Cluster Classification of Mycobacteriophages Isolated from Tropical Soils of Puerto Rico

Nicole Colón¹, Alberto Cintron¹, Carolina Montañez¹, Luzmari Reyes¹

University of Puerto Rico, Cayey campus¹

Abstract: Mycobacteriophages have been studied through time for a number of reasons. They have been

used as model systems for the study of biological processes, such as phage infection and the dogma

central. This study aims to analyze different unsequenced mycobacteriophages and classify them into

their respective clusters using PCR and Gel Electrophoresis. Each group of researchers received a specific

Mycobacteriophage. The bacteriophages’ DNA were amplified using the PCR method, and were later

analyzed by running an electrophoresis gel.

Introduction:

For many decades bacteriophages have been

know as viruses that infect bacteria. Each

Bacteriophage is composed of a head and a tail

(figure 1.1). The head, stores the genetic

material, while the tail is used to inject the

genetic material into the host bacterial cell.

Phages can be classified according to their

morphology and nucleic acids. The International

Comittee on Taxonomy on Viruses has study

and classified over 2,475 species in 2011.

“Mycobacteriophages are viruses that infect

bacteria belonging to the mycobacteria

genus”(Rubin 2012). Mycobacteriophages can

be found in a variety of soils. They can be

classified as harmless bacteria or disease causing

agents, such as tuberculosis. The size of a phage

depends on the average number of phage

particles liberated when an infected bacterium is

lysed. 

Bacteriophages can be isolated by enriching soil

samples in a nutrient media containing the

bacterial host. Doing this will let the phage

reproduce, increase in number, and form

plaques. The plaques represent the cycles of

infection and cell lysis which identify the phage.

After identification, the plaques are purified for

further characterization.

The Mycobacteriophages are classified into

clustesr using specific primers. Table 1 shows

the Mycobacteriophage Clusters in Phages data

base. For this experiment we only used from

cluster A to cluster I because the rest of the

cluster didn’t have design primers.

For many years, Mycobacteriophages

have been studied and analyzed in order to

understand biological processes. Around 2,400

Mycobacteriophages have been analyzed and

characterized. “Over 70 universities and collages

around the United States, have isolated, purified,

and characterized Mycobacteriophages from soil

samples” (Rubin 2012). This experiment

analyzes and examines specific phages in order

to assign them into clusters.

Figure 1.1 Shows the structure of a bacteriophage.

Head

Tail

Table 1 Shows the Mycobacteriophage Clusters In Phagesdb.

Materials and Methods:

During this experiment, four specific

Mycobacteriophages genomic DNA were

assigned. Different Genomic DNA were

designated from Mycobacteriophages classified

as Phagus_Maximus, Suave, Bloo and Wilie.

The preparation of the phage DNA’s and the

forward and reverse primers were previously

made. Test tubes were labeled from 1 to 15. To

each tube a certain amount of each reagent was

added. First, 5l of Nano Pure PCR Grade

Water (H2O) was added. Later, 5l of each

Mycobacteriophage genomic DNA was added to

each tube. Following this step, 1l of the

specific forward and reverse primers were also

incorporated. In addition, 12l of the PCR

Master Mix, which contained Taq Polymerase,

Buffer, Nucleotides, Mg2+, were added. The

tubes were placed in a thermocycler for

amplification. Once amplified, the

electrophoresis method was performed. The

agarose gel was prepared using 200mL of TAE

buffer and 4g of agarose. After that, 2l of

loading dye were added to the reagents. The

wells of the agarose gel were loaded and they

were left to run for one hour at 80 volts. This

whole procedure was repeated with five gels of

different Mycobacteriophages, including the

control gel.

Results:

The gel was analyzed and photographs were

taken using a gel documentation system. The

bands of some of the Mycobacteriophages

indicated that they were part of a specific

cluster. The thicker upper bands are bright,

because they contain the genome, and the ones

that ran towards the bottom of the wells are

the primer replications of a specific region of the

genome. Figure 1.2 shows the controls on an

agarose gel. In the control gel amplification of

Colbert and Puhltonio genomic DNAs resulted

in PCR products using B1 cluster specific

primers. Thus these phages are verified as

belonging to Cluster B1 and their size pair base

is 700. As well, Ghost and LRRHood resulted in

PCR products as belonging to Cluster C1. And

their size is 400 base pair. Also for Pumpkin,

which DNA resulted, as being part of Cluster E

and its size was 800 base pair. Figure 1.3 the

middle contains the results of the experimental

agarose gels. It contains the

Mycobacteriophages known as Bloo and Suave.

In the experimental gel neither Wilie nor Bloo

showed an amplified PCR product. The final gel

was named as class gel containing two

Mycobactheriophage. Figure 1.3 left bottom

contains the Mycobactheriophage Suave, which

did not show any amplified PCR product. While

in Figure 1.3, top left, contains the

Mycobactheriophage Phagus_Maximus, whose

amplification of its genomic DNA resulted in a

PCR product about 500 base pair using B2

clusters specific primers.

Figure 1.2 Shows the Control Gel.

Figure 1.3 Shows the three gels made during this Research Experience. From left to right are the Class Gel #4, Experimental Gel and Control Gel.

Discussion:

This study provides information about

the classification of each Mycobacteriophage.

The conclusion during this experiment was that

only Phagus_Maximus could be classified as

belonging to cluster B2. The results of the other

Mycobactheriophages were not clear; therefore

we cannot arrive to any conclusions. The

Mycobacteriophage named Wilie had

ambiguous results, because it did not show any

bands. Wilie had a low amount of DNA, that’s

the reason why there weren’t any amplify

products on the gel. In order to classify this

Mycobacteriophage it will be necessary to

prepare the phage with a greater amount of DNA

and if is necessary test the phage with a new set

of designed cluster primers. The

Mycobacteriophages classified as Wilie, Suave

and Bloo did not showed any amplified PCR

product. In order for them to be classified is

necessary to design new PCR cluster primers

from J trough Q to test on them.

Acknowledgements:

Special thanks to: our mentor Dr.Rubin, the TA’s, Melisa Medina and Valeria Rivera, Yadira Ortiz and RISE. Also to Dr. Eneida Díaz, and Dr. Elena González.

References:

• Hatfull, Graham F., Cresawn, Steven E., Hendrix, Roger, W. 2008. Comparative Genomics of the Mycobacteriophages: Insights into Bacteriophage Evolution. Research in Microbiology Volume 159, Issue 5. P. 332-339.

• Mycobacteriophage Database.  [unknown]. http://phagesdb.org/

• Ross, Robert. 2012. General Botany Study Guide. Department of Biology UPR Cayey. Puerto Rico pp xxvii, xxviii, xxix.

• Rubin. M, 2012. Experimental Classification of Mycobacteriophages: Theoretical Background on Important Concepts and Techniques.

Simmons, Michael J., Snustad, D. Peter. 2012. Principles of Genetics. John Wiley & Sons, Inc. New Jersey pp. 165, 167, 168.