A genetic approach to the study of mitomycin-induced lysis of Escherichia coli K-12 strains which produce colicin E2.

Abstract

A 2,250 base pair Sau3A restriction digest fragment of the colicin plasmid ColE2-P9 was shown to contain all the information necessary for colicin production, immunity to colicin E2 and mitomycin-induced lysis in host cells. Transposon Tn5 insertions in the colicin structural gene (ceaB) completely abolished colicin production and mitomycin-induced immunity protein production and lysis. This indicates the existence of a SOS-regulated colicin E2 operon in which ceaB:: Tn5 mutations exert a polar effect on genes concerened with lysis and production of immunity protein. However, cells harbouring these plasmids were immune to colicin E2, a phenomenon which is probably explained by the existence of an internal, SOS-independent promotor in front of the immunity gene (ceiB). A Tn5 insertion in a site (celB) removed from ceaB and ceiB produced a colicin+ immunity+ lysis- phenotype in host cells treated with mitomycin. These cells produced abnormally low amounts of colicin both in the presence and absence of mitomycin induction. Another difference between these cells and those harbouring wild-type ColE2 plasmids is that the vast majority of the colicin they produced remained within the cells. Cells carrying the wild-type ColE2 plasmid were shown to produce four additional polypeptides when grown in the presence of mitomycin. The two small polypeptides (Mr 5,000 and 3,000) were specifically absent from mitomycin-treated cells harbouring the plasmid with the celB:: Tn5 mutation, whereas all four polypeptides (the other two being the colicin and immunity proteins) were not detected in mitomycin-treated cultures of cells harbouring the plasmids with ceaB:: Tn5 mutations. The possible role of the Mr 5,0000 and 3,000 polypeptides in the regulation of colicin synthesis, cell lysis and colicin release is discussed.