## 2 Results
### P Expression Stimulates Selection for Rifampicin-Resistant (Rif\({}^{\text{R}}\)) CFU  
To examine if the influence of \(P\) expression could be duplicated using a different scheme, we switched from screening for auxotrophs to a direct forward selection for rifampicin resistant (Rif\({}^{\text{R}}\)) CFU. The influence of \(P\) expression was measured in cells derepressed for the (_clIII-ren_)\(\lambda\) fragment, or 594 cells transformed with plasmid pcIPR-_P_-timm (Figure 1C) in Table 4. When \(P\) is expressed in cells with a _dnaB\({}^{+}\)_ allele there is an enormous increase in the number of selected Rif\({}^{\text{R}}\) CFU. Inactivation of \(P\) by insertion, deletion, or alteration by a point mutation (_P\({}^{n39991}\)_) nullified the mutator effect of P expression, as did replacing _dnaB\({}^{+}\)_ with the _dnaB_grpD55 allele. Both the prophage and plasmid data suggest that a P-DnaB interaction is involved in the P-mutator phenotype. When the RK\({}^{+}\) phenotype is induced (_i.e._, combining _cis_-killing with _trans_-P-tethality) the frequency of Rif\({}^{\text{R}}\) mutants was stimulated by >20-fold over situations with _trans_-P-tethality, but lacking _cis_-killing. For example, in Table 4: divide the Rif\({}^{\text{R}}\) stimulation factors 88,727, 42,500, or 51,485 for the \(O^{+}\)\(P^{+}\) RK\({}^{+}\) strains Y836 and 594::(_clIII-cl857-O^{+}-P^{+}-ren_)\(\lambda\) by the factor of 2167 for the \(P^{+}\) strain defective in \(O\), _i.e._, Y836 O223a \(P^{+}\). Mutant \(O\)223a carries a deletion in \(O\) resulting in a frameshift, and a premature nonsense codon that may evoke polarity for the expression of downstream \(P\). The 20-fold enhancement in _rpoB_ mutagenesis when the RK\({}^{+}\) phenotype is induced requires further explanation. Do higher levels of P yield progressively higher mutagenic consequences? Additionally, further properties of the DnaBgrpD55 protein, _i.e._, beyond its ability to block _oriA_ replication initiation [71] and to nullify P- lethality [35], remain to be explored. Two fluctuation assays (FA) were employed to determine if the Rif\({}^{\text{R}}\) mutations arising in _rpoB_ preexisted the induction of \(P\) expression. In FA#1, we grew up 40 FA tubes of cells for 26-27 generations to saturation at 25 \({}^{\circ}\)C (without any \(P\) expression) [77]. From each of the 40 tubes, parallel aliquots were removed (each aliquot representing 1\(-\)2 \(\times\) 10\({}^{8}\) CFU) and spread on RM+100 \(\mu\)g/mL rifampicin plates (Table 5). One plate from the split aliquots was incubated at 25 \({}^{\circ}\)C and the other at 37 \({}^{\circ}\)C (which permitted partial expression of \(P\) from the plasmid). The 80 incubation plates yielded between 0 CFU (observed for 45 plates) to 11 Rif\({}^{\text{R}}\) CFU per plate. Aliquots taken from 23 of the 40 tubes yielded Rif\({}^{\text{R}}\)CFU at either 25 \({}^{\circ}\)C or 37 \({}^{\circ}\)C, or on both plates. Thirty individual Rift1 CFU were subcloned and the _rpoB_ gene from each mutant was sequenced. Eighteen of these CFU were selected from the 25 \({}^{\circ}\)C plates and 12 CFU were from the 37 \({}^{\circ}\)C plates. Twenty distinct _rpoB_ mutation sites were identified within the 30 Rift1 CFU. One mutation (1691, P564L) occurred four times, four arose two times, and the remainder were separate mutational events. Only one FA tube yielded the same _rpoB_ mutation (P564L) for the parallel 25 \({}^{\circ}\)C and 37 \({}^{\circ}\)C selections. Because different mutations occurred in the cells incubated at 37 \({}^{\circ}\)C compared to their counterparts at 25 \({}^{\circ}\)C in almost all cases, and because very few Rift2 colonies were recovered on each plate, the mutations have most likely occurred long after the culture was split into two aliquots and spread, _i.e._, the Rift2 mutations mostly arose during growth on the spread plates, where one set of cells was exposed to some P (plates incubated at 37 \({}^{\circ}\)C) and the other set was not (plates incubated at 25 \({}^{\circ}\)C).