Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Henry Potosnak Period 3 March 30, 2009 Independent Study Report Bacteriophage Discovery and Characterization Research Project I. Introduction Purpose The purpose of this investigation is to attempt to discover a never- before-classified bacteriophage. The research involves finding a novel bacteriophage and then characterizing it. The techniques employed to characterize the identified phage include electron microscopy; sequencing, annotating, and analyzing the genome of the phage; and comparing the genome of your phage to other sequenced phage genomes. Background The goal of this study is to discover a new species of bacteriophage. Bacteriophages are viruses that infect, feed off of, and destroy bacteria. They are much smaller than bacteria. A bacteriophage’s size is generally between 20 and 200 nanometers. Bacteriophages typically consist of an outer protein1 encasing genetic material. Their four genetic structure configurations are: ssRNA, dsRNA, ssDNA, or dsDNA. The “ss” or “ds” indicates whether the DNA/RNA is single-stranded or double-stranded. Bacteriophages have some of the largest populations of organisms on Earth. It is thought that they are the most amply dispensed and assorted organisms in our biosphere. Bacteriophages are practically universal. They can survive in almost any environment. If bacteria exist in a location, it is quite likely that bacteriophages exist there as well. The preeminent place to find bacteriophages (and other viruses) is the ocean. The population of bacteriophages in sea water can be so dense that some microbial mats have been found to contain up to 9x108 virions per milliliter. It is estimated that up to 70% of marine bacteria may be infected by bacteriophages.2 The group of scientists who classify bacteriophages is referred to as the International Committee on Taxonomy of Viruses (ICTV). The ICTV has found a few orders to classify bacteriophages, but most bacteriophages belong to just one order, Caudovirales. Caudovirales are the dsDNA tailed bacteriophages. 95% of all the recorded bacteriophage findings belong to the Caudovirales.3 It is possible that Caudovirales make up the majority of bacteriophages on our planet. Following is a list of the families of the Caudorvirales’ order: ICTV Classification of Bacteriophages Order Family Morphology Nucleic acid Myoviridae Non-enveloped, contractile tail Linear dsDNA Siphoviridae Non-enveloped, long non-contractile tail Linear dsDNA Podoviridae Non-enveloped, short noncontractile tail Linear dsDNA Tectiviridae Non-enveloped, isometric Linear dsDNA Corticoviridae Non-enveloped, isometric Circular dsDNA Lipothrixviridae Enveloped, rod-shaped Linear dsDNA Plasmaviridae Enveloped, pleomorphic Circular dsDNA Rudiviridae Non-enveloped, rod-shaped Linear dsDNA Fuselloviridae Non-enveloped, lemon-shaped Circular dsDNA Inoviridae Non-enveloped, filamentous Circular ssDNA Microviridae Non-enveloped, isometric Circular ssDNA Leviviridae Non-enveloped, isometric Linear ssRNA Cystoviridae Enveloped, spherical Segmented dsRNA Caudovirales Bacteriophages were discovered by a French-Canadian microbiologist Félix d'Hérelle. d'Hérelle was working at the Pasteur Institute in Paris when he announced on September 3, 1917 that he had found “an invisible antagonistic microbe of the dysentery bacillus.” He said, “in a flash I had understood: what caused my clear spots was in fact an invisible microbe … a virus parasitic on bacteria.” He named it a bacteriophage (from the Greek word phagein, which means: to eat4). The two cycles used by bacteriophages for replication are: 1) a lytic cycle, and 2) a lysogenic cycle.5 Most bacteriophages can only use one of these cycles. A few can use both. In the lytic cycle, a bacterial cell is burst agape (lysed) and eradicated after immediate cloning of the virion. After the bacterial cell is destroyed, the new bacteriophages can find another soonto-be-destroyed host. The less violent lysogenic cycle does not end in immediate lysing of the host cell. The bacteriophages that follow this cycle are known as temperate phages. In this cycle, bacteriophages integrate their viral genome with the host’s DNA and replicate with it.6 Sometimes, it may establish itself as a plasmid. The bacteriophage keeps the cell living and reproducing, because the virus is reproduced in every replicated cell. In the lysogenic cycle, bacteriophages do not destroy the host. They become long-term occupants. The bacteriophage will remain dormant until the host’s health weakens, which may be due to loss of nutrients. Then, it activates, begins the reproductive cycle, and eventually ends in the lysing of the host cell. Bacteriophages use several different adaptations to attach themselves to their bacterial host. The known adaptations are: Lipopolysaccharides Teichoic acids Proteins Flagella. Since different bacteriophages have specific adaptations to attach themselves to bacteria, each bacteriophage can attach to certain bacteria. The bacteria need the matching receptors to the bacteriophages’ adaptations. Bacteriophage virions cannot move independently, so they must rely on random encounters with matching receptors. After connecting with the matching receptor, the bacteriophages begin their assault on the bacteria. More complex bacteriophages use a motion that can be compared to that of a syringe to inject their genetic material into the bacteria cell. Other bacteriophages use their tail fibers to move closer to the bacteria cell’s surface. Next, the bacteriophages attach themselves to the surface completely, contract their tail (which could be possible due to the ATP in the tail), and inject their genetic material through the bacteria’s cell membrane. It only takes a few minutes to override and replace the bacteria’s RNA with the virus’s mRNA. The host’s ability to synthesize proteins and nucleic acids gets confused. It now must construct viral crops instead. The viral products and the helper proteins are used to create new virions within the cell. Some of the proteins are used in the cell for lysis. Once the bacterial cell has been used up, the bacteriophages will exit the cell via lysis, extrusion, or rarely by budding. Bacteriophages with tails use an enzyme known as endolysin. Endolysin breaks down and destroys the cell wall peptidoglycan. Another type of bacteriophage, the filamentous phage, forces the host cell to constantly excrete new virus particles. Budding is affiliated with specific Mycoplasma phages. Bacteriophages can and are being used to stop and prevent bacteria from infecting humans. In August, 2006, the United States Food and Drug Administration (FDA) approved the use of bacteriophages on cheese to eliminate the Listeria monocytogenes bacteria, giving them the status of GRAS (Generally Recognized as Safe). In July 2007, the very same bacteriophages were approved for use on every food product. Bacteriophage are even being used in hospitals as a preventative treatment for medical devices. New technology now allows for bacteriophages to be applied to dry surfaces. There is even research into their potential to serve as an offense to bio-terror events. II. Procedure Attachment 1 provides the detailed procedure for the experiments required for this investigation. I conducted these experiments using microbiology and molecular biology techniques as a volunteer participant in the Phagehunting independent research program in the Phage hunter lab of Dr. Graham Hatfull at the University of Pittsburgh’s Department of Biological Sciences and under the mentorship of Nicolle Gill. The project is funded by a Howard Hughes Medical Institute Professorship grant. III. Results Attachment 2 provides the results recorded during the weekly research sessions. The notebook pages contain details of each step performed as well the results and observations noted. IV. Conclusions Sample 00784 contains a phage. Sample 00784 was plated out from 10-1 to 10-10. A plaque was picked from plate 10-5. That pick was plated, and showed five different types of plaques. Plaque one was a larger, more opaque plaque. Plaque one was the plaque picked for purification. After four rounds of purification, only plaque one remained. Plaque one was identified as a bacteriophage. The following steps will be done in future lab sessions. The lysate will be filtered through a .22 micro liter filter. The next step will be to determine the concentration, or titer, of a lysate. After the titer is obtained, the phage will be amplified by using larger volumes and bigger plates. The goal to find a phage has been met, but the phage must still be characterized. 1 http://www.encyclopedia.com/topic/bacteriophage.aspx, The Columbia Encyclopedia, Sixth Edition, 2008 http://en.wikipedia.org/wiki/Bacteriophage 3 http://en.wikipedia.org/wiki/Bacteriophage 4 http://www.zeuscat.com/andrew/personal/info/tphage/ 5 http://www.phages.org/PhageInfo.html 6 http://www.phages.org/PhageInfo.html 2