Genomic and phenotypic characterization of novel bacteriophages infecting rhizobia Anupama Halmillawewa1 , Marcela Restrepo1 , Suriakarthiga Ganesan1, Rémy Gavard1, Christopher K. Yost2, Michael F. Hynes1 of Biological Sciences, University of Calgary, Calgary , AB, Canada T2N 1N4 2 Department Characteriza)on of Genomes Introduc)on Rhizobiophages are phages that infect members of the genera Rhizobium, Bradyrhizobium, Sinorhizobium, Azorhizobium Allorhizobium and Mesorhizobium, which are all capable of nodulating and fixing nitrogen inside the nodules of legume plants. . • PPF1 Genome sizes were estimated using pulsed field gel electrophoresis. Sanger sequencing and Ion-Torrent sequencing techniques were used to sequence the complete genomes of L338C and P106B, while PPF1 phage genome was sequenced using 454 pyrosequencing. P106B • of Biology, University of Regina, Regina, SK, Canada S4S 0A2 L338C 1 Department 109557 56024 54506 Average G+C content 59% 47.9% 61.9% No: of predicted ORFs 185 95 94 DNA packaging 2 1 2 Phage morphogenesis 11 13 10 DNA replication, transcription & repair 17 6 5 DNA methylation 1 0 2 Host cell lysis Lysogeny related 0 0 2 0 4 1 Hypothetical proteins 26 25 40 Other 11 1 7 No significant similarity 117 47 23 91.9 91.7 96.3 tRNAPro tRNALeu None We have isolated a number of rhizobiophages that infect different rhizobial hosts such as R. leguminosarum, R. gallicum and M. loti. Genome size (bp) • This study aims to expand our knowledge on rhizobiophage diversity by characterizing those phage isolates, including the whole genome sequencing of selected phages. Morphology A. C. B. Genome arrangement of vB_RleS-L338C . D. Genome arrangement of vB_RglS-P106B E. F. . G. % of genome coding for proteins Genome arrangement of vB_RleM-PPF1 Host Range Analysis A. vB_RleM-P10VF R.leguminosarum VF39 Family Myoviridae 85±6 122±7 Tail width (nm) Trapping Host Tail length (nm) Phage Name Head diameter (nm) 22±7 B. vB_RleM-P9VFCI R.leguminosarum VF39 Myoviridae 95±5 125±7 24±4 C. vB_RleM-AF3 R.leguminosarum F3 Myoviridae 114±3 126±6 18±5 D. vB_RglS-P106B R.gallicum S014B-4(6) Siphoviridae 61±6 109±8 12±3 E. vB_RleS-L338C R.leguminosarum 3841 Siphoviridae 76±11 298 8 F. vB_RleS-P11VFA R.leguminosarumVF39 Siphoviridae 81±6 217±15 12±2 G. vB_RleS-L338H R.leguminosarum 3841 Siphoviridae 83±6 ND 12±2 H. vB_RleM-PPF1 R.leguminosarum F1 Myoviridae 83±5 130±5 ND I. vB_RleS-P11VFC R.leguminosarum VF39 Siphoviridae 88±6 ND 12±2 J. vB_MloP-Lo5R7ANS M.loti R7ANS Podoviridae 60±2 ND 13 K. vB_MloP-Cp1R7ANSC2 M.loti R7ANS Podoviridae 60±4 ND ND ND- Not determined R. leguminosarum 3841 VF39 248 336 306 3855 F3 R. gallicum S014B-4(6) S019B-5(27) R602 M.loti R7A R7ANS R. tropicii CIAT899 CIAT299 R. etli CFN42 979 979 979 Cp1R7ANSC2 Lo5R7ANS L338H P11VFA L338C 228 228 486 486 228 486 439 439 + - + + + + - + + + + - + - + + + + - + + + + + + + - + - - + + + + + - - - 461 461 + - - - - - - - - - + + + + + + + + + + - + + - + - + + + + - - 190 503 503 270 270 213 173 503 213 270 88 213 88 1000 1000 461 984 984 1000 1000 1000 1000 992 1000 984 1000 1000 1000 1000 359 563 1000 1000 1000 1000 1000 1000 1000 1000 979 1000 190 190 842 1000 1000 1000 1000 173 439 + + + + 563 563 1000 1000563 1000 1000 173 173 P11VFC * Bars in A, B, C, D, F, G, H, I, J and K represent 100 nm, whereas bar in E represents 20 nm. P9VFCI Transmission electron micrographs of rhizobiophages* 1000 1000 1000 842 Phage Figure (B) 992 359 842 842 Fels-1 Fels-1 Gifsy-2 Gifsy-2 Fels-1 CP-933K CP-933K Gifsy-2 Agrobacterium Agrobacterium CP-933K 1000 vB Agrobacterium vitis S4 vB RleM-PPF1 RleM-PPF1 Agrobacterium RR1-A RR1-A vB RleM-PPF1 Pseudovibrio Pseudovibrio RR1-A N15 863 Pseudovibrio FO-BEG1 N15 Pseudovibrio lambda lambda N15 253 Gifsy-1 Gifsy-1 530 lambda VHML VHML Gifsy-1 VP16T VP16T VHML ES18 ES18 VP16T Fels-1 220 phiHSIC phiHSIC ES18 Gifsy-2 RHEph06 RHEph06 phiHSIC CP-933K 992 992 359 359 - No Lysis P10VF . Rhizobial strain + Lysis 1000 1000 Figure (A) All the phages were tested against an array of different rhizobial strains using plaque assays. K. J. P106B I. AF3 H. Presence of tRNA 991 9911000 461 1000 1000 1000 1000 1000 992 1000 991 1000 1000 359 563 1000 984 1000 1000 88 817 817 979 1000 228 486817 888 888 842 1000 461 888 439 673 673 820 820 673 190173820 833 833 998 998 833 356 1000 356 998 228 503 743 486 743 356 270 213 743745 889 889 745 88 1000 889 745 439 817 1000 1000 1000 1000 1000 1000 1000 984 1000 1000 1000 991 1000 1000 1000 1000 1000 1000 1000 1000 991 0.1 0.1 1000 1000 1000 1000 190 888 1000 1000 1000 1000 1000 RHEph06 Agrobacterium vB RlgS-P106B vB RlgS-P106B RleM-PPF1 RR1-A vB RlgS-P106B Pseudovibrio RB49 N15 RB49 T4 lambda Fels-1 T4 RB49 Aeh1 Gifsy-1 Gifsy-2 Aeh1 T4 1000 VHML CP-933K KVP40 1000 KVP40 Aeh1 VP16T Agrobacterium phi-pp2 KVP40 1000 phi-pp2 vB ES18 RleM-PPF1 vB RleS-L338C RleS-L338C phi-pp2 RHEph10 phiHSIC RR1-A RHEph10 vB RleS-L338C RHEph06 Pseudovibrio RHEph10 PBC5 N15 PBC5 vB RlgS-P106B lambda PBC5 Gifsy-1 VHML HK97 RB49 HK97VP16T ES18 HK022 T4 HK022 HK97 16-3 Aeh1 16-3phiHSIC HK022 RHEph06 phi-BT1 KVP40 1000 phi-BT1 16-3 phiC31 phi-pp2 phiC31 phi-BT1 vB RleS-L338C RlgS-P106B phiC31 vB RHEph10 bIL285 bIL285 bIL285 PBC5 RB49 T4 Aeh1 1000 1000 1000 782 426 1000 1000 279 Fels-1 Gifsy-2 phi 16-3 vB RleM-PPF1 D3 P22 Pseudovibrio Pseudovibrio FO-BEG1 1000 lambda 1000 HK97 CP-933K HK022 Agrobacterium vitis S4 Agrobacterium D29 PBC5 Sf6 Gifsy-1 bIL285 0.1 Neighbor-joining trees showing the phylogenetic relationship of 87 different amino acid sequences of large terminase subunit (Figure A) and 18 phage integrase (Figure B) sequences. The bootstrap values are indicated for 1000 trials. Rhizobiophages are indicated with a blue diamond, while phages used in this study are highlighted with a red box Acknowledgement: Ministry of Agriculture-­‐ Agriculture Development Fund