## 2 Result and Discussions
### Influence of P-alteration, or O O OOP RNA Co-Expression on P-Iethality and Plasmid Retention  
Linking P with a 66 amino acid SPA tag [72] at its C-terminal end did not suppress P lethality, nor plasmid loss, but the lethality of P\({}^{\text{SPA}}\) strongly decreased and plasmid retention sharply increased in the _grpD55_ host at 37-42 \({}^{\text{\text{\textdegree}}}\)C (Table 6). The 76 codon in frame N-terminal deletion, \(P^{\text{A76}}\), suppressed both plasmid loss and P-Iethality at 37 or 39 \({}^{\text{\texttextdegree}}\)C. Thus, amino acids 9-86 of P include a critical domain. Its removal knocks out P initiation of replication (our complementation data), the P-Iethality phenotype, and P-induced ColE1 plasmid loss. The allele \(P^{\text{\text{\textdegree}}39991}\) was used to make pcIpR-\(P^{\text{\text{\textdegree}}39991}\)-timm. The mutation was identified in \(\lambda c\Gamma 72\) mutants that were able to form plaques on the 594 _grpD55_ host, and is identical to the \(\pi\)A7 mutation previously sequenced [73]. This R137Q single base change partially suppressed P-Iethality and fully suppressed plasmid loss at 37-39 \({}^{\text{\textdegree}}\)C (but not at 42 \({}^{\text{\texttextdegree}}\)C where \(P^{\text{\textdegree}}\) was fully expressed) in the _dnaB\({}^{+}\)_ host. The lethality of \(P^{\text{\textdegree}}\) significantly increased at 37-39 \({}^{\text{\textdegree}}\)C in the _grpD55_ host even though plasmid retention remained near 100%. Cells with pcIpR-\(P^{\text{\textdegree}39991}\)-timm (or pcIpR-\(P^{\text{SPA}}\)-timm) could complement the amber mutation in \(\lambda imm434\)Pam3. We asked if co-expression of _O-P_, or if the introduction into the plasmid of the coding sequence for the CII antisense micro-RNA termed OOP, was able to suppress P-Iethality and plasmid loss (Table 7). \begin{table}
\begin{tabular}{l c c c c c} \hline
**Plasmids in 594** & \multicolumn{4}{c}{**Cell viability and (plasmid retention/cfu; \%)**} \\
**host cells** & \multicolumn{4}{c}{**at growth temperature (\({}^{\text{\textdegree}}\)C)**} \\  & **30** & **35** & **37** & **39** & **42** \\ \hline pcIpR-\(P\)-timm & 1.0 & 0.32 & 0.01 & 0.008 & 0.07 \\  & (35/35; 100) & (33/33; 100) & (0/35; 0) & (0/35; 0) & (0/35; 0) \\ pcIpR-\(O\)-\(P\)-timm & 1.0 & 0.345 & 0.12 & 0.12 & Nd \\  & (62/70; 89) & (28/70; 40) & (0/70; 0) & (0/70; 0) & \\ pcIpR-\(O\)-36\(P\)-timm & 1.0 & 0.825 & 0.793 & 0.012 & Nd \\  & (30/30; 100) & (30/30; 100) & (30/30; 100) & (1/30; 3) & \\ pcIpR-\(O\)-63P-timm & 1.0 & 0.895 & 0.895 & 0.055 & Nd \\  & 30/30; 100) & (30/30; 100) & (30/30; 100) & (14/40; 35) & \\ pcIpR-\(O\)-timm & 1.0 & 1.0 & 1.0 & 1.0 & 0.608 \\  & (30/30; 100) & (30/30; 100) & (30/30;100) & (30/30; 100) & (29/30; 97) \\ p434’pR-\(O\)-timm \({}^{\text{\text@underline{b}}}\) & 1.0 & 1.0 & 1.0 & 1.0 & 0.615 \\  & (26/30; 87) & (29/30; 97) & (28/30; 93) & (26/30; 87) & (26/30; 87) \\ pcIpR-\(oop\#1\)-\(O\)-\(P\)-timm \({}^{\text{\text@underline{c}}}\) & 1.0 & 0.938 & 0.20 & 0.005 & Nd \\  & (120/120; 100) & (120/120; 100) & (98/101; 97) & (115/120; 96) & \\ pcIpR-\(oop\#2\)\(O\)-\(P\)-timm & 1.0 & 0.988 & 0.055 & 0.048 & Nd \\  & (117/120; 98) & (60/60; 100) & (76/154; 49) & (62/120; 52) & \\ \hline \end{tabular}  
* As described for Table 6, except stationary phase cultures were grown up at 30 and not 25 \({}^{\text{\texttextdegree}}\)C.
* Plasmid has constitutive expression of \(O\).
* The viability results represent the average for four independent plasmid isolates, each single experiments. The results in parentheses, for plasmid retention by survivor cfu, sums results for all the cfu’s assayed from the four isolates. The cell viabilities at 30 and 35 \({}^{\text{\textdegree}}\)C were at or very near unity.  
\end{table} 
Table 7: Influence of \(O\), \(P\) and OOP-RNA expression on cell killing and plasmid loss. \({}^{\text{\text@underline{a}}}\)pclpR-_oop#1-O-P_-timm: 37'C (0.34, 0.49, 0.006, 0.008), 39'C (0.01, 0.006, 0.0006, 0.0015); pclpR-_oop#2-O-P_-timm: 37'C (0.01, 0.01, 0.12, 0.08), and 39'C (0.01, 0.01, 0.09, 0.08).