TABLE 3.
Appearance of colonies after transformation with ΔrodA, ΔmreCD, or ΔrodZ amplicons into D39 strainsa
| Recipient strain and condition | Genotype | Number of colonies at 22 h after transformation with deletion ampliconsb | ||
|---|---|---|---|---|
| ΔrodA | ΔmreCD | ΔrodZ | ||
| Strains of IU1945 (D39 Δcps) genetic background | ||||
| 1. IU1945 | WT | 0d | 0 | 0d |
| 2. IU10921 | ΔbgaA::PZn-rodA | 0d | ||
| 3. IU10921 + Znc | >500 | |||
| 4. IU9765 | ΔbgaA::PZn-rodZ | 0d | ||
| 5. IU9765 + Znc | >500 | |||
| Strains of IU1824 (D39 Δcps rpsL1) genetic background | ||||
| 6. IU1824 | pbp1a+ wild-type parent | 0 | 0 | 0 |
| 7. IU6741 | Δpbp1a | 0 | >500 | >500 |
| 8. IU8551 | Δpbp1a mltG(Y488D) | 0 | >500 | >500 |
| 9. IU9760e | pbp1a+ mltG(Y488D) | >500 | >500 | >500 |
| 10. IU9783 | pbp1a+ mltG(Y488D) Δ pbp2b | >500 | >500 | >500 |
| 11. IU8553 | Δpbp1a mltG(Δ488bp) | >500 | >500 | >500 |
| 12. IU10731 | Δpbp1a mltG(Δ488bp) ΔmreCD | >500 | >500 | |
| 13. IU10743 | Δpbp1a mltG(Δ488bp) Δpbp2b ΔmreCD | >500 | >500 | |
| Strain of IU1690 (D39 cps+) genetic background | ||||
| 14. IU1690 | WT | 0 | 0 | 0 |
Recipient strains were constructed as described in Table S1. Transformations and visualization of colonies normalized to 1 mL transformation mixture were performed as described in Experimental procedures. All transformants formed uniform colonies of nearly wild-type size, except for the quadruple and quintuple mutants (IU10731 and IU10743), which grew slower than the parent strain. The same results for each strain were obtained from two independent transformation experiments. Blank cells are non-applicable experiments.
rodZ was not named in the genome annotation and corresponds to spd_2050. Strains used to prepare amplicons are listed in Table S2.
0.4 mM ZnCl2 and 0.04 mM MnSO4 were added to all steps in the transformation procedure to induce RodA or RodZ expression in merodiploid strain IU10921 or IU9765, respectively (Table S1).
Fast-growing suppressors (<10 colonies per plate) were occasionally seen for IU1945 or the merodiploid strains under non-inducing condition.
Strain IU9895 (mltG(Y488D)-Pc-erm, which was independently constructed in the IU1945 Δcps background (Table S1), showed the same suppression of the ΔmreCD mutation (data not shown).