8/12/2019 Epoch - Poster Final

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ISOLATION, PURIFICATION, AND CHARACTERIZATION

OF THE MYCOBACTERIOPHAGE EPOCHGUSTAVO MIRALDA, DEMING, K.E., HUGHES, L.E. AND R.C. BENJAMIN

Universi ty of Nor th Texas

The novel mycobacteriophageEpochwas isolated fr om an environmental samplecollected under a tree in a residential front lawn through a process known as enrichment,

usingMycobacterium 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 studiesEpochhas

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 l arge 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.

Epochhas the characteristic features of the siphoviridae virus family, which includea 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 ofEpoch, 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 previousl y, 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, althoughEpoch

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 t he 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 DNA yield forEpochis on par with the results obtained with other

mycobacteriophages within the A cluster. Phages within the A cluster range from agenome size between 47 to 54 kilobases and this is again consistent with our

findings for Epoch. Other characteristics of t he A cluster 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 ofEpoch, it was concluded that the results best fit those of the

A2 subcluster. This includes a lack ofEcoRI andHindIII 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 ofEpochat 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

ofEpochor 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 phageEpochfrom 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. smegmatisculture, filter-sterilization

of the cultured lysate, serial dilutions and plaque purification.

A spot test was performed to verify putative plaques and a selected plaque was purified

by a series of phage-titer assays. A filter-sterilized sample medium titer lysate was created

from a web plate flooded with phage buffer. The final high titer lysate of the phageEpoch

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 DNA was prepared for restriction digestion and gelelectrophoresis. 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 r ound of screening yielded plaques of three different morphologies. Plaque A was 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 10-1dilution and 19 pfu

from 5 lof 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 104pfu/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 10-3ml. The final medium titer lysate was determined to have 1.69 x

1010pfu/ml. The medium titer lysate was used to produce a ten plate high titer lysate from

which genomic DNA was prepared and which was used as the phage source for electron

microscopy. The titer of the high titer lysate was 1.56 x 1013pfu/ml.

Electron microscopy of uranyl acetate s tainedEpoch reveal ed a siphoveri dae phage. The

tail length was determined to be 125 nm and its capsid diameter was 61 nm. (Figure 1)

The purification of phage genomic DNA yielded a total DNA volume of 86 lwith a 54.5ng/ul DNA concentration. Total DNA recovered was thus 4.7 g.

Gel electrophoresis of restriction digestions ofEpochDNA, 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 hasalready 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 combatLysteria

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 ofEpoch. 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.Epochis most

similar to the A cluster due to its lack of EcoRI andHindIII sites, which is a characteristics the A2

subcluster.