## 4 Materials and Methods
### Moving Chromosomal Fragments by PI and Complementation for P  
Every P1 vir lysate was serially passaged twice on the donor strain to avoid any carry-over of markers before being used to move gene fragments into recipient cells. Strain CAG12147 _nadA57::Tn10_ possesses a tetracycline resistance (Tet\({}^{\text{R}}\)) marker at 16.85 min on the _E. coli_ linkage map. [PERSON] moved into Y836 _his\({}^{+}\)_. A second step was to co-transduce the Tet\({}^{\rm R}\) marker plus the cryptic \(\lambda\) prophage genes (_cIII-ren_)\(\lambda\) into two recipient prototrophic strains 594 and W3101. Tet\({}^{\rm R}\) recipients were screened for the _imm_A fragment using the functional immunity (FI) recombination assay [71], Strain CAG12164 has a _malF:_Tr10 Tet\({}^{\rm R}\) marker at \(\sim\)91.5 min. The Tet\({}^{\rm R}\) marker was moved by transduction into DE407 _lexA3_[Ind] recipient and the Tet\({}^{\rm R}\) CFU were examined for increased sensitivity to UV to confirm that they retained the _lexA3_[Ind] allele. Both Tet\({}^{\rm R}\) and _lexA3_[Ind] alleles were transduced from DE407_malF:_Tr10_ into recipient strain Y836, which represents _bio275_[substituting for _int-kil_]_-_clIII-ren_-\(\Delta\)431 (Figure 1A). The Tet\({}^{\rm R}\) CFU were screened for acquisition of _lexA3_[Ind] by assaying for increased UV sensitivity. Note that the _bio275_ addition in Y836 confers UvrB\({}^{+}\) and Bio\({}^{+}\) phenotypes to cells with deletion \(\Delta\)431. The presence of _imm_\(\lambda\) phenotype was confirmed by the CFU being lysed by _xvir_ and resistant to \(\lambda\)_cl7_2 at 30 \({}^{\circ}\)C. Strain SF2006 has a _dimB:_Tr5 at 5 min and can form CFU on plates with kanamycin. A similar approach was used to introduce _umu_C112:Tr5 into strain Y836. The ethylmethanesulfonate (EMS) spot assay was performed on the Kan\({}^{\rm R}\) transductants to verify movement of the _dimB_ allele into Y836. Kan\({}^{\rm R}\) transductants were grown overnight, washed twice and 0.1 mL aliquots and spread onto MM+His plates in quadruplet. Two of the four plates were spotted with 5 \(\mu\)L concentrations of EMS, incubated 48 h at 30 \({}^{\circ}\)C, and compared. Cells defective for DinB mutator polymerase gave fewer auxotrophs in region of EMS spot (compared to DinB\({}^{+}\) cells) as the concentration of EMS was increased. The _rpoB\({}^{+}\)_ gene was moved by P1_vir_ transduction from Tet\({}^{\rm R}\) donor strain CAG18500 _thiC:_Tn10_ into Rif\({}^{\rm R}\) isolates of 594. Tet\({}^{\rm R}\) CFU were isolated and Tet\({}^{\rm R}\) Rif\({}^{\rm S}\) co-transductants (occurring at about 80%) were distinguished by stabbing on agar plates containing 100 \(\mu\)g/mL of rifampicin. The phage _\(\lambda\)imm_434 _P_am3 carries the amber mutation CAG(Gln)39786 TAG and can only form plaques on host cells that can complement for P, or on a host with a nonsense suppressor mutation, as TC600 \(supE\). For complementation assays the phage is diluted into \(\Phi\)80 buffer and aliquots were mixed with 0.3 mL of assayed host cells plus top agar and poured onto RM plates incubated between 25 \({}^{\circ}\)C to 42 \({}^{\circ}\)C. The efficiency of plating (EOP) for assay conditions is compared to parallel plates where TC600 is the permissive host (assumed to give a %EPO of 100) and 594 the non-permissive host, upon which \(P^{+}\)-revertant phages arise at a frequency of about 1 \(\times\) 10\({}^{-7}\). Complementation for \(P\) expression from plasmid pclpR-\(P\)-timm was described in reference [35]. Some examples include (%EOP at 38-39 \({}^{\circ}\)C/ 30 \({}^{\circ}\)C): Y836 (65/0.05), Y836 ir\(53\)c (41/0.02), Y836 ir\(O\)208b (67/0.001), Y836 ir\(O\)223a (35/0.008), and Y836 ir\(P\)::IS2 (0.0002/0.00008). \"ilr\" represents a characterized \(\lambda\) mutation conferring \"initiation of \(\lambda\) replication\" defective phenotype.