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Thrombospondin-1 (TSP1) is a large modular matrix protein containing three identical disulfide-linked 180-kD chains that inhibits neovascularization in vivo (Good et al., 1990). To determine which of the structural motifs present in the 180-kD TSP1 polypeptide mediate the anti-angiogenic activity, a series of protease-generated fragments were tested using several in vitro and in vivo assays that reflect angiogenic activity. The majority of the anti-angiogenic activity of TSP1 resides in the central 70-kD stalk region which alone could block neovascularization induced by bFGF in the rat cornea in vivo and inhibit both migration in a modified Boyden chamber and [~H]thymidine incorporation stimulated by bFGF in cultured capillary endothelial cells. Although TSP1 has been shown to bind active TGF/31, this cytokine could not account for the inhibitory effects of the stalk region of TSP1 on cultured endothelial cells.

We plan to print our own poster.

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.

We isolated and purified Tedro and BAjuniper which infect the host Microbacterium foliorium. Tedro is a lytic, cluster EF phage isolated from soil collected in Hawarden, Iowa. Its genome is 56,197 bp long, circularly permuted, and includes 83 protein-coding genes and no tRNA genes. We are examining two of Tedro’s genes, genes 56 and 57, both of which are predicted to encode a DnaE-like DNA polymerase III (alpha) in more detail. Tedro_57 is twice as large as Tedro_56 so we are using additional bioinformatic tools to understand these genes. BAjuniper was isolated from soil collected in a garden in Orange City, Iowa. Its genome is 41,985 bp long. It was assigned to cluster EB. BAjuniper’s genome includes one tRNA gene and we will finalize BAjuniper’s annotation shortly.

We discovered two novel Mycobacteriophagesfrom soil samples. JacoRen57 is a singleton, most closely related to cluster AB phages. DrLupois a member of the rare H2 sub-cluster containing only one other member. Both phages exhibit a siphoviridaemorphotype, double-stranded DNA genome, and a non-contractile tail. The genome of JacoRen57 is composed of 52,411 base pairs with a 56.7% GC content and includes genes and plaque morphology that suggest a lytic life cycle. The genome is organized according to the following order from the left to right arm: 33 forward genes followed by 16 reverse genes and 24 forward genes. We identified functions for 33 of the 73 protein coding genes using bioinformatics software. The 70,030 base-pairgenome of DrLupohas a 57.5% GC content and contains genes that support a lytic life cycle. We identified 110 forward genes and assigned functions to 25 of them.

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%..

We annotated the genomes of three recently discovered bacteriophages to learn more about their genetic composition. MScarn is a lytic bacteriophage that infects Gordonia terrae 3612. It was discovered and purified from soil collected in Iroquois, SD. MScarn is a cluster CT phage, one of only 37 discovered to date. Its genome is 45,677 base pairs long and has 10-nucleotide 3’ sticky overhanging ends. Its GC content is 60.3% which is typical of CT cluster members. Knocker is a cluster B9 phage that was isolated on the host Mycobacterium smegmatis mc²155 from soil collected in Watertown, SD. Its circularly permuted genome contains 71,459 base pairs, and it has a high GC content of 69.7%. Similar to the other three members of the B9 cluster, it exhibits a lytic life cycle. Neos5, a lytic bacteriophage, was also isolated on Mycobacterium smegmatis mc²155 from soil collected in Baltimore, MD. It is a cluster B3 phage with a circularly permuted genome of 68,886 base-pairs and a 67.5% GC content, synonymous to the other 37 members of the cluster. All three phages were discovered, purified, and annotated by Northwestern College students.

In 2018, the World Health Organization identified cancer as responsible for 1 in 6 deaths globally, encouraging the exploration of creative forms of cancer treatment. Our research interests include identifying anti-proliferative plant extracts and illuminating mechanisms of growth inhibition. In this study we measured the effects of Vitis vinifera (grapeseed), Juniperus communis (juniper berry), and Curcuma longa (turmeric) extracts on the proliferation of several normal and tumorigenic cell strains and lines (HFF-S2, HeLa, HepG2, MDA MB 231, and RAW 264.7). We performed dose response CyQUANT assays to analyze the effects on cell growth. We are using DNA analysis, recovery experiments, and DAPI staining to investigate whether our extracts induce apoptosis in these cells. Our results showed dose-dependent inhibition of cell growth in vitro with all three extracts but not with extracts from other plants. The results of this study suggest the potential for further study of components of grapeseed, juniper berry, and turmeric extracts as potential chemo preventive agents.

Island3 is a temperate Cluster I1 mycobacteriophage that infects Mycobacterium smegmatis mc2155. Its genome consists of 76 protein-coding genes, only 17 of which have known functions. Towards the goal of identifying additional gene functions, we amplified, cloned, and assayed 14 genes for host cytotoxicity and the ability to render the host resistant to infection by other phages (defense). We analyzed genes 10, 11, 12, 13, 14, 15, 21, 22, 25, 50, 51, 57, 60, and 61 and concluded that none of these genes exhibited either host cytotoxicity or defense against phage infection. We are in the process of assaying the remaining genes of Island3.

Plant extracts are an untapped source of medicinal potential. Even today they are used as standalone treatments and applied alongside conventional therapies. The focus of our laboratory is to identify plant extracts exhibiting antiproliferative activity in vitro, to determine which chemicals are responsible for this activity, and to elucidate mechanism(s) by which growth is slowed/inhibited by plant extracts. Specifically, we exposed five cell lines/strains to twenty-two plant extracts and measured cell proliferation. Extracts from Vinca, Juniper Berry, Guava, Grapeseed, and Yew slowed the growth of all five lines/strains in a dose dependent fashion. We are working to understand the mechanism of antiproliferation by measuring induction of apoptosis, effects on microtubule assembly, and wound healing.

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.

Phages are abundant particles that infect bacteria. For the SEA-PHAGES program, students discover phages and annotate their genomes. Throughout the annotation process, genes are identified based on bioinformatics evidence; however, little is known about mycobacteriophage promoters as they are not annotated. Promoters are necessary for gene expression, and in mycobacteriophages, a promoter typically precedes a series of genes that are expressed as a single transcript from which multiple proteins are translated. JacoRen57 is a singleton mycobacteriophage with a siphoviridae morphotype that possesses forward and reverse genes with gaps located at the transitions from forward to reverse genes. We hypothesized that these gaps contain promoters. We used BPROM and PePPER, prokaryotic promoter predictor software, which yielded matches to promoter consensus sequences in one of the gap regions. We cloned the putative promoter region into pLO86, a vector containing the mCherry reporter gene, to determine if the cloned region functions as a promoter by inducing mCherryexpression in Mycobacterium smegmatis. The putative promoter region did not function as a promoter in vivo under standard M. smegmatisgrowth conditions.

We plan to print our own poster., 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.

Mycobacteriophages are infectious particles that infect mycobacteria, and little is known about cis-regulatory elements that control their gene expression. In phage genomes, cis-regulatory elements commonly precede a series of genes that are expressed as an operon. JacoRen57 is a cluster AB mycobacteriophage that possesses forward and reverse genes with non-coding gaps interspersed throughout its genome. We assayed one of the gap regions of JacoRen57 (40644-40974 bps) for regulatory activity in the downstream direction when present in its host, Mycobacterium smegmatis, by cloning the region into pLO86, a vector containing the mCherry reporter gene. The putative regulatory region induced the expression of mCherry in vivo, indicating the presence of a promoter in this region of the JacoRen57 genome. Utilizing 5’ deletions analysis, we identified promoter and repressor elements within this regulatory region. We are conducting further experiments to understand the characteristics of the repressor region and which sigma factor/s binds to this promoter.

Sbash is a temperate bacteriophage, which was isolated on the host, Mycobacterium smegmatis mc2 155 from soil collected in South Africa. It is classified as cluster I and sub-cluster I2. Its genome consists of 55,832 base pairs and 89 protein-coding genes of which only 25 genes were assigned a function by bioinformatic analysis. We are using a genetic screen to uncover the functions of phage genes for which function is unknown. To begin to uncover the functions of the protein products of the genes in Sbash’s genome, we cloned each gene into the pExTra plasmid and assayed each phage gene for two phenotypes: cytotoxicity, the ability to interfere with host cell growth, and defense, the ability to protect the host cell from infection by other phages. In total, we successfully cloned approximately half of the genes in Sbash’s genome with sizes ranging from 90 bp to nearly 1500 bp. We identified two Sbash genes that defend host cells from infection by other mycobacteriophages. We identified six genes that reduced the growth of host cells when expressed. Here, we report our progress on this project. 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.

We annotated three newly discovered bacteriophages. TukTuk and Shamu were isolated on the host Microbacterium folorium and Megatron06 on Mycobacterium smegmatis. Based on based on gene-content similarity (GCS) of 35% or higher to sequenced bacteriophages present in the Actinobacteriophage database, phagesDB, TukTuk was assigned to cluster EB, Shamu to cluster EA, and Megatron06 to Cluster H1. Here we report a summary of our annotation findings along with one in-depth analysis of an aspect of our annotation for each phage.

We used three bacterial hosts: Mycobacterium smegmatis, Microbacterium foliorum, and Gordonia terrae, to isolate novel bacteriophages from soil samples. We named these phages, created high titer lysates, and purified their DNA genomes. We have archived the high titer lysates at Northwestern College and the University of Pittsburgh. The genomes of three of our phages were sequences at the University of Pittsburgh and we will be sequencing the remaining genomes this summer. Additionally, we are planning to image our phages with transmission electron microscopy at the University of Iowa or Nebraska yet this semester.

Bacteriophages (phages) are viruses that infect bacteria. They have a considerable effect on ecosystems (Focardi et al., 2020). Scientists believe phages are present virtually everywhere, although marine bacteriophages have remained largely undiscovered and understudied. Using ocean water samples from various parts of the United States (Figure 1), protocols adapted from the SEA-PHAGES program (site Discovery Guide), and a flocculation protocol (John, et al 2011) we are attempting to isolate bacteriophages that infect two species of marine bacteria, Marinobacter hydrocarbonoclasticus and Marinobacter atlanticus.