The immunity and lysis genes of ColN plasmid pCHAP4

Nucleotide sequencing of part of the plasmid pCHAP4, which encodes the ca. 42,000 Da putative poreforming colicin N, confirmed previous results indicating that the colicin N immunity gene (cni) and the colicin release or lysis gene (cnl) are located immediately downstream from the colicin N structural gene (cna) in the order cna-cni-cnl. The cni gene is transcribed in the opposite direction to cna and probably encodes an Mr 15239 Da protein. The putative immunity protein was detected among the [35S]methionine-labelled proteins produced by minicells carrying cni cloned under lac promoter control, and when the gene was subcloned into expression vectors under the control of a bacteriophage T7 promoter. Deletion of the region immediately upstream from cni completely abolished colicin N immunity, presumably because the natural promoter had been deleted. cnl is in the same operon as cna, and encodes a typical Col plasmid pro-lysis protein comprising a signal peptide and a 34 residue mature polypeptide with high homology to all but one of the other known Col lysis proteins, including the fatty acylated amino-terminal cysteine residue which was specifically labelled with 3H-palmitate. Cell fractionation studies indicated that the cnl gene product was located predominantly in the outer membrane.