which bands were missing. We then located the deletion more bacteriophages were then prepared for electron microscopy by precisely by accounting for the new bands that were produced by negative staining with 1.0% sodium phosphotungstate, pH 7.6, as the digestion of all restriction endonucleases used. previously done.9 Specimens were observed with a JEOL100CX To obtain single-base resolution, we designed primers and electron microscope in the Department of Pathology at The performed PCR across the suspected site of a deletion and University of Texas Health Science Center at San Antonio. compared the length of the PCR fragment and its digestion Fluorescence microscopy. To prepare a specimen for in situ products to the corresponding lengths obtained from the wild (in-plaque) fluorescence microscopy, part of the clear region of a type DNA. If this result confirmed deletion, we then Sanger 0305w8-36 plaque was excised and then mixed with a ~1:20 sequenced the PCR fragment to identify the site of the deletion amount of β-mercaptoethanol and a ~1:10 amount of 15 mg/ml with single-base resolution. DAPI (4',6-diamidino-2-phenylindole). Then, this mixture was Interpretation of DNA sequences. To advance beyond the incubated for at least 1 min at room temperature and 0.5–1.0 ml original annotation8 of bacteriophage 0305w8-36, we constructed extended protein families by iterative application of PsiBlast and SAM and an updated database.45,46 We also used HHpred47,48 was placed between cover glass and microscope slide, cleaned as described in reference 4. Specimens were visualized by use of an Olympus BX 60 upright fluorescence microscope with a short to address a common problem with iterative family building arc mercury vapor lamp and an oil immersion lens. The filter set methods, in which an unnamed member of a distant family is is described in reference 4. Real time analog video was recorded included at an early round with poor confidence, causing the inclusion of named members of the distant family at a later round and digitized as previously described.4 To prepare a specimen for observation of the contents of a with high confidence. In this case, the E value reported, when the fraction of a sucrose gradient, 5 ml of a fraction of a sucrose © named member 2012 of the distant family Landes appears, exaggerates the gradient was Bioscience. mixed with 0.2 ml of 15 mg/ml DAPI and 0.2 ml of confidence in the link between the query protein and the distant β-mercaptoethanol. This mixture was placed between a cover family. In cases where we suspected this problem, we verified the glass and microscope slide cleaned as described in the previous weak step of the linkage as follows. Sequences clearly related to the paragraph. Specimens were visualized by use of an Olympus query protein within a conservative E value were aligned and BX60 upright fluorescence microscope with a short arc mercury converted to an HHpred-style HMM. Sequences clearly related to vapor lamp and an oil immersion lens. The filter set was the NU the named member of the target family were similarly aligned set from Chroma Technology Corp. ~100,000 member HDHpred olibrary. TnheE ovalue tfor the dweakistrDiisclobsure of uPotenttial eConflic.ts of Interest and converted to an HMM. Both HMMs were inserted in the link joining the two families was then evaluated by HHpred No potential conflicts of interest were disclosed. during its search of this library with each family HMM used as the query. Acknowledgments Electron microscopy. Bacteriophages purified by centrifuga- This work was supported by grants from The Robert J. Kleberg, tion in a sucrose gradient were dialyzed against the following Jr. and Helen C. Kleberg Foundation and the Welch Foundation buffer: 0.01 M Tris-Cl, 0.01 M MgSO4, pH 7.4. The dialyzed (AQ-764). References 1. Clokie MRJ, Millard AD, Letarov AV, Heaphy S. Phages in nature. Bacteriophage 2011; 1:31-45; PMID: 21687533; http://dx.doi.org/10.4161/bact.1.1.14942 2. Summers WC. In the beginning…. Bacteriophage 2011; 1:50-1; PMID:21687535; http://dx.doi.org/10. 4161/bact.1.1.14070 3. Serwer P, Wang H. Single-particle light microscopy of bacteriophages. J Nanosci Nanotechnol 2005; 5:2014- 28; PMID:16430135; http://dx.doi.org/10.1166/jnn. 2005.447 4. Serwer P, Hayes SJ, Lieman K, Griess GA. In situ fluorescence microscopy of bacteriophage aggregates. J Microsc 2007; 228:309-21; PMID:18045325; http:// dx.doi.org/10.1111/j.1365-2818.2007.01855.x 5. Pais-Correia AM, Sachse M, Guadagnini S, Robbiati V, Lasserre R, Gessain A, et al. Biofilm-like extracellular viral assemblies mediate HTLV-1 cell-to-cell transmis- sion at virological synapses. Nat Med 2010; 16:83-9; PMID:20023636; http://dx.doi.org/10.1038/nm.2065 6. Jolly C. Cell-to-cell transmission of retroviruses: Innate immunity and interferon-induced restriction factors. Virology 2011; 411:251-9; PMID:21247613; http:// dx.doi.org/10.1016/j.virol.2010.12.031 7. Serwer P, Hayes SJ, Thomas JA, Demeler B, Hardies SC. Isolation of novel large and aggregating bacteriophages. Methods Mol Biol 2009; 501:55-66; PMID:19066810; http://dx.doi.org/10.1007/978-1-60327-164-6_6 8. Thomas JA, Hardies SC, Rolando M, Hayes SJ, Lieman K, Carroll CA, et al. Complete genomic sequence and mass spectrometric analysis of highly diverse, atypical Bacillus thuringiensis phage 0305phi8- 36. Virology 2007; 368:405-21; PMID:17673272; http://dx.doi.org/10.1016/j.virol.2007.06.043 9. Serwer P, Hayes SJ, Thomas JA, Hardies SC. Propagat- ing the missing bacteriophages: a large bacteriophage in a new class. Virol J 2007; 4:21; PMID:17324288; http://dx.doi.org/10.1186/1743-422X-4-21 10. Sutherland IW, Hughes KA, Skillman LC, Tait K. The interaction of phage and biofilms. FEMS Microbiol Lett 2004; 232:1-6; PMID:15061140; http://dx.doi. org/10.1016/S0378-1097(04)00041-2 11. Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol 2004; 2:95-108; PMID: 15040259; http://dx.doi.org/10.1038/nrmicro821 12. Abedon ST. Bacteriophages and Biofilms: Ecology, Phage Therapy, Plaques. Nova Science Publishers 2011, Hauppauge, New York. 13. Brady JM, Gray WA, Caldwell MA. The electron microscopy of bacteriophage-like particles in dental plaque. J Dent Res 1977; 56:991-3; PMID:270498; http://dx.doi.org/10.1177/00220345770560082901 14. Fujisawa H, Morita M. Phage DNA packaging. Genes Cells 1997; 2:537-45; PMID:9413995; http://dx.doi. org/10.1046/j.1365-2443.1997.1450343.x 15. Son M, Hayes SJ, Serwer P. Concatemerization and packaging of bacteriophage T7 DNA in vitro: deter- mination of the concatemers’ length and appearance kinetics by use of rotating gel electrophoresis. Virology 1988; 162:38-46; PMID:3336943; http://dx.doi.org/ 10.1016/0042-6822(88)90392-3 16. Studier FW, Rosenberg AH, Simon MN, Dunn JJ. Genetic and physical mapping in the early region of bacteriophage T7 DNA. J Mol Biol 1979; 135:917-37; PMID:231684; http://dx.doi.org/10.1016/0022-2836 (79)90520-5 17. Rhoades M, Schwartz J, Wahl JM. New deletion mutant of bacteriophage T5. J Virol 1980; 36:622-6; PMID:7431490 18. Séchaud J, Streisinger G, Emrich J, Newton J, Lanford H, Reinhold H, et al. Chromosome structure in phage T4, II. Terminal redundancy and heterozygosis. Proc Natl Acad Sci U S A 1965; 54:1333-9; PMID: 4222403; http://dx.doi.org/10.1073/pnas.54.5.1333 19. Casjens S, Wyckoff E, Hayden M, Sampson L, Eppler K, Randall S, et al. Bacteriophage P22 portal protein is part of the gauge that regulates packing density of intravirion DNA. J Mol Biol 1992; 224:1055-74; PMID:1569567; http://dx.doi.org/10.1016/0022-2836(92)90469-Z 20. Núñez B, De La Cruz F. Two atypical mobilization proteins are involved in plasmid CloDF13 relaxation. Mol Microbiol 2001; 39:1088-99; PMID:11251827; http://dx.doi.org/10.1046/j.1365-2958.2001.02308.x 34 Bacteriophage Volume 2 Issue 1