Anaeli Shockey López a, Nicolle Rosa Mercadob

a Chemistry Department, University of Puerto Rico-Cayeyb Biology Department, Univeristy of Puerto Rico-Cayey

Isolation and characterization of Mycobacteriophages from tropical soil of Puerto Rico

Abstract:Mycobacteriophages are viruses that infect bacteria from the genus Mycobacterium. They

are ubiquitous and are easily found in different types of soils. Phages are composed of a head, which contains the genetic material, and a tail. Bacteriophages have two possible life cycles: lytic or temperate. Most of them have a temperate life cycle in which they can cause immediate lysis or enter a state of dormancy within the host. The objective of this investigation was to iso-late a new phage using soil from Puerto Rico. This is necessary because there are too many undiscovered bacteriophages that can be of great use to mankind. The methodology for this in-vestigation consisted of isolating a phage using the protocols listed in the SEA-PHAGES re-source guide. Two phages were isolated from Gurabo, Puerto Rico and were taken up to the high-titter Assay protocol. Future work would include sequencing their DNA.

Introduction:Mycobacteriophages are viruses that infect

bacteria from the genus Mycobacterium. They are ubiquitous and are easily found in different types of soils. They can be isolated using a simple pro-cedure. Although phages are the most abundant life-form on earth, very few of them have been identified. Phages insert their genetic material into the bacteria and replicate within it provoking the lysis of its host. Bacteriophages have two possible life cycles: lytic or temperate. Most of them have a temperate life cycle in which they can cause im-mediate lysis or enter a state of dormancy within the host.

Hatfull et al. (2008), indicate the recogni-tion of the vast numbers of bacteriophages in the biosphere has prompted a renewed interest in un-derstanding their morphological and genetic di-versity, and elucidating the evolutionary mecha-nisms that give rise to them. This investigation has several important applications within the field of scientific research. An example of this within the biomedical field is the possible elimination of antibiotic resistant bacteria using phages. Phages can also help us understand certain aspects of the bacteria that they infect and the effects that they might have on them. Based on the immense diver-sity present in phages we can also obtain impor-tant information on the evolutionary line of these viruses. Some phages are an example of how viruses can be beneficial to humans. The objective of this investigation was to isolate a new phage using soil from Puerto Rico. This is necessary be-cause there are too many undiscovered bacterio-phages that can be of great use to mankind.

Mycobacteriophages can be found all around the world and are the most numerous bio-logical entities in the biosphere (Pope et al., 2011). This is a very promising research area since there is still much to be discovered concern-ing phages. There are yet many important undis-covered characteristics that may be helpful in the treatment of bacterial diseases. Their genetic di-versity provides a promising future in research. Phages help us get a better understanding of bac-teria as well.

Materials and Methods:As instructed by our mentor, for this ex-

periment, all of its materials and methods were recommended by Science Education Alliance (2012). - Sample Collection:

The first step of the experiment was the collection of a soil sample. In this step, as part of maintaining everything sterile, you used a pre-packed utensil to recollect the soil sample into a sealed and sterile test tube. After collecting the sample, the test tube was sealed and stored at room temperature. Data such as temperature, cli-mate, soil moisture, GPS site, soil depth, etc. was recorded. - Enrichment:

Afterwards, the second step of the experi-ment was the enrichment of the soil sample that was recollected. In this step, you added to a sterile 50ml test tube the following: 8ml of sterile water, 1ml of sterile 10x 7Hq/glycerol broth, 1ml of AD supplement, and 0.1ml of 1000mM CaCl2. To this enrichment solution, you also added 1ml of the bacteria M. smegmatis. In addition, you added 0.5g of the soil sample to the test tube with the en-richment solution and the bacteria. Lastly, you in-cubated the test tube at 37°C at 220rpm for 24 hours. - Harvesting:

Once 24 hours passed after the enrichment step, the test tube was centrifuged for 10 minutes. Then, you poured the supernatant into a new ster-ile 50ml test tube using sterile filtering tech-niques. Once successfully filtered, the test tube was capped and labeled. Next came the second part of the harvesting: to plaque. The plaque process was done on petri dishes and each plate was divided into three sections. This step con-sisted of using a wooden stick to streak, across the first section of the bottom agar of the petri dish, the supernatant that resulted after filtering (This sentence lacks clarity.). Afterwards, another wooden stick was used to streak from section one to section two and then, using a new wooden stick, we steaked from section two to section

three. After the streaking was completed, 4.5ml of top agar with 0.5ml of bacteria were added to the plate. Once the agar solidified, the plates were in-cubated at 37°C, for 24 hours, and after positive results appeared, the plates were refrigerated. - Plaque Purifications:

If positive results appeared. the phage(s) that you wanted to purify were to be circled at the bottom of plate so that you could view them clearly. Now to a labeled tube, we added 50µl of phage buffer. To the circled phage of the plate, we inserted a micropipette tip and then placed it in the tube with the phage buffer. Afterwards, a new petri dish was labeled and the plaque process described in the second part of the har-vesting was repeated. However, instead of using the filtering results to plaque, we used the tube with the mixture of the phage buffer and the phage inserted from the plate. Once again, we streaked section one, then from section one to sec-tion two, and section two to section three. Three rounds of plaque purifications were performed. - Second Enrichment:

The next step was to make another har-vesting. First of all, a phage was isolated with the tip of a micropipette and then added into the same enrichment solution as the first enrichment. After-wards, it was incubated for 24 hours like in the first enrichment. Next, the test tube was cen-trifuged for 10 minutes. Then, we poured the su-pernatant into a new sterile 50ml test tube using sterile filtering techniques. Once successfully fil-tered, the test tube was capped and labeled. - Medium Titer Assay:

This step consisted of creating serial dilu-tions. From the filtration obtained after the second filtration, four phage solutions were diluted in four tubes labeled -1 to -4, add 90µl of phage buf-fer. Then, we added 10µl of the filtration to the -1 tube and centrifuge it. Next, we added 10µl of the -1 tube to the -2 tube and centrifuge. This process was repeated for the 4 tubes. Afterwards, we added 10µl of each tube (filtration and -1 to -4 tubes) to a sample of 0.5ml of bacteria. Once the solution sat for 15-30 minutes we added 4.5ml of top agar to the bacteria solution and spread the so-

lution on a properly identified plaque. The plates wee incubated after solidifying and checked after 24 hours. Once the plate that was successful was identified (the one with the “web” pattern), we add 6ml of phage buffer to it and placed it in the refrigerator for 24 hours. Next, we extracted the phage buffer ___the plaque, filtered, and refriger-ated it. - High Titer Assay:

In this step, 10 plates were infected with the bacteriophage. First of all, we labeled 10 plates and labeled a sterile 50ml test tube. To the test tube, we added 5ml of bacteria culture and then infected it with 10µl of the dilution that com-pletely lysed the bacteria. The bacteria was incu-bate and shaken at 37°C for 30 minutes. After-wards, 45ml of top agar was addedto the test tube. Five ml of the mixture was distributed onto each plate and incubated at 37°C for 24 hours. Af-ter the time has passed and the web pattern was obtained, 6ml of phage buffer was added to each of the 10 plates, and using sterilized utensils the agar was broken and mixed with the buffer. Next, place the plates were placed in the incubator at 37°C for four hours. After the time has passed, the phage buffer was extracted from all of the plates and placed in a sterile 50ml test tube. As the last step, the tube was centrifuged and fil-tered. - Rapid Isolation, Separation, and Vi-sualization of Mycobacteriophages Cap-sid Proteins:

This step is performed with the extraction of phage buffer from the plate with the “web” pat-tern from the Medium Titer Assay step (Not clear!). One ml of Mycobacteriophage High Titer Phage Lysate (HTPL) was transferred to a clean sterile microtube and centrifuged at 10,000xg for one hour at 4°C. Afterwards, 950µl of the super-natant was aspirated. Next, a sample buffer was prepared by adding 25µl of Beta-mercaptoethanol (BME) to 475µl of Laemmli Sample Buffer (LSB) and vortexed completely. Later, 20µl of the LSB plus BME solution was added to the My-cobacteriophage virion coat protein pellet. After that was done, (from here on continue to use pas-

sive voice with past participle verbs for the rest of the report. You are reporting on the methodology that you used. It has to be past tense and to make it objective the voice must be passive.). boil the sample for two minutes, cool the protein sample for two minutes and centrifuge it briefly. Now you prepare the gel. This is done preparing 1x of run-ning buffer by adding 100ml of 10x Tris Glycine SDS buffer to 900ml of distilled water. Next, re-move the gel from the packaging, remove the tape from the bottom of the gel, carefully remove the comb using even pressure, and rinse the wells us-ing distilled water. Assemble the gel in the appa-ratus and add appropriate amounts of 1x running buffer. Afterwards, load the sample and the molecular weight markers. Now you run the gel at 200 volts for 30 minutes until the dye reaches, ap-proximately, 1cm from the bottom of the gel. Next, strain the gel in a plastic tray using Bio-Rad Biosafe Coomassie Blue G-250 strain. Wash the gel in distilled water for 5 minutes and remove the water (this step is repeated three times). Next, add 50ml of Coomassie Blue G-250 stain to the gel and stain for one hour with gentle shaking. After the staining is complete, rinse the gel with water for 30 minutes. Now you’re ready to photograph the gel on a white light box. The gel can be stored in water (in a zip lock plastic bag) or dried and the bands can be carefully excised using washed gloves and clean unused razor blades and placed in sterilized microtubes for subsequent protein identification by mass spectroscopy.

Results:The following results were found in the

experiment. First of all we have the soil recollec-tion data. The temperature at 8:00am on February 19, 2013 was 25.6°C and the day was sunny and clear. The sample was taken in Gurabo, Puerto Rico at these coordinates: 18°14'48.53"N  66° 0'6.55"W. The soil sample was taken from an ur-ban site next to trees and compost. In addition, the soil was dry and the depth from where it was taken was 5.74 inches.

After the first enrichment and harvesting, positive phage results were found when the soil

sample was used. From the plate with positive re-sults, three phages were identified because of their difference in sizes. Since they were treated as three different phages, each of them required three plaque purifications. The purification of the first phage resulted in morphologically small phages. The purification of the second and the third phage resulted in morphologically medium sized phages, both suspected to be the same size. After the sec-ond enrichment, filtration and -1 to -4 dilutions, each phage yielded a “web” pattern. For phage #1, the pattern was on the plate with the dilution -3. For phages #2 and #3, the pattern was on the plate with the dilution -4.

After extracting the phage buffer from each of the “web” pattern plates, a medium was created and analyzed with the SDS gel. The gel was loaded with a marker, other phages, and the three suspected phages. After the whole procedure was complete, the protein bands of all three phages could be seen and the bands of phages #2 and #3 were extremely similar.

Discussion: Positive results for phages obtained after

the enrichment and first plating was most proba-bly due to the sample depth and location (next to compost). With the positive phage results, three phages were identified because they had different sizes. The difference in sizes means that each phage is morphologically different from the other. Moreover, this would mean that each phage that is a different size would be a different phage.

After the plaque purifications were com-pleted, phages #2 and #3 were suspected to be the same phage because their sizes were relatively the same. However, they continued to be treated as different phages until the protein gel step was completed to determine if they were the same phage or not. Once the dilutions were completed, the “web” pattern of each phage was chosen based on the arrangement of plaques in which almost all of the bacteria was lysed. In addition, in the “web” pattern, all of the plaques must be in con-tact with each other.

A protein gel was run once a medium was created based on the “web” pattern. The protein bands of phage #1, now named Shockage, were different from the rest of the phages. The protein bands of phages #2 and #3 were practically the same; therefore, it is assumed that both phages are the same. Phage #2 is now named Zombage.

Conclusion:Through this experiment, we were able to

isolate Mycobacteriophages, which are viruses that infect bacteria. From a single soil sample, taken from Gurabo, Puerto Rico, two different phages have been isolated. Phage #1 is named Shockage and phage #2 is named Zombage. The next step for each of these phages would be to se-quence their DNA in order to finish the phage characterization and determine if the isolated phage is, in fact, unique. Furthermore, the isola-tion of phages has applications in the field of bio-medicine. As mentioned earlier, an example of this is the possible elimination of antibiotic resis-tant bacteria using phages. In addition, phages can also be helpful in understanding certain aspects of the bacteria that they infect and the effects that they might have on them.

References:

Science Education Alliance. 2012. SEA-PHAGES Resource Guide. Howard Hughes Medical Insti-tute; Chevy Chase, MA.

Hatfull G, Cresawn S, Hendrix R. 2008. Compar-ative genomics of the mycobacteriophages: in-sights into bacteriophage evolution. Research in Microbiology. 159(5): 332-339.

Pope WH, Jacobs-Sera D, Russell DA, Peebles CL, Al-Atrache Z, et al. 2011. Expanding the Di-versity of Mycobacteriophages: Insights into Genome Architecture and Evolution. [Internet] [Cited 2013 May 14] PLoS ONE 6(1) doi:10.1371/journal.pone.0016329 Available from: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0016329