Table 3.
Plasmids carrying rcbA or hda suppress the lethality caused by an oversupply of DnaA in a ΔrcbA straina
| Strain (relevant genotype) | Mean doubling time (min) ± SD | Cotransformed plasmid | Mean relative plating efficiency ± SDb |
|
|---|---|---|---|---|
| pLST435 M [dnaA(T435 M)] | pDS596 (dnaA+) | |||
| MG1655 (rcbA+) | 23 ± 1.7 | None | 0.9 ± 0.13 | 1.8 ± 0.60 |
| MF1341 (ΔrcbA) | 26 ± 1.7 | None | 0.6 ± 0.24 | <(1.1 ± 0.49) × 10−3 |
| MF1341 (ΔrcbA) | NA | pACYC184 | NA | (6.3 ± 0.42) × 10−3 |
| MF1341 (ΔrcbA) | NA | pMMF-D4 (rcbA) | NA | 1.2 ± 0.11 |
| MF1341 (ΔrcbA) | NA | pMMF41 (hda) | NA | 1.0 ± 0.18 |
The respective strains were grown in LB medium at 37°C to determine the generation time and to prepare electrocompetent cells, which were electroporated with pDS596 or pLST435 M (50 ng) (dnaA allele under araBAD promoter control) and either no second plasmid, pACYC184, or a plasmid carrying rcbA or hda, as described in Materials and Methods and Table 2. Transformants were obtained on LB medium supplemented with 100 μg/ml ampicillin (and 50 μg/ml kanamycin for MF1341), which either lacked or contained 0.5% (wt/vol) l-arabinose. For the strains bearing pACYC184 or its derivatives, the plates also contained 35 μg/ml chloramphenicol. The relative plating efficiency is defined in Table 2. NA, not applicable.
To observe a reduction in the plating efficiency caused by elevated dnaA expression levels in MF1341, kanamycin was required in the plating medium at a concentration of 50 μg/ml.