Table 1. Strains and plasmids used.
| Strains or plasmids | Relevant genotype, phenotype, descriptionsa | Reference |
|---|---|---|
| Vibrio alfacsensis | ||
| 04Ya249 | Aquaculture isolate harboring conjugative plasmid pSEA2; Tcr, Apr, Eryr Cmr | [27] |
| 04Ya108 | Aquaculture isolate harboring conjugative plasmid pSEA1; Tcr, Apr, Eryr Cmr | [27] |
| LN95 | pSEA2 free derivative of strain 04Ya249; Apr | This study |
| E. coli | ||
| DH5α | F-, Φ80dlacZΔM15, Δ(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rK-, mK+), phoA, supE44, λ-, thi-1, gyrA96, relA1 | Takara Bio Inc., Shiga, Japan |
| JW0452 | λ- rph-1 Δ(rhaD-rhaB)568 hsdR514 ΔlacZ4787(::rrnB-3) Δ(araD-araB)567 ΔacrA748::kan; Kmr | [68] |
| JW0452rif | Spontaneous rifampicin-resistant mutant of JW0452 | This study |
| JW0452ΔrecA | JW0452ΔrecA::cat; Kmr, Cmr | This study |
| JW0452ΔrecArif | Spontaneous rif resistant mutant of JW0452ΔrecA; Kmr, Cmr, Rifr | This study |
| Transconjugantsc | ||
| TJ249 | JW0452 insJ::SE-6945::pSEA2 yjjNt::SE-6945; Apr Tcr Eryr Cmr | This study |
| LN28, LN32, LN36, LN86 | JW0452 chr::pSEA2b; Apr Tcr Eryr Cmr | This study |
| LN29, LN30, LN82, LN88, LN91 | JW0452 yjjNt::SE-6945::pSEA2; Apr Tcr Eryr Cmr | This study |
| LN33, LN34, LN35, LN37, LN58, LN84, LN89, LN90 | JW0452 insJ::SE-6945::pSEA2; Apr Tcr Eryr Cmr | This study |
| LN52 | JW0452 yjjNt::SE-6945; Apr | This study |
| LN52rif | Spontaneous rifampicin-resistant mutant of LN52; Rifr, Apr | This study |
| Plasmids | ||
| pGEM-T | Cloning vector; pUC replicon, Apr | Promega, Madison, WI, USA |
| pGEM-attS | pGEM-T carrying the 206 bp attS fragment; pUC replicon, Apr | This study |
| pGEM-int | pGEM-T carrying the 103 bp intA fragment; pUC replicon, Apr | This study |
| pGEM-gyrB | pGEM-T carrying the 110 bp gyrB fragment from strain 04Ya249; pUC replicon, Apr | This study |
| pKD46 | Lambda-Red recombinase expression plasmid; pSC101ts replicon, Apr | [69] |
| pKD3 | Donor of the cat gene for the Lambda-Red recombination experiment; R6K replicon, Cmr | [69] |
a. Antimicrobial resistance phenotypes (r) listed were confirmed by experiments. Abbreviations are as follows: Ap, ampicillin; Tc, tetracycline: Ery, erythromycin; Cm, chloramphenicol; Rif, rifampicin; kan, kanamycin resistance gene. yjjNt denotes the putative transcription termination region for the yjjN gene. It is unclear whether the resistance genes on pSEA2 are still in the original locations for other transconjugants besides TJ249.
b. Cointegration of plasmid pSEA2 with chromosome, independent of SE-6945 transposition. Location of pSEA2 in the transconjugant genome could not be defined based on Southern hybridization results.
c. The 19 transconjugants listed (TJ249, LN28, LN29, LN30, LN32, LN33, LN34, LN35, LN36, LN37, LN52, LN58, LN82, LN84, LN86, LN88, LN89, LN90, LN91) were obtained from 19 independent mating experiments. Selection was done at 42°C. Antibiotic selection used was as follows: Tc for LN28, LN29 LN30, LN32, LN33, LN34, LN35, LN36, and LN37; Ap for LN52; Ap Tc for LN58 LN82, LN84, LN86, LN88, LN89, LN90, and LN91; Ery for TJ249.