1. Isolation and Annotation of Arthrobacteriophage
Stu Mair, Chrissy Sessa, Andrea Springman, Natalie Widdows
Department of Biology, Baylor University, Waco, Texas and HHMI Science Education
Introduction
In Vitro Methods
In Silico Methods
This dot plot demonstrates the similarity of the bacteriophage genomes within their cluster while still conveying the differences between the clusters. The same sequence, containing three genomes for each cluster, was used on both axes, with the significant out of cluster similarities circled.
DNA of Nubia, Shrooms, and Caterpillar were sequenced by Illumina sequencing at the Pittsburgh Bacteriophage Institute
Genomes were auto-annotated based on algorithms from Glimmer and Genemark
The calls from Glimmer and Genemark were verified or altered using after consulting the following programs:
NCBI Blast
PhagesDB BLAST
HHPred
Starterator
DNA Master ORF’s
82 soil samples were collected, filtered to isolate potential phages, and enriched with host bacteria, arthrobacter sp.
A series of plaque assays were performed and 25 plates were identified as positive
Single phages were then isolated from positive plates through multiple rounds of plaque assays, spot tests, and serial dilutions
These isolated phages were concentrated to a minimum titer of 10^7 and the DNA was extracted
Objective:
Isolate Arthrobacteriophages from soil and annotate their genomes according to the guidelines of the Science Education Alliance Phage Hunters Advancing and Evolutionary Science program.
Background:
Arthrobacteriophages are viruses that infect Arthrobacter bacteria, a bacteria commonly found in soil.
This project is a part of the SEA-PHAGE program of isolating and analyzing Arthrobacteriophages and their genomes to contribute to a national database and to increase understanding of genomics, evolution, and phage therapy.
Discussion
The discovery and characterization of these 3 arthrobacteriophages contributes valuable information to a growing body of information regarding phage genomics. Some of this information can be used to answer larger questions and some will be useful for characterizing future, such as the novel characterization of gene 52 as a NPPase, a new contribution to AL phages.
Nubia Slippage - YOU GO CHRISSY
The other element these phages demonstrate is the vast diversity that is present in phages that infect the same host. Despite all being found within 30 miles of each other, the phamerator map and the dot plot both help to demonstrate the vast differences in each of these bacteriophage genomes. This fact exemplifies why contributions to…
In Vitro Results
In Silico Results
Caterpillar
Nubia
Shrooms
Rounds of Purification 3
Plaques
.5mm, lytic plaques
1mm lysogenic plaques
.23 mm in diameter, lytic plaques
TEM
59.4nm, tail 137nm
Head .06nm, tail .220 nm
Head 55 nm, tail 120 nm
HTL (pfu/ml)
1.4 x 108
7.33 x 1010
1.2 x 109
Genome Characteristics
Caterpillar
Nubia
Shrooms
Genome Size
56622
44045
59707
Genes Annotated 88 60 98
Functions Found 19 26 31
%GC Content
51.1%
60.7%
64.5%
Phage Cluster AU AK AL
Overview
Example Annotation: Shrooms Gene 52
Location of soil samples for Caterpillar, Shrooms and Nubia
Soil Sample
The auto-annotation provided the best possible SD score for available start codons, with Glimmer and Genemark calling the same site.
The gene, in its initial form, covered the full coding potential present
NCBI gave a hit on a Hypothetical Protein from Laroye, with a Q1:S1 and E-Value of 1e-58. This hit was also seen on PhagesDB.
The function of MazG-NPPase was determined from a strong HHPred hit (E=3e-20) confirmed by a relatively weak conserved domain hit. (I want to use a photo for this)
Completed Gene Start:28395 Stop:28697 Function: MazG-NPPase
Phage Isolation
TEM Images
DNA Extraction
Acknowledgements
Caterpillar
Shrooms
Nubia
Genome Sequencing
HHMI Science Education Alliance-Phage
Hunters Advancing Genomics and Evolutionary Science program
Baylor University: Department of Biology
Phamerator Map of Caterpillar, Nubia and Shrooms
Annotation