ISOLATION, PURIFICATION, AND CHARACTERIZATION

OF THE MYCOBACTERIOPHAGE EPOCH

GUSTAVO MIRALDA, DEMING, K.E., HUGHES, L.E. AND R.C. BENJAMIN
University of North Texas
The novel mycobacteriophage Epoch was isolated from an environmental sample
collected under a tree in a residential front lawn through a process known as enrichment,
using Mycobacterium smegmatis as the host bacteria. The initial plate possessed plaques of
three different morphologies and Epoch was isolated as a unique phage following several
rounds of plaque purification. The mycobacteriophage was then characterized using several
different methods. These included plaque morphology, restriction digestion, gel
electrophoresis and electron microscopy. Based upon the results of these studies Epoch has
been given a preliminary assignment to mycobacteriophage group A5. This study was
conducted as part of the Howard Hughes-sponsored National Genomics Research Initiative.
The focus of this initiative is the isolation of a large numbers of unique mycobacteriophage
from the environment and an assessment of the diversity of characteristics, both physical and
at the genomic level, of mycobacteriophages worldwide. Long term goals of the project are
expected to include benefits to phage scientists as well as other researchers in the fields of
healthcare, environmental/ecological applications, as well as biomedicine.
Epoch has the characteristic features of the siphoviridae virus family, which include
a isometric head/capsid and a flexible noncontractile tail. As of early 2011, over
90% of known mycobacteriophages are categorized in the siphoviridae family. The
genome of Epoch, estimated to be 52 kilobases in length, likely contains at least 70
genes and this group has a guanine/cytosine content of at least 52%. Plaques
produced in the early protocols of serial dilutions did not demonstrate the same
characteristics as shown in Figure 2. As noted previously, the plaque type we
classified as A was chosen for purification from the original enrichment plate.
Interestingly, the size of the plaques produced by this phage increased as the phage
purification process progressed. What began as a uniformly turbid plaque attained
the characteristics of the halo plaque morphology (lytic inner circle, turbid outer
ring) by the high titer lysate stage of purification. Thus, although Epoch
demonstrated a traditional lysogenic life cycle at early stages, it appeared to be
more lytic when phage were present at high numbers on the plate. It is thought
that this change may have occurred due to the under favorable conditions for the
mycobacteriophages or host cells that would be found at the centers of large plaques
under web plate conditions.
The calculated DNAyield for Epoch is on par with the results obtained with other
mycobacteriophages within the Acluster. Phages within the Acluster range from a
genome size between 47 to 54 kilobases and this is again consistent with our
findings for Epoch. Other characteristics of the Acluster include a GC percentage
between 59 and 65, as well as a range of total genes from 75 to 104. Based upon the
restriction analysis of Epoch, it was concluded that the results best fit those of the
A2 subcluster. This includes a lack of EcoRI and HindIII sites as well as the
presence of numerous cutting sites for ClaI. Within the A2 subcluster, the phage L5
has characteristics very similar to Epoch, including a genome length of 52,297 base
pairs, a similar plaque morphology, a noncontractile tail and a isometric head.
Genome sequencing of Epoch at some future date would allow for a better
understanding of its lysogenic/lytic cycle, as well as its characteristic genes. It is not
known whether the observed change in plaque morphology that occurred during
phage purification/isolation was the result of actual phage physiology characteristic
of Epoch or a mutation that occurred in the phage line during purification. This
question cannot be readily answered without the full sequencing of Epoch.

Below is a short summary of the methods used in this study. These are taken from
The HHMI National Genomics Research Initiative Laboratory Manual.
The isolation of the novel phage Epoch from the environment was performed using an
enrichment protocol and a soil sample collected from a residential lawn in Irving, Texas. The
enrichment process consisted of soil inoculation of a M. smegmatis culture, filter-sterilization
of the cultured lysate, serial dilutions and plaque purification.
Aspot test was performed to verify putative plaques and a selected plaque was purified
by a series of phage-titer assays. Afilter-sterilized sample medium titer lysate was created
from a web plate flooded with phage buffer. The final high titer lysate of the phage Epoch
was obtained using the medium titer lysate to create 10 web plates that were again flooded
with phage buffer to collect phage. Final phage yields were determined using standard phage
dilution/assay techniques.
Epoch was then analysed by electron microscopy and its capsid diameter and tail length
were determined. Phage genomic DNAwas prepared for restriction digestion and gel
electrophoresis. The genome size of epoch was estimated from the resultant restriction data
and a comparison of the patterns obtained with each enzyme was used to make a preliminary
group assignment for the phage.
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The first round of screening yielded plaques of three different morphologies.
Plaque Awas roughly 1.4mm in diameter and was not visible unless it was held in direct
light due to its turbidity. This was chosen for the plaque streak protocol.
Plaque B had the largest diameter on average of 2.0mm and its plaque morphology was very
cloudy, often grouped in large clumps of other plaques. This was chosen for plaque
purification but not studied further beyond that point.
Plaque C was roughly about 0.7mm in diameter and showed minor turbidity in comparison
to the other plaques. This type of plaque was not chosen for further study.
Sample A, chosen for the phage purification/isolation, contained a 10
-1
dilution and 19 pfu
from 5 l of medium titer lysate. Plaques in average had a diameter of 2.5 mm and an area of
5.0 mm. The titer of the phage lysate was 3.8 x 10
4
pfu/ml. The total number of pfu needed to
create a lysed web pattern was estimated to be 1140 pfu, corresponding to phage lysate
volume of 3.0 x 10
-3
ml. The final medium titer lysate was determined to have 1.69 x
10
10
pfu/ml. The medium titer lysate was used to produce a ten plate high titer lysate from
which genomic DNAwas prepared and which was used as the phage source for electron
microscopy. The titer of the high titer lysate was 1.56 x 10
13
pfu/ml.
Electron microscopy of uranyl acetate stained Epoch revealed a siphoveridae phage. The
tail length was determined to be 125 nm and its capsid diameter was 61 nm. (Figure 1)
The purification of phage genomic DNAyielded a total DNAvolume of 86 l with a 54.5
ng/ul DNAconcentration. Total DNArecovered was thus 4.7 g.
Gel electrophoresis of restriction digestions of Epoch DNA, specifically digestions with
BamHI and ClaI, gave an estimated genome size of 52,000 bp. (Figure 3)
Although the focus of basic research on mycobacteriophages is fairly recent, it has
already yielded findings highly beneficial to scientists in a range of disciplines. One of the
useful properties of mycobacteriophages revolve around their bactericidal nature, which,
unlike antibiotics, only targets specific bacteria. The virus-host bacteria interaction is
unaffected by multi-drug-resistance of the target cells, which allows healthcare professionals
to use these phages to control specific disease-causing strains of bacteria without impacting
normal human bacterial flora. Since the 1930s, bacteriophages have been used as an
alternative to antibiotics when combating gangrene, leprosy and E. coli infections. These
phage regiments have also been completed without any known detrimental side-effects.
Although few western countries have adopted the use of mycobacteriophages in the field of
health, researchers at the Eliava Institute in Georgia have developed practical antibacterial
uses for the phages. Recently in the United States, however, the Food and Drug
Administration approved the spraying of meats with phages in order to combat Lysteria
monocytogenes without the risk of promoting drug-resistant strains of the pathogens. Overall,
the use of mycobacteriophages in medicine has benefitted individuals, but the research
required for broader application of the technique has been restricted because of its limited use
in western countries to this time. With additional research and new applications, the use of
mycobacteriophages will develop into a more mainstream alternative to the use of antibiotics,
which will be much more beneficial to target infectious diseases.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS CONCLUSIONS
REFERENCES
Figure 1. Electron microscopy image of
Epoch. The tail length is 125.35nm and
the capsid diameter is 61.29nm.
Figure 2. Plaque morphology of Epoch. The
plaques demonstrate halo-like
characteristics with lytic inner circles and
turbid outer rings. The average plaque
diameter was roughly 2.8mm. After 24
hours at 37C.
Figure 3. Gel electrophoresis of restriction digestions of Epoch. The estimated genome size averaged
from two restriction digestions (BamHI and ClaI) is estimated to be 52,000 base pairs. Epoch is most
similar to the Acluster due to its lack of EcoRI and HindIII sites, which is a characteristics the A2
subcluster.