Hits from Host to Host: Isolation, Characterization

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 Jumbo: Comparative Genomics

putative tail putative putative large terminase subunit putative tape measure protein putative lysin assembly protein lysin putative tail Relatively few phages infecting hosts other than Mycobacterium smegmatis have . putative major capsid putative major tail putative putative small terminase subunit assembly GMA3 protein protein holin protein 15816 (1) 15816 (8) 13008 (5) 16402 (5) 16296 15871 (4) 16175 (8) 12800 (8) 13323 (15) 10747 (18) 10679 (18) 15960 (2) 16261 (5) 17047 (14) 15922 (2) 13317 (6) 19133 (10) GMA3% putative helicase putative ATPase family protein

16165 (5) 16165 (4) 16130 putative 13185 (8) 2 9969 (17) 4 20495 (18) 6 10259 (18) 8 16299 (1) 10 20240 (4) 12 11016 (18) 13323 (15)15 16 10225 (18) 18 19064 (4) 20 16263 (1) 22 24 3171 (14) 26 16984 (10) 28 12742 (5) 30 32 putative von Willebrand factor protein integrase 1 3 5 7 9 11 13 14 17 19 21 23 25 27 29 31 been isolated and characterized (1). UWRF phage hunters set out to isolate phage 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

34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80 82 84 86 88 90 92 94 96 98 100 102 104

33 35 37 19091 (21) 39 41 16160 (158) 43 19904 (9) 45 47 49 13150 (8) 51 53 55 10269 (11) 57 10236 (18) 59 61 63 65 19110 (5)67 69 71 73 75 16427 (1) 77 13419 (8) 79 81 16223 (1) 83 85 87 10817 (5) 89 16224 (1) 91 18320 (9) 93 95 16148 (1) 97 15876 (1) 99 19078 (9) 101 15998 (1) 103

10439 (11) 16020 (5) 19303 (18) 16108 (5) 16366 (1) 9777 (9) 15920 (5) 16356 (1) 15945 (9) 15926 (5) 13247 (8) 16145 (2) 6186 (4) 16110 (11) 19862 (8) 15862 (1) 15862 (1) 16164 (1) 15807 (1) 15987 (5) 16071 (1) 16240 (1) 16101 (1) 16482 (1) 15988 (1) 15867 (5) 16038 (1) 16349 (1) 16461 (5) 16076 (1) 16300 (1) 16152 (1) 16265 (1) 15978 20253 (53) 20253 6916 (3) 6916

infecting another member of the phylum Actinobacteria, Gordonia terrae. Gordonia (1) 16047 (1) 16288 (1) 15809 (1) 15984 (1) 16306 (4) 16462 (5) 16404 (1) 16262 (8) 13282 (1) 15844 (1) 15854 (2) 16208 (2) 15821 (6) 12903 (8) 12885 (1) 15838 (1) 15857 (1) 15989 (1) 15818 (1) 16166 Jumbo_Draft

9775 (14) (1) 19087 16175 (8) (8) 13008 12800 (8) (5) 16402 13323 (15) 10747 (18) 10679 (18) 20509 (5) 7318 (54) (5) 16165 (5) 19046 9727 (3) 20292 (7)17875 (95) 19264 (1) (1) 19081 Lysin%%%%%%%%%%LysinA%%%%%%%%%%%%%%%%%%%Holin%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%Holin?% (1) 19156 (3) 19179 19179 (3) (3) 19179 13185 (8) 8 9969 (17) 10 20495 (18) 12 10259 (18) 14 19220 (4) 16 13451 (4) 18 11016 (18) 20 (1) 12991 22 10225 (18) 24 19064 (4) 26 (1) 19166 28 16261 (5) 30 17047 (14) 3216296 (5) 34 16984 (10) 36 38 15922 (2) 40 13317 (6) 42 19133 (10) (1) 19093 98 (1) 19107 10819179 (3) species are found in soil and wastewater, and are associated with foaming in 109 1 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 39 41 43 %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%Holin?%%%%%%%%Membrane%%%%%%%%%%% 97 99 109 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 2 4 6 44 46 48 50 52 54 56 58 60 62 64 %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%66 68 70 72 74 76 78 80 82 84 86 88 90 LysinB92 % 94 96 100 102 104 106 19078 (9) 3 5 19862 (8) 37 19091 (21) 45 19068 (1) 47 49 51 53 55 57 59 16108 (5) 61 63 19240 (1) 65 19110 (5) 67 19069 (57) 69 71 19189 (1) 73 75 13419 (8) 77 13360 (5) 79 81 19086 (1) 83 19101 (1) 85 19080 (1)87 89 19237 (1)91 93 13247 (8) 95 10817 (5) 101 16145 (2) 103 105 107

16110 (11) 19212 (1) 10439 (11) 19904 (9) 13150 (8) 16020 (5) 10269 (11) 10236 (18) 9777 (9) 15920 (5) 15945 (9) 19255 (1) 15926 (5) 19292 (1) 19150 (1) 19229 (1) 19228 (1)

19277 (1) 19277 morphogensis pre-TMP DNA (1) 19239 (5) 16404 (1) 19022 (5) 16071 (1) 19045 (8) 13282 (2) 15676 (1) 19233 (1) 19143 (1) 19291 (5) 12758 (1) 19265 (1) 19260 (1) 19205 (1) 19039 TerL tape measure protein lysin lysin A holin wastewater treatment. The same procedures used for isolating Arthrobacter and protein frameshift protein methylase pre-TMP (1) 19036 (1) 19082 (1) 19165 (5) 16038 (1) 19131 (2) 15821 (1) 19157 (5) 16076 (8) 12885 (1) 19217 (1) 19125 (1) 19073 (1) 19181 (4) 12660 (1) 19256 Gmala1 DUF935-like protein major capsid protein frameshift holin holin lysin B (53) 20253 protein

transcription DNA DUF732- 20126 (6) 20126 (8) 13008 (5) 16402 (4) 16130 thymidylate RusA-like 15871 (4) 16175 (8) 12800 (8) 13323 (15) 10747 (18) 10679 (18) 20183 (3) 16261 (5) 17047 (14) 16296 (5) 20292 (7) 9839 (8) factor DNA polymerase binding like ATPase DNA primase synthase ThyX ribonuclease WhiB protein protein 13185 (8) 2 9969 (17) 4 20495 (18) 6 10259 (18) 8 19220 (4) 10 13451 (4) 12 11016 (18) 13323 (15)15 16 10225 (18) 18 20422 (3) 20 1029 (242) 22 16165 (5) 24 (3) 20274 26 17023 (9) 28 (3) 20217 30 15963 (6) 32 19133 (10) Jumbo% ribonuclease helicase integrase methyltransferase von Willebrand factor domain protein Rhodococcus phage in previous years were used to test 44 soil samples: 1 3 5 7 9 11 13 14 17 19 21 23 25 27 29 31 33 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 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80 82 84 86 88 90 35 37 39 41 18393 (209) 43 45 47 13150 (8) 49 51 16020 (5) 53 55 16108 (5)57 20419 (3) 59 61 19110 (5) 63 65 67 20327 (3) 69 20186 (3) 71 13419 (8) 73 19128 (293)75 20315 (3) 77 20229 (3) 79 81 10817 (5) 83 20384 (3) 85 20254 (3) 87 19078 (9) 89 19862 (8)

19091 (21) 20147 (3) 19904 (9) 10942 (7) 10236 (18) 9777 (9) 15920 (5) 15945 (9) 20235 (3) 15926 (5) 13247 (8) 16110 (11) pre-TMP putative (3) 20352 (3) 20330 (3) 20277 (3) 20396 (3) 20060 (5) 16071 (8) 13282 (3) 20132 (5) 16038 (3) 20143 (2) 20398 Mu gp29-like protein frameshift tape measure protein holin holin (53) 20253 truncated holin protein pre- (3) 20426 (3) 20142 (4) 16462 (5) 16404 (3) 20332 (3) 20388 (3) 20465 (3) 20275 (3) 20199 (3) 20421 (5) 16076 (8) 12885 (3) 20356 (3) 20086 (3) 20156 (3) 20399 DocB7 TerL TMP LysA frameshift protein

• LB broth was used for bacteria culture and top agar, no antibiotics (1) 10035 (1) 9660 (143) 19429 (1) 10909 DNA HNH 10819 (1) 9969 (17) 9561 (2) 10332 (1) 20495 (18) 10259 (18) 10989 (1) 10594 (19) 10747 (18) 10679 (18) 16072 (7) WhiB LysB DnaN helicase vWFA methylase endonuclease tyrosine 19690 (1) 2 9775 (14) 4 10455 (1) 6 10582 (2) 8 (5) 19886 10 19211 (10) 12 (1) 10796 14 20289 (10) 16 9803 (1) 18 11016 (18) 21 10594 (19) 22 10225 (18) 24 9607 (1) 26 10772 (1) 289658 (1) 30 oligoribonuclease recombinase/ AAA protein integrase 1 3 5 7 9 11 13 15 17 19 20 23 25 27 29

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

32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80 82 84 86 88 90 92 94 96 98 100 102 104 • CaCl was added at 4.5 mM in culture broth and 2.25 mM in top agar 31 33 35 20685 (25) 37 18692 (2) 39 18847 (1)41 9996 (10) 43 15285 (8) 45 47 10236 (18) 49 10478 (1) 51 10412 (1) 53 55 9928 (1) 57 59 9508 (1) 61 63 15516 (3) 65 67 9833 (10) 69 10893 (1) 71 73 10196 (1) 75 77 9929 (1) 79 81 83 85 87 89 91 9659 (1) 93 10212 (1) 95 97 99 101 19078 (9) 103 105 19904 (9) 10942 (7) 9777 (9) 10411 (10) 15945 (9) 10176 (1) 10167 (10) 19124 (13) 10153 (1) 10878 (1) 10943 (10) 9546 (1) 9947 (1) 10441 (1) 19055 (10)

2 (1) 9700 (1) 9687 (1) 9874 (1) 9611 (1) 9892 (1) 9840 (1) 9488 (1) 9605 (1) 9808 (1) 9526 (1) 9988 19302 (9) 19302 (1) 10832 (1) 11017 (1) 10713 (1) 10248 (1) 10358 (1) 10119 20467 (10) 20467 9753 (1) 9753 (1) 9666 (1) 9609 (1) 9779 (1) 9752 10226 (1) 10226 (1) 15195 (1) 10904 (1) 10017 (1) 20061 (1) 10722 (1) 10864 (5) 10636 (1) 10760 (1) 10387 (1) 10548 (1) 11019 (9) 18320 (1) 10541 (1) 10257 20161 (10) 20161 (10) 13198 putative tail putative (108) 20265 putative large terminase subunit putative tape measure protein putative lysin assembly protein lysin putative tail putative major capsid putative major tail putative putative small terminase subunit assembly GMA3 protein o protein holin protein

15816 (1) 15816 (8) 13008 (5) 16402 (5) 16296 Gmala1% •Enrichments were15871 (4) cultured at16175 (8) 30 C 12800for (8) 2-513323 (15) days 10747 (18) 10679 (18) 15960 (2) 16261 (5) 17047 (14) 15922 (2) 13317 (6) 19133 (10) putative helicase putative ATPase family protein

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

34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80 82 84 86 88 90 92 94 96 98 100 102 104 33 35 37 19091 (21) 39 41 16160 (158) 43 19904 (9) 45 47 49 13150 (8) 51 53 55 10269 (11) 57 10236 (18) 59 61 63 65 19110 (5)67 69 71 73 75 16427 (1) 77 13419 (8) 79 81 16223 (1) 83 85 87 10817 (5) 89 16224 (1) 91 18320 (9) 93 95 16148 (1) 97 15876 (1) 99 19078 (9) 101 15998 (1) 103 10439 (11) 16020 (5) 19303 (18) 16108 (5) 16366 (1) 9777 (9) 15920 (5) 16356 (1) 15945 (9) 15926 (5) 13247 (8) 16145 (2) 6186 (4) 16110 (11) 19862 (8) 15862 (1) 15862 (1) 16164 (1) 15807 (1) 15987 (5) 16071 (1) 16240 (1) 16101 (1) 16482 (1) 15988 (1) 15867 (5) 16038 (1) 16349 (1) 16461 (5) 16076 (1) 16300 (1) 16152 (1) 16265 (1) 15978 20253 (53) 20253 6916 (3) 6916 Jumbo_Draft (1) 16047 (1) 16288 (1) 15809 (1) 15984 (1) 16306 (4) 16462 (5) 16404 (1) 16262 (8) 13282 (1) 15844 (1) 15854 (2) 16208 (2) 15821 (6) 12903 (8) 12885 (1) 15838 (1) 15857 (1) 15989 (1) 15818 (1) 16166

Results: Only five9775 (14) student(1) 19087 16175 (8) samples(8) 13008 12800 (8) (5) 16402 yielded13323 (15) plaques,10747 (18) along with 3 10679 (18) instructor 20509 (5) 7318 (54) (5) 16165 (5) 19046 9727 (3) 20292 (7)17875 (95) 19264 (1) (1) 19081 (1) 19156 (3) 19179

19179 (3) (3) 19179 13185 (8) 8 9969 (17) 10 20495 (18) 12 10259 (18) 14 19220 (4) 16 13451 (4) 18 11016 (18) 20 (1) 12991 22 10225 (18) 24 19064 (4) 26 (1) 19166 28 16261 (5) 30 17047 (14) 3216296 (5) 34 16984 (10) 36 38 15922 (2) 40 13317 (6) 42 19133 (10) (1) 19093 98 (1) 19107 10819179 (3)

109 1 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 39 41 43 97 99 109

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

2 4 6 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80 82 84 86 88 90 92 94 96 100 102 104 106 samples19078 (9) 3 5 piloted19862 (8) prior to class. Several phages were isolated from these samples. 37 19091 (21) 45 19068 (1) 47 49 51 53 55 57 59 16108 (5) 61 63 19240 (1) 65 19110 (5) 67 19069 (57) 69 71 19189 (1) 73 75 13419 (8) 77 13360 (5) 79 81 19086 (1) 83 19101 (1) 85 19080 (1)87 89 19237 (1)91 93 13247 (8) 95 10817 (5) 101 16145 (2) 103 105 107 16110 (11) 19212 (1) 10439 (11) 19904 (9) 13150 (8) 16020 (5) 10269 (11) 10236 (18) 9777 (9) 15920 (5) 15945 (9) 19255 (1) 15926 (5) 19292 (1) 19150 (1) 19229 (1) 19228 (1)

pre-TMP (1) 19036 (1) 19082 (1) 19165 (5) 16038 (1) 19131 (2) 15821 (1) 19157 (5) 16076 (8) 12885 (1) 19217 (1) 19125 (1) 19073 (1) 19181 (4) 12660 (1) 19256 Gmala1 DUF935-like protein major capsid protein frameshift holin holin lysin B (53) 20253 protein

transcription DNA DUF732- We selected phages15871 (4) for sequencing(6) 20126 16175 (8) (8) 13008 that12800 (8) (5) 16402 were13323 (15) likely to10747 (18) be different based10679 (18) on 20183 (3) 16261 (5) 17047 (14) 16296 (5) 20292 (7) 9839 (8) (4) 16130 thymidylate RusA-like factor DNA polymerase binding like ATPase DNA primase synthase ThyX ribonuclease DocB7% WhiB protein protein 13185 (8) 2 9969 (17) 4 20495 (18) 6 10259 (18) 8 19220 (4) 10 13451 (4) 12 11016 (18) 13323 (15)15 16 10225 (18) 18 20422 (3) 20 1029 (242) 22 16165 (5) 24 (3) 20274 26 17023 (9) 28 (3) 20217 30 15963 (6) 32 19133 (10) ribonuclease helicase integrase methyltransferase von Willebrand factor domain protein

1 3 5 7 9 11 13 14 17 19 21 23 25 27 29 31 33

34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80 82 84 86 88 90

plaque and EM morphologies (Fig. 1), restriction enzyme patterns (not shown), and 35 37 39 41 18393 (209) 43 45 47 13150 (8) 49 51 16020 (5) 53 55 16108 (5)57 20419 (3) 59 61 19110 (5) 63 65 67 20327 (3) 69 20186 (3) 71 13419 (8) 73 19128 (293)75 20315 (3) 77 20229 (3) 79 81 10817 (5) 83 20384 (3) 85 20254 (3) 87 19078 (9) 89 19862 (8)

soil source. Five unique phage sequences were(1) 10035 obtained(1) 9660 (Table 1). (143) 19429 (1) 10909 DNA HNH 10819 (1) 9969 (17) 9561 (2) 10332 (1) 20495 (18) 10259 (18) 10989 (1) 10594 (19) 10747 (18) 10679 (18) 16072 (7) WhiB LysB DnaN helicase vWFA methylase endonuclease tyrosine 19690 (1) 2 9775 (14) 4 10455 (1) 6 10582 (2) 8 (5) 19886 10 19211 (10) 12 (1) 10796 14 20289 (10) 16 9803 (1) 18 11016 (18) 21 10594 (19) 22 10225 (18) 24 9607 (1) 26 10772 (1) 289658 (1) 30 oligoribonuclease recombinase/ AAA protein integrase 1 3 5 7 9 11 13 15 17 19 20 23 25 27 29

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 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80 82 84 86 88 90 92 94 96 98 100 102 104 31 33 35 20685 (25) 37 18692 (2) 39 18847 (1)41 9996 (10) 43 15285 (8) 45 47 10236 (18) 49 10478 (1) 51 10412 (1) 53 55 9928 (1) 57 59 9508 (1) 61 63 15516 (3) 65 67 9833 (10) 69 10893 (1) 71 73 10196 (1) 75 77 9929 (1) 79 81 83 85 87 89 91 9659 (1) 93 10212 (1) 95 97 99 101 19078 (9) 103 105

19904 (9) 10942 (7) 9777 (9) 10411 (10) 15945 (9) 10176 (1) 10167 (10) 19124 (13) 10153 (1) 10878 (1) 10943 (10) 9546 (1) 9947 (1) 10441 (1) 19055 (10) 9700 (1) 9700 (1) 9687 (1) 9874 (1) 9611 (1) 9892 (1) 9840 (1) 9488 (1) 9605 (1) 9808 (1) 9526 (1) 9988 19302 (9) 19302 (1) 10832 (1) 11017 (1) 10713 (1) 10248 (1) 10358 (1) 10119 20467 (10) 20467 9753 (1) 9753 (1) 9666 (1) 9609 (1) 9779 (1) 9752 10226 (1) 10226 (1) 15195 (1) 10904 (1) 10017 (1) 20061 (1) 10722 (1) 10864 (5) 10636 (1) 10760 (1) 10387 (1) 10548 (1) 11019 (9) 18320 (1) 10541 (1) 10257 20161 (10) 20161 (10) 13198 A B C D E (108) 20265 Fig. 4. Lysis cassettes in Jumbo and related phages, which were isolated as potential biocontrol agents for reducing foaming in wastewater sludge by lysing Fig. 3. Homology between Jumbo and Gordonia singleton GMA3 (1), cluster DF phage Gmala1 (3), and bacteria. Rhodococcus singleton DocB7 (4), all isolated outside of the SEA-PHAGES program.

Jumbo summary: Fig. 7. Lysogeny in A15 phages. A. 30o C 35o C o • 42/107 orfs are orphams A. ACulture at 35 C yielded turbid • No tRNAs detected plaques. B. Cells from turbid plaques of JSwag, Remus, and Figaro (A15 by • Shared phams: structural genes, integrase, helicase, exonuclease, endolysins, holins PCR) were streaked, transferred to liquid • Phams shared with singleton GMA3, Gordonia cluster DF, Rhodococcus singleton DocB7 culture, and used in spot tests with Fig. 1. Plaque morphology and electron microscopy. There was a variety of plaque • 21 phams shared with phages in other hosts: Rhodococcus (15), Corynebacterium (2), lysates of these same phages. Lysogens morphologies observed. JSwag (A), had clear plaques of variable size (1-3 mm), sometimes with Streptomyces (4) were immune to all A15 phages tested, halos; Remus and Strosahl had a similar appearance. Jumbo (B) had smaller clear plaques (0.5 but not to Jumbo. mm) and Bantam (C) had very small (<0.5 mm), turbid plaques. All of these new phages are Phage Drake (A15 by PCR) Siphoviridae; Remus (D), JSwag and Strosahl had tails 100-125 nm in length, while Jumbo (E) and Cluster A15 phages B. Bantam had longer tails, 450-475 nm.

Tail Assembly Minor VIP2-like Tail Holin Portal Scaffolding Minor Tail Minor Tail ParB Chaperone Tail Capsid Tail Minor Tail Endolysin Terminase, large subunit Maturation Capsid Assembly Tapemeasure Minor Tail Lsr2 ParA KatherineG Tail Protease Chaperone HTH

17374 (254) 17374 (167) 4048 (7) 10868 (6) 11720 (198) 19198 (13) 19294 (6) 17791 (7) 11401 DNA DNA DnaB-like 18243 (31) 17737 (67) 2982 (414) 3257 (302) 17963 (313) 19254 (413) 18608 (314) 4899 (303) 12446 (406) 11297 (11) ThyX Ribonucleotide reductase phosphoesterase Endolysin SprT-like binding Primase helicase

4163 (31) 4163 (128) 18881 (91) 17868 (6) 11303 domain MazG- Transcription NrdH- DNA 5079 (124) 2 4 19250 (447) 6 11333 (6) 8 14746 (6) 13 17443 (281) 15 19266 (330) 17 3146 (348) 197209 (449)211827 (341) 23 2847 (449) 25 18608 (314)27 17995 (10) 29 19296 (432) 31 33 35 7133 (64) 37 39 DNA polymerase EndoVII RecB-like Repressor like regulator like Primase 1 3 5 7 12 14 16 18 20 22 24 26 28 30 32 34 36 38 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

41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 81 83 85 87 89 91 93 95 97 99 101

40 42 44 46 11528 (4) 48 50 52 18393 (192) 54 17822 (276)56 565 (367) 58 60 9297 (409) 62 64 6619262 (107)68 11548 (13) 70 7219104 (16)7418508 (6) 76 5065 (450) 78 80 19047 (4) 82 19123 (21) 84 86 1304 (171) 88 90 92 94 2322 (28) 96 6310 (15)98 100

18837 (412) 15909 (456) 13798 (118) 11277 (6) 4038 (448) 15729 (464) 11368 (1) 5238 (450) 19184 (182) 6789 (8) 11552 (1) 11552 (5) 19221 (6) 11167 (6) 11534 (6) 11582 (6) 11431 (6) 11588 (6) 11166 (7) 11719 (6) 11618 (6) 11615 (6) 11723 (6) 11363

gp2 gp4 gp6 gp8 gp13 gp15 gp17 gp19 gp21 gp23 gp25 gp27 gp29 gp31 gp33 gp35 gp37 gp39 (31) 19155 (33) 14286 17529 (215) 17529 11655 (1) 11655 (6) 11416 (6) 11443 (2) 11362 (6) 11442 (73) 5496 (6) 11553 (6) 11698 (6) 11505 (6) 11542 (6) 11640 (4) 11447 (9) 11355 (4) 11628 (6) 11450 (18) 6684 JSwag_Draftgp1 gp3 gp5 gp7 gp14 gp16 gp18 gp20 gp22 gp24 gp26 gp28 gp30 gp32 gp34 gp36 gp38 gp40 (16) 18805 (42) 18670 (65) 12571 (15) 17915 (15) 18656 17374 (254) 17374 (167) 4048 (128) 18881 (91) 17868 (6) 11303 Table 1. Sequencing Results 18243 (31) 17737 (67) 14746 (6) 17443 (281) 19266 (330) 3146 (348) 7209 (449) 1827 (341) 2847 (449) 18608 (314) 17995 (10) 19296 (432) 7133 (64) gp41 gp43 gp45 gp47 gp49 gp51 gp53 gp55 gp57 gp59 gp61 gp63 gp65 gp67 gp69 gp71 gp73 gp75 gp77 gp79 gp81 gp83 gp85 gp87 gp89 gp91 gp93gp95 gp97 gp99 gp101 gp103 gp105 5079 (124) 2 (31) 4163 4 19250 (447) 6 11333 (6) 8 gp10 13 (7) 10868 15 2982 (414) 17 3257 (302) 19 (6) 11720 21 (198) 19198 23 17963 (313) 25 19254 (413) 27 18608 (314) 29 4899 (303) 31 12446 (406) 33 11297 (11) 35 (13) 19294 37 (6) 17791 39 (7) 11401 gp42 gp44 gp46 gp48 gp50 gp52 gp54 gp56 gp58gp60 gp62 gp64 gp66 gp68 gp70 gp72 gp74 gp76 gp78 gp80 gp82 gp84 gp86 gp88 gp90 gp92 gp94 gp96 gp98 gp100 gp102 gp104

1 3 5 7 14 16 18 20 22 24 26 28 30 32 34 36 38 40

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

41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 81 83 85 87 89 91 93 95 97 99 101 103 105

10 18837 (412)42 44 46 15909 (456) 48 50 52 54 56 58 60 9297 (409) 62 64 66 19262 (107)68 11548 (13) 70 7219104 (16)7418508 (6) 76 5065 (450) 78 80 82 5238 (450) 84 19184 (182) 86 88 90 92 94 966789 (8) 98 100 102 104 578 (2)

11528 (4) 18393 (192) 17822 (276) 565 (367) 13798 (118) 11277 (6) 4038 (448) 15729 (464) 11425 (5) 19123 (21) 1304 (171) 2322 (28) 11416 (6) 11416 (6) 11443 (6) 11442 (73) 5496 (6) 11534 (6) 11582 (6) 11431 (6) 11588 (1) 19146 (6) 11542 (6) 11640 (7) 11719 (6) 11618 (9) 11355 (4) 11628 (6) 11450 (18) 6684

gp2 gp4 gp6 gp8 gp13 gp15 gp17 gp19 gp21 gp23 gp25 gp27 gp29 gp31 gp33 gp35 gp37 gp39 (33) 14286 (25) 13235 (42) 18670 17529 (215) 17529 19174 (1) 19174 (5) 19221 (6) 11167 (1) 19258 (6) 11553 (6) 11698 (6) 11505 (4) 19047 (6) 11166 (5) 11722 (4) 11447 (3) 18589 (6) 11615 (5) 11708 (6) 11723 (6) 11363 Remus_Draftgp1 gp3 gp5 gp7 gp12 gp14 gp16 gp18 gp20 gp22 gp24 gp26 gp28 gp30 gp32 gp34 gp36 gp38 (31) 19155 (16) 18805 (65) 12571 (15) 17915 (15) 18656 17374 (254) 17374 (167) 4048 (7) 10868 (6) 11720 (198) 19198 (13) 19294 (6) 17791 (7) 11401 18243 (31) 17737 (67) 2982 (414) 3257 (302) 17963 (313) 19254 (413) 18608 (314) 4899 (303) 12446 (406) 11297 (11) gp41 gp43 gp45 gp47 gp49 gp51 gp53 gp55 gp57 gp59 gp61 gp63 gp65 gp67 gp69 gp71 gp73 gp75 gp77 gp79 gp81 gp83 gp85 gp87 gp90 gp92 gp94 gp96 gp98 gp100 gp102

5079 (124) 2 (31) 4163 4 19250 (447) 6 11333 (6) 8 14746 (6) 13 17443 (281) 15 19266 (330) 17 3146 (348) 197209 (449)211827 (341) 23 2847 (449) 25 18608 (314) 27 17995 (10) 29 19296 (432) 31 (128) 18881 33 (91) 17868 35 7133 (64) 37 (6) 11303 39 gp40 gp42 gp44 gp46 gp48 gp50 gp52 gp54 gp56 gp58 gp60 gp62 gp64 gp66 gp68 gp70 gp72 gp74 gp76 gp78 gp80 gp82 gp84 gp86 gp89 gp91 gp93gp95 gp97 gp99 gp101

1 3 5 7 12 14 16 18 20 22 24 26 28 30 32 34 36 38

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

41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 81 83 85 87 90 92 94 96 98 100 102

40 42 44 46 15909 (456) 48 50 52 54 56 58 60 9297 (409) 62 64 66 68 15729 (464)70 72 74 11425 (5) 76 78 80 5238 (450) 82 19184 (182) 84 86 14542 (23) 89 91 93 95 97 2322 (28) 99 101

18837 (412) 18393 (192) 17822 (276) 565 (367) 13798 (118) 11277 (6) 4038 (448) 11548 (13) 19104 (16) 18508 (6) 5065 (450) 19123 (21) 6789 (8) 6310 (15)

HNH Tail Assembly Minor (2) 18265 (5) 19221 (6) 11167 (6) 11442 (73) 5496 (6) 11534 (6) 11582 (6) 11553 (6) 11698 (2) 18612 (6) 11588 (6) 11542 (5) 11722 (4) 11447 (3) 18589 (6) 11615 (6) 11450 (6) 11363 VIP2 Tail Holin Portal Scaffolding Major Tail Minor Tail Minor Tail ParB (33) 14286 (25) 13235 (42) 18670 (15) 17915 Endonuclease Chaperone Tail (215) 17529

Capsid Tail (3) 11533 (6) 11416 (6) 11443 (2) 18584 (6) 11431 (6) 11505 (6) 11166 (6) 11640 (7) 11719 (6) 11618 (9) 11355 (6) 11723

Minor (31) 19155 (16) 18805 (65) 12571 (15) 18656 (29) 18719 Tail Endolysin Terminase Maturation Capsid Assembly Tapemeasure Minor Tail Lsr2 ParA Rosalind Tail Protease Chaperone

17374 (254) 17374 (167) 4048 (7) 10868 (6) 11720 (198) 19198 (13) 19294 (6) 17791 (7) 11401 DNA DnaB-like 18243 (31) 17737 (67) 2982 (414) 3257 (302) 17963 (313) 19254 (413) 18608 (314) 4899 (303) 12446 (406) 11297 (11) ThyX Ribonucleotide reductase Phosphoesterase Endolysin HTH Primase helicase

4163 (31) 4163 (128) 18881 (91) 17868 (6) 11303 DNA NrdH- DNA 5079 (124) 2 4 19250 (447) 6 11333 (6) 8 14746 (6) 13 17443 (281) 15 19266 (330) 17 3146 (348) 197209 (449)211827 (341) 23 2847 (449) 25 18608 (314)26 17995 (10) 29 19296 (432) 31 33 35 7133 (64) 37 39 DNA polymerase MazG RDF EndoVII RecB Repressor binding like Primase 1 3 5 7 12 14 16 18 20 22 24 27 28 30 32 34 36 38 domain

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

41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 81 83 85 87 89 91 93 95 97 99 101

4018837 (412)42 44 11528 (4) 46 48 50 18393 (192) 52 17822 (276)54 565 (367) 56 58 9297 (409) 60 62 6419262 (107)66 11548 (13) 68 7019104 (16)7218508 (6) 74 5065 (450) 76 78 19047 (4) 80 19123 (21) 82 84 86 88 90 92 94 2322 (28) 96 6310 (15)98 100 102

15909 (456) 13798 (118) 11277 (6) 4038 (448) 15729 (464) 11425 (5) 5238 (450) 19184 (182) 6789 (8) Tail HNH Minor (6) 11167 (6) 11534 (6) 11582 (6) 11431 (6) 11588 (6) 11166 (6) 11640 (7) 11719 (6) 11618 (6) 11615 (6) 11723 (18) 6684 VIP2 Tail Holin Portal Scaffolding Major Tail Assembly Minor Tail Minor Tail ParB (31) 19155 (16) 18805 (29) 18719 Endonuclease Tail (215) 17529 Chaperone (5) 3693 Capsid (1) 12430 (6) 11416 (6) 11443 (6) 11442 (73) 5496 (6) 11553 (6) 11698 (6) 11505 (6) 11542 (1) 12429 (5) 11722 (2) 18689 (9) 11355 (4) 11628 (6) 11450 (6) 11363

Tail Assembly Minor (33) 14286 (25) 13235 (42) 18670 (65) 12571 (15) 17915 (15) 18656 Tail Endolysin Terminase Maturation Capsid Tapemeasure Minor Tail Lsr2 ParA Soups Chaperone Tail Protease

17374 (254) 17374 (167) 4048 (7) 10868 (6) 11720 (198) 19198 (13) 19294 (6) 17791 (7) 11401 DNA DnaB-like 18243 (31) 17737 (67) 2982 (414) 3257 (302) 17963 (313) 19254 (413) 18608 (314) 4899 (303) 12446 (406) 11297 (11) DNA Polymerase Ribonucleotide reductase phosphoesterase Endolysin Rec-B-like Repressor HTH Primase helicase

4163 (31) 4163 (128) 18881 (91) 17868 (6) 11303 DNA Transcription NrdH- DNA 5079 (124) 2 4 19250 (447) 6 11333 (6) 8 14746 (6) 13 17443 (281) 15 19266 (330) 17 3146 (348) 197209 (449)211827 (341) 23 2847 (449) 25 2618608 (314) 17995 (10) 29 19296 (432) 31 33 35 7133 (64) 37 39 ThyX MazG EndoVII binding regulator like Primase 1 3 5 7 12 14 16 18 20 22 24 27 28 30 32 34 36 38 domain

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

41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 81 83 85 87 89 91 93 95 97 99 101

4018837 (412)42 44 46 15909 (456) 48 50 52 54 17822 (276)56 565 (367) 58 60 9297 (409) 62 64 6619262 (107)68 11548 (13) 70 7219104 (16)74 18508 (6) 76 5065 (450) 78 80 5238 (450) 82 19184 (182) 84 86 14542 (23) 88 90 92 946789 (8) 96 98 100 102

11528 (4) 18393 (192) 13798 (118) 11277 (6) 4038 (448) 15729 (464) 11425 (5) 19123 (21) 2322 (28) 6310 (15) 11416 (6) 11416 (6) 11443 (2) 11362 (1) 11177 (6) 11534 (6) 11582 (6) 11431 (6) 11588 (6) 11542 (5) 11722 (2) 18689 (9) 11355 (4) 11628 (6) 11450 (18) 6684

gp2 gp4 gp6 gp8 gp13 gp15 gp17 gp19 gp21 gp23 gp25 gp27 gp29 gp31 gp33 gp35 gp37 gp39 (16) 18805 17529 (215) 17529 11533 (3) 11533 (5) 19221 (6) 11167 (6) 11442 (73) 5496 (6) 11553 (6) 11698 (6) 11505 (6) 11166 (6) 11640 (7) 11719 (6) 11618 (6) 11615 (6) 11723 (6) 11363 Strosahl_Draftgp1 gp3 gp5 gp7 gp12 gp14 gp16 gp18 gp20 gp22 gp24 gp26 gp28 gp30 gp32 gp34 gp36 gp38 (31) 19155 (33) 14286 (42) 18670 (65) 12571 (15) 17915 (15) 18656 (29) 18719 17374 (254) 17374 (167) 4048 (7) 10868 (6) 11720 (198) 19198 (13) 19294 (6) 17791 (7) 11401 18243 (31) 17737 (67) 2982 (414) 3257 (302) 17963 (313) 19254 (413) 18608 (314) 4899 (303) 12446 (406) 11297 (11) gp41 gp43 gp45 gp47 gp49 gp51 gp53 gp55 gp57 gp59 gp61 gp63 gp65 gp67 gp69 gp71 gp73 gp75 gp77 gp79 gp81 gp83 gp85 gp87 gp90 gp92 gp94 gp96 gp98 gp100 gp102

1 3 5 7 12 14 16 18 20 22 24 26 28 30 32 34 36 38

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

41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 81 83 85 87 90 92 94 96 98 100 102

40 42 44 46 15909 (456) 48 50 52 54 56 58 60 9297 (409) 62 64 66 68 15729 (464)70 72 74 11425 (5) 76 78 80 5238 (450) 82 19184 (182) 84 86 14542 (23) 89 91 93 95 97 2322 (28) 99 101

18837 (412) 18393 (192) 17822 (276) 565 (367) 13798 (118) 11277 (6) 4038 (448) 11548 (13) 19104 (16) 18508 (6) 5065 (450) 19123 (21) 6789 (8) 6310 (15) 18265 (2) 18265 (5) 19221 (6) 11167 (6) 11442 (73) 5496 (6) 11534 (6) 11582 (6) 11553 (6) 11698 (2) 18612 (6) 11588 (6) 11542 (5) 11722 (4) 11447 (3) 18589 (6) 11615 (6) 11450 (6) 11363 14286 (33) 14286 (25) 13235 (42) 18670 (15) 17915 17529 (215) 17529 11533 (3) 11533 (6) 11416 (6) 11443 (2) 18584 (6) 11431 (6) 11505 (6) 11166 (6) 11640 (7) 11719 (6) 11618 (9) 11355 (6) 11723 19155 (31) 19155 (16) 18805 (65) 12571 (15) 18656 (29) 18719 Fig. 5. Map of cluster A15 phages, showing extensive homology

Jswag, Remus, Strosahl summary: Summary of Results • 96% average nucleotide identity • Bantam is a singleton, with little nucleotide similarity to other phages but • 3 tRNA genes shared phams with other Gordonia phages (Fig. 2) • No orphams • Jumbo is also a singleton with similarities to Gordonia singleton GMA3 • 33/102 phams unique to A15 (isolated in Australia), Rhodococcus phage DocB7 (isolated in Texas), and Gordonia cluster DF (isolated in Texas) (Fig. 3) phages o 24/33 in all A15 phages • Jumbo and related phages may have multiple lysin and holin genes that could increase efficiency of cell lysis (Fig. 4) • 58 phams shared with other cluster • Remus, JSwag, Strosahl are cluster A15 phages, very similar to others in A phages that cluster (Fig. 5). Several other unsequenced class phages were A15 o Most are in A2 subcluster Fig. 6. Immunity regions of Gordonia cluster A15 and according to PCR results (not shown). Mycobacterium cluster A2 phages. Note the presence Bantam: Comparative Genomics • 14 phams shared with other of ParA and ParB genes present in A15 phages • JSwag and Remus are temperate, lysogens were isolated at 35o C (Fig. 7A) Gordonia clusters instead of integrase, suggesting a partitioning method o Partitioning system, similar to some A2 phages (Fig. 6) A • 21 phams shared with other hosts: of prophage maintenance (Stella et al 2013) o Immunity to other A15 phages (Fig. 7B) Rhodococcus (15), Streptomyces (4), Corynebacterium (2) Conclusions

B • 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 Exonuclease VII RecT ssDNA binding Nucleotidase large subunit protein Pham 11635 (6) different, based on GC content and shared phams Pham 18923 (7) Pham 13007 (7)

drpA- specificity like HNH Major tail tail assembly Peptidase Exonuclease VII large HNH NrdH- HNH Endonuclease Glycosyltransferase Portal protein Major capsid protein Tape measure protein Minor tail protein Lysin A Metallophosphatase Integrase Excise protein- ssDNA DNA helicase Single- Nucleotidyltransferase Lysin B endonuclease protein chaperone S1 subunit Exonuclease endonuclease Glutaredoxinendonuclease VII Capsid Head tail RecT ssDNA ThyX tyrosine binding stranded Endonuclease HNH VII HNH HNH endonuclease Glycosyltransferase Terminase large subunit morphogenesis F- decoration assembly Minor tail protein Lysin A Minor tail protein Virion protein RNA-splicing ligase RtcB RecE Exonuclease binding Thymidylate phosphatase protein DNA Yvonnetastic VII Endonuclease small endonuclease endonuclease VII Mu like protein protein chaperone protein synthase binding subunit protein 11075 (6) 11075 11530 (6) 11367 (10) 607 (2) 11683 (7) 6118 (84) 11463 (7) 11718 (7) (6) 11661 11274 (7) 17299 (43) 19582 (2) 11501 (7) 17144 (81) 19611 (24) 2740 (2) (1) 19738 19064 (4) (1) 19736 19889 (1) 19743 (1) 18923 (7) (1) 19748 (1) 19731 (1) 19893 8809 (20) 20513 (3) 860 (14) 10395 (20) 19787 (1) (1) 19627 (1) 19747 (31) 5284 (1) 19656 (1) 19853 (1) 19880 19766 (1) (2) 17161 16684 (107) (1) 19865 19804 (1) (1) 19592 (1) 19713 (1) 19790 (1) 19849 (1) 19868 (1) 19761 19789 (1) 20331 (3) 19555 (1) 19881 (1) 8133 (11) (3) 6916 19303 (18) 17427 (7) (19) 9885 16355 (21) (1) 19852 (6) 11381 (1) 19838 (1) 19724 15729 (490) (1) 19668 11524 (7) 11336 (7) (1) 19687 (2) 12789 (1) 19755 (1) 19670 (1) 19575 (1) 19561 11548 (15) (1) 19596 (1) 19912 • One or more of these phages may have significant environmental

11305 (6) 11305 14 (1) 19544 16 19552 (1) 18 8127 (81) 25 (6) 11512 27 17320 (7) 29 11376 (7) 31 7889 (7) 33 11459 (6) 35 11685 (7) 37 19268 (9) 39 (1) 19722 19582 (2)42 43 17168 (16) 45 8497 (2) 47 9537 (4) 49 19542 (1) 51 20503 (16) 53 (1) 19737 55 9475 (4) 57 11611 (4) 59 19649 (1) 61 (1) 19905 63 (1) 19832 65 (6) 11603 67 (1) 19927 69 5467 (70) 71 11445 (5) 73 19547 (1) 75 (1) 19910 77 (1) 19920 79 (1) 19604 81 (1) 19757 83 (5) 11169 85 19918 (1) 87 (1) 19839 89 (24) 4379 91 19541 (1) 93 (1) 19767 95 (1) 19857 97 11481 (7) 99 19625 (1) 101 (1) 19629 103 (1) 19935 105 13007 (7) 107 (1) 19543 109 18393 (209) 111 (7) 11220 113 16203 (3) 115 19921 (1) 117 (1) 19856 119 19778 (1) 121 (52) 6890 123 19583 (1) 125 2146 (2) 127 (1) 19888 129 (5) 19814 131 18544 (127) 133 (6) 11507 13515576 (17)137 7132 (10) 139 (1) 19774 141 (1) 19562 143 19776 (1) 145 19618 (7) 147 (1) 19576 149 702 (10) 151 (1) 19586 153 (1) 19891 155 (7) 11456 157 (1) 19703 159 (1) 19702 161 19623 (1) 163 (1) 19658 165 (3) 8516

13 15 17 24 26 28 30 32 34 36 38 40 41 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80 82 84 86 88 90 92 94 96 98 100 102 104 106 108 110 112 114 116 118 120 122 124 126 128 130 132 134 136 138 140 142 144 146 148 150 152 154 156 158 160 162 164 166

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

2 4 6 8 10 12 167 169 171 173 175 177 179 181 183 185 187 189 191 193 195 197 199 201 203

1 19540 (1) 3 5 19834 (1) 7 9 11 168 170 172 174 176 178 19694 (1) 180 182 184 186 188 190 192 19419556 (1)196 198 200 202 19180 (28)

11214 (6) 11197 (5) 19919 (1) 19798 (1) 19648 (1) 19648 (16) 1714 (1) 19536 (1) 19813 (1) 19734 (1) 19816 (1) 19594 (1) 19741 (1) 19712 (1) 19861 (6) 11717 (1) 19940 (1) 19784 (1) 19844 (7) 18816 (5) 11243 (1) 19571 (1) 19855 (1) 19842 (1) 19568 5281 (3) 5281 (8) 3726 19673 (1) 19673 (1) 19676 (1) 19732 (5) 11606 (14) 4542 (1) 19871 (1) 19560 (1) 19605 (1) 19929 (6) 11477 (1) 19796 (1) 19607 (1) 19783 (2) 17425 (1) 19890 (1) 19574 (1) 19545 18117 (76) 18117 Bantam_Draft applications, including treatment of wastewater sludge

6676 (55) 6676 4742 (35) (1) 19006 (1) 18809 (1) 19019 17320 (7) 11376 (7) 7889 (7) 17286 (5) (1) 19010 11274 (7) 1720 (6) 19840 (1) 11501 (7) 16400 (130) 19611 (24) 18867 (1) 20503 (16) (1) 18789 18895 (1) (1) 19001 (9) 13003 (1) 18843 (1) 18863 (1) 18962 18932 (1) (1) 18818 (1) 18808 18963 (1) (1) 18877 16684 (107) (1) 18734 (1) 18871 (1) 18998 20592 (52) (1) 18934 (1) 18937 2270 (13) (7) 11456 (1) 18862 (1) 18940 (1) 18979 19293 (15) (1) 18880 (1) 18984 (1) 18836 (1) 18889 7101 (5) 18853 (1) (2) 18753 (1) 18817

2 5941 (9) 4 7038 (35) 6 (1) 18830 8 (51) 206 11 11683 (7) 13 6118 (84) 15 11463 (7) 17 11718 (7) 19 18778 (1) 21 11685 (7) 23 19270 (11) 25 (1) 18961 27 (1) 17383 29 17168 (16) 31 10054 (8) 33 19276 (77) 35 18970 (1) 37 11263 (7) 39 11701 (6) 41 12598 (3) (1) 19013 45 (1) 18754 47 18923 (7) 49 (1) 18827 51 18995 (1) 53 (19) 19909 55 19128 (292)57 59 (1) 18752 61 (1) 18802 63 (1) 18885 66 (1) 18886 72 18972 (1) 11360 (6) 102 11336 (7) 104 481 (124) 106 18854 (1) 108 (2) 13458 110 (1) 18991 112 (1) 18770 114 10350 (2) 116 13620 (126) 118 (1) 18968 120 11635 (6) 122 (1) 18869 124 (10) 473 126 (1) 18829 128 (1) 18993 1301033 (5) 132 18917 (1) 134 19133 (10) 136 166

3 5 7 9 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 60 62 65 69 75 101 103 105 107 109 111 113 115 117 119 121 123 125 127 129 131 133 135

43 68 70 74 76 78 80 82 84 86 88 90 92 94 96 98 100 138 140 142 144 146 148 150 152 154 156 158 160 162 164 168 170 172 174

1 8416 (5) 58 67 18933 (1) 11524 (7) 71 73 18935 (1) 9393 (5) 77 18744 (1) 79 18851 (1) 81 18980 (1) 83 85 87 18835 (1) 89 17427 (7) 91 19388 (35) 93 95 97 13007 (7) 99 13718748 (1) 139 141 143 145 147 18771 (1) 149 151 18948 (1) 15318931 (1)155 157 159 161 163 165 167 169 6387 (68) 171 18953 (60) 173 175

18807 (1) 18800 (1) 18776 (1) 18866 (1) 18763 (1) 11481 (7) single 18788 (1) 19020 (1) 18943 (1) 19008 (1) FAD- head tail stranded capsid maturation exodeoxyribonuclease homing dependent (2) 18026 (1) 18840 (7) 11220 (1) 18992 nicotinate phosphoribosyl(1) 18736 HNH ribonucleotide (1) 18926 (1) 18883 (1) 18950 (1) 18997 (1) 18799 (1) 18884 (1) 18814 (1) 18939 (1) 18833 (1) 18740 (1) 18954 terminase decoration major tail assembly tape measure minor tail lysin A minor tail metallophosphatase RecT HTH DNA binding MazG DNA nucleotidyltransferase nucleotidase lysin B (12) 19035 protease VII, large subunit endonuclease thymidylate transferase endonuclease reductase protein chaperone exodeoxyribonuclease binding (3) 3014 18791 (1) 18791 (1) 18832 (1) 18813 (1) 19002 synthase (1) 18810 (1) 18743 HTH (1) 18812 (3) 20195 (1) 18780 (1) 18888 phosphoribosyl- (1) 18850 (1) 18792 (1) 19012 (1) 18964 (1) 18793 (7) 11401 (1) 18875 (1) 19007 (2) 17425 (1) 18909 (1) 18873 (1) 18904 (1) 18826 (1) 18765 (1) 18946 HNH Endo tail assembly VII, (25) 13235 NrdH Endonuclease portal scaffolding capsid minor tail endolysin holin minor tail RtcB-like integrase exonuclease DNA pyrophosphate DnaB helicase protein Endo VII OneUp endonuclease VII chaperone small redoxin VII binding synthase subunit 11305 (6) 11305 (6) 11512 (1) 20085 (6) 11661 (7) 20230 (6) 11603 (5) 11404 (6) 4091 (1) 20205 (1) 20069 (5) 11238 (1) 20271 (1) 20115 (5) 11682 (1) 20227 (45) 18670 (1) 20077 (5) 11641 (1) 20063 (1) 20232 (6) 11507 (5) 11272 (1) 20107 (3) 15271 (2) 18753 11530 (6) 11367 (10) 17320 (7) 11376 (7) 7889 (7) 11274 (7) 17299 (43) 11334 (10) 11501 (7) 17144 (81) 16093 (4) 2047 (13) 11297 (12) 20276 (1) Lsr2 18923 (7) 11702 (5) 6211 (6) 20407 (1) 13007 (7) 18393 (209) 11338 (5) 11385 (5) 20291 (19) 7038 (35) 19303 (18) 20080 (1) 18544 (127) 7132 (10) 11170 (5) 11336 (7) 11635 (6) 20146 (1) 1170 (10) 11548 (15)

8 (6) 11075 10 20309 (1) 12 (2) 13081 22 11683 (7) 24 6118 (84) 26 11463 (7) 28 11718 (7) 30 11459 (6) 32 11685 (7) 34 (9) 19268 36 (5) 11161 3811334 (10) 40 17168 (16) 42 11221 (5) 44 16984 (10) 46 (5) 11561 48 20503 (16) 50 11701 (6) 52 20443 (1) 56 (5) 11345 58 (5) 11663 60 (5) 11509 62 5467 (70) 64 (1) 20097 66 (1) 20379 68 (1) 20087 70 (5) 11403 72 16684 (107) 75 immunity 77 11481 (7) 79 11449 (5) 81 (2) 20337 83 (1) 20109 85 (1) 20251 87 (7) 11220 89 11249 (5) 91 (5) 11388 93 (5) 11518 95 20464 (1) 97 20167 (1) 99 4742 (35) 101 12974 (3) 103 (2) 13458 105 (91) 8838 107 17427 (7) 109 (5) 20449 111 (5) 11432 113 (6) 11381 115 (5) 11513 117 15729 (490) 119 11524 (7) 121 (1) 20148 123 (1) 20116 125 (1) 20112 127 2143 (4) 129 19223 (8) 131 (7) 11456 133 (6) 20355 135 repressor 9 11 19 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 57 59 61 63 65 67 69 71 74 78 80 82 84 86 88 90 92 94 96 98 100 102 104 106 108 110 112 114 116 118 120 122 124 126 128 130 132 134

2 4 6 54 137 139 141 143 145 147 149 151 153 155 157 159 161 163 165 167 169 171

1 3 5 7 55 76 136 11214 (6) 138 140 142 144 146 148 150 152 154 156 158 160 17594 (8) 162 164 20074 (2) 166 168 11347 (5) 170 172

20223 (1) 5179 (7) 19180 (28) 20372 (1) 3726 (8) 3726 20208 (1) 20208 (1) 20136 (4) 20224 (1) 20236 (5) 11633 (5) 11714 (1) 20413 (1) 20196 (1) 20138 (1) 20117 (7) 18816 (1) 20445 (5) 11236 (1) 20079 (1) 20184 (1) 20390 14993 (18) 14993 20151 (1) 20151 (5) 11606 (1) 20459 (6) 11360 (5) 11261 (1) 20137 (1) 20065 (6) 11717 (1) 20280 (6) 11477 (5) 11374 (5) 11420 (5) 11689 (1) 20244 (6) 11708 (2) 20312 (7) 11719 (1) 20506 (1) 20118 (1) 20444 Fig. 2. Synteny and homology between Bantam and other Gordonia(13) 18757 phages, Yvonnetastic (Singleton) References and OneUp (cluster CQ). A. Shared phams encoding structural genes. B. Shared genes involved in replication and regulation illustrate mosaic nature of these genomes. 1. Actinobacteriophage Database (2015), http://phagesdb.org 2. Dyson, ZA et al. (2015) PLoS One 10(8): e0134512. doi:10.1371/journal.pone.0134512 Bantam summary: 3. Liu, M. et al. (2015) Nature Scientific Reports 5:13754 | DOI: 10.1038/srep13754 • 108 / 172 orfs are orphams 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 • No tRNAs detected • Shared phams: structural genes, integrase, lysinA, exonucleases • 27 phams shared with Gordonia cluster CQ, singleton Yvonnetastic Acknowledgements RecE Exonuclease • 32 phams shared with phages in other hosts: Mycobacterium (23), Rhodococcus (10), Integrase Pham 11481 (7) We thank Rick Ellingworth and the Biology Department student lab assistants for preparing materials, Arthrobacter (3), Corynebacterium (2), Streptomyces (2) Pham 16684 (107) 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.