Structure and expression of the ColE2-P9 immunity gene

Abstract

The primary structure and expression of the ColE2-P9 immunity gene (imm) were investigated. The imm gene is located behind the colicin gene (col) in the same orientation with an intergenic space of two base pairs. Although the imm gene was transcribed primarily in response to the SOS function of the host cell as well as the col gene, the immunity phenotype also appeared to be expressed by only a slight level of leaky transcription without an evident promoter. On comparing the ColE2-P9 sequence with those of relevant plasmids, a highly homologous sequence with the immE2 gene was found downstream of the immE3 gene of ColE3-CA38, and thus, an evolutional relationships could be deduced among some E-group Col plasmids.

Colicin E8, a DNase which indicates an evolutionary relationship between colicins E2 and E3

“…These E2 and E3 operons have nearly identical nucleotide sequences except in the regions responsible for nuclease activities and immunities. Extensive sequence homology to the E3 operon was also shown for the cloacin operon of plasmid CloDF13 (3,16,25).We identified an open reading frame between the immE3 and lysis genes of ColE3-CA38 which is highly homologous to the ColE2-P9 immunity gene (12,17) To confirm these assumptions, we determined the nucleotide sequence of the downstream half of the colicin.E8 operon and showed that E8 is a DNase-type colicin that is highly homologous to E2. …”

“…Cells were grown at 37°C in L broth supplemented with 0.5% glucose and/or 50 pg of ampicillin per ml when required. Plasmid preparation, cell transformation, and restriction analysis were performed as described previously (16,17).…”

“…10,000). The immunity proteins are specific inhibitors of the respective colicin nucleases and protect the colicinogenic host cells from the killing action of both extra-and intracellular colicin molecules (16,17).The colicin (cob), immunity (imm), and lysis genes are integrated into an "SOS operon" in both ColE2-P9 and ColE3-CA38 plasmids. These E2 and E3 operons have nearly identical nucleotide sequences except in the regions responsible for nuclease activities and immunities.…”

“…Cells were grown at 37°C in L broth supplemented with 0.5% glucose and/or 50 pg of ampicillin per ml when required. Plasmid preparation, cell transformation, and restriction analysis were performed as described previously (16,17).DNA sequencing. The ClaI-PvuI DNA fragment of ColE8-J was first cloned into the BamHI sites of pUC18 and pUC19; after all the joining sites had been filled in with T4 DNA polymerase (Takara Shuzo, Kyoto, Japan), ligation of the fragments regenerated BamHI sites at both of the cloning ends (Fig.…”

“…10,000). The immunity proteins are specific inhibitors of the respective colicin nucleases and protect the colicinogenic host cells from the killing action of both extra-and intracellular colicin molecules (16,17).…”