Table 3.
Does O-P co-expression temper P-lethality and plasmid loss?
| Plasmid in 594 Host Cells | Cell Viability and (Plasmid Retention per CFU Assayed; %) at CFU Growth Temperature a | |||
|---|---|---|---|---|
| 30 °C | 37 °C | 39 °C | 42 °C | |
| Only P expression | ||||
| [P] | 1.0 (35/35; 100%) | 0.01 (0/35; 0%) | 0.008 (0/35; 0%) | 0.07 (0/35; 0%) |
| dnaB-GrpD55 [P] | 1.0 (35/35; 100%) | 1.0 (35/35; 100%) | 1.0 (35/35; 100%) | 0.98 (35/35; 100%) |
| Only O expression | ||||
| [O] | 1.0 (30/30; 100%) | 1.0 (30/30; 100%) | 1.0 (30/30; 100%) | 0.61 (29/30; 97%) |
| O-P expression combinations | ||||
| [O-P] | 1.0 (62/70; 89%) | 0.12 (0/70; 0%) | 0.12 (0/70; 0%) | 0.022 (0/36; 0%) |
| [oop#1-O-P] | 1.0 (120/120; 100%) | 0.20 (98/101; 97%) | 0.005 (115/120; 96%) | 0.002 (0/36; 0%) |
| [oop#2-O-P] | 1.0 (117/120; 98%) | 0.055 (76/154; 49%) | 0.048 (62/120; 52%) | 0.008 (0/36; 0%) |
| [O-36P] | 1.0 (30/30; 100%) | 0.79 (30/30; 100%) | 0.012 (1/30; 3%) | 0.0005 (0/36; 0%) |
| [O-63P] | 1.0 (30/30; 100%) | 0.90 (30/30; 100%) | 0.055 (14/40; 35%) | 0.0023 (0/36; 0%) |
a The 594 cultures with indicated plasmids were grown to stationary phase in LB (10 g Bacto-Tryptone, 10 g Bacto-Yeast Extract, 5 g NaCl per liter) plus 50 µg/mL ampicillin (=LBAmp50) for 48 h at 30 °C, diluted, spread on LB agar (includes the addition of 11 g/liter Bacto-Agar) plates (no ampicillin) that were incubated at 30, 37, 39, or 42 °C for 30 h and the average survivor titer for CFU per mL was determined for each plating temperature. Isolated survivor CFU were stabbed to LB and to LBAmp50 agar plates to estimate the proportion of CFU retaining the AmpR plasmid. We tried to assay all the CFU per plate to avoid colony size discrimination, and minimally 30 CFU. The cell viability results shown in each column entry were determined by dividing the cell titer obtained for each incubation by the cell titer at 30 °C, and each value represents the average of duplicate plasmid isolates for each single experiment, with plating variations of about 10%. The results in parentheses represent the sum of results for all the CFU’s assayed from isolates. These results represent independent determinations by KR to those reported in [25], where it was shown that 594[P] cells lost 100% of their plasmids when grown at 36 °C and higher, indicating that trace levels of P expression (where the CI[Ts] repressor still retains some ability to block pR-promoted transcription below about 38–39 °C) will cure cells of the plasmid.