hits from host to host: isolation, characterization, and … · 2017-06-02 · hits from host to...

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Hits from Host to Host: Isolation, Characterization, and Comparative Genomics of Five New Gordonia phages Alanna M Bram, Amanda J Weber, Katelyn R Chester, Kimberly E Doerr, Amanda A Drigans, Christopher O Dusek, Brenna K Franke, Allison K Granberg, Shiza S Idrees, Katie E Klinkhammer, Danielle M Kocina, Marissa L Leopold, Zachary A Lund, Theresa N Lusk, Kaitlyn J Matczynski, Abbey L Novotny, Kayla E Oberding, Allison N Potocnik, Chase A Quandt, Marissa Y Schmitz, Danielle E Schultz, Tiffany A Stocks, Willow M Stuart, Christian Thaoxaochay, Cami P Thomas, Tara N Toland, Samantha A Topel, Emily R Warren, Max R Wetzel, Rebecca J Welman, Katlyn M Williams, Olivia H Yue, Cody J Ziegler; J. Alfred Bonilla and Karen K. Klyczek, Instructors Biology Department, University of Wisconsin-River Falls, River Falls, WI 54022 Isolation of Gordonia phage Relatively few phages infecting hosts other than Mycobacterium smegmatis have been isolated and characterized (1). UWRF phage hunters set out to isolate phage infecting another member of the phylum Actinobacteria, Gordonia terrae. Gordonia species are found in soil and wastewater, and are associated with foaming in wastewater treatment. The same procedures used for isolating Arthrobacter and Rhodococcus phage in previous years were used to test 44 soil samples: •LB broth was used for bacteria culture and top agar, no antibiotics •CaCl 2 was added at 4.5 mM in culture broth and 2.25 mM in top agar •Enrichments were cultured at 30 o C for 2-5 days Results: Only five student samples yielded plaques, along with 3 instructor samples piloted prior to class. Several phages were isolated from these samples. We selected phages for sequencing that were likely to be different based on plaque and EM morphologies (Fig. 1), restriction enzyme patterns (not shown), and soil source. Five unique phage sequences were obtained (Table 1). Fig. 1. Plaque morphology and electron microscopy. There was a variety of plaque morphologies observed. JSwag (A), had clear plaques of variable size (1-3 mm), sometimes with halos; Remus and Strosahl had a similar appearance. Jumbo (B) had smaller clear plaques (0.5 mm) and Bantam (C) had very small (<0.5 mm), turbid plaques. All of these new phages are Siphoviridae; Remus (D), JSwag and Strosahl had tails 100-125 nm in length, while Jumbo (E) and Bantam had longer tails, 450-475 nm. References 1. Actinobacteriophage Database (2015), http://phagesdb.org 2. Dyson, ZA et al. (2015) PLoS One 10(8): e0134512. doi:10.1371/journal.pone.0134512 3. Liu, M. et al. (2015) Nature Scientific Reports 5:13754 | DOI: 10.1038/srep13754 4. Summer, EJ et al. Appl. Environ. Microbiol. (2011), 77(2):669 5. Stella, EJ et al. (2013) PLoS ONE 8(2): e56384. doi:10.1371/journal.pone.0056384 Acknowledgements We thank Rick Ellingworth and the Biology Department student lab assistants for preparing materials, the Hatfull lab, University of Pittsburgh, and HHMI SEA PHAGES program for supporting this work, and the entire SEA community, for sharing experiences and advice. Conclusions Phage Drake (A15 by PCR) Bantam: Comparative Genomics A A. 30 o C 35 o C Bantam is a singleton, with little nucleotide similarity to other phages but shared phams with other Gordonia phages (Fig. 2) Jumbo is also a singleton with similarities to Gordonia singleton GMA3 (isolated in Australia), Rhodococcus phage DocB7 (isolated in Texas), and Gordonia cluster DF (isolated in Texas) (Fig. 3) Jumbo and related phages may have multiple lysin and holin genes that could increase efficiency of cell lysis (Fig. 4) Remus, JSwag, Strosahl are cluster A15 phages, very similar to others in that cluster (Fig. 5). Several other unsequenced class phages were A15 according to PCR results (not shown). JSwag and Remus are temperate, lysogens were isolated at 35 o C (Fig. 7A) o Partitioning system, similar to some A2 phages (Fig. 6) o Immunity to other A15 phages (Fig. 7B) Cluster A15 phages Jumbo: Comparative Genomics . A B C D E Table 1. Sequencing Results 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 1 5079 (124) gp1 2 18243 (31) gp2 3 4163 (31) gp3 4 17374 (254) gp4 5 19250 (447) gp5 6 4048 (167) gp6 7 11333 (6) gp7 8 17737 (67) gp8 10 19174 (1) gp10 13 14746 (6) gp13 14 10868 (7) gp14 15 17443 (281) gp15 16 2982 (414) gp16 17 19266 (330) gp17 18 3257 (302) gp18 19 3146 (348) gp19 20 11720 (6) gp20 21 7209 (449) gp21 22 19198 (198) gp22 23 1827 (341) gp23 24 17963 (313) gp24 25 2847 (449) gp25 26 19254 (413) gp26 27 18608 (314) gp27 28 18608 (314) gp28 29 17995 (10) gp29 30 4899 (303) gp30 31 19296 (432) gp31 32 12446 (406) gp32 33 18881 (128) gp33 34 11297 (11) gp34 35 17868 (91) gp35 36 19294 (13) gp36 37 7133 (64) gp37 38 17791 (6) gp38 39 11303 (6) gp39 40 11401 (7) gp40 41 18837 (412) gp41 42 19221 (5) gp42 43 11416 (6) gp43 44 11167 (6) gp44 45 11443 (6) gp45 46 11528 (4) gp46 47 15909 (456) gp47 48 19155 (31) gp48 49 14286 (33) gp49 50 18805 (16) gp50 51 13235 (25) gp51 52 18393 (192) gp52 53 18670 (42) gp53 54 17822 (276) gp54 55 11442 (6) gp55 56 565 (367) gp56 57 5496 (73) gp57 58 19258 (1) gp58 59 17529 (215) gp59 60 13798 (118) gp60 61 9297 (409) gp61 62 11277 (6) gp62 63 11534 (6) gp63 64 12571 (65) gp64 65 11582 (6) gp65 66 4038 (448) gp66 67 19262 (107) gp67 68 11553 (6) gp68 69 11548 (13) gp69 70 15729 (464) gp70 71 11431 (6) gp71 72 11698 (6) gp72 73 19104 (16) gp73 74 17915 (15) gp74 75 18508 (6) gp75 76 11425 (5) gp76 77 5065 (450) gp77 78 11505 (6) gp78 79 11588 (6) gp79 80 18656 (15) gp80 81 19146 (1) gp81 82 19047 (4) gp82 83 5238 (450) gp83 84 19123 (21) gp84 85 19184 (182) gp85 86 11166 (6) gp86 87 11542 (6) gp87 88 1304 (171) gp88 89 11640 (6) gp89 90 11722 (5) gp90 91 11719 (7) gp91 92 11447 (4) gp92 93 11618 (6) gp93 94 18589 (3) gp94 95 11355 (9) gp95 96 11615 (6) gp96 97 6789 (8) gp97 98 2322 (28) gp98 99 11628 (4) gp99 100 11708 (5) gp100 101 11450 (6) gp101 102 11723 (6) gp102 103 6684 (18) gp103 104 11363 (6) gp104 105 578 (2) gp105 JSwag_Draft 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 1 5079 (124) 2 18243 (31) VIP2-like 3 4163 (31) 4 17374 (254) 5 19250 (447) Tail 6 4048 (167) Tail 7 11333 (6) 8 17737 (67) 12 14746 (6) Endolysin 13 10868 (7) Holin 14 17443 (281) Terminase, large subunit 15 2982 (414) Portal 16 19266 (330) Capsid Maturation Protease 17 3257 (302) Scaffolding 18 3146 (348) Capsid 19 11720 (6) 20 7209 (449) 21 19198 (198) 22 1827 (341) 23 17963 (313) 24 2847 (449) 25 19254 (413) 26 18608 (314) Tail Assembly Chaperone 27 18608 (314) Tail Assembly Chaperone 28 17995 (10) Tapemeasure 29 4899 (303) Minor Tail 30 19296 (432) Minor Tail 31 12446 (406) Minor Tail 32 18881 (128) Minor Tail 33 11297 (11) Minor Tail 34 17868 (91) Lsr2 35 19294 (13) 36 7133 (64) ParA 37 17791 (6) ParB 38 11303 (6) 39 11401 (7) 40 11655 (1) 41 11552 (1) 42 18837 (412) 43 19221 (5) 44 11416 (6) 45 11167 (6) 46 11443 (6) 47 11528 (4) 48 15909 (456) DNA polymerase 49 19155 (31) 50 11362 (2) 51 14286 (33) HTH DNA binding domain 52 18805 (16) 53 18393 (192) ThyX 54 18670 (42) MazG- like 55 17822 (276) 56 11442 (6) 57 565 (367) Ribonucleotide reductase 58 5496 (73) 59 17529 (215) 60 13798 (118) Transcription regulator 61 9297 (409) phosphoesterase 62 11277 (6) 63 11534 (6) 64 12571 (65) NrdH- like 65 11582 (6) 66 4038 (448) DNA Primase 67 19262 (107) DNA Primase 68 11553 (6) 69 11548 (13) Endolysin 70 15729 (464) EndoVII 71 11431 (6) 72 11698 (6) 73 19104 (16) 74 17915 (15) 75 18508 (6) 76 11368 (1) 77 5065 (450) DnaB-like helicase 78 11505 (6) 79 11588 (6) 80 18656 (15) 81 19047 (4) 82 5238 (450) RecB-like 83 19123 (21) 84 19184 (182) Repressor 85 11166 (6) 86 11542 (6) 87 1304 (171) SprT-like 88 11640 (6) 89 11719 (7) 90 11447 (4) 91 11618 (6) 92 11355 (9) 93 11615 (6) 94 6789 (8) 95 2322 (28) 96 11628 (4) 97 6310 (15) 98 11450 (6) 99 11723 (6) 100 6684 (18) 101 11363 (6) KatherineG 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 1 5079 (124) gp1 2 18243 (31) gp2 3 4163 (31) gp3 4 17374 (254) gp4 5 19250 (447) gp5 6 4048 (167) gp6 7 11333 (6) gp7 8 17737 (67) gp8 12 14746 (6) gp12 13 10868 (7) gp13 14 17443 (281) gp14 15 2982 (414) gp15 16 19266 (330) gp16 17 3257 (302) gp17 18 3146 (348) gp18 19 11720 (6) gp19 20 7209 (449) gp20 21 19198 (198) gp21 22 1827 (341) gp22 23 17963 (313) gp23 24 2847 (449) gp24 25 19254 (413) gp25 26 18608 (314) gp26 27 18608 (314) gp27 28 17995 (10) gp28 29 4899 (303) gp29 30 19296 (432) gp30 31 12446 (406) gp31 32 18881 (128) gp32 33 11297 (11) gp33 34 17868 (91) gp34 35 19294 (13) gp35 36 7133 (64) gp36 37 17791 (6) gp37 38 11303 (6) gp38 39 11401 (7) gp39 40 11533 (3) gp40 41 18265 (2) gp41 42 18837 (412) gp42 43 19221 (5) gp43 44 11416 (6) gp44 45 11167 (6) gp45 46 11443 (6) gp46 47 15909 (456) gp47 48 19155 (31) gp48 49 14286 (33) gp49 50 18805 (16) gp50 51 13235 (25) gp51 52 18393 (192) gp52 53 18670 (42) gp53 54 17822 (276) gp54 55 11442 (6) gp55 56 565 (367) gp56 57 5496 (73) gp57 58 18584 (2) gp58 59 17529 (215) gp59 60 13798 (118) gp60 61 9297 (409) gp61 62 11277 (6) gp62 63 11534 (6) gp63 64 12571 (65) gp64 65 11582 (6) gp65 66 4038 (448) gp66 67 11553 (6) gp67 68 11548 (13) gp68 69 15729 (464) gp69 70 11431 (6) gp70 71 11698 (6) gp71 72 19104 (16) gp72 73 17915 (15) gp73 74 18508 (6) gp74 75 11425 (5) gp75 76 5065 (450) gp76 77 18612 (2) gp77 78 11505 (6) gp78 79 11588 (6) gp79 80 18656 (15) gp80 81 5238 (450) gp81 82 19123 (21) gp82 83 19184 (182) gp83 84 11166 (6) gp84 85 11542 (6) gp85 86 18719 (29) gp86 87 14542 (23) gp87 89 11640 (6) gp89 90 11722 (5) gp90 91 11719 (7) gp91 92 11447 (4) gp92 93 11618 (6) gp93 94 18589 (3) gp94 95 11355 (9) gp95 96 11615 (6) gp96 97 6789 (8) gp97 98 2322 (28) gp98 99 6310 (15) gp99 100 11450 (6) gp100 101 11723 (6) gp101 102 11363 (6) gp102 Remus_Draft 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 1 5079 (124) 2 18243 (31) VIP2 3 4163 (31) 4 17374 (254) HNH Endonuclease 5 19250 (447) Tail 6 4048 (167) Tail 7 11333 (6) 8 17737 (67) 12 14746 (6) Endolysin 13 10868 (7) Holin 14 17443 (281) Terminase 15 2982 (414) Portal 16 19266 (330) Capsid Maturation Protease 17 3257 (302) Scaffolding 18 3146 (348) Capsid 19 11720 (6) 20 7209 (449) 21 19198 (198) 22 1827 (341) 23 17963 (313) 24 2847 (449) 25 19254 (413) Major Tail 27 18608 (314) Tail Assembly Chaperone 26 18608 (314) Tail Assembly Chaperone 28 17995 (10) Tapemeasure 29 4899 (303) Minor Tail 30 19296 (432) Minor Tail 31 12446 (406) Minor Tail 32 18881 (128) Minor Tail 33 11297 (11) Minor Tail 34 17868 (91) Lsr2 35 19294 (13) 36 7133 (64) ParA 37 17791 (6) ParB 38 11303 (6) 39 11401 (7) 40 12430 (1) 41 18837 (412) 42 11416 (6) 43 11167 (6) 44 11443 (6) 45 11528 (4) 46 15909 (456) DNA polymerase 47 19155 (31) 48 14286 (33) HTH DNA binding domain 49 18805 (16) 50 13235 (25) 51 18393 (192) ThyX 52 18670 (42) MazG 53 17822 (276) 54 11442 (6) 55 565 (367) Ribonucleotide reductase 56 5496 (73) 57 17529 (215) 58 13798 (118) RDF 59 9297 (409) Phosphoesterase 60 11277 (6) 61 11534 (6) 62 12571 (65) NrdH- like 63 11582 (6) 64 4038 (448) DNA Primase 65 19262 (107) DNA Primase 66 11553 (6) 67 11548 (13) Endolysin 68 15729 (464) EndoVII 69 11431 (6) 70 11698 (6) 71 19104 (16) 72 17915 (15) 73 18508 (6) 74 11425 (5) 75 5065 (450) DnaB-like helicase 76 11505 (6) 77 11588 (6) 78 18656 (15) 79 19047 (4) 80 5238 (450) RecB 81 19123 (21) 82 19184 (182) Repressor 83 11166 (6) 84 11542 (6) 85 18719 (29) 86 12429 (1) 87 11640 (6) 88 11722 (5) 89 11719 (7) 90 18689 (2) 91 11618 (6) 92 11355 (9) 93 11615 (6) 94 6789 (8) 95 2322 (28) 96 11628 (4) 97 6310 (15) 98 11450 (6) 99 11723 (6) 100 3693 (5) 101 6684 (18) 102 11363 (6) Rosalind 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 1 5079 (124) 2 18243 (31) VIP2 3 4163 (31) 4 17374 (254) HNH Endonuclease 5 19250 (447) Tail 6 4048 (167) Tail 7 11333 (6) 8 17737 (67) 12 14746 (6) Endolysin 13 10868 (7) Holin 14 17443 (281) Terminase 15 2982 (414) Portal 16 19266 (330) Capsid Maturation Protease 17 3257 (302) Scaffolding 18 3146 (348) Capsid 19 11720 (6) 20 7209 (449) 21 19198 (198) 22 1827 (341) 23 17963 (313) 24 2847 (449) 25 19254 (413) Major Tail 27 18608 (314) Tail Assembly Chaperone 26 18608 (314) Tail Assembly Chaperone 28 17995 (10) Tapemeasure 29 4899 (303) Minor Tail 30 19296 (432) Minor Tail 31 12446 (406) Minor Tail 32 18881 (128) Minor Tail 33 11297 (11) Minor Tail 34 17868 (91) Lsr2 35 19294 (13) 36 7133 (64) ParA 37 17791 (6) ParB 38 11303 (6) 39 11401 (7) 40 11533 (3) 41 18837 (412) 42 19221 (5) 43 11416 (6) 44 11167 (6) 45 11443 (6) 46 11528 (4) 47 15909 (456) DNA Polymerase 48 19155 (31) 49 11362 (2) 50 14286 (33) HTH DNA binding domain 51 18805 (16) 52 18393 (192) ThyX 53 11177 (1) 54 18670 (42) MazG 55 17822 (276) 56 11442 (6) 57 565 (367) Ribonucleotide reductase 58 5496 (73) 59 17529 (215) 60 13798 (118) Transcription regulator 61 9297 (409) phosphoesterase 62 11277 (6) 63 11534 (6) 64 12571 (65) NrdH- like 65 11582 (6) 66 4038 (448) DNA Primase 67 19262 (107) DNA Primase 68 11553 (6) 69 11548 (13) Endolysin 70 15729 (464) EndoVII 71 11431 (6) 72 11698 (6) 73 19104 (16) 74 17915 (15) 75 18508 (6) 76 11425 (5) 77 5065 (450) DnaB-like helicase 78 11505 (6) 79 11588 (6) 80 18656 (15) 81 5238 (450) Rec-B-like 82 19123 (21) 83 19184 (182) Repressor 84 11166 (6) 85 11542 (6) 86 18719 (29) 87 14542 (23) 88 11640 (6) 89 11722 (5) 90 11719 (7) 91 18689 (2) 92 11618 (6) 93 11355 (9) 94 11615 (6) 95 6789 (8) 96 2322 (28) 97 11628 (4) 98 6310 (15) 99 11450 (6) 100 11723 (6) 101 6684 (18) 102 11363 (6) Soups 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 1 5079 (124) gp1 2 18243 (31) gp2 3 4163 (31) gp3 4 17374 (254) gp4 5 19250 (447) gp5 6 4048 (167) gp6 7 11333 (6) gp7 8 17737 (67) gp8 12 14746 (6) gp12 13 10868 (7) gp13 14 17443 (281) gp14 15 2982 (414) gp15 16 19266 (330) gp16 17 3257 (302) gp17 18 3146 (348) gp18 19 11720 (6) gp19 20 7209 (449) gp20 21 19198 (198) gp21 22 1827 (341) gp22 23 17963 (313) gp23 24 2847 (449) gp24 25 19254 (413) gp25 26 18608 (314) gp26 27 18608 (314) gp27 28 17995 (10) gp28 29 4899 (303) gp29 30 19296 (432) gp30 31 12446 (406) gp31 32 18881 (128) gp32 33 11297 (11) gp33 34 17868 (91) gp34 35 19294 (13) gp35 36 7133 (64) gp36 37 17791 (6) gp37 38 11303 (6) gp38 39 11401 (7) gp39 40 11533 (3) gp40 41 18265 (2) gp41 42 18837 (412) gp42 43 19221 (5) gp43 44 11416 (6) gp44 45 11167 (6) gp45 46 11443 (6) gp46 47 15909 (456) gp47 48 19155 (31) gp48 49 14286 (33) gp49 50 18805 (16) gp50 51 13235 (25) gp51 52 18393 (192) gp52 53 18670 (42) gp53 54 17822 (276) gp54 55 11442 (6) gp55 56 565 (367) gp56 57 5496 (73) gp57 58 18584 (2) gp58 59 17529 (215) gp59 60 13798 (118) gp60 61 9297 (409) gp61 62 11277 (6) gp62 63 11534 (6) gp63 64 12571 (65) gp64 65 11582 (6) gp65 66 4038 (448) gp66 67 11553 (6) gp67 68 11548 (13) gp68 69 15729 (464) gp69 70 11431 (6) gp70 71 11698 (6) gp71 72 19104 (16) gp72 73 17915 (15) gp73 74 18508 (6) gp74 75 11425 (5) gp75 76 5065 (450) gp76 77 18612 (2) gp77 78 11505 (6) gp78 79 11588 (6) gp79 80 18656 (15) gp80 81 5238 (450) gp81 82 19123 (21) gp82 83 19184 (182) gp83 84 11166 (6) gp84 85 11542 (6) gp85 86 18719 (29) gp86 87 14542 (23) gp87 89 11640 (6) gp89 90 11722 (5) gp90 91 11719 (7) gp91 92 11447 (4) gp92 93 11618 (6) gp93 94 18589 (3) gp94 95 11355 (9) gp95 96 11615 (6) gp96 97 6789 (8) gp97 98 2322 (28) gp98 99 6310 (15) gp99 100 11450 (6) gp100 101 11723 (6) gp101 102 11363 (6) gp102 Strosahl_Draft 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 1 19690 (1) 2 10819 (1) 3 9775 (14) TerL 4 9969 (17) Mu gp29-like protein 5 10455 (1) 6 9561 (2) 7 10582 (2) 8 10332 (1) 9 19886 (5) 10 20495 (18) 11 19211 (10) 12 10259 (18) 13 10796 (1) 14 10035 (1) 15 20289 (10) 16 10989 (1) 17 9803 (1) 18 9660 (1) 19 11016 (18) 21 10594 (19) pre-TMP frameshift protein 20 10594 (19) truncated pre- TMP frameshift protein 22 10747 (18) tape measure protein 23 10225 (18) 24 10679 (18) 25 9607 (1) 26 19429 (143) putative holin 27 10772 (1) LysA 28 10909 (1) holin 29 9658 (1) 30 16072 (7) holin 31 9753 (1) 32 9700 (1) 33 19904 (9) 34 20467 (10) WhiB 35 10226 (1) 36 20685 (25) DNA methylase 37 15195 (1) 38 18692 (2) LysB DocB7 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 1 13185 (8) putative small terminase subunit 2 15871 (4) putative large terminase subunit 3 9969 (17) 4 15816 (1) 5 20495 (18) 6 16175 (8) 7 10259 (18) putative major capsid protein 8 13008 (8) 9 16299 (1) 10 12800 (8) 11 20240 (4) 12 16402 (5) 13 11016 (18) putative major tail protein 15 13323 (15) putative tail assembly protein 14 13323 (15) putative tail assembly protein 16 10747 (18) putative tape measure protein 17 10225 (18) 18 10679 (18) 19 19064 (4) 20 15960 (2) 21 16263 (1) 22 16261 (5) putative lysin 23 16165 (5) 24 17047 (14) 25 3171 (14) 26 16296 (5) putative lysin 27 16984 (10) putative holin 28 15922 (2) 29 12742 (5) 30 13317 (6) 31 16130 (4) 32 19133 (10) 33 16047 (1) 34 15862 (1) 35 16288 (1) 36 16164 (1) GMA3 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 1 19179 (3) 2 19078 (9) 3 16110 (11) 4 19277 (1) 5 19212 (1) 6 19862 (8) 7 13185 (8) 8 9775 (14) 9 9969 (17) 10 19087 (1) 11 20495 (18) 12 16175 (8) 13 10259 (18) 14 13008 (8) 15 19220 (4) 16 12800 (8) 17 13451 (4) 18 16402 (5) 19 11016 (18) 20 13323 (15) 21 12991 (1) 22 10747 (18) 23 10225 (18) 24 10679 (18) 25 19064 (4) 26 20509 (5) 27 19166 (1) 28 7318 (54) 29 16261 (5) 30 16165 (5) 31 17047 (14) 32 19046 (5) 33 16296 (5) 34 9727 (3) 35 16984 (10) 36 20292 (7) 37 19036 (1) 38 17875 (95) 39 15922 (2) 40 19264 (1) 41 13317 (6) 42 19081 (1) 43 19133 (10) 109 9179 (3) Jumbo_Draft 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 1 13185 (8) 2 15871 (4) TerL 3 9969 (17) DUF935-like protein 4 20126 (6) 5 20495 (18) 6 16175 (8) 7 10259 (18) major capsid protein 8 13008 (8) 9 19220 (4) 10 12800 (8) morphogensis protein 11 13451 (4) 12 16402 (5) 13 11016 (18) 15 13323 (15) pre-TMP frameshift protein 14 13323 (15) pre-TMP frameshift protein 16 10747 (18) tape measure protein 17 10225 (18) 18 10679 (18) 19 20422 (3) 20 20183 (3) 21 1029 (242) 22 16261 (5) lysin 23 16165 (5) 24 17047 (14) lysin A 25 20274 (3) 26 16296 (5) 27 17023 (9) holin 28 20292 (7) DNA methylase 29 20217 (3) 30 9839 (8) holin 31 15963 (6) holin 32 16130 (4) 33 19133 (10) lysin B Gmala1 Overall rates of phage isolation in Gordonia were fairly low. However, 5 new phages were identified, demonstrating the potential for successfully using this host All of the phages shared phams with phages from different hosts, suggesting old evolutionary connections The evolutionary histories of Jumbo, Bantam, and the A15 phages are different, based on GC content and shared phams One or more of these phages may have significant environmental applications, including treatment of wastewater sludge GMA3 Jumbo Gmala1 DocB7 Lysin LysinA Holin Holin? Holin? Membrane LysinB Integrase Pham 16684 (107) Nucleotidase Pham 11635 (6) RecE Exonuclease Pham 11481 (7) Exonuclease VII large subunit Pham 18923 (7) RecT ssDNA binding protein Pham 13007 (7) 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 38 11263 (7) 39 18789 (1) 40 11701 (6) 41 18895 (1) 42 12598 (3) 43 8416 (5) 44 19013 (1) 45 19001 (1) 46 18754 (1) 47 13003 (9) 48 18923 (7) 49 18843 (1) 50 18827 (1) 51 18863 (1) 52 18995 (1) 53 18962 (1) 54 19909 (19) 55 18932 (1) 56 19128 (292) 57 18818 (1) 58 18832 (1) 59 18808 (1) 60 18752 (1) 61 18963 (1) 62 18802 (1) 63 18877 (1) 65 18885 (1) 66 16684 (107) 67 18807 (1) 68 18933 (1) 69 18886 (1) 70 11524 (7) 71 18813 (1) 72 18734 (1) 73 18800 (1) 74 18935 (1) 75 18972 (1) 76 9393 (5) 77 19002 (1) 78 18744 (1) 79 13235 (25) 80 18851 (1) 81 18776 (1) 82 18980 (1) 83 18810 (1) 84 18026 (2) 85 18743 (1) 86 18840 (1) 87 18812 (1) 88 18835 (1) 89 20195 (3) 90 17427 (7) 91 18866 (1) 92 19388 (35) 93 18763 (1) 94 11220 (7) 95 18780 (1) 96 18992 (1) 97 18888 (1) 98 13007 (7) 99 11481 (7) 100 18736 (1) 101 11360 (6) 102 18871 (1) 103 11336 (7) 104 18998 (1) 105 481 (124) 106 20592 (52) 107 18854 (1) 108 18934 (1) 109 13458 (2) 110 18937 (1) 111 18991 (1) 112 2270 (13) 113 18770 (1) 114 11456 (7) 115 10350 (2) 116 18862 (1) 117 13620 (126) 118 18940 (1) 119 18968 (1) 120 18979 (1) 121 11635 (6) 122 19293 (15) 123 18869 (1) 124 18880 (1) 125 473 (10) 126 18984 (1) 127 18829 (1) 128 18836 (1) 129 18993 (1) 130 18889 (1) 131 1033 (5) 132 7101 (5) 133 18917 (1) 134 18853 (1) 135 19133 (10) 136 18753 (2) 137 18850 (1) 138 18748 (1) 139 18788 (1) 140 18926 (1) 141 19020 (1) 142 18883 (1) 143 18792 (1) 144 18950 (1) 145 19012 (1) 146 18997 (1) 147 18964 (1) 148 18771 (1) 149 18943 (1) 150 18799 (1) 151 18793 (1) 152 18948 (1) 153 11401 (7) 154 18931 (1) 155 18875 (1) 156 18884 (1) 157 19007 (1) 158 18814 (1) 159 17425 (2) 160 18939 (1) 161 18909 (1) 162 18833 (1) 163 18873 (1) 164 18740 (1) 165 19008 (1) 166 18817 (1) 167 18904 (1) 168 19035 (12) 169 18826 (1) 170 6387 (68) 171 3014 (3) 172 18953 (60) 173 18765 (1) 174 18954 (1) 175 18946 (1) 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 53 19064 (4) 54 19737 (1) 55 19736 (1) 56 9475 (4) Virion protein 57 19889 (1) 58 11611 (4) 59 19743 (1) Peptidase S1 60 19649 (1) 61 18923 (7) Exonuclease VII large subunit 62 19905 (1) Exonuclease VII small subunit 63 19748 (1) 64 19832 (1) 65 19731 (1) 66 11603 (6) 67 19893 (1) 68 19927 (1) 69 8809 (20) 70 5467 (70) RNA-splicing ligase RtcB 71 20513 (3) HNH endonuclease 72 11445 (5) 73 860 (14) 74 19547 (1) HNH endonuclease 75 10395 (20) Metallophosphatase 76 19910 (1) 77 19787 (1) 78 19920 (1) 79 19627 (1) 80 19604 (1) 81 19747 (1) 82 19757 (1) 83 5284 (31) 84 11169 (5) 85 19656 (1) 86 19918 (1) 87 19853 (1) 88 19839 (1) 89 19880 (1) 90 4379 (24) 91 19766 (1) 92 19541 (1) 93 17161 (2) 94 19767 (1) 95 16684 (107) Integrase 96 19857 (1) 97 19865 (1) 98 11481 (7) RecE Exonuclease 99 19804 (1) 100 19625 (1) 101 19592 (1) 102 19629 (1) 103 19713 (1) 104 19935 (1) 105 19790 (1) 106 13007 (7) RecT ssDNA binding protein 107 19849 (1) 108 19543 (1) 109 19868 (1) Excise 110 18393 (209) ThyX Thymidylate synthase 111 19761 (1) 112 11220 (7) 113 19789 (1) 114 16203 (3) HNH endonuclease 115 20331 (3) 116 19921 (1) 117 19555 (1) 118 19856 (1) 119 19881 (1) 120 19778 (1) 121 8133 (11) specificity protein- tyrosine phosphatase 122 6890 (52) 123 6916 (3) 124 19583 (1) 125 19303 (18) like ssDNA binding protein 126 2146 (2) 127 17427 (7) DNA helicase 128 19888 (1) 129 9885 (19) NrdH- Glutaredoxin 130 19814 (5) 131 16355 (21) HNH endonuclease 132 18544 (127) Single- stranded DNA binding protein 133 19852 (1) 134 11507 (6) 135 11381 (6) 136 15576 (17) 137 19838 (1) 138 7132 (10) 139 19724 (1) 140 19774 (1) 141 15729 (490) Endonuclease VII 142 19562 (1) 143 19668 (1) 144 19776 (1) 145 11524 (7) 146 19618 (7) 147 11336 (7) Nucleotidyltransferase 148 19576 (1) 149 19687 (1) 150 702 (10) 151 12789 (2) 152 19586 (1) 153 19755 (1) 154 19891 (1) 155 19670 (1) 156 11456 (7) 157 19575 (1) 158 19703 (1) 159 19561 (1) 160 19702 (1) 161 11548 (15) Lysin B 162 19623 (1) endonuclease VII 163 19596 (1) 164 19658 (1) 165 19912 (1) 166 8516 (3) 167 19734 (1) 168 19871 (1) 169 19816 (1) 170 19560 (1) 171 19594 (1) 172 5281 (3) 173 19741 (1) 174 19605 (1) 175 19712 (1) 176 19929 (1) 177 19861 (1) 178 11214 (6) 179 19694 (1) 180 11197 (5) 181 11717 (6) 182 11477 (6) 183 19940 (1) 184 19796 (1) 185 19784 (1) 186 19607 (1) 187 19844 (1) 188 19783 (1) 189 18816 (7) 190 17425 (2) 191 11243 (5) 192 19919 (1) 193 19571 (1) 194 19890 (1) 195 19556 (1) 196 19574 (1) 197 19855 (1) 198 3726 (8) 199 19842 (1) 200 19545 (1) 201 19568 (1) 202 19798 (1) 203 19180 (28) 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 50 11297 (12) 51 11701 (6) 52 20276 (1) 53 20443 (1) 54 14993 (18) Lsr2 55 20223 (1) 56 18923 (7) exodeoxyribonuclease VII, large subunit 57 11345 (5) exodeoxyribonuclease VII, small subunit 58 11603 (6) 59 11663 (5) 60 11702 (5) 61 11509 (5) 62 6211 (6) metallophosphatase 63 5467 (70) RtcB-like 64 20407 (1) homing endonuclease 65 20097 (1) 66 11404 (5) 67 20379 (1) 68 4091 (6) 69 20087 (1) 70 20205 (1) 71 11403 (5) 72 20069 (1) 74 16684 (107) integrase 75 11238 (5) 76 11360 (6) immunity repressor 77 20271 (1) 78 11481 (7) exonuclease 79 20115 (1) 80 11449 (5) 81 13007 (7) RecT 82 20337 (2) 83 18393 (209) FAD- dependent thymidylate synthase 84 20109 (1) 85 11682 (5) 86 20251 (1) HTH DNA binding 87 20227 (1) 88 11220 (7) 89 11338 (5) HTH DNA binding 90 11249 (5) 91 11385 (5) 92 11388 (5) 93 18670 (45) MazG 94 11518 (5) 95 20077 (1) 96 20464 (1) 97 11641 (5) 98 20167 (1) 99 20291 (19) 100 4742 (35) phosphoribosyl- pyrophosphate synthase 101 7038 (35) nicotinate phosphoribosyl transferase 102 12974 (3) 103 19303 (18) 104 13458 (2) 105 20063 (1) 106 8838 (91) 107 20232 (1) 108 17427 (7) DnaB helicase 109 20080 (1) HNH endonuclease 110 20449 (5) NrdH redoxin 111 18544 (127) single stranded DNA binding protein 112 11432 (5) 113 11507 (6) 114 11381 (6) 115 7132 (10) 116 11513 (5) 117 11272 (5) 118 15729 (490) Endonuclease VII 119 11170 (5) 120 11524 (7) 121 11336 (7) nucleotidyltransferase 122 20148 (1) 123 20107 (1) 124 20116 (1) 125 15271 (3) 126 20112 (1) 127 11635 (6) nucleotidase 128 2143 (4) Endo VII 129 20146 (1) 130 19223 (8) 131 1170 (10) ribonucleotide reductase 132 11456 (7) 133 11548 (15) lysin B 134 20355 (6) 135 18753 (2) 136 11261 (5) 137 11214 (6) 138 20137 (1) 139 20236 (1) 140 20065 (1) 141 11633 (5) 142 5179 (7) 143 11714 (5) 144 11717 (6) 145 20413 (1) 146 20280 (1) 147 20196 (1) 148 11477 (6) 149 20138 (1) 150 11374 (5) 151 20117 (1) 152 11420 (5) 153 18816 (7) 154 11689 (5) 155 20445 (1) 156 20244 (1) 157 3726 (8) 158 11708 (6) 159 11236 (5) 160 19180 (28) 161 17594 (8) 162 20372 (1) 163 20079 (1) 164 20312 (2) 165 20074 (2) 166 11719 (7) 167 20184 (1) 168 20506 (1) 169 11347 (5) 170 20118 (1) 171 20390 (1) 172 20444 (1) 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 38 92 (2) 39 10904 (1) 40 18847 (1) 41 10017 (1) 42 9996 (10) 43 20265 (108) 44 15285 (8) DnaN 45 10942 (7) oligoribonuclease 46 9687 (1) 47 9666 (1) 48 10236 (18) helicase 49 9609 (1) 50 10478 (1) 51 9777 (9) 52 10412 (1) 53 20061 (1) 54 9874 (1) HNH endonuclease 55 10411 (10) 56 9928 (1) 57 9779 (1) 58 19302 (9) 59 15945 (9) tyrosine recombinase/ integrase 60 9508 (1) 61 20161 (10) 62 10832 (1) 63 10176 (1) 64 15516 (3) 65 10722 (1) 66 9611 (1) 67 10167 (10) 68 9833 (10) vWFA 69 19124 (13) AAA protein 70 10893 (1) 71 10153 (1) 72 11017 (1) 73 10864 (1) 74 10196 (1) 75 10636 (5) 76 9892 (1) 77 10760 (1) 78 9929 (1) 79 10878 (1) 80 10713 (1) 81 10943 (10) 82 10248 (1) 83 9752 (1) 84 9840 (1) 85 10387 (1) 86 9488 (1) 87 10548 (1) 88 9605 (1) 89 11019 (1) 90 9808 (1) 91 18320 (9) 92 9659 (1) 93 9546 (1) 94 10212 (1) 95 10541 (1) 96 10358 (1) 97 9947 (1) 98 9526 (1) 99 10257 (1) 100 9988 (1) 101 13198 (10) 102 19078 (9) 103 10441 (1) 104 10119 (1) 105 19055 (10) 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 35 16288 (1) 36 16164 (1) 37 15809 (1) 38 19091 (21) 39 10439 (11) 40 15807 (1) 41 6916 (3) 42 16160 (158) 43 15984 (1) 44 19904 (9) 45 16306 (1) 46 15987 (1) 47 16462 (4) 48 20253 (53) 49 16404 (5) 50 13150 (8) 51 16262 (1) 52 16071 (5) 53 16020 (5) 54 16240 (1) 55 19303 (18) 56 10269 (11) 57 13282 (8) 58 10236 (18) putative helicase 59 16108 (5) 60 16101 (1) 61 16366 (1) 62 16482 (1) 63 9777 (9) 64 15988 (1) 65 15920 (5) 66 19110 (5) 67 15844 (1) 68 15867 (1) 69 15854 (1) 70 16038 (5) 71 16208 (2) 72 16349 (1) 73 16356 (1) 74 16461 (1) 75 15945 (9) putative integrase 76 16427 (1) 77 15821 (2) 78 13419 (8) 79 12903 (6) 80 16076 (5) 81 12885 (8) 82 16223 (1) 83 15926 (5) 84 16300 (1) 85 15838 (1) 86 16152 (1) 87 13247 (8) putative von Willebrand factor protein 88 10817 (5) putative ATPase family protein 89 15857 (1) 90 16224 (1) 91 16145 (2) 92 18320 (9) 93 6186 (4) 94 16265 (1) 95 15989 (1) 96 16148 (1) 97 15818 (1) 98 15876 (1) 99 16166 (1) 100 19078 (9) 101 16110 (11) 102 15998 (1) 103 19862 (8) 104 15978 (1) 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 44 19091 (21) 45 10439 (11) 46 19068 (1) 47 19904 (9) 48 19239 (1) 49 20253 (53) 50 16404 (5) 51 13150 (8) 52 19022 (1) 53 19082 (1) 54 16071 (5) 55 16020 (5) 56 19045 (1) 57 10269 (11) 58 13282 (8) 59 10236 (18) 60 16108 (5) 61 19165 (1) 62 15676 (2) 63 9777 (9) 64 19240 (1) 65 15920 (5) 66 19110 (5) 67 16038 (5) 68 19069 (57) 69 19131 (1) 70 19233 (1) 71 15945 (9) 72 19189 (1) 73 15821 (2) 74 19143 (1) 75 19255 (1) 76 13419 (8) 77 19157 (1) 78 13360 (5) 79 16076 (5) 80 19291 (1) 81 12885 (8) 82 19086 (1) 83 15926 (5) 84 19101 (1) 85 19292 (1) 86 19080 (1) 87 19217 (1) 88 12758 (5) 89 19125 (1) 90 19237 (1) 91 19073 (1) 92 19265 (1) 93 19150 (1) 94 13247 (8) 95 19181 (1) 96 10817 (5) 97 19093 (1) 98 19156 (1) 99 19107 (1) 100 19260 (1) 101 19229 (1) 102 16145 (2) 103 12660 (4) 104 19205 (1) 105 19256 (1) 106 19039 (1) 107 19228 (1) 108 19179 (3) 109 19179 (3) 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 34 20352 (3) 35 20426 (3) 36 20330 (3) 37 19091 (21) 38 20277 (3) 39 20142 (3) 40 20396 (3) 41 20147 (3) 42 18393 (209) thymidylate synthase ThyX 43 19904 (9) 44 20060 (3) 45 16462 (4) 46 20253 (53) transcription factor WhiB 47 16404 (5) 48 13150 (8) RusA-like ribonuclease 49 20332 (3) 50 16071 (5) 51 20388 (3) 52 16020 (5) DNA polymerase 53 10942 (7) ribonuclease 54 13282 (8) DNA binding protein 55 10236 (18) helicase 56 16108 (5) 57 20465 (3) 58 20419 (3) DUF732- like protein 59 9777 (9) 60 20132 (3) 61 15920 (5) 62 19110 (5) 63 20275 (3) 64 16038 (5) 65 20199 (3) 66 20143 (3) 67 15945 (9) integrase 68 20327 (3) 69 20421 (3) 70 20186 (3) 71 20235 (3) 72 13419 (8) 73 16076 (5) 74 19128 (293) 75 12885 (8) 76 20315 (3) 77 15926 (5) methyltransferase 78 20229 (3) 79 20356 (3) 80 20398 (2) 81 13247 (8) von Willebrand factor domain protein 82 10817 (5) ATPase 83 20086 (3) 84 20384 (3) 85 20156 (3) 86 20254 (3) 87 20399 (3) 88 19078 (9) DNA primase 89 16110 (11) 90 19862 (8) Summary of Results Fig. 2. Synteny and homology between Bantam and other Gordonia phages, Yvonnetastic (Singleton) and OneUp (cluster CQ). A. Shared phams encoding structural genes. B. Shared genes involved in replication and regulation illustrate mosaic nature of these genomes. A B Bantam summary: 108 / 172 orfs are orphams No tRNAs detected Shared phams: structural genes, integrase, lysinA, exonucleases 27 phams shared with Gordonia cluster CQ, singleton Yvonnetastic 32 phams shared with phages in other hosts: Mycobacterium (23), Rhodococcus (10), Arthrobacter (3), Corynebacterium (2), Streptomyces (2) Fig. 6. Immunity regions of Gordonia cluster A15 and Mycobacterium cluster A2 phages. Note the presence of ParA and ParB genes present in A15 phages instead of integrase, suggesting a partitioning method of prophage maintenance (Stella et al 2013) Fig. 5. Map of cluster A15 phages, showing extensive homology Fig. 3. Homology between Jumbo and Gordonia singleton GMA3 (1), cluster DF phage Gmala1 (3), and Rhodococcus singleton DocB7 (4), all isolated outside of the SEA-PHAGES program. Fig. 4. Lysis cassettes in Jumbo and related phages, which were isolated as potential biocontrol agents for reducing foaming in wastewater sludge by lysing bacteria. Jumbo summary: 42/107 orfs are orphams No tRNAs detected Shared phams: structural genes, integrase, helicase, exonuclease, endolysins, holins Phams shared with singleton GMA3, Gordonia cluster DF, Rhodococcus singleton DocB7 21 phams shared with phages in other hosts: Rhodococcus (15), Corynebacterium (2), Streptomyces (4) Jswag, Remus, Strosahl summary: 96% average nucleotide identity 3 tRNA genes No orphams 33/102 phams unique to A15 phages o 24/33 in all A15 phages 58 phams shared with other cluster A phages o Most are in A2 subcluster 14 phams shared with other Gordonia clusters 21 phams shared with other hosts: Rhodococcus (15), Streptomyces (4), Corynebacterium (2) Fig. 7. Lysogeny in A15 phages. A. Culture at 35 o C yielded turbid plaques. B. Cells from turbid plaques of JSwag, Remus, and Figaro (A15 by PCR) were streaked, transferred to liquid culture, and used in spot tests with lysates of these same phages. Lysogens were immune to all A15 phages tested, but not to Jumbo. B.

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Page 1: Hits from Host to Host: Isolation, Characterization, and … · 2017-06-02 · Hits from Host to Host: Isolation, Characterization, and Comparative Genomics of Five New Gordonia phages

Hits from Host to Host: Isolation, Characterization, and Comparative Genomics of Five New Gordonia phages

Alanna M Bram, Amanda J Weber, Katelyn R Chester, Kimberly E Doerr, Amanda A Drigans, Christopher O Dusek, Brenna K Franke, Allison K Granberg, Shiza S Idrees, Katie E Klinkhammer, Danielle M Kocina, Marissa L Leopold, Zachary A Lund, Theresa N Lusk, Kaitlyn J Matczynski, Abbey L Novotny, Kayla E Oberding, Allison N Potocnik, Chase A Quandt, Marissa Y Schmitz, Danielle E Schultz, Tiffany A Stocks, Willow M Stuart, Christian Thaoxaochay, Cami P Thomas, Tara N Toland, Samantha A Topel, Emily

R Warren, Max R Wetzel, Rebecca J Welman, Katlyn M Williams, Olivia H Yue, Cody J Ziegler; J. Alfred Bonilla and Karen K. Klyczek, Instructors Biology Department, University of Wisconsin-River Falls, River Falls, WI 54022

Isolation of Gordonia phage

Relatively few phages infecting hosts other than Mycobacterium smegmatis have been isolated and characterized (1). UWRF phage hunters set out to isolate phage infecting another member of the phylum Actinobacteria, Gordonia terrae. Gordonia species are found in soil and wastewater, and are associated with foaming in wastewater treatment. The same procedures used for isolating Arthrobacter and Rhodococcus phage in previous years were used to test 44 soil samples:

• LB broth was used for bacteria culture and top agar, no antibiotics • CaCl2 was added at 4.5 mM in culture broth and 2.25 mM in top agar • Enrichments were cultured at 30oC for 2-5 days Results: Only five student samples yielded plaques, along with 3 instructor samples piloted prior to class. Several phages were isolated from these samples. We selected phages for sequencing that were likely to be different based on plaque and EM morphologies (Fig. 1), restriction enzyme patterns (not shown), and soil source. Five unique phage sequences were obtained (Table 1). Fig. 1. Plaque morphology and electron microscopy. There was a variety of plaque morphologies observed. JSwag (A), had clear plaques of variable size (1-3 mm), sometimes with halos; Remus and Strosahl had a similar appearance. Jumbo (B) had smaller clear plaques (0.5 mm) and Bantam (C) had very small (<0.5 mm), turbid plaques. All of these new phages are Siphoviridae; Remus (D), JSwag and Strosahl had tails 100-125 nm in length, while Jumbo (E) and Bantam had longer tails, 450-475 nm.

References 1.  Actinobacteriophage Database (2015), http://phagesdb.org 2. Dyson, ZA et al. (2015) PLoS One 10(8): e0134512. doi:10.1371/journal.pone.0134512 3. Liu, M. et al. (2015) Nature Scientific Reports 5:13754 | DOI: 10.1038/srep13754 4. Summer, EJ et al. Appl. Environ. Microbiol. (2011), 77(2):669 5.  Stella, EJ et al. (2013) PLoS ONE 8(2): e56384. doi:10.1371/journal.pone.0056384

Acknowledgements

We thank Rick Ellingworth and the Biology Department student lab assistants for preparing materials, the Hatfull lab, University of Pittsburgh, and HHMI SEA PHAGES program for supporting this work, and the entire SEA community, for sharing experiences and advice.

Conclusions

Phage Drake (A15 by PCR)

Bantam: Comparative Genomics

A

A. 30o C 35o C

 •  Bantam is a singleton, with little nucleotide similarity to other phages but

shared phams with other Gordonia phages (Fig. 2) •  Jumbo is also a singleton with similarities to Gordonia singleton GMA3

(isolated in Australia), Rhodococcus phage DocB7 (isolated in Texas), and Gordonia cluster DF (isolated in Texas) (Fig. 3)

•  Jumbo and related phages may have multiple lysin and holin genes that could increase efficiency of cell lysis (Fig. 4)

•  Remus, JSwag, Strosahl are cluster A15 phages, very similar to others in that cluster (Fig. 5). Several other unsequenced class phages were A15 according to PCR results (not shown).

•  JSwag and Remus are temperate, lysogens were isolated at 35o C (Fig. 7A) o  Partitioning system, similar to some A2 phages (Fig. 6) o  Immunity to other A15 phages (Fig. 7B)

Cluster A15 phages

Jumbo: Comparative Genomics

.

A B C D E

Table 1. Sequencing Results 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

1

5079 (124)

gp1

2

18243 (31)

gp2

3

4163

(31

)

gp3

4

1737

4 (2

54)

gp4

5

19250 (447)

gp5

6

4048

(16

7)

gp6

7

11333 (6)

gp7

8

17737 (67)

gp8

10

1917

4 (1

)

gp10 13

14746 (6)

gp13

14

1086

8 (7

)

gp14

15

17443 (281)

gp15

16

2982 (414)

gp16

17

19266 (330)

gp17

18

3257 (302)

gp18

19

3146 (348)

gp19

20

1172

0 (6

)

gp20

21

7209 (449)

gp21

22

1919

8 (1

98)

gp22

23

1827 (341)

gp23

24

17963 (313)

gp24

25

2847 (449)

gp25

26

19254 (413)

gp26

27

18608 (314)

gp27

28

18608 (314)

gp28

29

17995 (10)

gp29

30

4899 (303)

gp30

31

19296 (432)

gp31

32

12446 (406)

gp32

33

1888

1 (1

28)

gp33

34

11297 (11)

gp34

35

1786

8 (9

1)

gp35

36

1929

4 (1

3)

gp36

37

7133 (64)

gp37

38

1779

1 (6

)

gp38

39

1130

3 (6

)

gp39

40

1140

1 (7

)

gp40

41

18837 (412)

gp41

42

1922

1 (5

)

gp42

43

1141

6 (6

)

gp43

44

1116

7 (6

)

gp44

45

1144

3 (6

)

gp45

46

11528 (4)

gp46

47

15909 (456)

gp47

48

1915

5 (3

1)

gp48

49

1428

6 (3

3)

gp49

50

1880

5 (1

6)

gp50

51

1323

5 (2

5)

gp51

52

18393 (192)

gp52

53

1867

0 (4

2)

gp53

54

17822 (276)

gp54

55

1144

2 (6

)

gp55

56

565 (367)

gp56

57

5496

(73

)

gp57

58

1925

8 (1

)

gp58

59

1752

9 (2

15)

gp59

60

13798 (118)

gp60

61

9297 (409)

gp61

62

11277 (6)

gp62

63

1153

4 (6

)

gp63

64

1257

1 (6

5)

gp64

65

1158

2 (6

)

gp65

66

4038 (448)

gp66

67

19262 (107)

gp67

68

1155

3 (6

)

gp68

69

11548 (13)

gp69

70

15729 (464)

gp70

71

1143

1 (6

)

gp71

72

1169

8 (6

)

gp72

73

19104 (16)

gp73

74

1791

5 (1

5)

gp74

75

18508 (6)

gp75

76

11425 (5)

gp76

77

5065 (450)

gp77

78

1150

5 (6

)

gp78

79

1158

8 (6

)

gp79

80

1865

6 (1

5)

gp80

81

1914

6 (1

)

gp81

82

1904

7 (4

)

gp82

83

5238 (450)

gp83

84

19123 (21)

gp84

85

19184 (182)

gp85

86

1116

6 (6

)

gp86

87

1154

2 (6

)

gp87

88

1304 (171)

gp88

89

1164

0 (6

)

gp89

90

1172

2 (5

)

gp90

91

1171

9 (7

)

gp91

92

1144

7 (4

)

gp92

93

1161

8 (6

)

gp93

94

1858

9 (3

)

gp94

95

1135

5 (9

)

gp95

96

1161

5 (6

)

gp96

97

6789 (8)

gp97

98

2322 (28)

gp98

99

1162

8 (4

)

gp99

100

1170

8 (5

)

gp100

101

1145

0 (6

)

gp101

102

1172

3 (6

)

gp102

103

6684

(18

)

gp103

104

1136

3 (6

)

gp104

105

578 (2)

gp105

JSwag_Draft

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

1

5079 (124) 2

18243 (31)

VIP2-like

3

4163

(31

)

4

1737

4 (2

54)

5

19250 (447)

Tail

6

4048

(16

7)

Tail

7

11333 (6) 8

17737 (67)

12

14746 (6)

Endolysin

13

1086

8 (7

)

Holin

14

17443 (281)

Terminase, large subunit

15

2982 (414)

Portal

16

19266 (330)

Capsid Maturation Protease

17

3257 (302)

Scaffolding

18

3146 (348)

Capsid

19

1172

0 (6

)

20

7209 (449)21

1919

8 (1

98)

22

1827 (341) 23

17963 (313)

24

2847 (449) 25

19254 (413)

26

18608 (314)

Tail Assembly Chaperone

27

18608 (314)

Tail Assembly Chaperone

28

17995 (10)

Tapemeasure

29

4899 (303)

Minor Tail

30

19296 (432)

Minor Tail

31

12446 (406)

Minor Tail

32

1888

1 (1

28)

Minor Tail

33

11297 (11)

Minor Tail

34

1786

8 (9

1)

Lsr2

35

1929

4 (1

3)

36

7133 (64)

ParA

37

1779

1 (6

)

ParB

38

1130

3 (6

)

39

1140

1 (7

)

40

1165

5 (1

)

41

1155

2 (1

) 42

18837 (412)

43

1922

1 (5

) 44

1141

6 (6

)

45

1116

7 (6

) 46

1144

3 (6

)

47

11528 (4) 48

15909 (456)

DNA polymerase

49

1915

5 (3

1) 50

1136

2 (2

)

51

1428

6 (3

3)

HTH DNA binding domain

52

1880

5 (1

6)

53

18393 (192)

ThyX

54

1867

0 (4

2)

MazG-like

55

17822 (276)56

1144

2 (6

)

57

565 (367)

Ribonucleotide reductase

58

5496

(73

)

59

1752

9 (2

15) 60

13798 (118)

Transcription regulator

61

9297 (409)

phosphoesterase

62

11277 (6)

63

1153

4 (6

) 64

1257

1 (6

5)

NrdH-like

65

1158

2 (6

) 66

4038 (448)

DNA Primase

67

19262 (107)

DNA Primase

68

1155

3 (6

)

69

11548 (13)

Endolysin

70

15729 (464)

EndoVII

71

1143

1 (6

)72

1169

8 (6

)

73

19104 (16)74

1791

5 (1

5)

75

18508 (6) 76

11368 (1)

77

5065 (450)

DnaB-like helicase

78

1150

5 (6

)

79

1158

8 (6

) 80

1865

6 (1

5)

81

19047 (4) 82

5238 (450)

RecB-like

83

19123 (21) 84

19184 (182)

Repressor

85

1116

6 (6

) 86

1154

2 (6

)

87

1304 (171)

SprT-like

88

1164

0 (6

)

89

1171

9 (7

) 90

1144

7 (4

)

91

1161

8 (6

) 92

1135

5 (9

)

93

1161

5 (6

) 94

6789 (8)

95

2322 (28) 96

1162

8 (4

)

97

6310 (15)98

1145

0 (6

)

99

1172

3 (6

) 100

6684

(18

)

101

1136

3 (6

)

KatherineG

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

1

5079 (124)

gp1

2

18243 (31)

gp2

3

4163

(31

)

gp3

4

1737

4 (2

54)

gp4

5

19250 (447)

gp5

6

4048

(16

7)

gp6

7

11333 (6)

gp7

8

17737 (67)

gp8

12

14746 (6)

gp12

13

1086

8 (7

)

gp13

14

17443 (281)

gp14

15

2982 (414)

gp15

16

19266 (330)

gp16

17

3257 (302)

gp17

18

3146 (348)

gp18

19

1172

0 (6

)

gp19

20

7209 (449)

gp20

21

1919

8 (1

98)

gp21

22

1827 (341)

gp22

23

17963 (313)

gp23

24

2847 (449)

gp24

25

19254 (413)

gp25

26

18608 (314)

gp26

27

18608 (314)

gp27

28

17995 (10)

gp28

29

4899 (303)

gp29

30

19296 (432)

gp30

31

12446 (406)

gp31

32

1888

1 (1

28)

gp32

33

11297 (11)

gp33

34

1786

8 (9

1)

gp34

35

1929

4 (1

3)

gp35

36

7133 (64)

gp36

37

1779

1 (6

)

gp37

38

1130

3 (6

)

gp38

39

1140

1 (7

)

gp39

40

1153

3 (3

)

gp40

41

1826

5 (2

)

gp41

42

18837 (412)

gp42

43

1922

1 (5

)

gp43

44

1141

6 (6

)

gp44

45

1116

7 (6

)

gp45

46

1144

3 (6

)

gp46

47

15909 (456)

gp47

48

1915

5 (3

1)

gp48

49

1428

6 (3

3)

gp49

50

1880

5 (1

6)

gp50

51

1323

5 (2

5)

gp51

52

18393 (192)

gp52

53

1867

0 (4

2)

gp53

54

17822 (276)

gp54

55

1144

2 (6

)

gp55

56

565 (367)

gp56

57

5496

(73

)

gp57

58

1858

4 (2

)

gp58

59

1752

9 (2

15)

gp59

60

13798 (118)

gp60

61

9297 (409)

gp61

62

11277 (6)

gp62

63

1153

4 (6

)

gp63

64

1257

1 (6

5)

gp64

65

1158

2 (6

)

gp65

66

4038 (448)

gp66

67

1155

3 (6

)

gp67

68

11548 (13)

gp68

69

15729 (464)

gp69

70

1143

1 (6

)

gp70

71

1169

8 (6

)

gp71

72

19104 (16)

gp72

73

1791

5 (1

5)

gp73

74

18508 (6)

gp74

75

11425 (5)

gp75

76

5065 (450)

gp76

77

1861

2 (2

)

gp77

78

1150

5 (6

)

gp78

79

1158

8 (6

)

gp79

80

1865

6 (1

5)

gp80

81

5238 (450)

gp81

82

19123 (21)

gp82

83

19184 (182)

gp83

84

1116

6 (6

)

gp84

85

1154

2 (6

)

gp85

86

1871

9 (2

9)

gp86

87

14542 (23)

gp87

89

1164

0 (6

)

gp89

90

1172

2 (5

)

gp90

91

1171

9 (7

)

gp91

92

1144

7 (4

)

gp92

93

1161

8 (6

)

gp93

94

1858

9 (3

)

gp94

95

1135

5 (9

)

gp95

96

1161

5 (6

)

gp96

97

6789 (8)

gp97

98

2322 (28)

gp98

99

6310 (15)

gp99

100

1145

0 (6

)

gp100

101

1172

3 (6

)

gp101

102

1136

3 (6

)

gp102

Remus_Draft

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

1

5079 (124) 2

18243 (31)

VIP2

3

4163

(31

)

4

1737

4 (2

54)

HNH Endonuclease

5

19250 (447)

Tail

6

4048

(16

7)

Tail

7

11333 (6) 8

17737 (67)

12

14746 (6)

Endolysin

13

1086

8 (7

)

Holin

14

17443 (281)

Terminase

15

2982 (414)

Portal

16

19266 (330)

Capsid Maturation Protease

17

3257 (302)

Scaffolding

18

3146 (348)

Capsid

19

1172

0 (6

)

20

7209 (449)21

1919

8 (1

98)

22

1827 (341) 23

17963 (313)

24

2847 (449) 25

19254 (413)

Major Tail

27

18608 (314)

Tail Assembly Chaperone

26

18608 (314)

Tail Assembly Chaperone

28

17995 (10)

Tapemeasure

29

4899 (303)

Minor Tail

30

19296 (432)

Minor Tail

31

12446 (406)

Minor Tail

32

1888

1 (1

28)

Minor Tail

33

11297 (11)

Minor Tail

34

1786

8 (9

1)

Lsr2

35

1929

4 (1

3)

36

7133 (64)

ParA

37

1779

1 (6

)

ParB

38

1130

3 (6

)

39

1140

1 (7

)

40

1243

0 (1

)

41

18837 (412)42

1141

6 (6

)

43

1116

7 (6

) 44

1144

3 (6

)45

11528 (4) 46

15909 (456)

DNA polymerase

47

1915

5 (3

1) 48

1428

6 (3

3)

HTH DNA binding domain

49

1880

5 (1

6) 50

1323

5 (2

5)

51

18393 (192)

ThyX

52

1867

0 (4

2)

MazG

53

17822 (276)54

1144

2 (6

)

55

565 (367)

Ribonucleotide reductase

56

5496

(73

)

57

1752

9 (2

15) 58

13798 (118)

RDF

59

9297 (409)

Phosphoesterase

60

11277 (6)

61

1153

4 (6

) 62

1257

1 (6

5)

NrdH-like

63

1158

2 (6

) 64

4038 (448)

DNA Primase

65

19262 (107)

DNA Primase

66

1155

3 (6

)

67

11548 (13)

Endolysin

68

15729 (464)

EndoVII

69

1143

1 (6

)70

1169

8 (6

)

71

19104 (16)72

1791

5 (1

5)

73

18508 (6) 74

11425 (5)

75

5065 (450)

DnaB-like helicase

76

1150

5 (6

)

77

1158

8 (6

) 78

1865

6 (1

5)

79

19047 (4) 80

5238 (450)

RecB

81

19123 (21) 82

19184 (182)

Repressor

83

1116

6 (6

) 84

1154

2 (6

)

85

1871

9 (2

9) 86

1242

9 (1

)

87

1164

0 (6

) 88

1172

2 (5

)

89

1171

9 (7

) 90

1868

9 (2

)

91

1161

8 (6

) 92

1135

5 (9

)

93

1161

5 (6

) 94

6789 (8)

95

2322 (28) 96

1162

8 (4

)

97

6310 (15)98

1145

0 (6

)

99

1172

3 (6

) 100

3693

(5)

101

6684

(18

) 102

1136

3 (6

)

Rosalind

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

1

5079 (124) 2

18243 (31)

VIP2

3

4163

(31

)

4

1737

4 (2

54)

HNH Endonuclease

5

19250 (447)

Tail

6

4048

(16

7)

Tail

7

11333 (6) 8

17737 (67)

12

14746 (6)

Endolysin

13

1086

8 (7

)

Holin

14

17443 (281)

Terminase

15

2982 (414)

Portal

16

19266 (330)

Capsid Maturation Protease

17

3257 (302)

Scaffolding

18

3146 (348)

Capsid

19

1172

0 (6

)

20

7209 (449)21

1919

8 (1

98)

22

1827 (341) 23

17963 (313)

24

2847 (449) 25

19254 (413)

Major Tail

27

18608 (314)

Tail Assembly Chaperone

26

18608 (314)

Tail Assembly Chaperone

28

17995 (10)

Tapemeasure

29

4899 (303)

Minor Tail

30

19296 (432)

Minor Tail

31

12446 (406)

Minor Tail

32

1888

1 (1

28)

Minor Tail

33

11297 (11)

Minor Tail

34

1786

8 (9

1)

Lsr2

35

1929

4 (1

3)

36

7133 (64)

ParA

37

1779

1 (6

)

ParB

38

1130

3 (6

)

39

1140

1 (7

)

40

1153

3 (3

)

41

18837 (412)42

1922

1 (5

)

43

1141

6 (6

) 44

1116

7 (6

)

45

1144

3 (6

) 46

11528 (4)

47

15909 (456)

DNA Polymerase

4819

155

(31)

49

1136

2 (2

) 50

1428

6 (3

3)

HTH DNA binding domain

51

1880

5 (1

6) 52

18393 (192)

ThyX

53

1117

7 (1

) 54

1867

0 (4

2)

MazG

55

17822 (276)56

1144

2 (6

)

57

565 (367)

Ribonucleotide reductase

58

5496

(73

)

59

1752

9 (2

15) 60

13798 (118)

Transcription regulator

61

9297 (409)

phosphoesterase

62

11277 (6)

63

1153

4 (6

) 64

1257

1 (6

5)

NrdH-like

65

1158

2 (6

) 66

4038 (448)

DNA Primase

67

19262 (107)

DNA Primase

68

1155

3 (6

)

69

11548 (13)

Endolysin

70

15729 (464)

EndoVII

71

1143

1 (6

)72

1169

8 (6

)

73

19104 (16)74

1791

5 (1

5)

75

18508 (6) 76

11425 (5)

77

5065 (450)

DnaB-like helicase

78

1150

5 (6

)

79

1158

8 (6

) 80

1865

6 (1

5)

81

5238 (450)

Rec-B-like

82

19123 (21)

83

19184 (182)

Repressor

84

1116

6 (6

)

85

1154

2 (6

) 86

1871

9 (2

9)

87

14542 (23) 88

1164

0 (6

)

89

1172

2 (5

) 90

1171

9 (7

)

91

1868

9 (2

) 92

1161

8 (6

)

93

1135

5 (9

) 94

1161

5 (6

)

95

6789 (8) 96

2322 (28)

97

1162

8 (4

) 98

6310 (15)

99

1145

0 (6

) 100

1172

3 (6

)

101

6684

(18

) 102

1136

3 (6

)

Soups

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

1

5079 (124)

gp1

2

18243 (31)

gp2

3

4163

(31

)

gp3

4

1737

4 (2

54)

gp4

5

19250 (447)

gp5

6

4048

(16

7)

gp6

7

11333 (6)

gp7

8

17737 (67)

gp8

12

14746 (6)

gp12

13

1086

8 (7

)

gp13

14

17443 (281)

gp14

15

2982 (414)

gp15

16

19266 (330)

gp16

17

3257 (302)

gp17

18

3146 (348)

gp18

19

1172

0 (6

)

gp19

20

7209 (449)

gp20

21

1919

8 (1

98)

gp21

22

1827 (341)

gp22

23

17963 (313)

gp23

24

2847 (449)

gp24

25

19254 (413)

gp25

26

18608 (314)

gp26

27

18608 (314)

gp27

28

17995 (10)

gp28

29

4899 (303)

gp29

30

19296 (432)

gp30

31

12446 (406)

gp31

32

1888

1 (1

28)

gp32

33

11297 (11)

gp33

34

1786

8 (9

1)

gp34

35

1929

4 (1

3)

gp35

36

7133 (64)

gp36

37

1779

1 (6

)

gp37

38

1130

3 (6

)

gp38

39

1140

1 (7

)

gp39

40

1153

3 (3

)

gp40

41

1826

5 (2

)

gp41

42

18837 (412)

gp42

43

1922

1 (5

)

gp43

44

1141

6 (6

)

gp44

45

1116

7 (6

)

gp45

46

1144

3 (6

)

gp46

47

15909 (456)

gp47

48

1915

5 (3

1)

gp48

49

1428

6 (3

3)

gp49

50

1880

5 (1

6)

gp50

51

1323

5 (2

5)

gp51

52

18393 (192)

gp52

53

1867

0 (4

2)gp53

54

17822 (276)

gp54

5511

442

(6)

gp55

56

565 (367)

gp56

57

5496

(73

)

gp57

58

1858

4 (2

)

gp58

59

1752

9 (2

15)

gp59

60

13798 (118)

gp60

61

9297 (409)

gp61

62

11277 (6)

gp62

63

1153

4 (6

)

gp63

64

1257

1 (6

5)

gp64

65

1158

2 (6

)

gp65

66

4038 (448)

gp66

67

1155

3 (6

)

gp67

68

11548 (13)

gp68

69

15729 (464)

gp69

70

1143

1 (6

)

gp70

71

1169

8 (6

)

gp71

72

19104 (16)

gp72

73

1791

5 (1

5)

gp73

74

18508 (6)

gp74

75

11425 (5)

gp75

76

5065 (450)

gp76

77

1861

2 (2

)

gp77

78

1150

5 (6

)

gp78

79

1158

8 (6

)

gp79

80

1865

6 (1

5)

gp80

81

5238 (450)

gp81

82

19123 (21)

gp82

83

19184 (182)

gp83

84

1116

6 (6

)

gp84

85

1154

2 (6

)

gp85

86

1871

9 (2

9)

gp86

87

14542 (23)

gp87

89

1164

0 (6

)

gp89

90

1172

2 (5

)

gp90

91

1171

9 (7

)

gp91

92

1144

7 (4

)

gp92

93

1161

8 (6

)

gp93

94

1858

9 (3

)

gp94

95

1135

5 (9

)

gp95

96

1161

5 (6

)

gp96

97

6789 (8)

gp97

98

2322 (28)

gp98

99

6310 (15)

gp99

100

1145

0 (6

)

gp100

101

1172

3 (6

)

gp101

102

1136

3 (6

)

gp102

Strosahl_Draft

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

1

19690 (1) 2

10819 (1)

3

9775 (14)

TerL

4

9969 (17)

Mu gp29-like protein

5

10455 (1) 6

9561 (2)

7

10582 (2) 8

10332 (1)

9

1988

6 (5

)

10

20495 (18)

11

19211 (10) 12

10259 (18)

13

1079

6 (1

)

14

1003

5 (1

)

15

20289 (10) 16

10989 (1)

17

9803 (1) 18

9660

(1)

19

11016 (18) 21

10594 (19)

pre-TMP frameshift protein

20

10594 (19)

truncated pre-TMP frameshift protein

22

10747 (18)

tape measure protein

23

10225 (18) 24

10679 (18)

25

9607 (1) 26

1942

9 (1

43)

putative holin

27

10772 (1)

LysA

28

1090

9 (1

)

holin

29

9658 (1) 30

16072 (7)

holin

31

9753

(1)

32

9700

(1) 33

19904 (9)

34

2046

7 (1

0)

WhiB

35

1022

6 (1

)

36

20685 (25)

DNA methylase

37

1519

5 (1

)

38

18692 (2)

LysB

39

1090

4 (1

)

40

18847 (1)41

1001

7 (1

)

42

9996 (10) 43

2026

5 (1

08)

44

15285 (8)

DnaN

45

10942 (7)

oligoribonuclease

46

9687

(1) 47

9666

(1)

48

10236 (18)

helicase

49

9609

(1)

50

10478 (1) 51

9777 (9)

52

10412 (1) 53

2006

1 (1

)

54

9874

(1)

HNH endonuclease

55

10411 (10)

56

9928 (1) 57

9779

(1)

58

1930

2 (9

) 59

15945 (9)

tyrosine recombinase/integrase

60

9508 (1) 61

2016

1 (1

0)

62

1083

2 (1

) 63

10176 (1)

64

15516 (3) 65

1072

2 (1

)

66

9611

(1) 67

10167 (10)

68

9833 (10)

vWFA

69

19124 (13)

AAA protein

70

10893 (1) 71

10153 (1)

72

1101

7 (1

) 73

1086

4 (1

)

74

10196 (1) 75

1063

6 (5

)

76

9892

(1) 77

1076

0 (1

)

78

9929 (1) 79

10878 (1)

80

1071

3 (1

) 81

10943 (10)

82

1024

8 (1

) 83

9752

(1)

84

9840

(1) 85

1038

7 (1

)

86

9488

(1) 87

1054

8 (1

)

88

9605

(1) 89

1101

9 (1

)

90

9808

(1) 91

1832

0 (9

)

92

9659 (1) 93

9546 (1)

94

10212 (1) 95

1054

1 (1

)

96

1035

8 (1

) 97

9947 (1)

98

9526

(1) 99

1025

7 (1

)

100

9988

(1) 101

1319

8 (1

0)

102

19078 (9) 103

10441 (1)

104

1011

9 (1

) 105

19055 (10)

DocB7

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77

1

13185 (8)

putative small terminase subunit

2

15871 (4)

putative large terminase subunit

3

9969 (17) 4

1581

6 (1

)

5

20495 (18) 6

16175 (8)

7

10259 (18)

putative major capsid protein

8

1300

8 (8

)

9

16299 (1) 10

12800 (8)

11

20240 (4) 12

1640

2 (5

)

13

11016 (18)

putative major tail protein

15

13323 (15)

putative tail assembly protein

14

13323 (15)

putative tail assembly protein

16

10747 (18)

putative tape measure protein

17

10225 (18) 18

10679 (18)

19

19064 (4) 20

15960 (2)

21

16263 (1) 22

16261 (5)

putative lysin

23

1616

5 (5

)

24

17047 (14)

25

3171 (14) 26

1629

6 (5

)

putative lysin

27

16984 (10)

putative holin

28

15922 (2)

29

12742 (5) 30

13317 (6)

31

1613

0 (4

)

32

19133 (10)

33

1604

7 (1

)

34

1586

2 (1

) 35

1628

8 (1

)

36

1616

4 (1

) 37

1580

9 (1

)

38

19091 (21) 39

10439 (11)

40

1580

7 (1

) 41

6916

(3)

42

16160 (158) 43

1598

4 (1

)

44

19904 (9) 45

1630

6 (1

)

46

1598

7 (1

) 47

1646

2 (4

)

48

2025

3 (5

3) 49

1640

4 (5

)

50

13150 (8) 51

1626

2 (1

)

52

1607

1 (5

) 53

16020 (5)

54

1624

0 (1

) 55

19303 (18)

56

10269 (11) 57

1328

2 (8

)

58

10236 (18)

putative helicase

59

16108 (5)

60

1610

1 (1

) 61

16366 (1)

62

1648

2 (1

) 63

9777 (9)

64

1598

8 (1

) 65

15920 (5)

66

19110 (5)67

1584

4 (1

)

68

1586

7 (1

) 69

1585

4 (1

)

70

1603

8 (5

) 71

1620

8 (2

)

72

1634

9 (1

) 73

16356 (1)

74

1646

1 (1

) 75

15945 (9)

putative integrase

76

16427 (1) 77

1582

1 (2

)

78

13419 (8) 79

1290

3 (6

)

80

1607

6 (5

) 81

1288

5 (8

)

82

16223 (1) 83

15926 (5)

84

1630

0 (1

) 85

1583

8 (1

)

86

1615

2 (1

) 87

13247 (8)

putative von Willebrand factor protein

88

10817 (5)

putative ATPase family protein

89

1585

7 (1

)

90

16224 (1) 91

16145 (2)

92

18320 (9) 93

6186 (4)

94

1626

5 (1

) 95

1598

9 (1

)

96

16148 (1) 97

1581

8 (1

)

98

15876 (1) 99

1616

6 (1

)

100

19078 (9) 101

16110 (11)

102

15998 (1) 103

19862 (8)

104

1597

8 (1

)

GMA3

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78

1

1917

9 (3

)

2

19078 (9) 3

16110 (11)

4

1927

7 (1

) 5

19212 (1)

6

19862 (8)

7

13185 (8) 8

9775 (14)

9

9969 (17) 10

1908

7 (1

)

11

20495 (18) 12

16175 (8)

13

10259 (18) 14

1300

8 (8

)

15

19220 (4) 16

12800 (8)

17

13451 (4) 18

1640

2 (5

)

19

11016 (18) 20

13323 (15)

21

1299

1 (1

)

22

10747 (18)

23

10225 (18) 24

10679 (18)

25

19064 (4) 26

20509 (5)

27

1916

6 (1

)

28

7318 (54)

29

16261 (5) 30

1616

5 (5

)

31

17047 (14) 32

1904

6 (5

)

33

16296 (5) 34

9727 (3)

35

16984 (10) 36

20292 (7)

37

1903

6 (1

)

38

17875 (95)

39

15922 (2) 40

19264 (1)

41

13317 (6) 42

1908

1 (1

)

43

19133 (10)

44

19091 (21) 45

10439 (11)

46

19068 (1) 47

19904 (9)

48

1923

9 (1

) 49

2025

3 (5

3)

50

1640

4 (5

) 51

13150 (8)

52

1902

2 (1

) 53

1908

2 (1

)

54

1607

1 (5

) 55

16020 (5)

56

1904

5 (1

) 57

10269 (11)

58

1328

2 (8

) 59

10236 (18)

60

16108 (5) 61

1916

5 (1

)

62

1567

6 (2

) 63

9777 (9)

64

19240 (1) 65

15920 (5)

66

19110 (5) 67

1603

8 (5

)

68

19069 (57) 69

1913

1 (1

)

70

1923

3 (1

) 71

15945 (9)

72

19189 (1) 73

1582

1 (2

)

74

1914

3 (1

) 75

19255 (1)

76

13419 (8) 77

1915

7 (1

)

78

13360 (5) 79

1607

6 (5

)

80

1929

1 (1

) 81

1288

5 (8

)

82

19086 (1) 83

15926 (5)

84

19101 (1) 85

19292 (1)

86

19080 (1)87

1921

7 (1

)

88

1275

8 (5

) 89

1912

5 (1

)

90

19237 (1)91

1907

3 (1

)

92

1926

5 (1

) 93

19150 (1)

94

13247 (8) 95

1918

1 (1

)

96

10817 (5)

97

1909

3 (1

)

98

1915

6 (1

)

99

1910

7 (1

)

100

1926

0 (1

) 101

19229 (1)

102

16145 (2) 103

1266

0 (4

)

104

1920

5 (1

) 105

1925

6 (1

)

106

1903

9 (1

) 107

19228 (1)

108

1917

9 (3

)

109

19179 (3)

109

19179 (3)

Jumbo_Draft

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

1

13185 (8) 2

15871 (4)

TerL

3

9969 (17)

DUF935-like protein

4

2012

6 (6

)

5

20495 (18) 6

16175 (8)

7

10259 (18)

major capsid protein

8

1300

8 (8

)

9

19220 (4) 10

12800 (8)

morphogensis protein

11

13451 (4) 12

1640

2 (5

)

13

11016 (18) 15

13323 (15)

pre-TMP frameshift protein

14

13323 (15)

pre-TMP frameshift protein

16

10747 (18)

tape measure protein

17

10225 (18) 18

10679 (18)

19

20422 (3) 20

20183 (3)

21

1029 (242) 22

16261 (5)

lysin

23

16165 (5) 24

17047 (14)

lysin A

25

2027

4 (3

)

26

16296 (5)

27

17023 (9)

holin

28

20292 (7)

DNA methylase

29

2021

7 (3

)

30

9839 (8)

holin

31

15963 (6)

holin

32

1613

0 (4

)

33

19133 (10)

lysin B

34

2035

2 (3

) 35

2042

6 (3

)

36

2033

0 (3

) 37

19091 (21)

38

2027

7 (3

) 39

2014

2 (3

)

40

2039

6 (3

) 41

20147 (3)

42

18393 (209)

thymidylate synthase ThyX

43

19904 (9)

44

2006

0 (3

) 45

1646

2 (4

)

46

2025

3 (5

3)

transcription factor WhiB

47

1640

4 (5

)

48

13150 (8)

RusA-like ribonuclease

49

2033

2 (3

)

50

1607

1 (5

) 51

2038

8 (3

)

52

16020 (5)

DNA polymerase

53

10942 (7)

ribonuclease

54

1328

2 (8

)

DNA binding protein

55

10236 (18)

helicase

56

16108 (5)57

2046

5 (3

)

58

20419 (3)

DUF732-like protein

59

9777 (9)

60

2013

2 (3

) 61

15920 (5)

62

19110 (5) 63

2027

5 (3

)

64

1603

8 (5

) 65

2019

9 (3

)

66

2014

3 (3

) 67

15945 (9)

integrase

68

20327 (3) 69

2042

1 (3

)

70

20186 (3) 71

20235 (3)

72

13419 (8) 73

1607

6 (5

)

74

19128 (293)75

1288

5 (8

)

76

20315 (3) 77

15926 (5)

methyltransferase

78

20229 (3) 79

2035

6 (3

)

80

2039

8 (2

) 81

13247 (8)

von Willebrand factor domain protein

82

10817 (5)

ATPase

83

2008

6 (3

)

84

20384 (3) 85

2015

6 (3

)

86

20254 (3) 87

2039

9 (3

)

88

19078 (9)

DNA primase

89

16110 (11)

90

19862 (8)

Gmala1

•  Overall rates of phage isolation in Gordonia were fairly low. However, 5 new

phages were identified, demonstrating the potential for successfully using this host

•  All of the phages shared phams with phages from different hosts, suggesting old evolutionary connections

•  The evolutionary histories of Jumbo, Bantam, and the A15 phages are different, based on GC content and shared phams

•  One or more of these phages may have significant environmental applications, including treatment of wastewater sludge

GMA3%

Jumbo%

Gmala1%

DocB7%

Lysin%%%%%%%%%%LysinA%%%%%%%%%%%%%%%%%%%Holin%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%Holin?%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%Holin?%%%%%%%%Membrane%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%LysinB%

Integrase Pham 16684 (107)

Nucleotidase Pham 11635 (6)

RecE Exonuclease Pham 11481 (7)

Exonuclease VII large subunit

Pham 18923 (7)

RecT ssDNA binding protein

Pham 13007 (7)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92

1

1879

1 (1

)

2

6676

(55

)

3

5941 (9) 4

4742 (35)

5

7038 (35) 6

1900

6 (1

)

7

1883

0 (1

)

8

1880

9 (1

)

9

206

(51)

11

1901

9 (1

)

12

11683 (7) 13

17320 (7)

14

6118 (84) 15

11376 (7)

16

11463 (7) 17

7889 (7)

18

11718 (7) 19

17286 (5)

20

18778 (1) 21

1901

0 (1

)

22

11685 (7) 23

11274 (7)

24

19270 (11) 25

1720 (6)

26

1896

1 (1

)

27

19840 (1)

28

1738

3 (1

)

29

11501 (7)

30

17168 (16) 31

16400 (130)

32

10054 (8) 33

19611 (24)

34

19276 (77) 35

18867 (1)

36

18970 (1) 37

20503 (16)

38

11263 (7) 39

1878

9 (1

)

40

11701 (6) 41

18895 (1)

42

12598 (3)

43

8416 (5)

44

1901

3 (1

)

45

1900

1 (1

)

46

1875

4 (1

)

47

1300

3 (9

)

48

18923 (7) 49

1884

3 (1

)

50

1882

7 (1

)

51

1886

3 (1

)

52

18995 (1) 53

1896

2 (1

)

54

1990

9 (1

9)

55

18932 (1)

56

19128 (292)57

1881

8 (1

)

58

1883

2 (1

)

59

1880

8 (1

)

60

1875

2 (1

)

61

18963 (1)

62

1880

2 (1

)

63

1887

7 (1

)

65

1888

5 (1

)

66

16684 (107)

67

18807 (1)

68

18933 (1)

69

1888

6 (1

)

70

11524 (7) 71

1881

3 (1

)

72

1873

4 (1

)

73

18800 (1)

74

18935 (1)

75

18972 (1)

76

9393 (5) 77

1900

2 (1

)

78

18744 (1) 79

1323

5 (2

5)

80

18851 (1) 81

18776 (1)

82

18980 (1) 83

1881

0 (1

)

84

1802

6 (2

) 85

1874

3 (1

)

86

1884

0 (1

) 87

1881

2 (1

)

88

18835 (1) 89

2019

5 (3

)

90

17427 (7) 91

18866 (1)

92

19388 (35) 93

18763 (1)

94

1122

0 (7

) 95

1878

0 (1

)

96

1899

2 (1

) 97

1888

8 (1

)

98

13007 (7) 99

11481 (7)

100

1873

6 (1

)

101

11360 (6) 102

1887

1 (1

)

103

11336 (7) 104

1899

8 (1

)

105

481 (124) 106

20592 (52)

107

18854 (1) 108

1893

4 (1

)

109

1345

8 (2

)

110

1893

7 (1

)

111

1899

1 (1

)

112

2270 (13)

113

1877

0 (1

)

114

1145

6 (7

)

115

10350 (2) 116

1886

2 (1

)

117

13620 (126) 118

1894

0 (1

)

119

1896

8 (1

)

120

1897

9 (1

)

121

11635 (6) 122

19293 (15)

123

1886

9 (1

)

124

1888

0 (1

)

125

473

(10)

126

1898

4 (1

)

127

1882

9 (1

)

128

1883

6 (1

)

129

1899

3 (1

)

130

1888

9 (1

)

131

1033 (5) 132

7101 (5)

133

18917 (1) 134

18853 (1)

135

19133 (10) 136

1875

3 (2

)

137

1885

0 (1

)

138

18748 (1) 139

18788 (1)

140

1892

6 (1

) 141

19020 (1)

142

1888

3 (1

) 143

1879

2 (1

)

144

1895

0 (1

) 145

1901

2 (1

)

146

1899

7 (1

) 147

1896

4 (1

)

148

18771 (1) 149

18943 (1)

150

1879

9 (1

) 151

1879

3 (1

)

152

18948 (1) 153

1140

1 (7

)

154

18931 (1)155

1887

5 (1

)

156

1888

4 (1

) 157

1900

7 (1

)

158

1881

4 (1

) 159

1742

5 (2

)

160

1893

9 (1

) 161

1890

9 (1

)

162

1883

3 (1

) 163

1887

3 (1

)

164

1874

0 (1

) 165

19008 (1)

166

1881

7 (1

)

167

1890

4 (1

)

168

1903

5 (1

2) 169

1882

6 (1

)

170

6387 (68) 171

3014

(3)

172

18953 (60) 173

1876

5 (1

)

174

1895

4 (1

) 175

1894

6 (1

)

Bantam_Draft

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98

1

1967

3 (1

)

2

19540 (1) 3

1811

7 (7

6)

4

1964

8 (1

) 5

1967

6 (1

)

6

19834 (1) 7

1973

2 (1

)

8

1714

(16

) 9

1160

6 (5

)

10

1953

6 (1

) 11

4542

(14

)

12

1981

3 (1

)

13

1130

5 (6

)

14

1107

5 (6

)

HNH endonuclease

15

1954

4 (1

)

16

11530 (6)

17

19552 (1) 18

11367 (10)

24

8127 (81)

Glycosyltransferase

25

607 (2)

Glycosyltransferase

26

1151

2 (6

)

27

11683 (7)

28

17320 (7)

Terminase large subunit

29

6118 (84)

Portal protein

30

11376 (7)

Capsid morphogenesis F-Mu like protein

31

11463 (7)

32

7889 (7)

Head decoration protein

33

11718 (7)

Major capsid protein

34

11459 (6) 35

1166

1 (6

)

36

11685 (7) 37

11274 (7)

38

19268 (9)

Endonuclease VII

39

17299 (43)

Major tail protein

40

1972

2 (1

)

42

19582 (2)

tail assembly chaperone

41

19582 (2)

tail assembly chaperone

43

11501 (7)

Tape measure protein

44

17168 (16)

Minor tail protein

45

17144 (81)

Minor tail protein

46

8497 (2)

HNH Endonuclease

47

19611 (24)

Lysin A

48

9537 (4)

Lysin A

49

2740 (2)

50

19542 (1) 51

1973

8 (1

)

52

20503 (16)

Minor tail protein

53

19064 (4)

54

1973

7 (1

)

55

1973

6 (1

)

56

9475 (4)

Virion protein

57

19889 (1)

58

11611 (4) 59

19743 (1)

Peptidase S1

60

19649 (1) 61

18923 (7)

Exonuclease VII large subunit

62

1990

5 (1

)

Exonuclease VII small subunit

63

1974

8 (1

)

64

1983

2 (1

)

65

1973

1 (1

)

66

1160

3 (6

)

67

1989

3 (1

)

68

1992

7 (1

)

69

8809 (20)

70

5467 (70)

RNA-splicing ligase RtcB

71

20513 (3)

HNH endonuclease

72

11445 (5) 73

860 (14)

74

19547 (1)

HNH endonuclease

75

10395 (20)

Metallophosphatase

76

1991

0 (1

)

77

19787 (1)

78

1992

0 (1

)

79

1962

7 (1

)

80

1960

4 (1

)

81

1974

7 (1

)

82

1975

7 (1

)

83

5284

(31

)

84

1116

9 (5

)

85

1965

6 (1

)

86

19918 (1) 87

1985

3 (1

)

88

1983

9 (1

)

89

1988

0 (1

)

90

4379

(24

)

91

19766 (1)

92

19541 (1) 93

1716

1 (2

)

94

1976

7 (1

)

95

16684 (107)

Integrase

96

1985

7 (1

)

97

1986

5 (1

)

98

11481 (7)

RecE Exonuclease

99

19804 (1)

100

19625 (1) 101

1959

2 (1

)

102

1962

9 (1

)

103

1971

3 (1

)

104

1993

5 (1

)

105

1979

0 (1

)

106

13007 (7)

RecT ssDNA binding protein

107

1984

9 (1

)

108

1954

3 (1

)

109

1986

8 (1

)

Excise

110

18393 (209)

ThyX Thymidylate synthase

111

1976

1 (1

)

112

1122

0 (7

)

113

19789 (1)

114

16203 (3)

HNH endonuclease

115

20331 (3)

116

19921 (1) 117

19555 (1)

118

1985

6 (1

)

119

19881 (1)

120

19778 (1) 121

8133 (11)

specificity protein-tyrosine phosphatase

122

6890

(52

)

123

6916

(3)

124

19583 (1) 125

19303 (18)

drpA-like ssDNA binding protein

126

2146 (2) 127

17427 (7)

DNA helicase

128

1988

8 (1

)

129

9885

(19

)

NrdH-Glutaredoxin

130

1981

4 (5

)

131

16355 (21)

HNH endonuclease

132

18544 (127)

Single-stranded DNA binding protein

133

1985

2 (1

)

134

1150

7 (6

)

135

1138

1 (6

)

136

15576 (17)137

1983

8 (1

)

138

7132 (10) 139

1972

4 (1

)

140

1977

4 (1

)

141

15729 (490)

Endonuclease VII

142

1956

2 (1

)

143

1966

8 (1

)

144

19776 (1) 145

11524 (7)

146

19618 (7) 147

11336 (7)

Nucleotidyltransferase

148

1957

6 (1

)

149

1968

7 (1

)

150

702 (10) 151

1278

9 (2

)

152

1958

6 (1

)

153

1975

5 (1

)

154

1989

1 (1

)

155

1967

0 (1

)

156

1145

6 (7

)

157

1957

5 (1

)

158

1970

3 (1

)

159

1956

1 (1

)

160

1970

2 (1

)

161

11548 (15)

Lysin B

162

19623 (1)

endonuclease VII

163

1959

6 (1

)

164

1965

8 (1

)

165

1991

2 (1

)

166

8516

(3)

167

1973

4 (1

) 168

1987

1 (1

)

169

1981

6 (1

)170

1956

0 (1

)

171

1959

4 (1

) 172

5281

(3)

173

1974

1 (1

) 174

1960

5 (1

)

175

1971

2 (1

) 176

1992

9 (1

)

177

1986

1 (1

) 178

11214 (6)

179

19694 (1) 180

11197 (5)

181

1171

7 (6

) 182

1147

7 (6

)

183

1994

0 (1

) 184

1979

6 (1

)

185

1978

4 (1

) 186

1960

7 (1

)

187

1984

4 (1

) 188

1978

3 (1

)

189

1881

6 (7

) 190

1742

5 (2

)

191

1124

3 (5

) 192

19919 (1)

193

1957

1 (1

) 194

1989

0 (1

)

195

19556 (1)196

1957

4 (1

)

197

1985

5 (1

) 198

3726

(8)

199

1984

2 (1

) 200

1954

5 (1

)

201

1956

8 (1

) 202

19798 (1)

203

19180 (28)

Yvonnetastic

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93

1

1875

7 (1

3)

2

2020

8 (1

) 3

2015

1 (1

)

4

2013

6 (1

) 5

1160

6 (5

)

6

2022

4 (4

) 7

2045

9 (1

)

8

1130

5 (6

)

9

1107

5 (6

)

HNH endonuclease

10

11530 (6)

11

20309 (1) 12

11367 (10)

19

1308

1 (2

)

22

1151

2 (6

)

23

11683 (7) 24

17320 (7)

terminase

25

6118 (84)

portal

26

11376 (7)

capsid maturation protease

27

11463 (7)

scaffolding

28

7889 (7)

head decoration protein

29

11718 (7)

capsid

30

2008

5 (1

)

31

11459 (6) 32

1166

1 (6

)

33

11685 (7) 34

11274 (7)

35

1926

8 (9

)

Endo VII

36

17299 (43)

major tail

37

1116

1 (5

)

38

11334 (10)

tail assembly chaperone

39

11334 (10)

tail assembly chaperone

40

11501 (7)

tape measure

41

17168 (16)

minor tail

42

17144 (81)

minor tail

43

11221 (5)

endolysin

44

16093 (4)

lysin A

45

16984 (10)

holin

46

2023

0 (7

)

47

1156

1 (5

)

48

2047 (13)

49

20503 (16)

minor tail

50

11297 (12)

minor tail

51

11701 (6) 52

20276 (1)

53

20443 (1)

54

1499

3 (1

8)

Lsr2

55

20223 (1)

56

18923 (7)

exodeoxyribonuclease VII, large subunit

57

1134

5 (5

)

exodeoxyribonuclease VII, small subunit

58

1160

3 (6

)

59

1166

3 (5

)

60

11702 (5)

61

1150

9 (5

)

62

6211 (6)

metallophosphatase

63

5467 (70)

RtcB-like

64

20407 (1)

homing endonuclease

65

2009

7 (1

)

66

1140

4 (5

)

67

2037

9 (1

)

68

4091

(6)

69

2008

7 (1

)

70

2020

5 (1

)

71

1140

3 (5

)

72

2006

9 (1

)

74

16684 (107)

integrase

75

1123

8 (5

)

76

1136

0 (6

)

immunity repressor

77

2027

1 (1

)

78

11481 (7)

exonuclease

79

2011

5 (1

)

80

11449 (5) 81

13007 (7)

RecT

82

2033

7 (2

)

83

18393 (209)

FAD-dependent thymidylate synthase

84

2010

9 (1

)

85

1168

2 (5

)

86

2025

1 (1

)

HTH DNA binding

87

2022

7 (1

)

88

1122

0 (7

)

89

11338 (5)

HTH DNA binding

90

11249 (5) 91

11385 (5)

92

1138

8 (5

)

93

1867

0 (4

5)

MazG

94

1151

8 (5

)

95

2007

7 (1

)

96

20464 (1) 97

1164

1 (5

)

98

20167 (1) 99

20291 (19)

100

4742 (35)

phosphoribosyl-pyrophosphate synthase

101

7038 (35)

nicotinate phosphoribosyl transferase

102

12974 (3) 103

19303 (18)

104

1345

8 (2

)

105

2006

3 (1

)

106

8838

(91

)

107

2023

2 (1

)

108

17427 (7)

DnaB helicase

109

20080 (1)

HNH endonuclease

110

2044

9 (5

)

NrdH redoxin

111

18544 (127)

single stranded DNA binding protein

112

1143

2 (5

)

113

1150

7 (6

)

114

1138

1 (6

)

115

7132 (10)

116

1151

3 (5

)

117

1127

2 (5

)

118

15729 (490)

Endonuclease VII

119

11170 (5)

120

11524 (7) 121

11336 (7)

nucleotidyltransferase

122

2014

8 (1

)

123

2010

7 (1

)

124

2011

6 (1

)

125

1527

1 (3

)

126

2011

2 (1

)

127

11635 (6)

nucleotidase

128

2143 (4)

Endo VII

129

20146 (1)

130

19223 (8) 131

1170 (10)

ribonucleotide reductase

132

1145

6 (7

)

133

11548 (15)

lysin B

134

2035

5 (6

)

135

1875

3 (2

)

136

1126

1 (5

)

137

11214 (6) 138

2013

7 (1

)

139

2023

6 (1

) 140

2006

5 (1

)

141

1163

3 (5

) 142

5179 (7)

143

1171

4 (5

) 144

1171

7 (6

)

145

2041

3 (1

) 146

2028

0 (1

)

147

2019

6 (1

) 148

1147

7 (6

)

149

2013

8 (1

) 150

1137

4 (5

)

151

2011

7 (1

) 152

1142

0 (5

)

153

1881

6 (7

) 154

1168

9 (5

)

155

2044

5 (1

) 156

2024

4 (1

)

157

3726

(8) 158

1170

8 (6

)

159

1123

6 (5

) 160

19180 (28)

161

17594 (8) 162

20372 (1)

163

2007

9 (1

) 164

2031

2 (2

)

165

20074 (2) 166

1171

9 (7

)

167

2018

4 (1

) 168

2050

6 (1

)

169

11347 (5) 170

2011

8 (1

)

171

2039

0 (1

) 172

2044

4 (1

)

OneUp

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

1

19690 (1) 2

10819 (1)

3

9775 (14)

TerL

4

9969 (17)

Mu gp29-like protein

5

10455 (1) 6

9561 (2)

7

10582 (2) 8

10332 (1)

9

1988

6 (5

)

10

20495 (18)

11

19211 (10) 12

10259 (18)

13

1079

6 (1

)

14

1003

5 (1

)

15

20289 (10) 16

10989 (1)

17

9803 (1) 18

9660

(1)

19

11016 (18) 21

10594 (19)

pre-TMP frameshift protein

20

10594 (19)

truncated pre-TMP frameshift protein

22

10747 (18)

tape measure protein

23

10225 (18) 24

10679 (18)

25

9607 (1) 26

1942

9 (1

43)

putative holin

27

10772 (1)

LysA

28

1090

9 (1

)

holin

29

9658 (1) 30

16072 (7)

holin

31

9753

(1)

32

9700

(1) 33

19904 (9)

34

2046

7 (1

0)

WhiB

35

1022

6 (1

)

36

20685 (25)

DNA methylase

37

1519

5 (1

)

38

18692 (2)

LysB

39

1090

4 (1

)

40

18847 (1)41

1001

7 (1

)

42

9996 (10) 43

2026

5 (1

08)

44

15285 (8)

DnaN

45

10942 (7)

oligoribonuclease

46

9687

(1) 47

9666

(1)

48

10236 (18)

helicase

49

9609

(1)

50

10478 (1) 51

9777 (9)

52

10412 (1) 53

2006

1 (1

)

54

9874

(1)

HNH endonuclease

55

10411 (10)

56

9928 (1) 57

9779

(1)

58

1930

2 (9

) 59

15945 (9)

tyrosine recombinase/integrase

60

9508 (1) 61

2016

1 (1

0)

62

1083

2 (1

) 63

10176 (1)

64

15516 (3) 65

1072

2 (1

)

66

9611

(1) 67

10167 (10)

68

9833 (10)

vWFA

69

19124 (13)

AAA protein

70

10893 (1) 71

10153 (1)

72

1101

7 (1

) 73

1086

4 (1

)

74

10196 (1) 75

1063

6 (5

)

76

9892

(1) 77

1076

0 (1

)

78

9929 (1) 79

10878 (1)

80

1071

3 (1

) 81

10943 (10)

82

1024

8 (1

) 83

9752

(1)

84

9840

(1) 85

1038

7 (1

)

86

9488

(1) 87

1054

8 (1

)

88

9605

(1) 89

1101

9 (1

)

90

9808

(1) 91

1832

0 (9

)

92

9659 (1) 93

9546 (1)

94

10212 (1) 95

1054

1 (1

)

96

1035

8 (1

) 97

9947 (1)

98

9526

(1) 99

1025

7 (1

)

100

9988

(1) 101

1319

8 (1

0)

102

19078 (9) 103

10441 (1)

104

1011

9 (1

) 105

19055 (10)

DocB7

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77

1

13185 (8)

putative small terminase subunit

2

15871 (4)

putative large terminase subunit

3

9969 (17) 4

1581

6 (1

)

5

20495 (18) 6

16175 (8)

7

10259 (18)

putative major capsid protein

8

1300

8 (8

)

9

16299 (1) 10

12800 (8)

11

20240 (4) 12

1640

2 (5

)

13

11016 (18)

putative major tail protein

15

13323 (15)

putative tail assembly protein

14

13323 (15)

putative tail assembly protein

16

10747 (18)

putative tape measure protein

17

10225 (18) 18

10679 (18)

19

19064 (4) 20

15960 (2)

21

16263 (1) 22

16261 (5)

putative lysin

23

1616

5 (5

)

24

17047 (14)

25

3171 (14) 26

1629

6 (5

)

putative lysin

27

16984 (10)

putative holin

28

15922 (2)

29

12742 (5) 30

13317 (6)

31

1613

0 (4

)

32

19133 (10)

33

1604

7 (1

)

34

1586

2 (1

) 35

1628

8 (1

)

36

1616

4 (1

) 37

1580

9 (1

)

38

19091 (21) 39

10439 (11)

40

1580

7 (1

) 41

6916

(3)

42

16160 (158) 43

1598

4 (1

)

44

19904 (9) 45

1630

6 (1

)

46

1598

7 (1

) 47

1646

2 (4

)

48

2025

3 (5

3) 49

1640

4 (5

)

50

13150 (8) 51

1626

2 (1

)

52

1607

1 (5

) 53

16020 (5)

54

1624

0 (1

) 55

19303 (18)

56

10269 (11) 57

1328

2 (8

)

58

10236 (18)

putative helicase

59

16108 (5)

60

1610

1 (1

) 61

16366 (1)

62

1648

2 (1

) 63

9777 (9)

64

1598

8 (1

) 65

15920 (5)

66

19110 (5)67

1584

4 (1

)

68

1586

7 (1

) 69

1585

4 (1

)

70

1603

8 (5

) 71

1620

8 (2

)

72

1634

9 (1

) 73

16356 (1)

74

1646

1 (1

) 75

15945 (9)

putative integrase

76

16427 (1) 77

1582

1 (2

)

78

13419 (8) 79

1290

3 (6

)

80

1607

6 (5

) 81

1288

5 (8

)

82

16223 (1) 83

15926 (5)

84

1630

0 (1

) 85

1583

8 (1

)

86

1615

2 (1

) 87

13247 (8)

putative von Willebrand factor protein

88

10817 (5)

putative ATPase family protein

89

1585

7 (1

)

90

16224 (1) 91

16145 (2)

92

18320 (9) 93

6186 (4)

94

1626

5 (1

) 95

1598

9 (1

)

96

16148 (1) 97

1581

8 (1

)

98

15876 (1) 99

1616

6 (1

)

100

19078 (9) 101

16110 (11)

102

15998 (1) 103

19862 (8)

104

1597

8 (1

)

GMA3

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78

1

1917

9 (3

)

2

19078 (9) 3

16110 (11)

4

1927

7 (1

) 5

19212 (1)

6

19862 (8)

7

13185 (8) 8

9775 (14)

9

9969 (17) 10

1908

7 (1

)

11

20495 (18) 12

16175 (8)

13

10259 (18) 14

1300

8 (8

)

15

19220 (4) 16

12800 (8)

17

13451 (4) 18

1640

2 (5

)

19

11016 (18) 20

13323 (15)

21

1299

1 (1

)

22

10747 (18)

23

10225 (18) 24

10679 (18)

25

19064 (4) 26

20509 (5)

27

1916

6 (1

)

28

7318 (54)

29

16261 (5) 30

1616

5 (5

)

31

17047 (14) 32

1904

6 (5

)

33

16296 (5) 34

9727 (3)

35

16984 (10) 36

20292 (7)

37

1903

6 (1

)

38

17875 (95)

39

15922 (2) 40

19264 (1)

41

13317 (6) 42

1908

1 (1

)

43

19133 (10)

44

19091 (21) 45

10439 (11)

46

19068 (1) 47

19904 (9)

48

1923

9 (1

) 49

2025

3 (5

3)

50

1640

4 (5

) 51

13150 (8)

52

1902

2 (1

) 53

1908

2 (1

)

54

1607

1 (5

) 55

16020 (5)

56

1904

5 (1

) 57

10269 (11)

58

1328

2 (8

) 59

10236 (18)

60

16108 (5) 61

1916

5 (1

)

62

1567

6 (2

) 63

9777 (9)

64

19240 (1) 65

15920 (5)

66

19110 (5) 67

1603

8 (5

)

68

19069 (57) 69

1913

1 (1

)

70

1923

3 (1

) 71

15945 (9)

72

19189 (1) 73

1582

1 (2

)

74

1914

3 (1

) 75

19255 (1)

76

13419 (8) 77

1915

7 (1

)

78

13360 (5) 79

1607

6 (5

)

80

1929

1 (1

) 81

1288

5 (8

)

82

19086 (1) 83

15926 (5)

84

19101 (1) 85

19292 (1)

86

19080 (1)87

1921

7 (1

)

88

1275

8 (5

) 89

1912

5 (1

)

90

19237 (1)91

1907

3 (1

)

92

1926

5 (1

) 93

19150 (1)

94

13247 (8) 95

1918

1 (1

)

96

10817 (5)

97

1909

3 (1

)

98

1915

6 (1

)

99

1910

7 (1

)

100

1926

0 (1

) 101

19229 (1)

102

16145 (2) 103

1266

0 (4

)

104

1920

5 (1

) 105

1925

6 (1

)

106

1903

9 (1

) 107

19228 (1)

108

1917

9 (3

)

109

19179 (3)

109

19179 (3)

Jumbo_Draft

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

1

13185 (8) 2

15871 (4)

TerL

3

9969 (17)

DUF935-like protein

4

2012

6 (6

)

5

20495 (18) 6

16175 (8)

7

10259 (18)

major capsid protein

8

1300

8 (8

)

9

19220 (4) 10

12800 (8)

morphogensis protein

11

13451 (4) 12

1640

2 (5

)

13

11016 (18) 15

13323 (15)

pre-TMP frameshift protein

14

13323 (15)

pre-TMP frameshift protein

16

10747 (18)

tape measure protein

17

10225 (18) 18

10679 (18)

19

20422 (3) 20

20183 (3)

21

1029 (242) 22

16261 (5)

lysin

23

16165 (5) 24

17047 (14)

lysin A

25

2027

4 (3

)

26

16296 (5)

27

17023 (9)

holin

28

20292 (7)

DNA methylase

29

2021

7 (3

)

30

9839 (8)

holin

31

15963 (6)

holin

32

1613

0 (4

)

33

19133 (10)

lysin B

34

2035

2 (3

) 35

2042

6 (3

)

36

2033

0 (3

) 37

19091 (21)

38

2027

7 (3

) 39

2014

2 (3

)

40

2039

6 (3

) 41

20147 (3)

42

18393 (209)

thymidylate synthase ThyX

43

19904 (9)

44

2006

0 (3

) 45

1646

2 (4

)

46

2025

3 (5

3)

transcription factor WhiB

47

1640

4 (5

)

48

13150 (8)

RusA-like ribonuclease

49

2033

2 (3

)

50

1607

1 (5

) 51

2038

8 (3

)

52

16020 (5)

DNA polymerase

53

10942 (7)

ribonuclease

54

1328

2 (8

)

DNA binding protein

55

10236 (18)

helicase

56

16108 (5)57

2046

5 (3

)

58

20419 (3)

DUF732-like protein

59

9777 (9)

60

2013

2 (3

) 61

15920 (5)

62

19110 (5) 63

2027

5 (3

)

64

1603

8 (5

) 65

2019

9 (3

)

66

2014

3 (3

) 67

15945 (9)

integrase

68

20327 (3) 69

2042

1 (3

)

70

20186 (3) 71

20235 (3)

72

13419 (8) 73

1607

6 (5

)

74

19128 (293)75

1288

5 (8

)

76

20315 (3) 77

15926 (5)

methyltransferase

78

20229 (3) 79

2035

6 (3

)

80

2039

8 (2

) 81

13247 (8)

von Willebrand factor domain protein

82

10817 (5)

ATPase

83

2008

6 (3

)

84

20384 (3) 85

2015

6 (3

)

86

20254 (3) 87

2039

9 (3

)

88

19078 (9)

DNA primase

89

16110 (11)

90

19862 (8)

Gmala1

Summary of Results

Fig. 2. Synteny and homology between Bantam and other Gordonia phages, Yvonnetastic (Singleton) and OneUp (cluster CQ). A. Shared phams encoding structural genes. B. Shared genes involved in replication and regulation illustrate mosaic nature of these genomes.

A

B

Bantam summary: •  108 / 172 orfs are orphams •  No tRNAs detected •  Shared phams: structural genes, integrase, lysinA, exonucleases •  27 phams shared with Gordonia cluster CQ, singleton Yvonnetastic •  32 phams shared with phages in other hosts: Mycobacterium (23), Rhodococcus (10),

Arthrobacter (3), Corynebacterium (2), Streptomyces (2)

Fig. 6. Immunity regions of Gordonia cluster A15 and Mycobacterium cluster A2 phages. Note the presence of ParA and ParB genes present in A15 phages instead of integrase, suggesting a partitioning method of prophage maintenance (Stella et al 2013)

Fig. 5. Map of cluster A15 phages, showing extensive homology

Fig. 3. Homology between Jumbo and Gordonia singleton GMA3 (1), cluster DF phage Gmala1 (3), and Rhodococcus singleton DocB7 (4), all isolated outside of the SEA-PHAGES program.

Fig. 4. Lysis cassettes in Jumbo and related phages, which were isolated as potential biocontrol agents for reducing foaming in wastewater sludge by lysing bacteria.

Jumbo summary: •  42/107 orfs are orphams •  No tRNAs detected •  Shared phams: structural genes, integrase, helicase, exonuclease, endolysins, holins •  Phams shared with singleton GMA3, Gordonia cluster DF, Rhodococcus singleton DocB7 •  21 phams shared with phages in other hosts: Rhodococcus (15), Corynebacterium (2),

Streptomyces (4)

Jswag, Remus, Strosahl summary:

•  96% average nucleotide identity

•  3 tRNA genes

•  No orphams

•  33/102 phams unique to A15 phages

o  24/33 in all A15 phages

•  58 phams shared with other cluster A phages o  Most are in A2 subcluster

•  14 phams shared with other Gordonia clusters

•  21 phams shared with other hosts: Rhodococcus (15), Streptomyces (4),

     Corynebacterium (2)

Fig. 7. Lysogeny in A15 phages. A. Culture at 35o C yielded turbid plaques. B. Cells from turbid plaques of JSwag, Remus, and Figaro (A15 by PCR) were streaked, transferred to liquid culture, and used in spot tests with lysates of these same phages. Lysogens were immune to all A15 phages tested, but not to Jumbo.

B.