Article

Abstract

Mycobacteriophages are being studied to

comprehend bacterial pathogenesis, to

perform phage therapy, and for a better

understanding of basic molecular

biology. The research question of this

experiment is whether or not novel and

interesting mycobacteriophages can be

isolated from the tropical soils of Puerto

Rico. It is hypothesized that unique

mycobacteriophages with useful properties

will be isolated and characterized

successfully. The purpose of this

investigation is to isolate

mycobacteriophages from soil samples so

they can be characterized using genomic and

proteomic approaches. A

mycobacteriophage was discovered in the

seventh soil sample obtained from Caguas

P.R. The sample was then enriched and

filtrated. The filtrate was then streaked on a

Petri dish and incubated. After obtaining the

mycobacteriophages, plaques were extracted

and purified 4 times. The first purification

had to be repeated with a different phage

plug since no plaques were present on the

plate. The first two purifications showed a

greater concentration of plaques on the

second and third streak regions when

compared to the first region, unlike the third

purification. The third purification had to be

repeated as well due to contamination. The

isolated phage, named Musamodel, seems to

have a lytic life cycle since the plaques are

circular and completely clear, characteristic

of a virulent phage. This investigation

proves that mycobacteriophages can be

effectively isolated from the soils of Puerto

Rico. Since the investigation was not

finished, some techniques and procedures

still remain to be fulfilled. These procedures

include the spot test, making phage stocks,

the empirical test and the 10 plate

preparation, and finally the analysis and

bioinformatics/ DNA sequencing of the

mycobacteriophage.

Introduction

Mycobacteriophages are viruses that

infect a specific type of bacteria belonging

to the mycobacteria genus. “These

bacteriophages are the most ample life forms

in the biosphere and possess genomes

characterized by highly diverse genetic

designs” (Hatfull et al. 2006). The goal is to

characterize novel mycobacteriophages

using genomic and proteomic approaches,

therefore the following research questions

will be answered: Can novel and interesting

mycobacteriophages be isolated from the

tropical soils of Puerto Rico? If so, could

these be characterized? It is hypothesized

that unique mycobacteriophages with useful

Isolation and Characterization of Mycobacteriophage

Musamodel from Tropical Soils of Puerto Rico

Mónica C. Del Moral, Félix J. Vallés, Dr. Michael Rubin, RISE Program, Department of Biology, University of

Puerto Rico at Cayey

2

properties will be isolated and characterized

successfully.

Mycobacteriophages could be

identified as virulent or lysogenic. Virulent

phages follow a lytic life cycle by lysing all

bacteria they infect. On the other hand,

temperate phages follow a lysogenic life

cycle in which they either enter a dormant

state by incorporating their genetic material

into the DNA of the host bacteria, or

replicate and lyse the host bacteria like

virulent phages. Mycobacteriophages

consist of a capsid, which contains the

genetic material, the genetic material or

DNA, and a tail, which serves to attach to

bacteria and functions as a passageway for

DNA from tail to bacterium.

Mycobacterium can be used for

hosting or infection in labs to obtain these

mycobacteriophages for later

characterization, annotation and

classification. In this case, the two

mycobacteria used for infection are

Mycobacterium smegmatis (M. smegmatis)

and Bacillus cereus (B. cereus). M.

smegmatis, an aerobic organism usually

found in the soil, water, and plants. They are

commonly used in research due to their fast

growing and non-pathogenic behavior. B.

cereus, on the other hand, can be obligate

aerobes or facultative anaerobes, commonly

found in the soil. They are one of the best-

understood prokaryotes and are currently

used as a model for differentiation, gene

regulation, and cell cycle events in bacteria.

Both bacteria are Gram-positive, acid-fast,

and spore forming, with a rod-shaped

morphology and a strepto-cell arrangement.

If the infection of the mycobacterium

is successful, a mycobacteriophage should

be obtained from the soil sample. The

mycobacteriophage discovered will be

characterized using morphologic, genomic,

and proteomics techniques. Genomic

sequences from the mycobacteriophages will

be identified, annotated using

Bioinformatics tools, and then submitted

into an international DNA database. The

mycobacteriophage are usually classified

into clusters using comparative techniques,

genomic DNA restriction digestion patterns,

polymerase chain reaction and separation of

proteins using polyacrylamide gel

electrophoresis. Finally, once discovered

the mycobacteriophages are added to the

other 1000 mycobacteriophages that have

been sequenced, annotated and analyzed by

the scientific community. “Genome

clustering facilitates the identification of

genes that are more likely to genetically

mutate and to have been exchanged in recent

evolutionary times” (Hatfull et al. 2010).

According to Pope et al. 2011,

“mycobacteriophages provide extremely

useful tools for the study and manipulation

of their host”, hence, bacteria could be better

understood by the study of

mycobacteriophages, and could be used for

different research and medicine application.

Since bacteria cause animal diseases, and

bacteriophages kill bacteria, bacteriophages

could be used in substitution of antibiotics.

Mycobacteriophages are being studied to

understand bacterial pathogenesis, to

perform phage therapy, and for a better

understanding of basic molecular biology.

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Materials and methods

The methodology used has four

major steps: collection of the environmental

sample, isolation of the phage from the

environmental sample, purification of the

phage, and characterization of the phage.

During the sample collection phase, the

objective was to capture a phage to then

isolate it. For the collection, it was

necessary to use a sterile spoon and deposit

at least one gram of soil into a sterile bag.

For each sample, the location (by GPS o

Google Earth), date and time of the

collection, the temperature, the depth at

which the sample was obtained, soil

description, and site description was

recorded. The sample was then taken to the

lab to extract the phage from it and tested on

two hosts: Bacillus cereus (B. cereus) and

Mycobacterium smegmatis (M. smegmatis).

First, 0.500 grams were added into two

weight boats. Then, two 50mL conical

tubes were labeled with the initials, date, the

collection location, sample number, and

respective host name. Afterwards, 10mL of

the enrichment mix were pipetted. In the

case of the M. smegmatis labeled tube, the

enrichment mix was Master Mix, made with

H2O, 10x 7H9/glycerol broth, AD

supplement, and 100 mM CaCl2. On the

other hand, for the B. cereus labeled tube,

the enrichment mix was tryptic soy broth

(TSB). In addition, 1000µL of the

correspondent bacteria and the 0.500 grams

of soil to the tubes were added. At last,

these tubes were left incubating at 37˚C and

shaken at 220 rpm for 24 hours.

The following day the enriched

sample was ready for isolation. In this step,

first, the enrichments were centrifuged at

3,000 rpm for 10 minutes to pellet the

particulate matter. Then, two new 15mL

conical tubes were labeled with the initials,

the date, the collection, the sample number,

and the name of the corresponding bacteria

hosts. Further on, 5mL of the B. cereus

enrichment supernatant were pipette into an

assembled filtration unit, with a 0.22-µm

filter and a 5mL syringe, and the filtrate was

added into the 15mL tube. This step was

repeated with the M. smegmatis enrichment.

If these filtrates were not to be used

immediately, we stored them at 4˚C.

An alternative way of “filtering” the

enrichment was by centrifugation. First, the

enrichments were centrifuged. Secondly,

two centrifugation microtubules were

labeled with the sample information. Then,

1,000µL of the corresponding enrichment

supernatant were added into each

microtubule. Thirdly, these microtubules

were centrifuged at 10,000rpm for 10

minutes. Subsequently, 500µL of the

corresponding microtubules supernatant

were added into two new microtubules

labeled as filtrate, along with the other

information.

The filtrate was then used to streak

on agar plates and assess if the sample had

phages. In order to perform the streak, one

plate prepared for B. cereus was labeled

with tryptic soy agar (TSA) and another

plate prepared for M. smegmatis prepared

with Luria base agar (LB) with the sample

information. Once the streak was performed

with both filtrates, 4.5mL TSA top agar was

mixed with .5mL of B. cereus, and deposited

it on the correspondent plate from the most

diluted region. This was repeated with the

M. smegmatis bacteria, but with the LB top

4

Table 1. The Seven Soil Samples Collection Data

agar. After about 30 minutes of wait for the

top agar to harden, the inverted plates were

incubated at 37˚C for 24 hours.

The day after, the plates were

assessed to see if they had phage plaques. If

the plates did not have any plaques, a new

soil sample needed to be collected and

repeat the procedure until a phage was

found. When one of the plates had phage

plaques, the purification stage of the

experiment was begun. First, a

centrifugation microtubule was labeled.

Secondly, 50µL of phage

buffer was added to the

microtubule. Then, using a

1,000µL micropipette, a plaque

plug was extracted and

deposited it in the phage

buffer. After waiting a few

minutes for it to dissolve, a M.

smegmatis plate was labeled,

because that was our phages

host, as the first purification, along with the

usual information. Additionally, a streak

was performed using the mix we prepared.

Then 4.5mL of LB top agar and .5mL of M.

smegmatis mix were pipetted to the plate, it

was allowed to harden, and it was incubated

inverted at 37˚C for 24 hours. Further on,

two more purifications were done, each

made with a phage plug from the previous

purification plate. If at any of the steps the

purification did not result with any plaques,

the same purification had to be repeated.

Once the three purifications were

obtained, a second enrichment was done, but

using a phage plug from the third

purification. First, one 50mL conical tube

was labeled with our initials, the phages

name, the date, and “second enrichment”.

Secondly, 10mL of Master Mix, 1,000µL of

M. smegmatis were added using a

micropipette, and a phage plug from the

third purification was added. Then, this tube

was left incubating at 37˚C and shaking at

220rpm for 24 hours.

Results

None of the first six soil samples that

were enriched, filtered and streaked on the

Petri dishes had positive results. It was not

until the seventh soil sample that a phage

was found. The Petri dish displayed almost

no plaques on the first streak region, a large

concentration of plaques on the second

region, and less concentration of plaques on

the third region. The first purification

attempt had negative results since no

plaques were present on the plate. Therefore,

the first purification was done with another

phage plug, and positive results were

obtained.

Figure 1. The Seventh Soil Sample Phage Plate

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The first and second purification

plates showed a greater concentration of

plaques on the second and third streak

regions when compared to the first region.

This is not supposed to occur. This might

have been due to adding the top agar and

bacteria mix from one of the less diluted

regions or moving the plate by mistake

before the top agar solidified. The third

purification had to be repeated since it had

gotten contaminated.

The repeated third purification had positive

results.

However, it had the same pattern of phages

concentrations as the first two purifications.

The second region was more diluted than the

third region.

Discussion

After six unsuccessful attempts to

find a mycobacteriophage, successful results

were obtained with the seventh soil sample

(Table 1). Interestingly, this sample was

collected right next to the roots of a plantain

plant. This was the only sample that was

collected that close to a plant. The sample

probably had phages, because the soil was

more fertile there and it uses M. smegmatis

as bacterial host to reproduce. Once the

presence of a phage was confirmed it was

named Musamodel. The name comes from

the word “Musa”, meaning inspiration, and

the word “model” in honor of one of the

researchers and due to its function in the

study.

The novel phage, Musamodel,

appears to be virulent and to have a lytic life

cycle based on the plaques found on the

latest purification (Figure 5). These plaques

seem to be perfectly circular and completely

clear, characteristic of a virulent phage.

However, in order to know for certain the

characteristics of our phage. Other steps

Figure 2. First Phage Purification Plate

Figure 3. Second Phage Purification Plate

Figure4.Third Phage Purification Plate (First Attempt)

Figure 5. Third Phage Purification Plate

(Second Attempt)

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need to be carried out. Therefore, future

plans include doing the spot test, phage

stocks, empirical test, the ten-plate

preparation, the phage analysis, and the

bioinformatics/sequencing portion of the

characterization.

Ultimately, the hypothesis unique

bacteriophages with useful properties will be

isolated and characterized successfully was

partially proven. Musamodel was isolated,

but not characterized. Currently work is

being done towards achieving that goal. It is

inferred that phages can be isolated from the

tropical soils of Puerto Rico, because of the

richness of the soils. There is no doubt that

a lot needs to be learned from these phages

and that there are many yet to be discovered.

Acknowledgements

Dr. Michael Rubin- Howard Hughes

Program director at the University of

Puerto Rico at Cayey and mentor

Eduardo Correa- teaching assistant and

mentor

Giovanni Cruz- laboratory technician

Gustavo Martínez- teaching assistant

Literature cited

Hatfull GF, Pedulla ML, Jacobs-Sera D,

Cichon PM, Foley A, et al. (2006)

Exploring the Mycobacteriophage

Metaproteome: Phage Genomics as an

Educational Platform. PLoS Genet 2(6):

e92.

Pope WH, Ferreira CM, Jacobs-Sera D,

Benjamin RC, Davis AJ, et al. (2011)

Cluster K Mycobacteriophages: Insights

into the Evolutionary Origins of

Mycobacteriophage TM4. PLoS ONE

6(10): e26750.

Hatfull GF, Jacobs-Sera D, Lawrence

JG, Pope WH, Russell DA, et al. (2010)

Comparative Genomic Analysis of 60

Mycobacteriophage Genomes: Genome

Clustering, Gene Acquisition, and Gene

Size. Journal of Molecular Biology

397(1): e119-43