## 2 Result and Discussions  
### Transformation of Cells by Plasmids Expressing P, Complementation and Immunity Assays  
Plasmid pHB30 (Table 10), encoding an intact lambda _P-ren_ sequence whose expression is regulated by the CI[Ts] repressor, yielded similar transformation results as those reported by [PERSON] _et al._[24], i.e. no transformants were observed at 42 \({}^{\circ}\)C for _dnaB\({}^{+}\)_ hosts, or for several host strains isolated as being defective for \(\lambda\) replication, i.e., _groPA_15, _groP_558, _grpE_280, _grpC_[31]. However two mutants employed in this study, _grpA_80 and _grpD_55 [32] supported pHB30 transformation at 42 \({}^{\circ}\)C at about the same efficiency as at 30 \({}^{\circ}\)C [31]. The _grpD_55 mutation (initial reported _E. coli_ linkage-map position -71.5 min) was re-mapped as an allele of _dnaB_ and was co-transduceable with malF3089::Tn10 at \(\sim\)91.5 min [33]. (We were unable to move the grpA80 mutation into another host.) These results were considered preliminary because pHB30 included several genes (a _cro-O_ gene fusion, \(P\) and _ren_), and because the _grpA80_ and _grpD_55 mutations were not then confirmed to fall within _dnaB_. Table 1 shows that both _grpA_80 and _grpD_55 mutations each possess two missense changes within _dnaB_, one being in common, E426K. The ability of the _dnaB-grpD55_ allele within _E. coli_ strain 594 to inhibit \(\lambda\) vegetative growth is shown in Table 2.  
Plasmid pcIpR-_P_-timm (Figure 1A) includes \(\lambda\) gene _cl_[Ts]857, encoding a thermolabile repressor that is expressed from promoter _pM_, and a precise copy of wild type gene \(P\) DNA, situated downstream from \(\lambda\) promoter _pR_ in exactly the same configuration as is occupied by gene _cro_ in the \(\lambda\) genome. The expression of \(P\) from this plasmid is regulated by the CI[Ts] repressor binding to the _oR_ sequences overlapping _pR_. The expression of \(P\) from pcIpR-_P_-timm was initially assumed similar to that shown for D-CAP or D-SPA gene fusions, as in pcIpR-D-CAP-timm (Figure 4 in [20]), where there was no detected expression from the non-induced gene fusion. The expression of genes \(P\) and _cl_[Ts]857 from pcIpR-_P_-timm was assessed by complementation, measuring the biological activity of \begin{table}
\begin{tabular}{l l l l} \hline _dnaB allele_ & **bp _E. coli_ mutation** & **AA changed** \\ \hline _grpD55_ & 4,263,102 G to A & V256I \\  & 4,263,612 G to A & E426K \\ _grpA80_ & 4,263,349 G to A & G338E \\  & 4,263,612 G to A & E426K \\ \hline \end{tabular}
\end{table} 
Table 1: Sequencing analysis of alleles of _dnaB_. \begin{table}
\begin{tabular}{l|l l l l} \hline \multirow{2}{*}{Host strains} & \multicolumn{3}{c}{EOP on host cells incubated at temperature \({}^{\mathrm{a}}\)} \\ \cline{2-5}  & 30 & 37 & 39 & 42 \({}^{\circ}\)C \\ \hline
594 & 1.0 & nd\({}^{\mathrm{b}}\) & nd & 0.94 \\
594 _grpD55_ & 0.0147 & \(4.4\times 10^{-7}\) & \(4.4\times 10^{-7}\) & \(4.6\times 10^{-7}\) \\ \hline \end{tabular} \({}^{\mathrm{a}}\): Efficiency of plating (EOP) of \(\lambda\)_cl_72 was determined by dividing the average phage titer on 594 _dnaB-grpD55_ host cells by the titer on 594 host at 30 \({}^{\circ}\)C (\(=6.8\times 10^{9}\) pfu/ml). \({}^{\mathrm{b}}\): nd = not determined.  
\end{table} 
Table 2: Inhibition of \(\lambda\)_cl_72 vegetative growth on _dnaB-grpD55_ host. P, and a CI-immunity assay to assess CI[Ts] repression of _imm\(\lambda\)_ phage plating. The complementation assay determined whether P expressed from the plasmid could support the plating of a phage with an amber mutation in \(P\), i.e., the heteroimmune infecting phage \(\lambda\)_imm_434_P_am3, which is insensitive to repression by CI\({}^{\lambda}\). The ability of cells with pclpR-\(P\)-timm to produce CI[Ts] at a sufficient level to turn off \(pR\) transcription from both plasmid or an infecting phage was assessed by its inhibition of \(\lambda\)_cI72_ plating.