Introduction
Materials and methods
Brucellaphages and bacterial strains
Phage specificity and purification
Phages
|
Isolation source
|
Time of adsorption (min)
|
% adsorption
|
Latent period (min)
|
Lysis time (min)
|
Average burst size (PFU/ml)
|
---|---|---|---|---|---|---|
Tb_141 | liquid manure | 190 | 90 | 200 | 460 | 40-46 |
Tb_19 | liquid manure | 180 | 81 | 210 | 460 | 50-53 |
110 |
B. melitensis
| 120 | 70 | 180-220 | 440 | 18-20 |
141 |
B. abortus
| 120 | 76 | 240 | 450 | 27-30 |
11sa |
B. suis
| 120 | 53 | 210-240 | 410 | 28-30 |
1066 |
B. canis
| 150 | 98 | 180 | 425 | 110-120 |
/02 |
B. ovis
| 120 | 82 | 240 | 400 | 36-40 |
544 |
B. abortus
| 90-100 | 82 | 270-300 | 480-490 | 30-35 |
281 |
B. ovis
| 90-100 | 70 | 250-280 | 450-460 | 60-65 |
177 |
B. melitensis
| 180 | 78 | 240 | 460 | 40-45 |
V |
B. abortus
| 180 | 37 | 240 | 480 | 25-30 |
DNA isolation, genome sequencing and assembly
DNA isolation and sequencing
Nucleotide sequences
Comparative genomics, phylogenetic structure, and selection
Results and discussion
Phage lytic activity and host specificity
Genomic comparison and phylogenetic structure
a
ORF
|
7
|
8
|
12
|
12
|
13
|
17
|
20
|
21
|
21
|
21
|
21
|
21
|
21
|
21
|
21
|
23
|
23
|
23
|
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Locus | 2868 | 3357 | 7313 | 7799 | 8715 | 11046 | 13192 | 15576 | 15578 | 15774 | 15775 | 15996 | 16003 | 16012 | 16064 | 16700 | 16952 | 16984 |
Coding Δ | C/T(D) | G(R) T(L) | T(I) G(S) | A(K) C(T) | A/G(Q) | C(L) A(I) | C(T) A(N) | T(L) G(R) | C(D) A(E) | C(P) T(S) | C(S) T(L) A(H) | G(A) A(T) C(P) | T(V) GG) | A(E) C(A) | A(K) T(N) | C/T (N) | A/G (S) | C(S) A(Y) |
Codon p. | 3rd | 2nd | 2nd | 2nd | 1st | 1st | 2nd | 1st | 3rd | 1st | 2nd | 1st | 2nd | 2nd | 3rd | 2nd | 2nd | 1st |
TbM | C | G | G | A | A | C | C | G | C | C | C | G | G | C | T | C | G | C |
Pr | C | G | G | A | A | C | C | G | C | C | C | G | G | C | T | C | G | C |
TbW | C | G | G | A | A | C | C | G | C | C | C | G | G | C | T | C | A | C |
TbE _141 | T | T | G | A | A[G] | C | C | G[T] | C[A] | T | C | A | T | A | A | C | A | C |
TbE _19 | T | T | G | A | A[G] | C | C | G | C | T | C | A | T | A | A | C | A | C |
02_141 | T | T | G[T] | A | A | A | A | G | C | T | C | A | T | A | A | T | A | A |
02_19 | T | T | G[T] | A | A | A | A | G | C | T | C | A | T | A | A | T | A | A |
11sa_141 | T | G | G | A | A | C | C | G | C | C | T | G | T | A | A | C | A | C |
11sa_19 | T | T | T | A | A | A | A | G | C | T | C | A | T | A | A | T | A | A |
141_141 | T | G[T] | G | C | A | C | C | G | C | C | C | G | T | A | A | C | A | C |
141_19 | T | G[T] | G | C | A | C | C | G | C | C | C | G | T | A | A | C | A | C |
177_141 | T | T | G[T] | A | A[G] | C[A] | C[A] | G | C | T | C | A | T | A | A | C | A | C |
177_19 | T | T | G[T] | A | A | C[A] | C[A] | G | C | T | C | A | T | A | A | C | A | C |
281_141 | T | T | G[T] | A | A[G] | C[A] | C[A] | G | C | T | C | A | T | A | A | C | A | C |
281_19 | T | G | G | C | A | C | C | G | C | C | C[A] | C | T | A | A | C | A | C |
544_141 | T | G[T] | G | A | A[G] | C | C | G | C | C[T] | C | G[A] | T | A | A | C | A | C |
544_19 | T | G[T] | G | A | A[G] | C | C | G | C | C[T] | C | G[A] | T | A | A | C | A | C |
1066_141 | T | T | G | C | A | C | C | G | C | C | C[A] | G | T | A | A | C | A | C |
1066_19 | T | G[T] | G[T] | A[C] | A | C[A] | C[A] | G | C | C[T] | C[T] | G[A] | T | A | A | C[T] | A | C[A] |
V_141 | T | G | G | A | A | C | C | G | C | T | T | A | T | A | A | C | A | C |
V_19 | T | T | G[T] | A | A | A | A | G | C | T | C | A | T | A | A | T | A | A |
110_141 | T | T | G[T] | A | A | A[C] | A | G | C | T | C | A | T | A | A | T | A | A |
110_19 | T | T | G[T] | A | A[G] | A[C] | C[A] | G | C | T | C | T | T | A | A | C | A | C |
ORF | 25 | 26 | 27 | 27 | 27 | 27 | 27 | 27 | 27 | 27 | 27 | 39 | 42 | 43 | 58 | NC | NC | |
Locus | 19780 | 21544 | 21769 | 21815 | 22178 | 22364 | 22409 | 22466 | 22469 | 22470 | 22472 | 28322 | 29512 | 29915 | 39320 | 40644 | 41105 | |
Coding Δ | C(P) A(H) | G (C ) T(S) | C(A) A(E) | G(D) T(Y) | G(D) T(Y) | G(D) T(Y) | G(V) T(F) | A(S) C(A) | A(K) G(A) | A(K) C(A) | A(N) T(Y) | G(L) T(M) | T(N) C(D) | C(L) A(Y) | C(I) A(Y) | NA | NA | |
Codon p. | 3rd | 1st | 3rd | 1st | 1st | 1st | 1st | 1st | 1st | 2nd | 1st | 3rd | 3rd | 3rd | 3rd | NA | NA | |
TbM | C | G | C | G | G | G | G | C | A | A | A | G | T | C | A | A | C | |
Pr | C | G | C | G | G | T | G | A | A | G | A | G | T | A | C | A | T | |
TbW | C | G | C | G | G | T | G | A | A | C | A | G | T | C | C | T | T | |
TbE _141 | C | G | C | G[T] | T | T | T | A | G | C | T | G | C | C | C | T | T | |
TbE _19 | C[A] | G | C | G | T[G] | T | T | A | G | C | T | G | C | C | C | T | T | |
02_141 | C | G | C | G | G | T | G | A | G | C | T | G[T] | C | C | C | T | T | |
02_19 | C | G | C | G | G | T | G | A | G | C | T | G[T] | C | C | C | T | T | |
11sa_141 | C | G | C | G | G | G[T] | G | A | A | A | T | G | T | C | C | T | T | |
11sa_19 | C | G | C | G | G | T | G | A | G | C | T | G[T] | C | C | C | T | T | |
141_141 | C | G | C[A] | G | G | T | G | A | A | A | T | G | C | C | C | T | T | |
141_19 | C | G | C[A] | G | G | T | G | A | A | A | T | G | C | C | C | T | T | |
177_141 | C[A] | G | C | G | T | T | T | A | G | C | T | G | C | C | C | T | T | |
177_19 | C[A] | G | C | G | T | T | T | A | G | C | T | G | C | C | C | T | T | |
281_141 | C[A] | G | C | G | T | T | T | A | G | C | T | G | C | C | C | T | T | |
281_19 | C | T | C[A] | G | G | G[T] | G | A | A | A | T | G | C | C | C | T | T | |
544_141 | C | G | C | G | G | T | T | A | A[G] | A[C] | T | G | T[C] | C | C | T | T | |
544_19 | C | G | C | G | G | T | T | A | A[G] | A[C] | T | G | T[C] | C | C | T | T | |
1066_141 | C | G | C[A] | G | G | G[T] | G | A | A | A | T | G | C | C | C | T | T | |
1066_19 | C[A] | G | C[A] | G | G[T] | G[T] | G[T] | A | A[G] | A[C] | T | G[T] | T[C] | C | C | T | T | |
V_141 | C | G | C | G | G | G[T] | T | A | A | A | T | G | T | C | C | T | T | |
V_19 | C | G | C | G | T | T | G | A | G | C | T | G[T] | C | C | C | T | T | |
110_141 | C | G | C | G | G | T | G | A | G | C | T | T | C | C | C | T | T | |
110_19 | A | G | C | G | T | T | T | A | G | C | T | G | C | C | C | T | T |
Candidate loci for host specificity
Annotation
|
ORF#
|
Start
|
End
|
Length
|
Direction
|
Coding Δ
|
# Loci
b
|
Passage
c
|
---|---|---|---|---|---|---|---|---|
Putative HNH endonuclease | 8 | 3,170 | 3,856 | 687 | forward | S | 1 | N |
Putative portal protein | 12 | 6,370 | 8,694 | 2,325 | forward | N | 2 | Y |
Hypothetical protein | 13 | 8,694 | 8,912 | 219 | forward | S | 1 | Y |
Structural protein | 16 | 9,822 | 11,003 | 1,182 | forward | N | 1 | N |
Major head protein | 17 | 11,013 | 12,023 | 1,011 | forward | N | 1 | Y |
Structural protein | 20 | 13,164 | 13,823 | 660 | forward | N | 1 | Y |
Structural protein (neck motif) | 21 | 13,827 | 16,211 | 2,385 | forward | N | 5 | Y/N |
Structural protein (peptidase_S74)a
| 23 | 16,660 | 17,646 | 987 | forward | N | 2 | Y/N |
Putative peptidoglycan hydrolase | 25 | 18,062 | 20,251 | 2189 | forward | N | 1 | Y |
Structural protein | 26 | 20,251 | 21,753 | 1,503 | forward | N | 1 | Y |
Putative tail collar protein | 27 | 21,755 | 22,612 | 858 | forward | S-N | 7 | Y/N |
Hypothetical protein | 39 | 28,262 | 28,382 | 121 | reverse | N | 1 | Y |
Hypothetical protein | 42 | 28,727 | 29,683 | 957 | reverse | S-N | 2 | Y |
Restriction sites
| ||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
HaeII
|
HhaI
|
Cfol
|
AluI
|
HinfI
|
MseI
|
HaeIII
|
TaqI
|
SalI
|
HincII
|
AccI
|
MspI
|
HpaII
|
MboI
|
Sau3AI
|
NdeI
| |
3357 | G | G | G | - | - | - | - | - | - | - | - | - | - | - | - | - |
11046 | - | - | - | a | A | - | - | - | - | - | - | - | - | - | - | |
13192 | - | - | - | - | - | A | - | - | - | - | - | - | - | - | - | - |
15576-8 | - | - | - | - | - | - | - | t | t | t | t | - | - | - | - | - |
15774 | - | - | - | - | - | - | C | - | - | - | - | - | - | - | - | - |
15775 | - | - | - | - | - | T | - | - | - | - | - | - | - | - | - | - |
15996 | - | G | G | - | - | - | - | - | - | - | - | C | C | - | - | - |
19780 | - | - | - | - | - | - | a | - | - | - | - | - | - | - | - | - |
22178 | - | - | - | - | - | - | - | - | - | - | - | - | - | t | t | - |
22470 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | a |
28322 | - | - | - | - | - | - | t | - | - | - | - | - | - | - | - | - |