# Implementation Studies of Bacteriophage \(\lambda\) O
## 2 Results
### Influence of O-P Co-Expression on Cell Growth, P-Lethality, and Plasmid Loss  
The expression of P, or N-terminal fragments of P, block O complementation (Table 2). If O and P expression can influence O complementation, does their co-expression negate P-lethality? Figure 4 shows that \(O\) expression alone, or the co-expression of WT genes \(O\) and \(P\) over a span of four doublings in culture absorbance (with 45 min per doubling) did not perturb cell growth for cultures shifted from growth at 30 \({}^{\circ}\)C to 42 \({}^{\circ}\)C. In contrast, expressing \(P\) alone, or constructs expressing _oop_-\(O\)-\(P\), or constructs that were WT for \(O\) but could express a portion of the N-terminal region of \(P\) were each highly inhibitory to cell growth. Table 3 shows the effect of \(O\)-\(P\) constructs on cell viability and plasmid loss. We re-examined several of the observations reported in [25], where the expression of \(P\), even in trace levels at 37 \({}^{\circ}\)C, kills about 99% of the transformed cells and all plasmids were lost in survivor CFU's. The lethality of P is completely suppressed by two missense mutations in \(dnaB\) that comprise the allele \(dnaB\)-GrpD55. Expressed by itself, O is not toxic and does not cause plasmid loss. Co-expression of \(O\)-\(P\) reduces the cellular toxicity of \(P\) expression alone by 12 to 15-fold at 37 and 39 \({}^{\circ}\)C. Co-expression of _oop_-\(O\)-\(P\) significantly prevents plasmid loss at 37 and 39 \({}^{\circ}\)C but exerts a minimal effect on cell viability. The inclusion of portions of the N-terminal end of \(P\) plus \(O\) significantly reduces cell viability and plasmid retention at 39 and 42 \({}^{\circ}\)C, compared to the expression of only \(O\).  
Figure 4: Influence of induced \(O\), \(P\) gene expression on cell growth at 42 \({}^{\circ}\)C. All the strains were made by transforming hosts 594 or 594 \(dnaB\)-GrpD55 with pclpR-(..)-timm plasmids that included the cloned \(O\), \(P\) DNA fragment, and selecting the transformants on LBAmp50 agar (medium composition is described in footnote to Table 3). The plasmid inserts in each CFU employed were verified by DNA sequence analysis. Cells were inoculated from overnight cultures grown up overnight in LBAmp50 broth and then 0.4 mL of culture was added to triplicate 20 mL fresh LB cultures that were incubated at 30 \({}^{\circ}\)C for 30 min to reach an A575 = 0.1. Upon reaching an absorbance of 0.1 the cultures were transferred to a shaking 42 \({}^{\circ}\)C water bath. Aliquots were sampled every 30 min for 3 h. The average absorbance is shown, with a standard error for each culture time of less than 5% the averaged absorbance value.  
The _oop_ DNA sequence encodes a 77-base noncoding small RNA that is transcribed in an antisense orientation to \(O\). Part of the _oop_ sequence overlaps the sequence for gene _clI_ preceding \(O\), with the _pO_ promoter for _oop_ transcription overlapping the N-terminal end of the \(O\) sequence. The #1 and #2 _oop-O_ or _oop-O-P_ constructs are deleted for the N-terminal end of cII. Since \(O\) or \(O\)-\(P\) are directly transcribed from _pR_ on the plasmid, we asked if the antisense _oop_ transcript would influence O or P activity expressed from the plasmid. Except for its ability to support some low-level suppression of \(\lambda O\)am905 plating, OOP RNA expression does not significantly influence O complementation (Table 2); however, it exhibits a profound effect on the ability of P expression to evoke plasmid loss (Table 3), over and above the quenching influence of \(O\)-\(P\) co-expression on cellular P-Iethality. A hypothesis is that OOP RNA hybridization to the _pR-oop_-O-P mRNA expressed from the induced plasmid reduces downstream \(P\) translation/accumulation within the cell.