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