Isolation, Purification, and Characterization of Gemeos, a novel phage isolated from tropical soils.

Amanda Icazatti Burtell, Fernando Pacheco Ocasio, and RISE Program University of Puerto Rico- Cayey Campus

Abstract

Viruses are the most abundant organisms on the Earth and are found in a great number of environments. Bacteriophages are viruses that infect bacteria. The purpose of this research is to isolate a bacteriophage (also abbreviated as phage) in tropical soils using soil as the environmental sample and doing enrichment to see if any phage can be obtained directly from soil. Since phages are viruses and viruses need a living host in order to reproduce, two host bacteria are being used in this experiment: Mycobacterium smegmatis and Baccilus cerous. These bacteria do not represent a threat to health since they are nonpathogenic and safe to use in a learning laboratory. The soil sample were enriched with bacteria and placed at 37˚C for 24 hours to stimulate ideal conditions for phage growth. The culture would then be centrifuged and filtrated. The filtration will be used to do Petri dishes and check if any phages were present. If phages do appear, a single plaque would be isolated and purified using streaking techniques with the purpose of isolating a single phage colony. After the fourth purification, a plaque would be isolated that has already been purified to perform dilutions. The dilutions are used to show which concentration of phages is the best for obtaining a web pattern, which is the phase where the optimums concentration of the phage is found. Different soil samples were analyzed from tropical soils of Puerto Rico and a false positive was obtained. An phage adoption was made. The phage was obtained from soil samples from Sao Pablo, Brazil. This phage was named Gêmeos and its host is Baccilus cereus. From initial observations the page appears to be a lysogenic phage. Further experimentations, like genome sequencing, will give more details about the type of phage that has been isolated.

Key words: Virus, bacteriophages, Mycobacterium smegmatis, Baccilus cereus, lytic and lysogenic cycle, and electrophoresis.

Introduction

Viruses are the most abundant infectious agents on Earth. They straddle between the living and nonliving. Therefore, viruses need to infect a living host in order to replicate and reproduce themselves. Bacteriophages are viruses that infect bacteria (Snustad and Simmons 2012). These agents infect a variety of bacteria, which can be either harmful or helpful to human beings. Bacteriophages have many useful characteristics. They can be used to manipulate the genetic constitution of bacteria and even to create antibiotics resistant to bacteria.

Bacteriophages are identified according the type of life cycle they have. There are two ways that a virus can replicate

itself in a bacteria and that is through the lytic cycle or through the lysogenic cycle. In the lytic cycle the bacteriophage infects the host by attaching itself to the membrane of the bacteria cell. The virus’s DNA is then injected into the bacteria and the bacteria’s DNA will be degraded. The bacteriophage then replicates its genetic information inside the bacteria until, the phage ruptures or “lysis” the bacteria, liberating mature bacteriophages that can infect more bacteria (Pendleton 2013).

In the lysogenic cycle, the phage’s DNA is integrated into the bacteria chromosome. The phage infects the bacterial host in the same way as the lytic cycle. Once the phage’s DNA is injected into the bacteria, the replication of its genetic material can take one of

two pathways. The phages can choose to stay attached to the bacteria chromosome and it can replicate itself along with the bacteria’s genetic material. In this stage of integration, the combined DNA is called a prophage. If the environmental conditions become unfavorable for bacterial cell growth, the phage genome exists the host chromosome by a recombination process called excision. Once separated from the host chromosomes the phage genome begins to arm itself like in the lytic process, resulting in cell lysis and the production of new mycobacteriophages.

In this study, bacteriophages where isolated from soil samples. An enrichment technique was used with two host bacteria with the purpose of expanding the possibilities of finding bacteriophages and to study the similarities between bacteriophages. The host bacteria used were Mycobacterium smegmatis and Baccilus cereus. A mycobacteriophage infects specifically the bacteria genre Mycobacterium and a bacilophage infects strictly the bacteria genre Baccilus. In addition to the enrichment technique, purifications and dilutions of a phage colony were carried out to identify and characterize a novel phage.

Materials and Methods

Mycobacteriophage and bacilophage were used as models to study how a virus infects bacteria. These bacteriophages have the capacity to infect specific bacteria such as Mycobacterium smegmatis and Bacillus cerous, respectively. Both bacteria are nonpathogenic, therefore, they are safe

to use for a laboratory experiment. The use of techniques in microbiology is fundamental in the process of studying and characterizing isolated bacteriophages. Also, it is important to understand the importance of the aseptic technique, which helps to minimize the chance of contamination (SEA PHAGE Part 1).

Procedures Since viruses are the most abundant agents on Earth, they can be found in any kind of environment. The finding of bacteriophages was carried out through the collection of 1.000 g of soil samples from a tropical environment. Specific information such as coordinates, temperature, hour, and address from the place where the soil sample was collected is needed. After the collection of the soil samples, approximately 0.5000 g were used for the enrichment culture technique. This was performed to create conditions that favor replication of the specific bacterial phages. By seeding the sample with host bacteria (0.5-mL) and adding a medium broth to the mixture (5.0-mL) conditions would be optimized for bacterial growth and the phages that are specific to that bacterial species infect the bacterial cells and replicate in higher concentrations (SEA PHAGES, 2012). When the enrichment culture technique was done, incubation of the soil samples at 37˚C for 24 hours was required.

The incubated enrichment cultures were ready for a plaque assay. A requirement for performing this process was to centrifuge to obtain a supernatant (~ 5.0- mL) with the bacteria and phages. In addition, a filtration had to be performed to isolate the bacteria from the phages (if any present) by the removal of liquid

from the supernatant that was centrifuged. Afterwards, the filtrate obtained was diluted in petri dishes by the streaking process, which consisted in making rifling through the petri dish three times to obtain different concentration of phages. Petri dishes were incubated at 37˚C for 24 hours.

After the incubation period, plaques may be found in the petri dishes. If different morphologies can be identified, it indicates that a variety of phages were found. In order to isolate a single phage population from samples with potentially mixed a purification process had to be done. In this process a single plaque was isolated from the petri dish and a dilution process through the streaking technique was performed again. This process was performed four times, to obtain accurate results. When the purification process was completed,

another enrichment culture technique was performed to amplify the quantity of the phages that were purified.

The third purification stock filtrate, obtained from the second enrichment culture technique, was used to perform a polyacrylamide gel. The purpose of performing such a process was to separate the phage’s capsid proteins in order to understand its composition with a chemical denaturant, SDS. The protein sample was obtained from the supernatant of a centrifugation performed for the third purification stock filtrate.

A series of eight dilutions were performed with the purpose of obtaining the purified phage in different concentrations. After the dilutions, a Spot test was performed (Image 3).

Results

Sample Coordinates Details Depth Temperature Date PlaceSample #1 AMIB

Lat: 18.215651Lon: -66.044790

Humid and Dry soil

3’’ 25˚C 02/02/14 Caguas, PR

Sample #2 AMIB

Lat: 18.234378Lon: -66.061491

Dry soil 2’’ 25˚C 02/17/14 Caguas, PR

Sample #3 AMIB

Lat: 18.234604Lon: -66.06138

Humid and dry soil

1’’ 27˚C 02/24/14 Caguas, PR

Sample #4 AMIB

Lat: 18.213103Lon: -66.041709

Humid and damp Soil

3’’ 26˚C 04/03/14 Caguas, PR

Sample #5 AMIB

Lat: 18.215715Lon: -66.044879

Dry soil 1’’ 25˚C 11/03/14 Caguas, PR

Sample #6AMIB

Lat: 18.234378Lon: -66.061491

Dry soil 1’’ 26˚C 25/03/2014 Caguas, PR

Sample #1 FPO

Lat: 18.11’ 15’’Lon: 65.57’ 54’’

Dry soil 3’’ 24˚C 02/04/14 San Lorenzo, PR

Sample #2 FPO

Lat: 18.11’ 15’’Lon: 65.57’ 53’’

Dry soil 4’’ 26˚C 02/18/14 San Lorenzo, PR

Sample #3 FPO

Lat: 18.7’ 6’’Lon: 66.9’ 42’’

Humid soil 3’’ 28˚C 02/18/14 Cayey, PR

Sample #4 FPO

Lat: 18.11’ 15’’Lon: 65.57’ 53’’

Humid soil 4’’ 22˚C 02/26/14 San Lorenzo, PR

Sample #5 FPO

Lat: 18.2’ 26’’Lon: 66.35’ 35’’

Humid soil 6’’ 30˚C 02/03/14 Ponce, PR

Table 1. Soils samples collected in Tropical soils

Eleven soil samples were analyzed for the phages hunting process (five samples one person and six samples one person).

A phage was found in one partner’s 5th

soil sample after 3 days of incubation. The enrichment culture technique

process had to be repeated twice for the soil sample to retrieve the obtained result. No positive results were obtained after the process repeated.

A soil sample collected from Sao Paolo, Brazil gave positive results for five phage plaques. The phage was found on April 2nd, 2024 in a populated area near a river at a 19˚C temperature. The phage was named Gemeos. Process was performed until the Third Purification Stock Filtrate (Image 1).

A polyacrylamide gel was performed to separate the phage’s capsid proteins in order to understand is composition by analyzing how proteins migrate though the gel according to the

macromolecule’s length. The protein sample was loaded into the EM Grids in channel 9C for both gels. (Image 2). Protein migration was evident and could be appreciated through the gels, which indicates that the samples had a great quantity and potential proteins, and that the procedure was successfully performed.

Eight dilutions were done to obtain the purified phage in different concentrations and a Spot test was performed. In the Spot test it was appreciated that the concentrations where the purified phage is at its highest concentration are at the dilutions number one, two, three, and four (Image 2).

Image 1. Bacteriophage photo through a Dissecting microscope

Image 2. Electrophoresis (37 minutes)

Discussion

The results of this experiment demonstrate that bacteriophages can be isolated form tropical soils. In addition, that the place where soil samples are collected have a great influence in the finding of a phage.

Throughout the experiment a total of ten soil samples were analyzed. Problems with contamination, the availability of the bacteria, and deficient in the bacteria function were reasons that the soil samples did not give a positive result on phages. On the gel made for the capsid’s protein composition time, voltage and how samples were administered with pipets could be

reasons for the resolution of the gel not to be precise.

The highest concentration of purified phage was found on dilutions the first four dilutions, which indicates that the phage has a great potential and development.

These kinds of experiments help scientific students enhance Scientific Research. It also helps understand how science can impact and contribute to a better society.

Acknowledgments

Research supported by the RISE Program & PHAGES Program sponsored by the Howard Hughes Medical Institute.

Special thanks to Dr. Michael Rubin, Gustavo Martinez (TA), Giovanni Cruz (TA), and Christopher Quintanal (TA).

References

Science Education Alliance, Howard Hughes Medical Institute. 2012. SEA PHAGES Laboratory Manual. 2012.

Image 3. Results of Spot test

(Before you start: Part I; Capture: Part I)

Snustad and Simmons. 2012. Principles of Genetics, 6th edition. John Wiley &

Sons, Inc. Hoboken, New Jersey, USA. (p. 165-169)

Dr. Michael Rubin and Dr. Edwin Vázquez. 2012. Mycobacteriophage Proteomics: From Genotype to Phenotype (There and Back Again!). Howard Hughes Medical Institute.

Pendleton JN, Gorman SP, Gilmore BF. 2013. Clinical relevance of the ESKAPE pathogens. Expert Rev Anti Infect Ther 2013; 11:297-308; PMID:23458769;