Table 3. Bacterial strains and plasmids.
| Plasmids | Genetic Characteristics | Properties | Source or Reference |
| Strains | |||
| E. coli | |||
| DH5α | F− endA1 hsdR17(rk −mk)thi-1λ−recA1 gyrA96relA1 rhoA supE44 deoRФ80dlacZΔM15Δ(lacZYA argF)U169 | Invitrogen | |
| RosettaGami(DE3). pLysS | Δ(ara-leu)7697 ΔlacX74 ΔphoA PvuII phoR araD139 ahpC galE galK rpsL (DE3) F'[lac+ lacIq pro] gor522::Tn10 trxB pLysSRARE (CamR, StrR, TetR) | Novagen | |
| D. nodosus | Protease produced | ||
| VCS1703A | D. nodosus serogroup G wild-type, virulent | AprV2, AprV5, BprV | J. Egerton, University of Sydney |
| CS101 | D. nodosus serogroup G wild-type, benign | AprB2, AprB5, BprB | D. Stewart, CSIRO Livestock Industries |
| JIR3740*, JIR3743* | VCS1703AaprV2Ωtet(M) | AprV5, BprV | Double crossover from pJIR2097 |
| JIR3756*, JIR3757* | VCS1703AaprV5Ωbla | AprV2, BprV | Double crossover from pJIR2309 |
| JIR3766*, JIR3767* | JIR3740bprVΩerm(B) | AprV5 | Double crossover from pJIR2275 |
| JIR3768*, JIR3769* | JIR3757aprV2Ωbla | BprV | Double crossover from pJIR2097 |
| JIR3883 | JIR3756rrnAΩaprV5+ | AprV2, AprV5, BprV | Double crossover from pJIR2545 |
| JIR3900 | JIR3743Δerm(B)ΩaprV2+tet(M) | AprV2, AprV5, BprV | Double crossover from pJIR3044 |
| JIR3903*, JIR3905* | JIR3743ΔaprV5bprVΩerm(B) | None | Double crossover from pJIR3060 |
| JIR3907*, JIR3908* | VCS1703AΔaprV5bprVΩerm(B) | AprV2 | Double crossover from pJIR3060 |
| JIR3923 | JIR3743Δerm(B)ΩaprB2+tet(M) | AprB2, AprV5, BprV | Double crossover from pJIR3268 |
| JIR3928 | VCS1703AbprVΩerm(B) | AprV2, AprV5 | Double crossover from pJIR3159 |
| JIR3930 | JIR3928Δerm(B)ΩbprV+KanR | AprV2, AprV5, BprV | Double crossover from pJIR3148 |
| Plasmids | Properties | ||
| pUC18 | Apr lacZ cloning vector | [54] | |
| pWSK29 | Apr lacZ low copy number cloning vector | [55] | |
| pWKS30 | Apr lacZ low copy number cloning vector | [55] | |
| pWSK129 | Kmr lacZ low copy number cloning vector | [55] | |
| pET22b | Apr lacI cloning vector harbouring T7 promoter and terminator with pBR322 and f1 origin. | Novagen | |
| pJIR2097 | pWSK29 harbouring 5.2 kb fragment containing aprV2 Ω tetM | aprV2 suicide plasmid | Recombinant |
| pJIR2275 | pUC18 harbouring 3.4 kb fragment containing bprV Ω erm (B) | bprV suicide plasmid | Recombinant |
| pJIR2309 | pWSK129 harbouring 3.5 kb fragment containing aprV5Ω bla | aprV5suicide plasmid | Recombinant |
| pJIR2545 | pWKS30 harbouring 4.8 kb fragment containing D. nodosus rrnA promoter, kanamycin resistance, aprV5, rrnA terminator | aprV5 complementation plasmid | Recombinant |
| pJIR3044 | pWSK29 harbouring 5.9 kb fragment containing aprV2, erm(B) and 2.3 kb chromosomal region downstream of aprV2 | aprV2 complementation plasmid | Recombinant |
| pJIR3060 | pWSK29 harbouring 3.75 kb fragment containing 5′fragment of aprV5, erm(B) and 3′fragment of bprV | aprV5/bprV suicide plasmid | Recombinant |
| pJIR3148 | pWSK29 harbouring 4.3 kb fragment containing 3′ fragment of aprV5, kanamycin resistance, bprV | bprV complementation plasmid | Recombinant |
| pJIR3159 | pWSK29 harbouring 3.5 kb fragment containing bprV Ω erm(B) | bprV suicide plasmid | Recombinant |
| pJIR3268 | pWSK29 harbouring a 5.9 kb fragment containing aprB2, erm(B) and 2.3 kb chromosomal region downstream of aprV2 | aprB2 complementation plasmid | Recombinant |
| pET22b-AprV2 | pET22b harbouring 1.4 kb fragment encoding 1–474 residues of the AprV2 precursor. | AprV2 expression | Recombinant |
| pET22b-AprB2 | pET22b harbouring 1.4 kb fragment encoding 1–474 residues of the AprB2 precursor. | AprB2 expression | Recombinant |
| pET22b-AprV2.Y92F | pET22b harbouring 1.4 kb fragment encoding 1–474 residues of the AprV2 precursor with a Y92F substitution. | AprV2.Y92F expression | Recombinant |
| pET22b-AprV2.Y92D | pET22b harbouring 1.4 kb fragment encoding 1–474 residues of the AprV2 precursor with a Y92D substitution. | AprV2.Y92D expression | Recombinant |
| pET22b-AprV2.Y92L | pET22b harbouring 1.4 kb fragment encoding 1–474 residues of the AprV2 precursor with a Y92L substitution. | AprV2.Y92L expression | Recombinant |
| pET22b-AprV2.Y92A | pET22b harbouring 1.4 kb fragment encoding 1–474 residues of the AprV2 precursor with a Y92A substitution. | AprV2.Y92A expression | Recombinant |
| pET22b-AprV2.C141S | pET22b harbouring 1.4 kb fragment encoding 1–474 residues of the AprV2 precursor with a C141S substitution. | AprV2.C141S expression | Recombinant |
*Independently derived mutants.