Article

Abstract

Mycobacteriophages are viruses that infect a

specific type of bacteria belonging to the

mycobacteria genus. Mycobacteriophages

can be obtained by infecting mycobacterium

in a controlled environment. The two types

of mycobacterium used in the lab were

Mycobacterium smegmatis, suitable for

investigation due to its fast growing and

non-pathogenic behavior, and Bacillus

cereus, currently used as a model organism

for different live events in bacteria.

Mycobacteriophages are being studied to

comprehend bacterial pathogenesis, to

perform phage therapy and for a better

understanding of basic molecular biology.

The purpose of this investigation is to isolate

novel and interesting bacteriophages from

the tropical soils of Puerto Rico so they can

be characterized using genomic and

proteomic approaches. A

mycobacteriophage was discovered in our

seventh soil sample 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 3 times.

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 GF, et al. 2006).

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 that will be used for infection

Isolation and Characterization of Mycobacteriophage

Musamodel from Tropical Soils of Puerto Rico

Mónica C. Del Moral and Félix J. Vallés, RISE Program, Howard Hughes Medical Institute, Department of

Biology, University of Puerto Rico at Cayey

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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 its 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

bacteria; form spores and have 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 will be classified into

clusters using comparative techniques,

genomic DNA restriction digestion patterns,

polymerase chain reaction separation of

proteins using polyacrylamide gel

electrophoresis. Finally, the

mycobateriophage will be 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 GF, et al. 2010).

According to (Pope WH, 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 implications on research and

medicine. Since bacteria cause animal

diseases, and bacteriophages kill bacteria,

bacteriophages could be exploited to

function as antibiotics. Mycobacteriophages

are being studied to understand bacterial

pathogenesis, to perform phage therapy and

for a better understanding of basic molecular

biology.

Our goal is to characterize novel

bacteriophages using genomic and

proteomic approaches. However, can novel

and interesting bacteriophages be isolated

from the tropical soils of Puerto Rico? If so,

could these be characterized? We believe

that unique bacteriophages with useful

properties will be isolated and characterized

successfully.

Materials and methods

This experiments methodology is

composed of 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 is to

capture a phage to then isolate it. For the

collection it is necessary to use a sterile

spoon and deposit at least one gram of soil

into a sterile bag. For each sample, we

recorded the location (by GPS o Google

Earth), date and time of the collection, the

temperature, the depth at which the sample

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was obtained, soil description, and site

description. The sample was then taken to

the lab to extract the phage from it and

tested it with two hosts: Bacillus cereus (B.

cereus) and Mycobacterium smegmatis

(M.smegmatis). Once in the lab after

cleaning the work area aseptically, we first

added 0.500 grams into two weight boats.

Then, we labeled two 50mL conical tubes

with our initials, date, the location where the

sample was collected, sample number, and

respective host name. Afterwards, we

pipette 10mL of the enrichment mix. In the

case of the M. smegmatis labeled tube, the

enrichment mix is Master Mix, made with

H2O, 10x 7H9/glycerol broth, AD

supplement, and 100 mM CaCl2. On the

contrary, for the B. cereus labeled tube, the

enrichment mix is tryptic soy broth (TSB).

In addition, we added 1000µL of the

correspondent bacteria and the 0.500 grams

of soil to the tubes. At last, these tubes are

left incubating at 37˚C and shaking at 220

rpm for 24 hours.

The following day the enriched

sample is ready for the next step: isolation.

In this step, we first centrifuge the

enrichments at 3,000 rpm for 10 minutes to

pellet the particulate matter. Then, after

aseptically cleaning the work area, we

labeled two new 15mL conical tubes with

our initials, the date, the location where the

sample was collected, the sample number,

and the name of the corresponding bacteria

hosts. Further on, we pipette 5mL of the B.

cereus enrichment supernatant into an

assembled filtration unit, with a 0.22-µm

filter and a 5mL syringe, and added the

filtrate into the 15mL tube. This step was

repeated with the M. smegmatis enrichment.

If these filtrates were not to be used

immediately, we could store them at 4˚C.

An alternative way of “filtering” the

enrichment is by centrifugation. First, we

centrifuged the enrichments. Secondly, we

aseptically clean the work area and we

labeled two centrifugation microtubules with

our initials, the date, the location where the

sample was collected, the sample number,

and the correspondent bacteria host. Then,

we added 1,000µL of the corresponding

enrichment supernatant into each

microtubule. Thirdly, we centrifuged these

microtubules at 10,000rpm for 10 minutes.

Subsequently, we added 500µL of the

corresponding microtubules supernatant into

two new microtubules labeled as filtrate,

along with the other information.

The filtrate is then used to streak on

agar plates and assess if the sample had

phages. In order to perform the streak, we

labeled one plate prepared for B. cereus with

tryptic soy agar (TSA) and another plate

prepared for M. smegmatis prepared with

luria base agar (LB) with our initials, the

date, the location where the sample was

collected, the sample number, and the

respective bacteria host name. Once we

performed the streak in three quadrants with

both filtrates, we mixed 4.5mL TSA top

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

it on the correspondent plate from the most

diluted region. This is repeated with the M.

smegmatis bacteria, but with the LB top

agar. We waited about 30 minutes for the

top agar to harden and incubated the plates

inverted at 37˚C for 24 hours.

The day after, we assessed if any of

the plates had phage plaques. If the plates

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did not have any plaques, we needed to

collect a new soil sample and repeat the

procedure until a phage is found. When one

of the plates had phage plaques, we

continued to the next step of the experiment:

the purification of the phage. First, we

labeled a centrifugation microtubule with

our initials, the date, and original phage

plug. Secondly, we added 50µL of phage

buffer to the microtubule. Then, using a

1,000µL micropipette, we extracted a plaque

plug and deposited it in the phage buffer.

After waiting a few minutes for it to

dissolve, we labeled a M. smegamtis plate,

because that is our phages host, as the first

purification, along with the usual

information. Additionally, we performed a

streak in three regions using the phage plug

and buffer mix we prepared. We then

pipette the 4.5mL of LB top agar and .5mL

of M. smegmatis mix to the plate, allowed it

to harden, and incubated it 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, we had to

repeat that same purification.

Once we had our three purifications,

we did a second enrichment, but using a

phage plug from the third purification. First,

we labeled one 50mL conical tube with our

initials, the phages name, the date, and

“second enrichment”. Secondly, we pipette

10mL of Master Mix, 1,000µL of M.

smegmatis using a micropipette, and added a

phage plug from the third purification.

Then, we left this tube incubating at 37˚C

and shaking at 220rpm for 24 hours.

This is as far as we have reached in

the methodology. Later on, we will do a

Spot test to see at which dilution we should

obtain a web pattern with the phages. Then,

we will do an Empirical test to see and

confirm our web pattern. Subsequently, we

will prepare a medium titer phage lysate

(MTPL) using the plate that shows the most

clear web pattern. This MTPL will enable

us to replicate the dilution and prepare the

high titer phage lysate (HTPL). With this

step we would conclude our purification

segment and move on to the characterization

portion of our work. First, we will observe

our phages using a scanning electron

microscope. Afterwards we will isolate the

DNA, amplify it with PCR, run a

polyacrylamide gel electrophoresis, send our

phage DNA for sequencing, and at last, use

bioinformatics to characterize the phages

genome.

Results

Neither 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 and we saw phage plaques. 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, we tried doing the first

purification with another phage plug, with

which we obtained positive results. 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, which is not

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supposed to occur. This might have been

due to adding the top agar and bacteria mix

from one of the less dilute regions or

moving the plate before the top agar solidify

by mistake. 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, that instead of the third region

being the most dilute it was the second.

Discussion

After six unsuccessful attempts to

find a mycobacteriophage, we obtained the

wanted results with our seventh soil sample.

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. We believe that this sample

had phages because the soil was more fertile

there. In addition, we know that this phage

uses M. smegmatis as bacterial host to

reproduce. Once we had confirmed that we

found a phage, we named it Musamodel.

The name is made up of “Musa”, meaning

inspiration, and “model” because of the

name of the researcher who found it and as

the model to be studied.

Up to this point, we believe that our

phage, Musamodel, is virulent and has a

lytic life cycle because of the plaques on the

latest purification. These plaques seem to be

perfectly circular and completely clear,

characteristic of a virulent phage. However,

we need to continue with the steps of the

research in order to know for certain the

characteristics of our phage. Therefore, our

future plans include doing the empirical test,

the ten plate preparation, the phage analysis,

and the bioinformatics/sequencing portion of

the characterization.

Ultimately, we can say that the part

of our hypothesis that stated that we would

find a phage was accepted. Although, we

have not been able to characterize

Musamodel yet, we are currently working

towards achieving that goal. Still, we have

confirmed that phages can be isolated from

the tropical soils of Puerto Rico because of

the richness of the soils.

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

6

Size. Journal of Molecular Biology

397(1): e119-43

Appendices

Appendix 1: Soil sample

collection data

Appendix 2: Seventh soil sample

plate

Appendix 3: First purification

Appendix 4: Second purification

Appendix 5: Third purification

Appendix 6: Third purification-

second attempt

Appendix 1. Soil sample collection data

Appendix 3. First purification Appendix 2. Seventh soil sample plate

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Appendix 4. Second purification Appendix 5. Third purification

Appendix 6. Third purification- second attempt