Genetic Analyses and Annotations of Two Newly Discovered C1 Mycobacteriophages

Poster presentation abstract

Isolation and Characterization of Previously Undiscovered Bacteriophages

Poster presentation abstract

Isolation and Characterization of an A4 Mycobacteriophage from Central Illinois

Sixteen mycobacteriophages were isolated by students at Illinois Wesleyan University in Bloomington IL using a soil enrichment technique and a Mycobacterium smegmatis host. Each student created and archived a high titer lysate of his or her mycobacteriophage, and of these sixteen, two were selected to be sent in for sequencing, Eidsmoe and Morrow. Morrow was found just outside the Morrow Plots at the University of Illinois at Urbana-Champaign in 2014, and was found to be one of 64 members of the A4 subcluster. Its 51,411 base pair genome is comparable to the average A4 genome of 51,395 base pairs. However, Morrow has 94 genes, which is eight more genes than the average A4 genome. Morrow was then annotated using BLASTp, Phamerator, Starterator, and DNA Master, and was found to be 98% identical to Abdiel, which was found in Missouri in 2011. The identification of the sixteen mycobacteriophages and the sequencing and annotation of two of them expands our knowledge, as well as the online database, where they are contributing to scientific research

Antiviral Properties of Fruit Juices on Bacteriophages

Viruses utilize many different mechanisms to infect their hosts and spread through populations. Because of this humans have long sought ways to treat viral infections and develop antiviral medications to stop these parasites or at least alleviate symptoms. Historically, a number of plant chemicals have been shown to have antimicrobial properties that have shown promise for these purposes. Prior to this study, cranberry juice has been demonstrated to be inhibitory for certain viruses. Over the last several years students at Illinois Wesleyan University have amassed a collection of about thirty different bacteriophages, viruses that infect bacteria. The aim of this research is to examine how widespread the effects of cranberry and other juices are on a variety of these bacteriophages. Bacteriophages from the Illinois Wesleyan University collection were exposed to different fruit juices to see how their replication was affected. The effects of juice on viral replication on a variety of virus types were examined and the results of these experiments will be discussed. This research has contributed to the overall knowledge of antivirals, and provides a new starting point for further research on the antiviral properties of juices

Exploring Phages Affecting Rhodobacter capsulatus: RcSwan and RcKeef

Bacteriophages, viruses that feed on bacteria, are recognized as the most common biological entities on the planet, with numbers believed to be as high as 1031 particles. They play a significant role in the ecological balance of microbial life. There are almost 150 clusters of known phages, with multiple sub-clusters and even singletons, each categorized based on the bacteria they infect and their genomic similarities. Through our research, we hoped to understand how two phages that infect the same bacteria, obtained under similar conditions, such as temperature, food source, and incubation time, showed various structural and functional differences. We isolated two phages infecting the freshwater bacteria Rhodobacter capsulatus, one was found to be a member of the RcC cluster, RcSwan, and the other was found to be a member of the RcD cluster, RcKeef. We noticed stark differences between the two phages in the Transmission Electron Microscopy results of both phages as RcKeef had a tail length of 221.0 nm and a capsid diameter of 74.5 nm, while RcSwan had a much smaller tail length of 120.9 nm and a slightly smaller capsid diameter of 65.1 nm. Additional differences were seen in the sequenced genome of both phages, as RcKeef had 68,260 base pairs and 102 genes, while RcSwan had only 41,417 base pairs and 70 genes. These differences in the genomes between the two phages directly influence the bacterial infection rate. Overall, the morphological and genetic differences between the two phages display some of the diversity exhibited in bacteriophages and provide some insight into the unseen pathogens found throughout our local community

World of Bacteriophages: The Discovery and Genomic Analysis of Microbacterium foliorum Phages Big4 and Potty

Bacteriophages (“phages”) are diverse and abundant viruses that infect bacteria. There are approximately 1031 phage particles on Earth with less than 2 x 103 having been sequenced. In the fall of 2021, IWU’s SEA-Lab students found 12 phages that infect Microbacterium foliorum. We will discuss two sequenced M. foliorum phages, Big4 and Potty. MfBig4 and MfPotty were discovered using soil samples collected from the IWU campus and a potted plant in a neighborhood in Bloomington, IL, respectively. MfBig4 and MfPotty both formed small and clear plaques, which are some properties of lytic phages. After comparing the capsid diameters and tail lengths from the Transmission Electron Microscopy (TEM) images and analyzing genomic sequences from the phages’ DNA samples, we predicted that MfPotty is from the EF cluster and MfBig4 is from the GD cluster. The genome of MfBig4 is significantly larger than that of MfPotty at 192,363 base pairs, with MfPotty having 56,617 base pairs. We assigned functions and annotated genes in these genomes in order to further interpret this sequence information. After analyzing the gene patterns for both phages, we compared them to others that belonged in the same cluster. MfBig4 was found to be from a rare cluster with only 5 known phages, whereas MfPotty is more common, being in a cluster with 22 known phages. Overall, identifying patterns in genomic sequences of phage genomes is essential for predicting where genes are located and understanding phage genetics and evolution

Aliso and Jasper: Analysis of an Extraordinary Marine Phage and a Fun-Size Freshwater Phage

Bacteriophages, viruses that infect bacterial cells, are thought to be the most abundant and diverse biological entities on the planet. Due to an estimated 1031 bacteriophages in the world, they can be found in nearly every environment occupied by bacteria. Their great diversity allows for new phages to be discovered each time these environments are sampled. Through the isolation and propagation of individual specimens, our research group was able to characterize two distinct phages using polymerase chain reactions, transmission electron microscopy, and genomic sequencing. Bacteriophage Aliso was found to be notably unique due to its “corn dog” shaped capsid, long tail, and marine bacterial host Ruegeria pomeroyi. This phage also forms distinctly small plaques when cultured on its bacterial host, which is likely a result of having a relatively large genome size of 149,362bp. Our other phage, Jasper, infects the host Rhodobacter capsulatus, a freshwater bacterium. It is unique amongst phages isolated this past semester due to its small capsid size and short tail length. Unlike Aliso, Jasper has a relatively small genome of 41,414bp. Through analyzing phage genomes, new strains and adaptations can be uncovered, allowing for new phages to be clustered. Aliso however is an exception to this norm and is considered to be a singleton, as it does not fit into a cluster. Interestingly, it is most closely related to another phage named Xuper which was isolated on a freshwater host. Unlike Aliso, Jasper does cluster with other known phages. It most closely demonstrates similarities with one of the more divergent members of the RcC group, Kemmy. The similarities these phages share with each other and previously isolated phages will be presented

The Isolation and Characterization of Rhodobacter capsulatus Phages Lkye and Aqua

Bacteriophages, commonly referred to as phages, are viruses that infect bacteria for the purpose of reproduction and the expansion of their species. The Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science Program, acronymed SEA-PHAGES Program, aims to increase the knowledge of the bacteriophage population through isolation and characterization of individual bacteriophages. The two phages studied were procured from water samples using the bacterial host known as Rhodobacter capsulatus. The isolated phages were identified as RcLkye and RcAqua, or simply Lkye and Aqua. Lkye and Aqua were discovered from enriched samples of water collected along Bloomington-Normal’s Sugar Creek in Illinois. The samples were collected five days apart and in separate locations. The phages were then purified via plaque assay and amplified for further analysis. It was discovered through Transmission Electron Microscopy (TEM) imaging that Lkye and Aqua were both siphoviridae phages, with similar capsid diameters and tail lengths. Based on comparisons of the sequenced genomes and TEM images of phages with confirmed clusters, it has been determined that Aqua and Lkye belong to the RcD cluster. The discovery and analysis of unique phages provide advancements in the field of microbiology and virology while potentially providing insight into combating viral diseases and infections

Exploring the World of Phages with Dora and Explorer

Bacteriophages, commonly known as phages, are viruses that infect bacteria cells. Although they are invisible to the naked eye, phages are thought to outnumber bacterial cells by a factor of ten creating a wide diversity among them. This large population of phages provides us with ample opportunities to explore the diversity of phages locally. For this project we sought to determine whether aqueous phages that infect Rhodobacter capsulatus can be found in Bloomington and if so, how are they related to one another. To test this, we acquired water samples from Miller and White Oak Parks and were able to both isolate phages that infect R. capsulatus. Both phages, RcDora and RcExplorer, underwent several tests to determine the characteristics of each virus. Transmission Electron Microscopy (TEM) analysis allowed us to calculate the sizes of their capsids and tail lengths which were found to be similar for both phages. Polymerase Chain Reaction (PCR) tests further suggested that both may be members of the RcD cluster. Ultimately full sequencing of their genomic DNA revealed that these two phages have a 99% match rate between their genomes indicating that while they are highly related, they are not identical. The research we performed has now added two new unique RcD phages to the growing collection at IWU. This continues to make this cluster the most highly represented in the collection but also helps to provide insight into viral diversity and evolution