Spring 2015 BNFO 252: Phage Discovery Laboratory II

Comparative genomics of Bacillus infecting phages
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
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
164443
164227
163345
161552
26504
# predicted ORFs
288
293
296
296
48
# predicted tRNAs
0
0
3
3
0
% GC
Genome Length, bp
The major genomic features of the five phage genomes annotated this
spring semester are displayed (Table 1). Average Nucleotide Identity
comparison for the Bacillus phages from VCU allows us to form clusters
based on these values (Table 2).
Table 2. Average Nucleotide Identity of Bacillus phages
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
Host range of Bacillus phages and searching for protein determinates of host range
Twenty Bacillus phages were
tested for their ability to lyse 16
different strains and species of
Bacillus.
All
phages
were
discovered using B. thuringiensis
as a host, and lysis on that bacteria
is shown on the right as an
example of our testing. The phages
were able to lyse between 3 and 12
species/strains.
Sequenced
phages are labeled with stars in the
bacterial lysis graph.
Cell Wall Hydrolase
Phage Claudi is a podovirus with a
stub of a tail. Podovirus genomes
are ~27,000 bp long, with less than
50 genes.
Functional annotation of phage Claudi.
Holin
Predicting phage sigma factor function based on sequence homology
iTasser was used to predict structure of predicted holin proteins.
Class 1 holins are typically
● 90 to 125 amino acid residues in length
● form three potential transmembrane helices
● have residues with charged or hydrophilic side chains
Endosialidase
We compared holin and endolysin proteins from a group of Bacillus phages.
Endolysin and holin are usually found in a ‘lytic casette’ with the two proteins
located close to each other in the genome. Instead, many of our phages
contain endolysin and holin at opposite ends of the genome.
In analyzing these genomes, students hoped to find the
link between gene expression in this region that related
to host specificity. In the phylogeny trees depicted above
as well as the gene map, it is evident that while these
novel phages are quite similar to one another on a
genomic level (see dot plot), a change in protein or
mutations in the amino acid sequence of one or more
genes in each genome slightly affects the host range of
a bacillus phage.
We are continuing to characterize these proteins and to
look for recombination or mutation as a clue to the
genetic determinants of host range.
endolysin
Phrodo
has
an
Polymerase gene.
intact
These DNA Polymerase
genes do not contain an
intron
DNA
B. cereus=blue
B. subtilis=yellow
B. pumilus=green
B. anthracis=orange
B. thuringiensis=purple
=Endolysin & Holin close
Endolysin
Carbohydrate Binding Domains
Typical lytic casette
Atypical lytic gene localization
We found:
● All holin proteins compare 3 transmembrane domains.
● Sequences group by the host bacteria that was used to find the
phage.
● Holins from a lytic casette are grouped with a common ancestor.
● Endolysins from a lytic casette are interspersed with endolysin
groups with distal proteins.
D-alanyl-D-alanine carboxypeptidase
Nigalana’s DNA polymerase is
interrupted by an intron encoding an
HNH endonuclease
Peptidoglycan Peptidase
holin, gp174
holin
endolysin, gp59
Exploring evolution of a DNA Polymerase and an intron encoding an HNH endonuclease
Cysteine Peptidase
Holin
Holin permeabilizes the bacterial membrane
by accumulating and forming pores. Holin
sequences were collected and compared by
multiple sequence alignment and phylogeny.
Phage sigma factors control phage and host bacteria gene expression. Our phages each contain two sigma factors. We compared our proteins to others
documented in the literature to try to predict the role of our sigma factors in regulating transcription.
Combined phylogenetic trees of potential sigma factors
● The top cluster was a collection of sigma factors believed
to be responsible for stopping sporulation, and the larger
left cluster was a collection of potential late gene
regulation sigma factors.
● SPO1_34, BCP1_199, and BCP1_200 which were collected
from the literature are relatively distant from all groups of
sigma factors on the tree, but had strong blast matches
with phrodo homologs.
● We will continue to explore variability in the -10 and -35
promoter binding regions.
Lysis of B. thuringiensis by Fall 2014 phages
We analyzed the gene families of the proteins predicted
to interact with the bacterial cell wall: hydrolase,
endosialidase, cysteine peptidase, peptidoglycan
peptidase, and a carbohydrate binding domain.
Phage genome sequences were compared by dot plot (10 bp
word size). Dot plot comparison of genome sequences shows
● Phrodo and Jugalone have high identity across their
genomes.
● DIGNKC, NotTheCreek, Nigalana, Zuko and Sage Fayge
form a second group of strong identity.
● Vinny is relatively unique with diffuse identity across the
genome.
● Claudi shares no sequence identity with the other
phages.
Phage Phrodo is a myovirus with a
long, contractile tail. Myovirus
genomes are ~ 160,000 bp, with
almost 200 genes.
DNA encapsidation
protein
Megatron
Endolysin
Troll
Lower Collar
Protein
Troll
Best Blast Match
Functional annotation of phage Phrodo.
Upper Collar
Protein
Claudi
Tail protein
DIGNKC
Capsid
Protein
Zuko
Terminal
protein
Jugalone
DNA pol type B
Phrodo
Glutaredoxin
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. Upon compiling the data 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 are now
completing comparative genomics projects examining both small and large
scale genome characteristics. These research projects focus primarily on
exploring genome features related to host range (tail fibers and base plate
proteins), understanding the unique location of endolysin and holin in these
genomes compared to a canonical lytic cassette, examining points of
recombination in order to better understand genomic diversity, and analyzing
promoter sequences and sigma factor proteins to understand regulation of
phage gene expression.
Characteristic
dUTPase
Abstract: While phage therapy might not be widely used in the United States
it has been proven successful where traditional antibiotics, such as penicillin,
have failed (Svoboda, 2009). One of the biggest benefits to studying phages
is the fact that they could be used for future medical practice to end the
“superbug” dilemma that is being caused today with many antibiotic
resistances forming. It would be beneficial to use these amazing viruses for
medical purposes because they have the ability to infect specific bacterial
cells without harming a human cell, possibly negating concerns about phages
infecting the wrong kinds of cells and causing more harm than good.
Table 1. Characteristics of Bacillus Phage Genomes
NotTheCreek’s DNA Polymerase
gene is also interrupted by an intron
encoding an HNH endonuclease,
with little similarity to Nigalana’s
intron.
Lytic transglycolylase
These HNH
endonuclease genes
are “Bastille” like, and form protein phamily
2023.
These DNA
Polymerase genes
contain an intron
N-acetylmuramoylL-alanine amidase
We are continuing to characterize the introns by Class
based on their mechanism of cleavage, and to explore
whether the DNA Polymerase might be functional.
N-acetylmuramoyl-L-alanine amidase
Endolysin
These HNH
endonuclease genes are
more diverged, and form
protein phamily 2288.
Acknowledgements
We are thankful for support from:
VCU Life Sciences for supporting the course, the VCU
Nucleic Acids Research Core and Hatfull Lab at University
of Pittsburgh for genome sequencing, and the VCU
Nanomaterials Characterization Core for electron
microscopy.
N-acetylmuramoyl-L-alanine amidase
Endolysin degrades the bacterial cell wall by cleaving peptidoglycan
bond. Endolysin sequences were collected and compared by
multiple sequence alignment and phylogeny.
Blastp was used to identify conserved domains. Endolysins contain a variety of:
● Cell wall binding domain- SH3 or PGRP
● Peptidoglycan cleaving domain (diagrammed above)