## 2 Results
### Sensitivity of ColE1 Plasmid Replication to P-inhibition  
Culture cells with a _grpD55_ allele of _dnaB_ have a P\({}^{\mathrm{R}}\) phenotype and are refractory to curing of the ColE1-based pclpR-_P_-timm plasmid when grown at 37 \({}^{\circ}\)C or 42 \({}^{\circ}\)C [35], whereas P\({}^{\mathrm{S}}\)_dnaB\({}^{+}\)_ culture cells are cured of pclpR-_P_-timm when incubated at or above 35 \({}^{\circ}\)C. The influence of Rif\({}^{\mathrm{R}}\) mutations on P-dependent ColE1 curing is shown in Figure 5. The Rif\({}^{\mathrm{R}}\) mutants were transformed with pclpR-_P_-timm and examined for retention of the plasmid at 30 \({}^{\circ}\)C, 37 \({}^{\circ}\)C and 42 \({}^{\circ}\)C. The P\({}^{\mathrm{R}}\) Rif\({}^{\mathrm{R}}\) mutants 3-37-C1 and T325C10 were cured of pclpR-_P_-timm during cell growth at 37 \({}^{\circ}\)C or 42 \({}^{\circ}\)C as was the Rif\({}^{\mathrm{S}}\) 594 parent. The remaining P\({}^{\mathrm{R}}\) Rif\({}^{\mathrm{R}}\) mutants 3-37-B1, -B8, -C4, -D2 and -D6 showed varying levels of plasmid retention at 37 \({}^{\circ}\)C and 42 \({}^{\circ}\)C. These mutants were sequenced through _dnaA_ to  
Figure 4: Location of selected Rif\({}^{\mathrm{R}}\) mutations in _rpoB_. Mutants designated \(P^{\mathrm{S}}\) are transformable at 30 \({}^{\circ}\)C but not 37 \({}^{\circ}\)C by pclpR-_P_-timm, whereas the \(p^{\mathrm{R}}\) mutants were transformable at both temperatures. The _rpoB_ mutant designated with \({}^{\star}\) is identical to mutant rpo*148 (see Discussion). The missense mutations (designated by sideways triangle) each fall within codons for amino acids found in the Rif binding pocket of _rpoB_. determine if they possessed an _rpl_ (resistance to P-Iethality) mutation shown to map within _dnaA_[56]. Each P\({}^{\text{R}}\) Rift\({}^{\text{R}}\) mutant was wild type for _dnaA_. The results show that some alleles of _rpoB_, _i.e._, the same ones conferring P\({}^{\text{R}}\), can suppress ColE1 plasmid curing by P.  
The original non-transformed Rif\({}^{\text{R}}\) mutants (shown left column, Figure 5) were transduced to Rif\({}^{\text{S}}\) by P1 transduction of Tet\({}^{\text{R}}\) Rif\({}^{\text{S}}\) from donor cells. The Tet\({}^{\text{R}}\) transductants were screened for replacement of the Rif\({}^{\text{R}}\)_rpoB_ allele with a Rif\({}^{\text{S}}\)_rpoB\({}^{+}\)_ allele and loss of the Rif\({}^{\text{R}}\) phenotype. Each of the Tet\({}^{\text{R}}\) Rif\({}^{\text{S}}\) transductants were sequenced for _rpoB_ and shown to have lost the _rpoB_ mutation originally conferring the Rif\({}^{\text{R}}\) phenotype. All of the sequenced Tet\({}^{\text{R}}\) Rif\({}^{\text{S}}\) transductants were then transformed with pcIpR-_P_-timm at 25 \({}^{\circ}\)C and then examined for plasmid loss after culture cell growth at 30, 37, or 42 \({}^{\circ}\)C. Each of the Tet\({}^{\text{R}}\) Rif\({}^{\text{S}}\) transductants retained the pcIpR-_P_-timm plasmid when grown at 30, but completely lost the plasmid during culture growth at 37 \({}^{\circ}\)C or 42 \({}^{\circ}\)C, Figure 5. These results show that replacement of the Rif\({}^{\text{R}}\) allele with the _rpoB\({}^{+}\)_ allele restores the sensitivity of ColE1 to P.  
The sequenced P\({}^{\text{R}}\) Rif\({}^{\text{R}}\) isolates and their Tet\({}^{\text{R}}\) Rif\({}^{\text{S}}\) transductants (see Figure 5) were also transformed with pcIpR-_P_-timm\(\Delta\)_rpo_ to increase selective pressure from the higher copy plasmid (Table 7 and Figure S1). These results further support the results in Figure 5 showing that some Rif\({}^{\text{R}}\)_rpoB_ alleles, especially those that show a P\({}^{\text{R}}\) phenotype, interfere with the sensitivity of ColE1 to P.  
Figure 5: pcIpR-_P_-timm retention in P\({}^{\text{S}}\) and P\({}^{\text{R}}\) Rif\({}^{\text{R}}\) isolates and for Rif\({}^{\text{S}}\) transductants.