## 2 Result and Discussions
### P-Lethality Suppression  
The results in Figure 3 do not account for P-lethality, only plasmid loss, and so their interconnection was examined (Table 6). Whereas cells within a starting culture that become cured of a plasmid expressing P can grow and eventually fully populate a culture, isolating individual cells by spreading dilutions on an agar plate will permit determination of cell viability at the incubation temperature. Less than 1% viability was observed for spread 594[pclpR-_P_-timm] cells incubated at or above 37 \({}^{\circ}\)C, and any surviving cfu had lost the plasmid. \(P\) expression was not lethal in spread 594 _grpD55_[pclpR-_P_-timm] cells incubated between 37 and 42 \({}^{\circ}\)C, where the cfu arising at or above 37 \({}^{\circ}\)C retained the plasmid. We examined whether the plasmids extracted from these survivor cfu retained P-lethality and plasmid-loss properties, when moved back into 594 _dnaB\({}^{+}\)_ cells. The reclaimed plasmids retained both the P-lethality and plasmid loss phenotypes (bottom lines, Table 6). Figure 3: _Cont._A stark contrast was noted between the \(O\) and \(P\) versions of pcIpR-timm plasmids regarding their ability to prevent the plating of _imm\(\lambda\)_ phage \(\lambda\)_cI72_. The _O_-version retained the _imm\(\lambda\)_ phenotype between 25 and 39 'C, losing it when the CI[Ts] repressor became denatured; the _P_-version lost it between 30 and 35 'C and supported plaque formation on the cell lawns at 35 and 36 'C. The simplest explanation is that the _P_-version plasmid was lost from the cells grown above 30 'C, and coordinately, _cI_ repressor gene expression was lost. Gel analysis showed that 100% of cfu from cultures grown at 30 'C retained the _P_-version plasmid, but cultures grown at 34 and 35 'C were reduced in plasmid copies, and cultures grown at or above 36 'C were fully cured. Since cultures grown at 30 'C could complement for \(P\), plasmid maintenance was very sensitive to trace \(P\) expression, which apparently increased with an increase in culture temperature from 30 to 36 'C. In support of this claim, we obtained \(1.5\times 10^{5}\) transformants per \(\mu\)g of pcIpR-_P_-timm DNA at 25 'C and none at 37 'C. The partial to full loss between \(34-37\) 'C (Figure 3) of _P_-encoded plasmids, argues for trace levels of P causing \begin{table}
\begin{tabular}{l c c c} \hline
**Host cells and plasmids** & \multicolumn{2}{c}{**Cell viability 1 and (plasmid retention/cfu; \%) 2**} \\  & \multicolumn{2}{c}{**at growth temperature (’C)**} \\  & **25** & **Ave 37 \& 39** & **42** \\ \hline
594[pcIpR-_P_-timm] & 1.0 & 0.0049 & 0.001 \\  & (195/196; \(\sim\)100) & (0/57; 0) & (0/28; 0) \\
594[pcIpR-_P_SA-timm] & 1.0 & 0.006 & 0.004 \\  & (14/14; 100) & (0/150; 0) & (0/39; 0) \\
594[pcIpR-_P_SA-timm] & 1.0 & 0.635 & 0.003 \\  & (103/103; 100) & (789/793; \(\sim\)100) & (0/294; 0) \\
594[pcIpR-_P_SA-timm] & 1.0 & 0.96 & 0.12 \\  & (166/168; 99) & (334/336; 99) & (166/168; 99) \\
594 _grpD55_ [pcIpR-_P_-timm] & 1.0 & 0.99 & 1.0 \\  & (35/35; 100) & (87/88; 99) & (14/14; 100) \\
594 _grpD55_ [pcIpR-_P_SA-timm] & 1.0 & 0.75 & 0.57 \\  & (42/42; 100) & (88/94; 94) & (18/19; 95) \\
594 _grpD55_ [pcIpR-_P_SA-timm] & 1.0 & 0.08 & 0.01 \\  & (54/54; 100) & (434/435; \(\sim\)100) & (5/108; 5) \\
594 _grpD55_ [pcIpR-_P_SA-timm] & 1.0 (nd) & 0.45 (nd) & 0.001 (nd) \\ Re-claim pcIpR-_P_-timm from 594 _grpD55_ cultures and transform into 594 _dnaB 1_ cells \\  & 1.0 & 0.08 & 0.01 \\  & (54/56; 96) & (0/119; 0) & (0/91; 0) \\ Re-claim from 42’C cultures 2  & 1.0 & 0.059 & 0.026 \\  & (130/130; 96) & (1/230; 0.4) & (0/201; 0) \\ \hline \end{tabular}
\end{table} 
Table 6: Influence of modifying \(P\) or _dnaB_ on P lethality and plasmid loss. ColE1 plasmid curing. This result suggests a new dimension to the concept of P-lethality. It appears that ColE1 plasmid replication is extremely sensitive to P\({}^{\lambda}\) protein and that P-inhibition of plasmid replication prevents plasmid establishment upon transformation.