TABLE 1.
Transformation efficiencies with a ΔgpsB<>aad9 amplicon and colony sizes of recipient and transformant strainsa
| Recipient strain and condition |
Genotype of recipient (colony size relative to wild-type (WT) parent strain) |
Number of ΔgpsB<>aad9 transformants at 24 h (colony size after streaking; strain) |
|---|---|---|
| IU1945 (D39 Δcps) genetic background | ||
| 1. IU1945 | WT | 0b |
| 2. IU4846 − fucose | ΔbgaA::PfcsK-gpsB (WT) | <20 (variable; Land et. al, 2013) |
| 3. IU4846 + fucosec | ΔbgaA::PfcsK-gpsB (WT) | >500 (WT; Land et. al, 2013) |
| 4. IU11442 | ΔphpP::Pc-erm (small) | 20–100 (medium; IU11508)d |
| 5. IU7922 | ΔstkP::Pc-[kan-rpsL+] (medium) | 20–100 (medium: IU10109)d |
| 6. IU11460 | ΔstkP::Pc-erm (medium) | 20–100 (medium; IU11546)d |
| 7. IU11462 | Δ[phpP-stkP]::Pc-erm (medium) | 20–100 (medium; IU11512)d |
| 8. K739 | Δ[phpP-stkP]::Pc-[kan-rpsL+] (medium) |
20–100 (medium; IU10107)d |
| IU1824 (D39 Δcps rpsL1) genetic background | ||
| 9. IU1824 | WT | 0 |
| 10. IU7685 |
phpP(G229D) stkP(G10stop) (medium) |
≈ 200 (medium; IU7733) |
| 11. IU10423 | phpP(G229D) (tiny) | ≈ 100 (medium; IU11346) |
| 12. IU11223 | phpP(D192A) (tiny) | >500 (medium; IU11348) |
| IU1690 (D39 cps+) genetic background | ||
| 13. IU1690 | WT | 0e |
| 14. IU11183 | ΔphpP::Pc-erm (small) | 16–20 (medium; IU11350) |
| 15. IU11456 | ΔstkP::Pc-erm (medium) | ≈ 30 (medium; IU11504) |
| 16. IU11458 | Δ[phpP-stkP]::Pc-erm (medium) | ≈ 30 (medium; IU11506) |
| IU1781 (D39 cps+ rpsL1) genetic background | ||
| 17. IU1781 | WT | 0 |
| 18. IU11195 | phpP(G229D) (tiny) | ≈ 30 (small; IU11352) |
| 19. IU11227 | phpP(D192A) (tiny) | ≈ 30 (small; IU11354) |
| R6 or Rx1 genetic background | ||
| 20. R6 (EL59) | WT | >500 (medium; IU8224) |
| 21. Rx1 (IU9256) | WT | ≈ 300 (tiny; IU11574)f |
Recipient strains are described in Table S1. Transformations and visualization of colonies were performed as described in Experimental procedures. The numbers of colonies are normalized to 1 mL of transformation mixture. Similar results were obtained for each D39 Δcps, Rx1, or R6 strain from at least two independent transformation experiments in which multiple isolates were examined. Transformation experiments of D39 cps+ strains was performed once.
0 to <10 colonies were visible after 24 h of incubation from >20 independent transformations. Suppressor strain IU5845 appeared ≈ 24 h after transformation, and IU6441 and IU6442 appeared after ≈ 40 h (see text and Table S1). >500 colonies were obtained for transformations of IU1945 with a ΔpurR<>aad9 control amplicon.
0.8% (wt/vol) L-fucose was added to all steps in the transformation procedure to induce gpsB+ expression in merodiploid strain IU4846 (Land et al., 2013).
Numbers of transformants obtained for ΔphpP or ΔstkP mutants were similar for the ΔgpsB<>aad9 test and ΔpurR<>aad9 control amplicons within experiments, but varied in independent transformations. This variability is consistent with lower and variable transformation efficiency and recovery of ΔphpP or ΔstkP mutants reported previously (Echenique et al., 2004, Saskova et al., 2007).
≈ 80 colonies were obtained for transformation of strain IU1690 with control amplicon ΔhtrA::Pc-erm.
Strain IU11574 (Rx1 ΔgpsB<>aad9) was isolated from a tiny colony 24 h after transformation of strain Rx1.