1. Phenotypic & Genomic Characterization of a novel isolate
Mycobacteriophage Horchata
Iger Ostreni1
, Eric Zhou1
, Eduardo Silva1
, Elizabet Cabrera1
, Khristina Ipapo1
, and Jordan Moberg Parker1
1
Department of Microbiology, Immunology, & Molecular Genetics University of California Los Angeles CA 90024
BACKGROUND METHODSABSTRACT
DISCUSSION
FUTURE DIRECTIONS
REFERENCES
ACKNOWLEDGEMENTS
HYPOTHESES
1 2 3 4 5
RESULTS
6
7
8
9
DOT PLOT
1) Soil Sample attained
2) Spot test done to find putative
phage.
3) Serial Plaque Assay done to isolate and
purify phage.
4) Midi-Lysate was produced for
higher concentration in Electron Mi
croscopy imaging and phage DNA puri
fication.
5) Electron Microscopy done to look at
phage morphology
6) Phage DNA was isolated
7) PCR for multiplication of DNA.
8) DNA sent for pyrosequencing
9) Genomic Sequence attained
10)Annotation of phage genome done
through DNA Master.
10A) Comparative genomics analysis
(Dot Plot and Tree Maps).
10B)Produced annotated genome
showing localization and function of
genes.
10A
10B
PHYSICAL CHARACTERISTICS
PHYLOGENETIC TREE
A B
C D
10
• The Phage Hunters project aim is to discover and characterize novel bacterio-
phages via experimental and analytical methods.
• Bacteriophages are viruses that infect bacteria
• Lytic phages infect and lyse bacteria; Lysogenic phages integrate into
host genome and may adapt the lytic cycle
• Abundance and ubiquity of phages affects environment stability and hu-
man health
• Potential use of bacteriophages as alternative therapeutic agents
• Model Organism: Mycobacteria smegmatis
• Non-pathogenic, gram-positive, fast growing
• Closely related to pathogenic strains, M. tuberculosis and M. leprae
Part 1: If a novel mycobacteriophage can be isolated and cultivated from a soil
sample in a laboratory setting, then the plaque morphology, phage morphotype
and viral genome will demonstrate the phage’s novelty.
Part 2: If a novel phage is successfully isolated, then annotating the genome
will reveal lysogenic, lytic, functional and potentially virulent genes that can be
used to assess the phage’s potential to be used in phage therapy.
• Evidence of evolution in phage Horchata is evident in the transient plaque mor-
phology.
• Optimal growth conditions are attained with the presence of cations, especially
Ca2+
.
• Electron microscopy confirms isolation of a single phage as consistent phage
morphology is observed.
• Mycobacteriophage Horchata exhibits Siphoviridae morphotype with an icosa-
hedral head and extended non-contractile tail.
• Horchata genome is 67,863 bp, including 30 genes with putative functions and
71 unknown genes.
• Unique genes, such as Tor inhibition protein, suggest Horchata is a temperate or
lysogenic phage.
• Phage Horchata belongs to subcluster B1 and holds strong resemblance to other
phages in the subcluster.
Genome annotation using non-curated databases and wet lab experimentation
• Curate phage genome through wet lab experimentation
• Knockout experiments to confirm gene function calls
• Study Horchata genes in other genomes via knock-in experiments
• Investigate possible acquisition of pathogenicity genes
Phage Therapy
• Test combined use of antibiotics and phage Horchata on M. smegmatis in vivo
• Investigate infectivity of phage Horchata on M. tuberculosis
A special thanks to Dr. Jordan Parker for her patience and help throughout the
project. The project would have been lost without her constant dedication and
support. We greatly appreciate Khristina Ipapo and help and her astute ability to
guide us through the struggles and challenges we faced in the lab. Thank you Dr.
Kris Reddi for your support throughout the isolation of our phage. This work was
supported by HHMI Education Grant Award (No. 52006944).
Ackermann HW. 2007. 5500 Phages examined in the electron microscope. Arch Virol 152:
227-243.
Lu TK and Koeris MS. 2011. The next generation of bacteriophage therapy. Current Opinion in
Microbiology 14: 524-531.
Stella EJ, Franceshelli JJ, Tasselli SE, and Morbidoni HR. 2013. Analysis of Novel Mycobacte
riophages Indicates the Existence of Different Strategies for Phage Inheritance in My
cobacteria. PloS ONE. 8: 56384
Williamson KE, Schnitker JB, Radosevich M, Smith DW and Wommack KE. 2008. Cultivation-
Based Assessment of Lysogeny Among Soil Bacteria. Mi crob. Ecol. 56: 437-447.
PLAQUE ASSAYS CATION EXPERIMENT
FIGURE 1: Purification plaque assays develop lysogenic phage. Plaque size de-
creases throughout each plaque assay and slowly adapt bulls-eye morphology.
A)Turbid/Cloudy Plaques B) Cloudy Plaques C) Bulls-Eye/Cloudy D) Bulls-Eye
3.70E+10
6.10E+10
4.20E+10
0
0.00E+00
1.00E+10
2.00E+10
3.00E+10
4.00E+10
5.00E+10
6.00E+10
7.00E+10
MgCl2,
CaCl2
CaCl2
MgCl2
No
Ca5on
Average
Titer
(PFU/ml)
Effect
of
Different
Ca5ons
on
Average
Titer
FIGURE 3: Efficient Horchata proliferation depends on Ca2+
. Plates
containing Ca2+
produce significantly higher titer. Slightly lower titers ob-
served in plates containing both cations. No plaques present in the ab-
sence of cations.
TRANSMISSION ELECTRON MICROGRAPH
FIGURE 2:
TransmissionElectronMicros-
copy (TEM) taken at CNSI at
UCLA by Philips CM120 re-
veals siphoviridae morpho-
type. TEM image of a single
phage is taken at 27,000X
magnification showing consis-
tent phage particle structures.
Phage seems to have an ico-
sahedral head and a long
non-contractile tail. Estimated
head diameter and tail length
are 65.881 nm and 305.989
nm, respectively. Many de-
capitated and broken phage
as a result of rough handling.
GENOME MAP GENE COMPOSITION
FIGURE 6:
Dot Plot Analysis of Horchata ge-
nome reveals similarity among the
B1 Cluster. Comparison with these
phages in subcluster B1 shows
strong similarity. Small breaks at
the end of the genome may be
due to unique smaller genes with
unknown function. This unique ge-
nomic region differentiates phage
Horchata from the other phages in
the B1 subcluster.
FIGURE 7: Phylogenetic Tree Map utilizing RuvC Resolvase showing it’s essential for phage proliferation. Phylogenetic analysis of RuvC Resolvase
gene indicates gene conservation. Resolvase gene is found and conserved in Phage Horchata, Soto, JAMal and SDCharge11.
FIGURE 5:Genomic composition of phage Horchata. Horchata genome
contains 70% unknown genes and 30% putative genes.
Pathogenic mycobacteria, such as M. tuberculosis and M. smegmatis, are
becoming more resistant to antibiotic treatments. Bacteriophage therapy is
a novel approach that is being investigated in treating these diseases which
uses phages to lyse pathogenic bacteria. To contribute to this research, phage
Horchata was isolated and its genomic and morphological properties were
analyzed to determine its potential use as a treatment phage. Phage obser-
vation under transmission electron microscopy revealed Horchata to have
an icosahedral head and long non-contractile tail characteristic of Siphoviri-
dae morphology. Genome sequencing by 454 Pyrosequencing and subse-
quent gene location calls by using Glimmer and GeneMark revealed Horcha-
ta to have 99 putative functional genes. Analysis of the putative functions of
these genes were made using software which were PhagesDB, HHPRED,
and NCBI revealing the possible function of 30% of these genes. The catego-
ries of these genes were DNA packaging/synthesis, structural, lysins, mRNA/
tRNA modifications as well as others with unknown benefit to phage prolif-
eration such as genes relating to galactose metabolism. PCR analysis re-
vealed association to B1 cluster which was further supported by Dot Plot
analysis comparing genomes to other B1 cluster phages. To gain a better
understanding of conditions controlling phage life cycle, growth under differ-
ent cation conditions was done which revealed Ca2+
to play an important role
in converting lysogenic to lytic cycle. Following this research, future direc-
tions would be to start analyzing host range as well as determining which un-
known genes contribute to host lysis to better fight pathogenic mycobacteria.
FIGURE 4: Annotated Horchata genome map. Genome consists of 67, 863 bp encompassing 99 total genes (2 were deleted from total). Most genes with unknown function
are smaller and lie at the end of the genome.