Directed in Vitro Evolution of Therapeutic Bacteriophages: The Appelmans Protocol
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial and Bacteriophage Strains
2.2. Analysis of Host Range
2.3. DNA Sequencing and Annotation
2.4. Mitomycin C Induction and Southern Blot Analysis of Temperate Phages
3. Results
3.1. Appelmans ProtocolUusing a Phage Cocktail Expands Bacteriophage Host Ranges
3.2. Appelmans Protocol Using Single Phages Generates Little Host-Range Expansion
3.3. Phi176 is a Recombinant Derivative of Phages Pa2 and RWG
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Appendix A
References
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Bacterial/Phage Strain | Source | Reference | Notes |
---|---|---|---|
PAO1 | Dr A. N. Hamood (TTUHSC) | [1] | Host strains used for the Appelmans protocol |
PAK | [2] | ||
PA14 | Dr K. Rumbaugh (TTUHSC) | [3] | |
WCC176 | Dr R. D. Wolcott MD Southwest Regional Wound Care Center. (Lubbock, TX) | This study | |
WCC199 | |||
WCC201 | |||
WCC205 | |||
WCC222 | |||
WCC229 | |||
WCC232 | |||
Pa2 | ATCC [14203-B1] | [1] | 73,008 bp. Plaques 1–2 mm, clear |
ΦKZ | Felix d’Herelle Reference Center for Viruses | [4] | 280,334 bp. Plaques <1 mm, clear |
RWG | This laboratory | This study | 72,646 bp. Plaques 2–3 mm, clear |
Test Strain | Phage | |||||
---|---|---|---|---|---|---|
Pa2 | ΦKZ | RWG | Round 10 Cocktail | Round 20 Cocktail | Round 30 Cocktail | |
PAO1 | + | + | + | + | + | + |
PA14 | + | + | + | |||
PAK | + | + | + | + | + | |
WCC176 | + | + | ||||
WCC199 | + | + | + | |||
WCC201 | + | + | + | |||
WCC205 | + | + | + | |||
WCC222 | + | |||||
WCC229 | + | |||||
WCC232 | + |
Test Strain | Phage 1 | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
phiPAO1.1 | phiPAO1.2 | phiPAO1.3 | phiPA14.1 | phiPA14.2 | phiPAK.1 | phiPAK.2 | phiPAK.3 | phi176 | phi201 | phi229.1 | phi229.2 | |
PAO1 | + | + | + | + | + | + | + | + | + | + | + | |
PA14 | + | + | + | + | + | + | ||||||
PAK | + | + | + | + | + | + | + | + | + | + | + | |
WCC176 | + | + | + | |||||||||
WCC199 | + | + | ||||||||||
WCC201 | + | + | + | + | ||||||||
WCC205 | + | + | + | |||||||||
WCC222 | + | + | + | |||||||||
WCC229 | + | + | + | |||||||||
WCC232 | + | + | + |
Test Strain | phi176 | phi201 | phi229.1 | phi229.2 | ||||
---|---|---|---|---|---|---|---|---|
EOP | SD | EOP | SD | EOP | SD | EOP | SD | |
PAO1 | 73.6 | 5.9 | 0.7 | 0.1 | 6.7 | 2.0 | 51.2 | 11.0 |
PA14 | 9.6 × 10−5 | 5.4 × 10−4 | 1.0 × 10−9 | 2.4 × 10−8 | 8.3 × 10−5 | 4.6 × 10−5 | 1.1 × 10−3 | 4.2 × 10−4 |
PAK | 2.7 × 10−5 | 2.0 × 10−6 | 0.1 | 1.8 × 10−2 | 1.3 × 10−6 | 4.6 × 10−7 | 2.0 × 10−4 | 4.6 × 10−5 |
WCC176 | 1.0 | 0.1 | - | - | 1.5 | 0.5 | 7.9 | 2.7 |
WCC199 | 4.3 | 1.1 | - | - | 3.3 × 10−2 | 1.2 × 10−2 | - | - |
WCC201 | 1.8 × 10−6 | 1.2 × 10−6 | 1.0 | 0.2 | - | - | - | - |
WCC205 | 3.3 | 0.4 | - | - | 1.4 | 0.5 | 2.6 | 0.6 |
WCC222 | 18.2 | 1.6 | - | - | 1.1 | 0.3 | 2.6 | 0.8 |
WCC229 | 50.0 | 6.9 | - | - | 1.0 | 0.4 | 1.0 | 0.3 |
WCC232 | 39.3 | 6.7 | - | - | 0.1 | 4.7 × 10−2 | 7.9 | 1.9 |
Phi176 ORF | Start | Stop | ORF Size (bp) | Cross-Overs | Crossover Location(s) in phi176 Genome (bp) 1 | Putative Gene Function |
---|---|---|---|---|---|---|
02 | 872 | 1063 | 192 | 1 | 1020 | |
03 | 1078 | 1308 | 231 | 1 | 1210 | |
08 | 2394 | 2597 | 204 | 1 | 2581 | |
10 | 2832 | 3065 | 234 | 1 | 2884 | |
17 | 5053 | 5493 | 441 | 1 | 5352 | |
18 | 5533 | 5880 | 348 | 1 | 5840 | |
23 | 8042 | 9283 | 1242 | 1 | 8764 | RNA polymerase 2 |
23a | 9380 | 9532 | 153 | 1 | 9384 | |
35 | 16,639 | 17,976 | 1167 | 1 | 16,721 | DNA helicase |
37 | 18,503 | 21,118 | 2616 | 2 | 19,203 19,359 | DNA polymerase |
42 | 22,534 | 25,050 | 2517 | 1 | 22,811 | rIIA-like |
43 | 25,062 | 26,840 | 1779 | 5 | 25,434 26,071 26,254 26,321 26,473 | rIIB-like |
IG44-45 | N/A | N/A | N/A | 1 | 27,137 | N/A |
45 | 27,196 | 27,513 | 318 | 1 | 27,326 | |
51 | 29,943 | 29,464 | 480 | 1 | 29,758 | |
52 | 33,215 | 29,943 | 3273 | 6 (+ 1 missense) | 30,115 30,122 31,070 31,073 31,118 33,095 | Tail fiber/tailspike; lipase/esterase domain |
53 | 33,925 | 33,254 | 672 | 2 | 33,375 33,379 | |
59 | 37,720 | 39,888 | 2169 | 2 | 38,669 39,095 | DNA primase P4 type |
66 | 43,308 | 43,712 | 405 | 1 | 43,324 | |
67 | 44,118 | 44,354 | 237 | 1 | 44,210 | |
70 | 55,093 | 44,897 | 10,197 | 1 | 54,100 | Virion RNA polymerase |
71 | 56,659 | 55,094 | 1566 | 3 | 55,210 55,751 56,313 | Structural protein |
72 | 57,126 | 56,659 | 468 | 1 | 56,747 | |
73 | 59,329 | 57,107 | 2223 | 1 | 59,203 | |
75 | 61,019 | 60,354 | 666 | 1 | 60,455 | |
77 | 63,503 | 62,310 | 1194 | 3 | 62,768 63,143 63,333 | |
79 | 66,091 | 63,911 | 2181 | 1 | 64,015 | |
82 | 67,614 | 66,880 | 735 | 1 | 67,134 | |
83 | 69,263 | 67,611 | 1653 | 3 | 68,333 68,417 68,774 | |
85 | 70,055 | 70,486 | 432 | 1 | 70,398 |
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Burrowes, B.H.; Molineux, I.J.; Fralick, J.A. Directed in Vitro Evolution of Therapeutic Bacteriophages: The Appelmans Protocol. Viruses 2019, 11, 241. https://doi.org/10.3390/v11030241
Burrowes BH, Molineux IJ, Fralick JA. Directed in Vitro Evolution of Therapeutic Bacteriophages: The Appelmans Protocol. Viruses. 2019; 11(3):241. https://doi.org/10.3390/v11030241
Chicago/Turabian StyleBurrowes, Ben H., Ian J. Molineux, and Joe A. Fralick. 2019. "Directed in Vitro Evolution of Therapeutic Bacteriophages: The Appelmans Protocol" Viruses 11, no. 3: 241. https://doi.org/10.3390/v11030241
APA StyleBurrowes, B. H., Molineux, I. J., & Fralick, J. A. (2019). Directed in Vitro Evolution of Therapeutic Bacteriophages: The Appelmans Protocol. Viruses, 11(3), 241. https://doi.org/10.3390/v11030241