## 2 Result and Discussions
### Contrasting _trans_ P-lethality / inhibition, and _cis_ Replicative Killing  
We showed that shifting culture cells transformed with pcIpR-_P_-timm and grown at 25 'C up to 37 'C for two hrs reduced cell viability to about half, and thus cells are capable of tolerating or metabolizing some level of P, with a reported half-life of up to an hr [79, 80]. However, initiating irreversible _ori_\(\lambda\) replication from a trapped cryptic prophage, Figure 1C, reduced cell viability between 4 and 10-fold after only 20 min of induction. The Replicative Killing (RK\({}^{+}\) phenotype) survivor frequency of 10\({}^{\text{-}6}\) to 10\({}^{\text{-}8}\) is distinguished by rare RK' mutants in host or prophage (not merely in _P_) that block some aspect of \(\lambda\) replication initiation [78, 81-83]. We suggest the terms _cis_ Replicative Killing and _trans_ P lethality to distinguish mechanistically these the two ideas. The RK\({}^{+}\) phenotype seems uniquely dependent upon multiple, non-repairable \(\lambda\) replication forks arising from _ori_\(\lambda\) (drawn in Figure 1C), but there are likely multiple possibilities for P-lethality. Several experiments suggest that the encoded gene _ren_ downstream of \(P\) is not responsible for P-lethality. A modified version of pHB30 (i.e., pHB31) encoding _P-ren,_ but deleted for bases 39609-39836 within \(P\) (equivalent to _P_\({}^{\text{A76}}\) used herein) was fully capable of transforming 594 cells at 42 'C, suggesting that P, not Ren blocked transformation at 42 'C [31]. RK' mutants of Y836 with insertions or deletions (recombinant or natural) within/inactivating \(O\) (ilr208b, ilr223a, ilr541c, ilr200b, ilr203b, ilr207d, ilr201b), or \(P\) (ilr566a, Bib11t), yet which sequenced to be _ren_\({}^{+}\), lost the Replicative Killing competence phenotype when shifted from 30 to 42 'C, suggesting that replication initiation from _ori_\(\lambda\), and not _ren_ expression, \begin{table}
\begin{tabular}{l l l l} \hline \hline
**A.** & **Cells tolerate short-term exposure to P (short-term interference, not lethality) a** \\ \hline Strain with plasmid & Incubation & Cell viability & Outcome of P expression \\  & at 37  °C & (Amp\({}^{\text{R}}\) cfu & from plasmid \\  & & / total cfu) & \\ \hline
594[pcIpR-_P_-timm] & 1 hr & 0.87 (232/242) & most cells recover \\  & 2 hr & 0.49 (258/281) & many cells recover \\  & 6 hr & 0.13 (2/173) & high plasmid loss \\ \hline
**B.** & **Inducing replication from a trapped cryptic prophage causes Replicative Killing a** \\ \hline Strains with cryptic prophage & Prophage & Cell viability & Outcome of prophage \\  & Induction time & & Induction \\ \hline Y836[\(\
eg\)_cIII-cl857-O-P-ren_] & 5 min & 0.33 & rapid cell killing \\  & 20 min & 0.13 & rapid cell killing \\  & 3 hr & 0.00018 & extensive cell killing \\  & 5 hr & 0.00008 & extensive cell killing \\ \hline
594[\(\
eg\)_cIII-cl857-O-P-ren_] & 5 min & 0.55 & rapid cell killing \\  & 20 min & 0.25 & rapid cell killing \\  & 3 hr & 0.0022 & extensive cell killing \\  & 5 hr & 0.0022 & extensive cell killing \\ \hline Y836[\(\
eg\)_cIII-cl857-O-P::kan-ren_] & 3 hr & 5.1 b  & cell growth \\ _ren_] & 5 hr & 6.1 b  & cell growth \\ \hline \hline \end{tabular} a  
\end{table} 
Table 8: Contrasting _trans_ P-lethality / inhibition and _cis_ Replicative Killingis responsible for Replicative Killing; transduction of the \(\lambda\) fragments into 594 did not alter the phenotype observed for Y836 [RK'], or the RK' mutants. Ren is not required for P-lethality phenotype, nor for Replicative Killing, i.e., in the absence of _orin_ replication initiation, _ren_ expression will not produce the RK' phenotype, however, our experiments do not rule out some ancillary role for Ren.