Searching for Atlanticus: Isolating Bacteriophages Infecting Marinobacter

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

Analysis of Alternative Storage Conditions for DNA Recovery from Field Samples

As ecologists increasingly employ molecular methods, they find that tried and true preservation solutions (e.g. ethanol or formalin) may not be optimal when samples are targeted for genetic analyses. Before traveling to remote sample sites, researchers need to consider which preservation methods are likely to yield the largest quantity and highest quality of DNA based on their travel times and field conditions. They also need to consider whether they will have access to preservatives at remote sites and whether those preservatives can be safely transported. To determine which preservation methods would most reliably preserve tissue for genetic analysis under a range of field conditions, we examined total DNA recovery from female fruit flies (Drosophila melanogaster) individually held in various solutions (70% ethanol; 2% SDS, 100 mM EDTA; 1% SDS, 50 mM EDTA; 0.66% SDS, 33 mM EDTA; Zymo© lysis buffer; Zymo Xpedition© lysis buffer) at three different temperatures (22oC, 4oC and -20oC) for varying lengths of time (1 day, 4 weeks, and 8 weeks). We predicted that insects held in Zymo Xpedition© buffer would yield the overall highest DNA recovery since this buffer was designed for field collected animal tissue. We also predicted that variation in DNA recovery from insects held in different solutions would increase with preservation time and holding temperature. Although we observed significant differences in total DNA recovery from some of our samples, no trends were identified. Preliminary band quality analyses of PCR products utilizing stored DNA as template for amplification of the mCOI gene generally indicated decline in product quality as storage time increased. Future work will focus on better quantifying stored DNA quality and examining the relationship between total DNA recovered and overall DNA quality

Investigating Gene Functions in Mycobacteriophage Island3

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

Exploring a Transcriptional Regulatory Region in Mycobacteriophage JacoRen57

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

Discovering Gene Functions in Mycobacteriophage Sbash Using a Genetic Screen

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

Exploring a Putative Promoter Region in Mycobacteriophage JacoRen57

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

Annotation of Two Soil Mycobacteriophages: JacoRen57 and DrLupo

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

Elucidating Antiproliferative Mechanisms of Grapeseed, Guava, and Juniper Berry Extracts

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

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

Phun With Phages: Discovering Novel Bacteriophages in the Soil

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