## 3 Discussion  
The initiation of replication from a trapped \(\lambda\) fragment (Figure 1B) results in backward, or wrong-orientation replication forks that can be problematic for cell viability (44; 48; 80; 81). The leftward fork from _ori\(\lambda\)_ can undergo head-on collisions with the rightward replication fork arising from _oriC_, or with rightward-directed transcription arising, e.g., from five of the seven _rrn_ operons: _rrn_, \(A\), \(B\), \(E\), and \(H\), positioned between _oriC_ and _ori\(\lambda\)_. Such head-on collisions are likely responsible for the powerful RK\({}^{+}\) phenotype. We previously compared the kinetics of cell death resulting from \(P\) expression from pclpR-_P_-timm or Replicative Killing by initiation from a trapped _ori\(\lambda\)_(35). The _cis_-RK\({}^{+}\) phenotype became irreversible by 20 min while _trans_-P lethality/inhibition was slower and reversible for several hours. The RK\({}^{+}\) phenotype (Figure 1B) seems uniquely dependent upon unresolved collisions between replication arising from _ori\(\lambda\)_ and the rightward fork from _oriC_ or with cellular transcripts. Of course, when the trapped \(\lambda\) fragment is induced, both _cis_-RK\({}^{+}\) and _trans_-P-lethality/inhibition phenotypes are jointly produced. Normal \(\lambda\) induction would likely escape the _cis_-RK\({}^{+}\) phenotype because the prophage excises from the chromosome, which is not possible for the defective prophage. The combined lethal effects may amplify the selective pressure for survivors and be responsible for the 20-fold increase in mutants arising compared to situations where only \(P\) is expressed. This extreme selective pressure may account for the very high frequency of co-selected auxotrophs among the selected RK\({}^{-}\) mutants, especially for the RK\({}^{-}\) CFU acquiring a mutation conferring Ts auxotrophy. For example, any co-selected Ts auxotrophic mutation arising in an amino acid synthesis or utilization pathway would inhibit protein synthesis at the limiting temperature and reduce \(\lambda\) fragment gene expression, in turn lowering the buildup of P, and help such RK\({}^{-}\) mutants be selected for colony formation during 42 \({}^{\circ}\)C selection period. As previously noted, RK\({}^{-}\) mutants arising on RM at 42 \({}^{\circ}\)C are defective for _ori\(\lambda\)_ replication and can form a CFU at 42 \({}^{\circ}\)C. Any host mutation that facilitates colony formation during cell growth at 42 \({}^{\circ}\)C could be co-selected. While never previously explored in the literature, co-selected Ts auxotrophs might be particularly supportive in situations where the RK\({}^{-}\) mutation does not inactivate \(P\), for example mutations nullifying \(O\) activity. However, none of the original characterized _O_-defective _P\({}^{+}\)_ RK\({}^{-}\) starting strains employed herein had acquired an auxotrophic mutation when originally isolated (and all were then maintained at non-inducing temperatures to prevent further selective pressure). Nevertheless, a high proportion of auxotrophs appeared from these strains when re-isolated CFU were obtained from 42 \({}^{\circ}\)C plates when _P\({}^{+}\)_ was expressed. Given the subsequent results suggesting that P conferred a mutator effect, we explain the high proportion of auxotrophs (refer to Table 3 section \"Induced defective \(\lambda\) lysogens\") as representing a combination of P mutagenesis combined with a powerful unrealized selection for cellular mutations that limited \(P\) expression toxicity during cell growth at 42 \({}^{\circ}\)C. It is also entirely possible that some of the Rif\({}^{R}\)_rpoB_ CFU selected at 37 \({}^{\circ}\)C (cells with plasmid) or 42 \({}^{\circ}\)C served to reduce cellular toxicity to \(P\) expression. Further investigation is required to explore potential manifestations of P toxicity on alternative mutation selection; however, this may simply be an inherent limitation in all studies wherea mutagenic agent is also toxic, and where toxicity can influence selection.