No abstract

Colicin E8-J and its immunity protein were characterized with regard to their activities and gene structures. Colicin E8 is a complex of proteins A and B; protein A (the naked E8) exhibits an apparently nonspecific DNase activity that is inhibited by protein B (the immunity protein), as in the case of colicin E2. The nucleotide sequence of the downstream half of the colicin operon of ColE8-J was determined to be highly homologous to that of ColE2-P9, with the exception of the hot spot region of the 3'-terminal segment of the colicin gene and the adjacent immunity gene. The immE2-like gene of ColE3-CA38 was, as assumed previously, extensively homologous to the immE8 gene of ColE8-J, and thus, CoIE8-J was shown to be situated between CoIE2-P9 and ColE3-CA38 in the evolution of the E-group Col plasmids. E-group colicins bind to a common surface receptor, BtuB, when attacking sensitive Escherichia coli cells. A colicin-producing strain is not killed by its own colicin or a closely related colicin, a phenomenon referred to as immunity. Based on their immunity specificities, E-group colicins are divided into 10 subclasses, i.e., colicins A and El to E9 (4,14,18,26).The modes of action for some E-group colicins have been elucidated in vitro; colicins A and El are ionophores, E2 is a DNase, and E3 is a special kind of RNase which effectively acts on the 16S RNA of the 70S ribosome. All these in vitro activities are located on the C-terminal portions of the respective proteins. In both E2 and E3, the C-terminal active fragments obtained by tryptic digestion are referred to as T2A (5,15,20,21). Serological and physiological comparisons have suggested that colicins E4 to E7 resemble either E2 or E3 (14, 18, 26). Both E2 and E3, but not A or El, are purified as a complex of a colicin molecule (Mr, ca. 60,000) and an immunity protein (Mr, ca. 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. 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. MATERIALS AND METHODSStrains and plasmids. All the strains used in this work were E. coli K-12 derivatives. The original E8 colicinogenic strain, W3110(CoIE8-J), was obtained from R. James (13). RR1 was used as both a colicin-sensitive indicator strain and a recipien...

The lysis genes of plasmids ColE2-P9 and ColE3-CA38 were identified by DNA sequencing and electrophoretic analysis of the products of both wild type and artificially introduced ochre mutant genes. The E2 and E3 lysis genes had identical primary structures and were shown to encode 47 amino acids with a calculated molecular weight of 4,861, which is much smaller than that proposed previously for the ColE3-CA38 lysis protein. They are homologous with ColDF13 gene H, except in their 3'-portions. The nine C-terminal amino acids of the E2 and E3 lysis proteins proved to be non-essential for the lysis phenotype.

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.