# Implementation Studies of Bacteriophage \(\lambda\) O
## 1 Introduction
Most all the RK\({}^{-}\) mutants were obtained before the possibility for PCR amplification of a mutated region of the chromosome, which enables direct sequence determination of the RK\({}^{-}\) mutation. Hence, those RK\({}^{-}\) mutations that have been characterized depended mainly upon genetic analysis using phage mapping and complementation. Figure 1: Replicative Killing, RK\({}^{+}\) phenotype and selection for RK\({}^{-}\) mutants. **(A)** Defective prophage strains were made where the _int-kil_ or _int-ral_ genes of \(\lambda\) were substituted with the _bio_275 or _bio_10 regions of specialized transducing phage to remove a phenotype termed “killing to the left”, dependent on _kil_[23]. The starting cells included the _chlA_ deletion \(\Delta\)434 that removed all of the late genes, i.e., cell lysis, head and tail for \(\lambda\)[11]. These constructs include (i) an active _immt_\(\lambda\) region with gene _cl_[Ts]857  
encoding a repressor that blocks transcription from promoters \(pL\) and \(pR\) along with the _cro_ repressor just right of \(pR\), and (ii) the _repA_ region that includes genes \(O\) and \(P\) and the _oriA_ target for replication initiation from the \(\lambda\) genome. The genome for strain Y836, shown, has the _bio\({}^{+}\)_ operon to the left of the \(\lambda\) fragment and \(\Delta 431\)deletion to the right; (**B**) As long as strain Y836 maintains CI repressor activity the cells can grow normally without gene expression from the repressed \(\lambda\) fragment; (**C**) When the cells are shifted to growth conditions where the CI[Ts] repressor loses its ability to block transcription from \(pL\) and \(pR\) the remaining \(\lambda\) genes become derepressed, the phage replication initiation genes \(O\) and \(P\) are expressed and rounds of replication initiation arise from _oriA_. The \(\lambda\) replication forks extend bidirectionally into the adjacent regions of the _E. coli_ genome, likely colliding with _E. coli_ replication forks. The event is highly lethal to the cell because the \(\lambda\) fragment has no mechanism for excision from the genome and was termed Replicative Killing [18]; (**D**) When cells with a conditionally repressible defective \(\lambda\) prophage are shifted from growth at 30 \({}^{\circ}\)C to 42 \({}^{\circ}\)C the Replicative Killing, RK\({}^{+}\), phenotype is triggered, resulting in cell death. Rare mutations that suppress the loss of \(\lambda\) replication control are selected as RK\({}^{-}\) clones capable of colony formation at 42 \({}^{\circ}\)C. These survivor CFU have lost the capacity for \(\lambda\) replication. This strategy is based on the PDS selection [15], where an intact prophage is made \(N\)-defective, so that expression of _int-xis_ and late/cell lysis gene expression is limited without \(N\)-antitermination of \(pL\) and \(pR\) transcription upon prophage induction. There are many possibilities for RK\({}^{-}\) mutants; (**E**) Cells acquiring defects in host genes participating \(\lambda\) replication are termed RK\({}^{-}\) Hd\({}^{-}\). For example, the GrpD55 mutation in _dnaB_ is of this type, though not isolated as shown [24; 25]; (**F**) A marker rescue recombination assay is used to determine if the _innmA_ regions genes and target sites remain functional (i.e., FI\({}^{+}\)) when substituted for the _innm_434 region of a hybrid phage. The FI assay scores for the activity of the \(pR\) promoter, but in practice it is a good indication of whether the \(\lambda\) fragment in Y836 cells was partially or fully deleted. An example of the deletion endpoints of RK\({}^{-}\) FI\({}^{-}\) mutants from Y836 is shown [26; 27; 28]; (**G**) It was found that brief pretreatment RK\({}^{+}\) of cells held at 30 \({}^{\circ}\)C with a mutagenic substance, prior to shifting them to 42 \({}^{\circ}\)C increases the frequency of RK\({}^{-}\) mutants. This assay, termed the RK Mutatest, proved very sensitive due to the rather large target potential for RK\({}^{-}\) mutants [29; 30; 31].