Table 3.
Analysis of spontaneous ΔstkP suppressor mutations that arose in unencapsulated (Δcps) and encapsulated D39 S. pneumoniae D39a
| ΔstkP suppressor designation | Strain number | Genotype | Doubling time (min)b | Growth yield (OD620)b | |
|---|---|---|---|---|---|
| 1 | WT parent of sup stkP-1 | IU1824 | D39 Δcps rpsL1 | 36 ± 2 | 1.0 ± 0 |
| 2 | sup stkP-1 | IU16883 | D39 Δcps rpsL1 ΔstkP::Pc-erm Δ[spd_1024’-spd_1037’] (≈13.4 kb, 14 genes) Ω[spd_0889’-spd_1023] (≈147 kb, 135 genes) Amplification of murZ |
49 ± 2 | 1.0 ± 0 |
| 3 | WT parent of sup stkP-2, -3, -5, -6 | IU1945 | D39 Δcps | 31 ± 0.1 | 0.9 ± 0.0 |
| 4 | sup stkP-2 | E740c | D39 Δcps Δ[phpP-stkP]::Pc-erm
Ω[spd_0966’-spd_0986’] (≈18 kb, 21 genes) Amplification of murZ |
39 ± 1 | 0.9 ± 0.1 |
| 5 | sup stkP-3 | IU11912d | D39 Δcps ΔstkP::Pc-cat Ω[spd_1703-spd_1803] (≈91.3 kb, 101 genes) Amplification of murA |
51 ± 2 | 0.6 ± 0.1 |
| 6 | WT parent of sup stkP-4 | IU1690 | D39 cps+ | 44 ± 4 | 0.9 ± 0.1 |
| 7 | sup stkP-4 | IU11456 | D39 ΔstkP::Pc-erm Ω[spd_1703-spd_1878] (≈153 kb, 176 genes) Amplification of murA |
57 ± 1 (n=2) | 0.7 (n=1) |
| 8 | sup stkP-5 | E739 | D39 Δcps Δ[phpP-stkP]::Pc-erm Δ[spd_1032’-spd_1036’] detected, indicative of adjacent duplication |
41 (n=1) | 1.0 (n=1) |
| 9 | sup stkP-6 | K740 | D39 Δcps Δ[phpP-stkP]::Pc-erm 14 bp-duplication detected in the RBS of ireB(Spn); duplication status not determined |
45 (n=1) | 1.1 (n=1) |
WT D39 and its derivatives (D39 Δcps, or D39 Δcps rpsL1) were transformed with a ΔstkP::Pc-erm, ΔstkP::Pc-cat, or Δ[phpP-stkP]::Pc-erm amplicon as described in Experimental procedures. 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. The larger colonies were stored and analyzed by whole-genome sequencing.
Doubling times and maximal growth yields obtained within 8 h of growth in BHI broth were determined as described in Experimental procedures. Values (means ± SEM) were obtained from 2 independent biological experiments, except for sup4. Representative growth curves are shown in Fig. S4C, S4D, and S20A.
Additional mutation detected with whole genome sequence of E740 includes a sun(A324D, GCT → GAT). sun encodes rRNA small subunit methyltransferase B.
Additional mutation detected with whole genome sequence of IU11912 includes spd_0921(K420M, AAG → ATG). spd_0921 encodes a site-specific recombinase family protein.