Table 2.
Suppression of ΔgpsB or ΔstkP mutation in S. pneumoniae Δcps D39a
| Recipient strains | Zn | Number of and appearance of colonies 20 to 22 h after transformation b | |
|---|---|---|---|
| ΔgpsB<>aad9 | ΔstkP::Pc-erm | ||
| 1. WT (IU1824)c | - | 0d | >500 fainte |
| + | 0 | >500 faint | |
| 2. gpsB+//PZn-gpsB+(IU15877)c | - | 0 | >500 faint |
| + | >500 WTf | >500 faint | |
| 3. stkP+//PZn-stkP+(IU14974)c | - | 0 | >500 faint |
| + | 0 | >500 WTf | |
| 4. murZ+//PZn-murZ+(IU13393)c | - | 0 | >500 faint |
| + | >500 small | >500 WTf | |
| 5. murA+//PZn-murA+(IU13395)c | - | 0 | >500 faint |
| + | >500 WTf | >500 WTf | |
| 6. murZ(D280Y) (IU13438) | - | >500 small | >500 WTf |
| 7. murZ(I265V, R6 allele) (IU14210) | - | >500, smallg | >500 WTf |
| 8. murZ(E259A) (IU17627) | - | >500 small | >500 WTf |
| 9. ΔkhpA (IU9036) | - | >500, small | >500, WTf |
| 10. ΔkhpB (IU10592) | - | >500, small | >500, WTf |
| 11. ΔclpP (IU17138)h | - | 0 | >500 faint |
| 12. ireB(Q84(STOP)) (IU13606) | - | >500 small | >500 WT |
| 13. ΔireB (markerless) (IU13604) | - | >500 small | >500 WT |
Recipient strains in D39 Δcps rpsL1 (IU1824) background and amplicons were obtained as described in Table S1. Transformations and visualization of colonies normalized to 1 mL of transformation mixture were performed as described in Experimental procedures. All transformation experiments were performed with Δpbp1b amplicons containing the same antibiotic selections as the positive control for detection of colonies and colony size comparison. The volumes of transformation mixture plated were adjusted to provide ≈150–500 colonies for the Δpbp1b control amplicon. Transformations with control Δpbp1b amplicons yielded >500 colonies per 1 mL of transformation mixture. Transformants were confirmed by PCR reactions. Each transformation experiment was performed 2 or more times independently with similar results.
Unless indicated, transformed colonies were generally uniform in size and of similar size as the recipient strain transformed with a control Δpbp1b amplicon.
0.4 mM (Zn2+/(1/10)Mn2+) (IU15877, IU14974, and IU13395) or 0.2 mM (Zn2+/(1/10)Mn2+) (IU13393) were added to transformation mixes and in subsequent steps to induce expression of gpsB, stkP, murZ or murA under control of the PZn zinc-inducible promoter in the ectopic bgaA site. 1/10 concentrations of Mn2+ was added to eliminate toxicity caused by addition of Zn2+ (Jacobsen et al., 2011, Martin et al., 2017, Tsui et al., 2016). The wild-type parent strain (IU1824) was transformed with the same Zn-containing condition to control for possible effects of Zn2+ on transformation efficiency.
Occasional suppressor mutants were present.
Typically only faint colonies appeared on TSAII-BA plates in the first 20 h after transformation (Fig. S3). However, upon re-streaking, these mutants show heterogeneous colony sizes.
Colonies are described as WT when the colony size and appearance are similar to the recipient strain transformed with the control Δpbp1b amplicon.
Colonies remained very small, but uniform-sized upon re-streaking on antibiotic selection plates. This strain was stored as IU14234 and verified to be ΔgpsB.
Similar results were obtained with ΔclpC (IU12462), ΔclpL (IU17136), and ΔclpE (IU17134) strains as with the ΔclpP strain after transformation with ΔgpsB<>aad9 and ΔstkP::Pc-erm amplicons.