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Characterization of Bacteriophages of Pseudomonas Syringae Pv. Tomato Sara E

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Characterization of Bacteriophages of Pseudomonas Syringae Pv. Tomato Sara E Journal of the Arkansas Academy of Science Volume 61 Article 14 2007 Characterization of Bacteriophages of Pseudomonas syringae pv. tomato Sara E. Prior University of Arkansas at Monticello Amanda J. Andrews University of Arkansas at Monticello Russell O. Nordeen University of Arkansas at Monticello, [email protected] Follow this and additional works at: http://scholarworks.uark.edu/jaas Part of the Plant Biology Commons, and the Plant Pathology Commons Recommended Citation Prior, Sara E.; Andrews, Amanda J.; and Nordeen, Russell O. (2007) "Characterization of Bacteriophages of Pseudomonas syringae pv. tomato," Journal of the Arkansas Academy of Science: Vol. 61 , Article 14. Available at: http://scholarworks.uark.edu/jaas/vol61/iss1/14 This article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able to read, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. This Article is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academy of Science by an authorized editor of ScholarWorks@UARK. For more information, please contact [email protected], [email protected]. Journal of the Arkansas Academy of Science, Vol. 61 [2007], Art. 14 Characterization ofBacteriophages ofPseudomonas syringae pv. tomato I 1 SARA E. PRIORI, AMANDA 1. ANDREWS , AND RUSSELL O. NORDEEN ,2 an WI 'School ofMathematical and Natural Sciences, University ofArkansas at Monticel1o, 397 University Drive, Monticel1o, AR 71656 11 ac 2Correspondence: [email protected] S(I to Abstract.-Bacteriophages from supernatants ofthe plant pathogenic bacteria Pseudomonas syringae pv. tomato (P. tomato) were isolated, enriched, and purified by density block centrifugation in cesium chloride (CsCl) step gradients. The DNA from purified O. phage was isolated and digested with the restriction endonucleases EcoRl or HindIII. Three different DNA fingerprint patterns were iJJ determined indicating 3 unique phage isolates. Genome sizes ofthe phage ranged from 40 to 52 kilobases (kB). Buoyant densities of N phage particles in CsCI varied from 1.36 to 1.51 glml. Electron microscopy revealed a single morphological type with an elongated polyhedral head and a long tail indicating the family Siphoviridae. Key words:- Bacteriophages, Pseudomonas syringae pv. tomato, DNA, endonucleases, EeoR1, HindlII, Siphoviridae. CI e: Introduction the .suitability of phage in controlling bacterial plant diseases o (Flaherty et al. 2000, Flaherty et aJ. 2001, Obradovic et al. a Bacterial speck, first described and identified in Taiwan and 2004). Further genetic knowledge ofphage used inthese studies F the United States in 1933, caused a major outbreak in 1978, and would be beneficial and might lead to a more efficient use ofthis v the disease has become a major problem for tomato growers in disease-control strategy. certain regions ofNorth America. Characterized by small brown The objectives of this study were to enrich for, then to black lesions on various tomato tissues, this disease is caused characterize bacteriophages ofP. tomato by electron microscopy by the bacterial pathogen Pseudomonas tomato (Jones et al. and restriction endonuclease digestion. This approach should 1991). Bacterial speck is not significantly harmful to the plant reveal unique phage isolates for further transduction and itself, but severe spotting on the fruit can reduce marketable possible DNA sequencing studies. The infonnation from these yield. studies should clarify the role these phage play in host-pathogen The taxonomic status ofP. tomato is unclear. It is difficult relationships between P. tomato strains and appropriate host to distinguish morphologically and physiologically from other plants. pathovars ofthe species, particularly Pseudomonas syringae pv. s}Tingae (P. syringae). Pathovars ofP. syringae cause disease on different plant hosts, but P. syringae has also been characterized Materials and Methods from necrotic lesions on infected tomato fruit (Jones et al. 1981). Bacterial Strains and Media.-Three P. tomato strains, Pseudomonas tomato is also of interest as it has been used B19, Pst6, and PDDCC3647, which had proven to be suitable as a model pathogen in studies with Arabidopsis tha/iana, a hosts for phage propagation and enrichment (Minor et al. 1996), common lawn weed ofearly spring that has proven amenable to were used for the isolation and characterization ofphages in this molecular genetic manipulations. Researchers are studying P. study. NBY media was used for all bacterial strains (Vidaver tomato infections ofA. thaliana to gain a better understanding 1967). NBYsoft agar (0.7g1100 rnl) was used to create overlays ofthe molecular basis of bacterial disease and bacterial-disease for phage enrichment andtiter(Adams 1959). Media components resistance (Whalen and Staskawicz 1990). The genomes ofA. as well as all reagents for phage enrichment and DNA isolation thaliana and P. tomato strain De3000 have been sequenced; were obtained from Fisher (St. Louis, MO). however, there is limited knowledge of P. tomato temperate Propagation and Enrichment ofBacteriophages.-NBY phage (Cuppels 1983, Minor et at. 1996). thick agar plates containing 35 to 40 ml offreshly prepared NBY Temperate phages capable of lysogeny probably account agar were overlaid with 3 ml ofmolten NBY soft agar at 50° C for most transduction-mediated gene flow among bacteria in containing approximately 5 x 107 cells per ml ofthe appropriate nature and provide genetic systems that are most easily studied propagating strain. These strains were grown to stationary phase (Gerhardt et at. 1984, Kidambi et al. 1993). Genomic sequencing in NBY broth at 28° C and diluted in phage buffer to an optical of a number of plant pathogenic bacteria has revealed that a denslty of 0.15 at 600 nm. The phage buffer consisted of 10 • substantial portion of these genomes contain mobile genetic roM Tris HCI. pH 7.5, and IO roM MgS04 Following overnight elements, including up to 7% prophage sequences (Buell et al. incubation at 28° C (I5 to 18 hours), 5 ml ofphage buffer was 2003, Simpson et aI. 2000). The role of phage in the disease added to the surface ofthe overlay, and the plates were incubated process caused by these bacterial pathogens is unclear at at room temperature for 30 minutes to 2 hours or 4° C overnight. present. Studies have recently been performed to investigate The phage buffer was then drawn offwith a sterile Pasteur pipet Published by Arkansas Academy of Science,Journal 2007 oftheArkansas Academy ofScience, VoL 61, 2007 84 Journal of the Arkansas Academy of Science, Vol. 61 [2007], Art. 14 Characterization ofBacteriophages ofPseudomonas s)'ringae pv. tomato and placed into 30-ml Oak Ridge centrifuge tubes. The tubes gels in TAE buffer at a constant voltage of 5 to 6 volts per cm. 56 were centrifuged at 10,000 rpm at 4° C for at least 10 minutes. EcoRI and HindlII endonucleases were purchased from Promega The supernatants were filtered through a 0.2-mm cellulose (Milwaukee, WI). Size standards for gel electrophoresisconsisted acetate filter into sterile 1.5-ml Eppendorf tubes. These phage ofbacteriophage lambda DNA digested with HindU!. Following solutions were stored at 4° C until they were used in experiments staining of gels with ethidium bromide (4 mg/mI), gels were to determine phage titer. destained in distilled water and documented with Polaroid film were Phage enrichments for DNA isolation were prepared using or an Alpha Innotech imaging system. Sizes of DNA fragments ilied 0.35% (w/v) of the synthetic gelling agent gelrite to avoid were determined by plotting the log of the molecular weight of were inhibition of restriction endonucleases (Bouche 198I ). The DNA fmgments observed against the distance traveled in mOl. :s of NBY thick-plate underlay contained 0.74% (w/v) gelrite. The ated enrichment plates were incubated overnight (15-I 8 hours) at 28° Cand overlaid with 5 ml ofphage buffer for 30 min at room Results temperature. The enriched bacteriophage preparations were centrifuged and filtered as previously described. Titers of the Three strains of P. tomato (819, Pst6, and PDDCC3647) enriched bacteriophage isolates were determined by the plate­ were used to enrich bacteriophages. These strains were selected overlay method (Adams 1959). Following overnight incubation on the basis ofa preliminary survey for bacteriophage production lSe5 al. at 28° C, the plates were checked and the phage titer determined. by 44 P. tomato stmins (Minor et al. 1996). Plate enrichments 9 10 lies High-titer phage isolates, >10 pfu (plaque forming units) perml, using these 3 hosts produced titers of>10 pfu (plaque forming 10 this were further concentrated to >10 pfu per ml by polyethylene units) per ml after concentmtion with PEG6000. (Table I). glycol 6000 precipitation (Yamamoto et al. 1970). High-titer Initial attempts to purify phage using CsCI block gradients len phage stocks were purified by CsCI block density gradients containing three 2.4-ml layers of 1.7 glml, 1.5 g/ml, and 1.45 lpy (Davis et al. 1980). glml caused the phage to band in the upper third of the 12-ml tJ1d Electron Microscopy.-Phages purified from CsC! gradients tubes used. Centrifugations were performed in a SW41 rotor Uld were dialyzed in two changes ofphage buffer overnight at 4° C at 23,000 rpm for 2 hours at room tempemture. Subsequent :se to remove CsCI. The phage titers were determined and samples purifications used five 2-ml layers of 1.7 glml, 1.5 glml, 1.45 with a titer of >I0 10 pfu per ml were spread onto grids with glml, 1.3 glml, and 1.2 glml and 1.0 ml of enriched phage in :en )st carbon-eoated Fonnvar support films, negatively stained with phage buffer. Using this protocol, phage banded near the middle 1% uranyl acetate, and examined with a Joel 1200EX electron of the centrifugation tubes; although both upper diffuse and microscope.
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