Clustering of VCU Bacillus Phage Genomes - VCU Wiki

OutPHAGEous Discoveries
Alina Afzal, Nasser Alali, Robert Allison, Rahaf AlQahtani, Demetrius Carter, Erin Cochran, Kaivalya Dandamudi, Jordan Davis, Ryan Duong,
Shelby Edling, Samantha Foltz, Sailasya Gundlapudi, Livia Horton, Damien Islek, Deeksha Jain, Monica Jeyasankar, Michael Livingston,
Amanda Luong, Tom Mathew, Andrew Miller, Rachel Miller, Herleen Mokha, Natalia Olszewski, Bharath Peddibhotla, Aarthi Prakash, Lucas
Rizkalla, Christopher Rowe, Neha Sehgal, Yash Singh, Morgan Van Driest, Emma Weber, Erik Wolfsohn, Kristen Wade and 1Allison Johnson
Spring 2015 BNFO 252: Phage Discovery Laboratory II and 1Center for the Study of Biological Complexity
Virginia Commonwealth University, Richmond VA 23284
Abstract: Over the course of the year, our class discovered 20 bacteriophages infecting Bacillus thuringiensis subspecies Kurstaki.
These phages were purified and tested for their ability to infect and lyse (host range) 16 different strains of Bacillus bacteria. We
observed a broad spectrum of host range, with some phages infecting only a few hosts and other phages infecting many host
bacteria. We sequenced the genomic DNA of 6 of these novel phages. The genes in each of the genomes were annotated to
compare and contrast genome features. We used dot plot and average nucleotide identity analysis to form two groups of myovirus
phages in our collection. We completed comparative genomics projects examining both small and large scale genome
characteristics. We explored genome features related to host range looking for the long tail fiber and receptor binding proteins. We
examined the unique location of endolysin and holin in these genomes compared to a canonical lytic cassette, confirmed each
predicted holing has three transmembrane domains, and examined potential promoter elements to explore regulation of expression
of these proteins. A DNA Polymerase containing a Bastille-like HNH endonuclease in some phages, and an unrelated HNH
endonuclease in other phages highlights a unique recombination event in these phages. Other topics to better understand genomic
diversity include analyzing promotor sequences and sigma factor proteins to understand regulation of phage gene expression, and
comparison of tape measure proteins. Finally, we’ve characterized one podovirus low sequence similarity to any published phages.
Combined, our results show these phages represent a dynamic and and diverse collection of Bacillus phages.
Testing phage host range
Clustering of VCU Bacillus Phage Genomes
Table 1. Characteristics of 2014-2015 Bacillus Phage Genomes
Characteristic
Phrodo
Jugalone
Zuko
DIGNKC
Claudi
Troll
Troll
Megatron
Hakuna
MG-B1
% Query Cover
94
96
91
92
33
% Identity
98
99
88
87
74
37.8
37.80
38.60
38.70
30.3
164227
163345
161552
26504
293
296
296
48
0
3
3
0
0
2
?
16
Best Blast
Match
% GC
Genome Length,
164443
bp
# predicted
288
ORFs
# predicted
0
tRNAs
# novel genes
2
Twenty Bacillus phages were spot tested for their
ability to lyse 16 different strains and species of
Bacillus. All phages were discovered using B.
thuringiensis as a host. Lysis on our host bacteria is
shown on the right as an example of our testing. The
number of bacteria species/strains infected by each
virus, and the number of phages that were able to
infect and lyse each bacteria are shown below. We
found:
• The phages were able to lyse between 3 and 12
species/strains.
• Almost all bacteria were lysed by more than one
virus.
• Bacterial susceptibility to phage infection was strain
dependent.
We will use this testing to guide the future use of
bacterial strains in our course.
The major genomic features of the VCU Bacillus phage genomes are
displayed (Table 1). Dot plot, Average Nucleotide Identity
calculation and phage genome map analysis allowed us to form
clusters based on these comparisons (Table 2, Figures 1 and 2):
● Phrodo and Jugalone have high identity across their genomes
● DIGNKC, NotTheCreek, Nigalana, Zuko and Sage Fayge form a
second group of identity.
● Phrodo and Jugalone have high identity across their genomes
● Vinny is relatively unique with diffuse identity across the
genome.
● Claudi shares no sequence identity with the other phages.
Table 2. Average Nucleotide Identity of Bacillus phages from VCU
Figure 1. Dot plot comparision of VCU phage genome
sequences. Gepard was used to analyze a
concatenated file containing the genome sequences.
Can we predict phage determinates of host range?
We analyzed (by multiple sequence alignment and phylogeny) five protein families that might interact with the bacterial cell wall
based on HHPred functional prediction: Tail lysin, cysteine peptidase, tail fiber, and carbohydrate binding domain protein.
• Multiple sequence alignment showed these novel phages are quite similar to one another.
• We used horizontal branch length as a measure of difference.
• Carbohydrate binding domain protein and Tape measure/tail fiber protein were the only phamilies with significant sequence
difference between Phrodo and Jugalone, two phages with 95% identical genome sequences but very different host range.
We compared this pham to identified
homologs, and couldn’t find
conservation of chaperone cleavage site.
Claudi
DIGNKC
Jugalone
Nigalana
NotTheCreek
Phrodo
SageFayge
Vinny
Zuko
Claudi
100
55.5
55.6
54.7
54.8
55.9
54.8
55
55.2
DIGNKC
55.5
100
61.2
87.1
87.1
61.3
87.7
62.8
90.6
Jugalone
55.6
61.2
100
61.4
61.3
95.7
60.8
66.8
61.2
Nigalana
54.7
87.1
61.4
100
92.3
61.6
91.9
62.1
87
NotTheCreek
54.8
87.1
61.3
92.3
100
61.5
92.9
62.6
87.8
Phrodo
55.9
61.3
95.7
61.6
61.5
100
61
66.8
61.2
SageFayge
54.8
87.7
60.8
91.9
92.9
61
100
62.8
88
Vinny
55
62.8
66.8
62.1
62.6
66.8
62.8
100
62.4
Zuko
55.2
90.6
61.2
87
87.8
61.2
88
62.4
100
Figure 2. Genome map comparision of VCU phages. Phamerator was used to display genome maps, where colored rectangles represent protein coding genes, and shading between genomes
indidates blastn similarity.
Characterization of typical and atypical endolysin and holin casettes
Typical lytic casette
endolysin
Atypical lytic gene localization
holin
endolysin, gp59
Tape measure/Tail fiber
Tail lysin 1
holin,
gp174
Endosialidase & chaperone
A few Bacillus phages have
a canonical lytic casette
Vinny
All of our myoviridae Bacillus phages have
this atypical localization of lytic genes
Zuko
Phrodo
Cysteine Peptidase
D-alanyl-Dalanine
carboxypeptidase
Carbohydrate binding domain
Endolysin phylogeny shows
Endolysin from phages with
holin/endolysin close are
interspersed on tree
Lytic transglycolylase
Holin phylogeny shows holin from phages
with holin/endolysin close are separated
from others by common ancestor.
Their promoters are different! We
predicted the distal holin is transcribed by
a sigma 70 promoter, while the typical
lytic casette is transcribed by a middle
gene expression sigma factor.
Tail Fiber
N-acetylmuramoyl-L-alanine
amidase
NacetylmuramoylL-alanine amidase
N-acetylmuramoyl-L-alanine
amidase
B. cereus = blue
B. subtilis = yellow
B. pumilus = green
B. anthracis=orange
B. thuringiensis=purple
= Endolysin & Holin close
Thanks to
our phage friends at VCU,
VCU Life Sciences,
University of Pittsburgh
HHMI SEA PHAGES Program