TABLE 1.
Strains used in this study and their abilities to act as virulence plasmid conjugative donors
| Strain | Descriptiona | Construction, source, or referenceb | Donor abilityc |
|---|---|---|---|
| W3110 | E. coli K-12 | CGSCd | NA |
| BA769 | W3110 Nalr | Selection for spontaneous nalidixic acid resistance | NA |
| LT2 (ATCC23565) | S. typhimurium | ATCCd | NA |
| 14028 (ATCC14028) | S. typhimurium | ATCCd | NA |
| SR-11 | S. typhimurium | Susanne Lindgren | NA |
| SL1344 | S. typhimurium | 16, Cathy Lee | NA |
| IR715 | 14028 Nalr | 31 | NA |
| 14028 −pSLT | 14028, cured of the virulence plasmid | 3 | NA |
| BA770 | 14028 −pSLT Nalr | Selection for spontaneous nalidixic acid resistance | NA |
| BA901a,b,c | LT2 spv1541::MudJ | LT2 × P22/BA1541 | Yes |
| BA902a,b,c | SR-11 spv1541::MudJ | SR-11 × P22/BA1541 | Yes |
| BA903a,b,c | SL1344 spv1541::MudJ | SL1344 × P22/BA1541 | No |
| BA1504 | 14028 traC1504::MudJ (antisense) | 2, this study | No |
| BA1505 | 14028 traG1505::MudJ (antisense) | 2, this study | No |
| BA1523 | 14028 orfG1523::MudJ (antisense) | 2, this study | Noe |
| BA1527 | 14028 traW1527::MudJ (sense) | 2, this study | No |
| BA1532 | 14028 trbC1532::MudJ (antisense) | 2, this study | Noe |
| BA1533 | 14028 pefC1533::MudJ (sense) | 2, this study | Yes |
| BA1534 | 14028 traP1534::MudJ (sense) | 2, this study | No |
| BA1535 | 14028 traC1535::MudJ (antisense) | 2, this study | No |
| BA1539 | 14028 tra1539::MudJ (MudJ is between traR and traC; antisense) | 2, this study | No |
| BA1541 | 14028 spv1541::MudJ (sense) | 2, this study | Yes |
| BA1542 | 14028 orfG1542::MudJ (antisense) | 2, this study | Noe |
| BA1544 | 14028 traD1544::MudJ (antisense) | 2, this study | No |
| BA1545 | 14028 traD1545::MudJ (antisense) | 2, this study | No |
| BA1546 | 14028 traG1546::MudJ (antisense) | 2, this study | No |
| BA1550 | 14028 hilA1550::MudJ (sense) | 2 | NA |
| BA1569 | 14028 tlpA1569::MudJ (antisense) | 2, this study | Yes |
| BA1571 | 14028 trbI1571::MudJ (antisense) | 2, this study | No |
| BA1572 | 14028 traG1572::MudJ (antisense) | 2, this study | No |
| BA1576 | 14028 traW1576::MudJ (antisense) | 2, this study | No |
| BA1741 | 14028 spv1541::MudJ sirA::cam | 2 | Yesf |
The DNA surrounding the MudJ insertions was amplified by inverse PCR, cloned, and sequenced as described previously (1). Three MudJ insertions were within previously sequenced genes. The pefC1533 insertion is before nucleotide 4740 of GenBank accession no. L08613. The spv1541 insertion is upstream of spvA, before nucleotide 2440 of GenBank accession no. X56727. The tlpA1569 insertion is before nucleotide 216 of GenBank accession no. M88208. When the MudJ insertion is within a gene that has not been sequenced, the name of the closest homolog is given. The orientations of the promoterless lacZY genes of the MudJ element are listed with respect to the orientation of the gene disrupted (sense or antisense).
Transductions were performed with phage P22HTint, followed by streaking to isolation in the presence of 10 mM EGTA and confirming smooth lipopolysaccharide and lack of pseudolysogeny by cross-streaking transductants against P22vir on Evans blue-uranine plates (21). As a side note, we observed that P22 can be used to introduce genes into strain SR-11, but it cannot form plaques on SR-11. Therefore, it always appears rough in the Evans blue-uranine test, presumably because it is lysogenized with a phage conferring P22 immunity. We have found that phage KB1int-1 (22) works well for transducing markers out of SR-11.
Ability to act as a donor of the virulence plasmid in filter matings on M9 glucose plates is indicated by Yes, and failure to act as a donor is indicated by No. NA, not applicable.
CGSC, E. coli Genetic Stock Center; ATCC, American Type Culture Collection.
Donor ability was dramatically reduced but not completely eliminated by mutations in orfG or trbC.
Donor ability was slightly reduced (two- to threefold) by a sirA mutation.