## 2 Results
### Hypothesis of P as Mutator, Evaluating Using Screen for Auxotrophy  
Table 2 reveals that the RM/MM CFU ratio remains at unity for all strains where \(\lambda\)-fragment derepression was blocked by pCI\({}^{+}\) but was elevated when _ori\(\lambda\)_ replication initiation was blocked but transcription of the \(\lambda\) fragment was allowed at 42 \({}^{\circ}\)C. This suggests that the putative mutator effect accounting for the reduction in colony formation on MM plates at 42 \({}^{\circ}\)C is linked to the induction of gene expression from the (_clll-ren_)\(\lambda\) fragment but that it does not require actual replication initiation from _ori\(\lambda\)_. The conclusions from Table 2 are limited to the suggestion that any of the inducible \(\lambda\) fragment gene products could be responsible for the increased recovery of auxotrophs on RM _vs._ MM. However, it is important to insert prior information that is relevant to this experiment: we previously found (Figure 2C in reference [71]) that the addition to strain Y836 of plasmid pCI\({}^{+}\) expressing wild-type _imm\(\lambda\) cl_ repressor, or the addition by transduction of the _dnaBgrpD55 allele, each prevented replication initiation and blocked Replicative Killing when cells with a \(\lambda\) prophage fragment were shifted from 30 \({}^{\circ}\)C to 42 \({}^{\circ}\)C. The plasmid pCI\({}^{+}\) blocked both derepression of \(\lambda\) fragment transcription and replication initiation from _ori\(\lambda\)_. The _dnaBgrpD55 allele fully suppressed Replicative Killing of Y836 cells shifted from 30 \({}^{\circ}\)C to 42 \({}^{\circ}\)C (Figure 2C in [71]), even better than with the pCI\({}^{+}\) plasmid, but did not block the induced expression of the \(\lambda\) fragment genes. In addition, we previously compared cell viability (Table 6 in reference [35]) for 594 _dnaBgrpD55_[pclpR-P-timm] cells grown up in culture at 25 \({}^{\circ}\)C by plating them at 25 \({}^{\circ}\)C (cell viability assumed 1.0), 37 \({}^{\circ}\)C, 39 \({}^{\circ}\)C (viabilities averaged 0.99), and 42 \({}^{\circ}\)C (viability 1.0). This showed that the _dnaBgrpD55 allele did not impart cellular toxicity over this temperature range, and that it could completely suppress the toxicity resulting from \(P\) expression from this plasmid. In comparison, 594[pclpR-P-timm] cells had a viability of 0.001 when plated at 42 \({}^{\circ}\)C. Therefore, it can obliquely be argued from Table 2 that the increase in the RM/MM CFU ratio for the _dnaBgrpD55 cells is more complex than simply equating it to a manifestation of cell toxicity._  
To examine the suggestion that \(\lambda\) gene expression could contribute to the mutator effect, a stab assay was evoked to screen for the proportion of acquired auxotrophic mutations within selected RK\({}^{-}\) CFU, Table 3. This permitted a direct analysis of the linkage and potential co-selection betweenrophe induction and the appearance of auxotrophs within selected RK\({}^{-}\) mutants. Using strain Y836 _his_ as an example, the frequency of RK\({}^{-}\) CFU selected at 42 \({}^{\circ}\)C on RM (for ten independent selections, all values \(\times\) 10\({}^{-6}\)) was: 16.9, 11.2, 13.5, 16.5, 2, 3.5, 1.1, 5.6, 1.3, and 0.9. Individual CFU arising on RM plates incubated at 30 \({}^{\circ}\)C were stabbed to MM plates incubated at 30 \({}^{\circ}\)C to check whether any spontaneous auxotrophs arose within the starting cells. Similarly, _all_ the selected CFU (large or tiny) arising per RM plate(s) that were incubated at 42 \({}^{\circ}\)C and yielded \(\sim\)10 to \(\sim\)100 CFU/selection plate were picked with sterile toothpicks onto two MM+Histidine plates. One stab plate was incubated at 30 \({}^{\circ}\)C and another at 42 \({}^{\circ}\)C to determine if the CFU possessed a Ts auxotrophic mutation, and a parallel set were stabbed to an RM plate incubated at 30 \({}^{\circ}\)C to ensure transfer of cells had occurred.