## 3 Discussion
One possibility is that the selected mutants are resistant to the toxic agent. We explored this difficulty in FA#2 by accounting for both toxicity and recovered RifR mutants for each selection temperature. We could demonstrate that many RifR mutants were sensitive to P-toxicity, and, for selections at 36 \({}^{\circ}\)C, which were less toxic than those at 37 \({}^{\circ}\)C, there was a significant increase in the appearance of RifR mutant CFU (see below). A separate manuscript is in preparation on the complementation and examined toxicity of cloned _vs._ defective prophage (single copy) expressed \(\lambda\)_imm-rep_ gene products. Fluctuation assays were used to determine if the RifR mutations arising in _rpoB_ preexisted the expression of \(P\). As noted, this was complicated by observations that 80-fold cellular toxicity occurred in cells with pclpR-\(P\)-timm incubated at 37 \({}^{\circ}\)C, and that most of the RifR CFU were P\({}^{\text{S}}\). On lowering the incubation temperature to 36 \({}^{\circ}\)C, P-Iethality was reduced by half but the recovered RifR mutants increased by nine-fold compared to 30 \({}^{\circ}\)C. With incubation lowered yet another degree to 35 \({}^{\circ}\)C, the toxicity of P was minimal (1.3-fold), ColE1 curing of cells was slight (5%), yet the RifR mutants recovered were 3.9-fold higher than at 30 \({}^{\circ}\)C. These observations suggest that the increase in RifR mutants was dependent upon P expression, rather than selection conditions favoring preexisting PR RifR mutants. Seven of the RifR mutants acquired a PR phenotype. Two of these mutations, G537C and L571Q map close to the Rif binding pocket in RpoB [79], but the remaining mutations Q148L, Q148P, \(\Delta\)GEV440-442V, R451S, and S509R fell outside of the pocket. These mutations may help define a contact point(s) between RpoB and P that has long been suggested [64]. Our PR mutation Q148P in _rpoB_ is identical to rpo*148 [82]. A small collection of rpo* mutations, which possess some level of RifR [82], were isolated based on the discovery [83] that mutations in RNAP alleviate the UV-sensitive phenotype of _ruv_ strains, e.g., rpo*148 suppresses the extreme sensitivity of UV treatment to a \(\Delta\)_rela_\(\Delta\)_spoTruv_ strain. This property was linked to the rpo* mutation destabilizing RNAP open complexes and stalled elongation complexes, thereby reducing the occurrence of stalled RNAP(s) at lesions in the DNA template [82; 83]. Co-directional collisions between the replisome and RNAP in an arrested (backtracked) elongation complex can lead to DNA double strand breaks [84]. Transcription pausing and stalling regulators include ppGppp which destabilizes open complexes, and the RNAP modulators Dska, GreA, GreB, and Mfd [81; 84; 85]. The failure of rpo* RNAP to pause and backtrack, and their ability to reduce the accumulation of RNAP arrays [81], helps explain why rpo* mutations can suppress the formation of DSB [84]. Assuming that our PR Q148P RifR mutation will reduce the accumulation of RNAP arrays, can this explain a PR phenotype and the suppression of P-dependent ColE1 plasmid curing? Future studies require determining if the other PR mutants share an rpo* phenotype. Several unanswered problems arise in relation to the observed mutator effect caused by \(P\) expression. Based on the low \(\lambda\) requirement for P, coupled with P sequestration of the low cellular amount of DnaB [18; 19], either \(P\) expression from \(\lambda\) requires a high degree of negative regulation to prevent P-Iethality/inhibition (probably not fully appreciated), or there is an unknown mechanism for limiting the P\({}^{\text{S}}\) phenotype. The apparent complete escape of \(\lambda\) replication from P is contrasted with the exquisite sensitivity of ColE1 plasmid replication to P, and its mutagenic effect on _E. coli._ One possible explanation is that late \(\lambda\) rolling circle replication may be independent of replication restart.