. Wrigley was isolated using an enrichment protocol from soil collected in Johnston, IA. It is a cluster CY phage, one of only 17 discovered to date. It is a temperate phage whose genome is 51,878 bp long and includes 81 protein-coding genes. It has 10 base pair 3’ sticky overhanging ends and a GC content of 66.3%.