# Implementation Studies of Bacteriophage \(\lambda\) O
## 1 Introduction  
Bacteriophage \(\lambda\) prophage is maintained within the chromosome of _Escherichia coli_ cells by its CI repressor protein, which prevents the transcription of \(\lambda\) genes positioned leftward and rightward from promoters _pL_ and _pR_ that straddle _cl_ (Figure 1A). CI binds to operator sites that overlap these promoters. Upon inactivation of CI, the derepressed prophage genes _N-int_ are expressed from _pL_, and genes _cro-clI-O-P-Q_ are expressed from _pR_. Transcription initiated from _pR_ requires gpN activity to proceed effectively past the rho-dependent \(t_{R1}\) termination site positioned between _cro_ and _clI_ (reviewed in [1; 2; 3; 4]). The mechanism for bi-directional initiation of \(\lambda\) DNA replication involves a complex interaction of phage proteins gpO and gpP (designated herein as O and P) with _E. coli_ host DNA replication proteins. In brief, O acts to bind the replicator site, _ori\(\lambda\)_, or origin for replication initiation that is situated midway within the \(O\) sequence [5; 6]. The P protein recruits DnaB, the major replicative helicase for unwinding double-stranded DNA, bringing it to _ori\(\lambda\)_-bound O to form a DnaB:P:O_ori\(\lambda\)_ preprimosomal complex. P can commandeer DnaB away from its cellular equivalent, DnaC [7]. Since the interaction of P with DnaB inactivates the helicase activity of DnaB, the dissociation of P bound to DnaB in the preprimosomal complex is required to restore DnaB activity, which involves _E. coli_ heat shock proteins DnaK, DnaJ, and GrpE [8; 9]. By inhibiting transcription from _pR_, CI blocks a _cis_ requirement for replication initiation described as transcriptional activation, explaining why providing O and P from a superflecting heteroimmune phage will not stimulate replication initiation from an integrated resident _imm\(\lambda\)_ prophage [10; 11]. This requirement for _pR_ transcription can be suppressed by _ri\({}^{\text{C}}\)_ (replicative-inhibition constitutive) mutations which lie outside of _ori\(\lambda\)_[12; 13]. The excision of a \(\lambda\) prophage from the host chromosome between B.P' and P.B' sites (Figure 1A) is dependent upon \(\lambda\) genes _int_ and _xis_ (reviewed in [1; 4; 14]. Their expression requires that gpN antiterminate transcription at \(t_{L}\) terminator signals positioned between _pL-N_ and ahead of _xis_. The PDS selection (refer to Abbreviations, Unique \(\lambda\) Terminology) for cell survivors of \(\lambda\)_Ncl_ ([Ts], temperature sensitive) prophage, named for its inventor [15], takes advantage of the induced prophage being unable to excise from the chromosome or lyse its host cell. In addition, the \(N\) null mutation reduces late \(\lambda\) gene expression and cell lysis, which depend on N for transcriptional antitermination at several \(t_{R}\) sites upstream of the late genes. Examinations for _E. coli_ cell survival using the PDS selection led to the suggestion that mutations preventing the initiation of \(\lambda\) replication suppress cell killing [15; 16; 17; 18; 19; 20; 21]. The induction lethality phenotype for non-excisable prophage was termed Replicative Killing [18]. Accordingly, starting cells with de-repressible, but non-excisable prophage, as in Figure 1A,B, are termed RK\({}^{+}\) (Replicative Killing competent) cells and the selected survivor cells that form CFU at 42 \({}^{\circ}\)C were named Replicative Killing defective (RK\({}^{-}\)) mutants (Figure 1C-G). The concept evolved that the starting cells possess the capacity for \(\lambda\) replication initiation upon prophage induction, whereas the survivor clones do not. The results of these early studies were reviewed [22]. Only a few mutations conferring the RK\({}^{-}\) phenotype for survivor CFU derived from the PDS selection or those from the N\({}^{+}\)\(\lambda\) fragment strains (an example is shown in Figure 1) have been characterized by DNA sequence analysis.