## 4 Discussion
When it was examined whether an _hflA_::kannull mutant would support enhanced CII degradation, 30-fold _less_ CII toxicity was observed at 39 \({}^{\circ}\)C. This suggested that inactivating the _lftA_ locus reduces CII toxicity and its interference with lytic phage growth, which is more in keeping with its effect on FtsH and that CII toxicity can be a factor in \(\lambda\) development. We propose that by reducing some form of CII toxicity, e.g., via enhanced CII degradation, the frequency of \(\lambda\) lysogeny is increased. This is an alternative explanation for the HHA phenotype to one based on enhancing CII activity. What is responsible for CII toxicity? It was shown that the single base substitution G537C responsible for _rpoB_ D2 [37] can completely suppress CII toxicity and plasmid loss at the highest level of _clI_ expression at 42 \({}^{\circ}\)C (with cell viability of 0.95 at 39 \({}^{\circ}\)C and no plasmid loss), while reducing but not eliminating CII complementation at 39 \({}^{\circ}\)C. This suggests that both CII toxicity and plasmid loss depend upon a binding interaction between CII and _E. coli_ RNA polymerase holoenzyme, or a direct interaction with the \(\beta\)-subunit encoded by _rpoB_. The _rpoB_ D2 mutation also confers resistance to cell killing by the highly toxic \(\lambda\) gpP. Even trace expression of \(P\) significantly exceeds the replication-inhibition and plasmid curing levels conferred by CII. For example, minimally expressed \(P\) from plasmid pclpR-P-timm in cells grown at 36 \({}^{\circ}\)C [38] cured the cells of ColE1 plasmids. [PERSON] et al. previously isolated _E. coli rpoB_ mutants S531F and P564L resistant to killing by CII protein [55]. One mutant used, _rpoB_ C1 (P564L), shared an identical mutation as reported by [PERSON] et al. [55] but was not nearly so resistant to CII as were the _rpoB_ D2 cells. [PERSON] et al. [31] found that _E. coli_ growth inhibition was independent of mutations in CII (A30T) [56] or in _rpoA_ (L271E) [57] that abolish transcriptional activation by CII [58] at several positively regulated promoters [59]. They suggested that CII toxicity is independent of its transcription activation function, even given the potential for 294 CII binding sites noted to be present in the _E. coli_ genome [31]. They rationalized, because of the almost immediate effect of CII overproduction, that its inhibitory effect to both _E. coli_ and \(\lambda\) replication was linked to interference with the DNA replication process. Herein, an assay for cellular loss of ColE1 plasmids was employed as an indicator for replication inhibition. The _rpoB_ mutations D2 (R451S) and C4 (\(\Delta\):GEV440-442V) completely abolished plasmid loss when _clI_ was constitutively expressed at 42 \({}^{\circ}\)C and mutation D6 (G537C) was almost as effective. These results suggest that the inhibitory effect of CII on plasmid maintenance involves its interaction and interference at a stage of RNA polymerase participation in replication. RNA transcripts terminated by the _oop_ terminator, _tO_ are reported very labile, compared to transcripts from other terminators [60]. The influence of polyadenylation on _clI_ and _clI-oop_ expression was examined, since it was suggested that polyadenylation by _E. coli_ poly (A) polymerase I (PAP I) leads to destabilization of RNA in bacterial cells [61; 62]. Reports suggest that OOP RNA is polyadenylated by PAP I, that this is impaired in a _pcnB_ mutant [35] and that polyadenylated OOP RNA is rapidly degraded [34] in comparison to non-polyadenylated OOP. CII activity in cells with a _pcnB_ null mutation was examined. [Note that _clI_-mRNA expression terminates at _tlmm_ from [clI] missing _oop-pO_]. Surprisingly, all CII complementation activity was suppressed, the _imm_434_clI_ phages plated efficiently (suggesting any CII made was not inhibiting phage growth) and yet high-level cellular CII toxicity and plasmid loss were evident. It would appear, that cellular capacity for polyadenylation is essential for some aspect of CII complementation.