TABLE 2.
Appearance of colonies after transformation with a Δpbp2b<>aad9 amplicon into D39 strainsa
| Recipient strain and condition | Genotype | Number of colonies at 20 h after transformation with Δpbp2b<>aad9 amplicon |
|---|---|---|
| Strains of IU1945 (D39 Δcps) genetic background | ||
| 1. IU1945b | WT | 0 |
| 2. IU7337 | ΔbgaA::PfcsK-pbp2b | 0 |
| 3. IU7337 + fucosec | ΔbgaA::PfcsK-pbp2b | >500 |
| Strains of IU1824 (D39 Δcps rpsL1) genetic background | ||
| 4. IU1824 | pbp1a+ wild-type parent | 0 |
| 5. IU6741 | Δpbp1a | 0 |
| 6. IU7325 | Δpbp1a ΔmltG::Pc-[kan-rpsL+] | >500 |
| 7. IU7327 | Δpbp1a ΔmltG::Pc-erm | >500 |
| 8. IU8549 | Δpbp1a mltG(Δ5bp) (sup2) | >500 |
| 9. IU8551 | Δpbp1a mltG(Y488D) (sup3) | 0 |
| 10. IU9760d | pbp1a+ mltG(Y488D) (sup3) | >500 |
| 11. IU8553 | Δpbp1a mltG(Δ488bp) (sup4) | >500 |
| 12. IU8555 | Δpbp1a mltG(Ω45bp)2 (sup5) | >500 |
| 13. IU8873 | Δpbp1a mltG(E428Q) | >500 |
| 14.IU8982 | Δpbp1a mltG(E428A) | >500 |
| 15. IU8910 | Δpbp1a mltG(ΔDUF_1346) | 0 |
| 16. IU9025 | pbp1a+ mltG(ΔDUF_1346) | 0 |
| 17. IU10919 | pbp1a+ mltGSpn-Eco | >500 tiny colonies |
| 18. IU10965 | Δpbp1a mltGSpn-Eco | >500 tiny colonies |
| 19. IU11007e | pbp1a+ mltGEco | >500 tiny colonies |
| 20. IU11009e | Δpbp1a mltGEco | >500 tiny colonies |
| Strain of IU1690 (D39 cps+) genetic background | ||
| 21. IU1690 | WT | 0 |
Recipient strains were constructed as described in Table S1. Transformations and visualization of colonies normalized to 1 mL of transformation mixture were performed as described in Experimental procedures. The same results for each strain were obtained from two independent transformation experiments. Normal wild-type colony size was observed, except for the last two strains. Full MltG activity in a pbp1a+ or Δpbp1a strain gave 0 transformants, whereas no MltG activity in a Δpbp1a strain gave >500 normal sized colonies (see text for details).
<10 colonies were visible after 40 h of incubation. Transformants of IU1945 with the Δpbp2b<>aad9 amplicon that arose after 40 h of incubation were characterized as the original sup2-sup5 suppressor mutations described in Table 1 (see text and Table S1).
1% (wt/vol) L-fucose was added to all steps in the transformation procedure to induce PBP2b+ expression in merodiploid strain IU7337 (Table S1).
Strain IU9895 (mltG(Y488D)-Pc-erm, which was independently constructed in the IU1945 Δcps background (Table S1), showed the same suppression of the Δpbp2b mutation (data not shown).
mltGSpn reading frame in the pneumococcal chromosome is replaced with the intact mltGEco reading frame; the promoter, ribosome binding site, and downstream regions are from S. pneumoniae.