Isolation, characterization, and action of colicin M

Abstract

Colicin M was isolated from Escherichia coli K-12 32T 19F/T1. The purified, biologically active protein had a molecular weight of 27,000. It contained phosphatidyl ethanolamine. The molecular weight found for the polypeptide chain by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was 18,000. Colicin M was found to be firmly integrated in the membrane of the producing strain. The action of the colicin seems to be on the membrane, since cells of the susceptible strain E. coli K-12 ROW/V/22.1 lyse rapidly. Using the phase contrast microscope, lysis was followed by decrease in turbidity of the cell culture and release of protein into the medium. Lysis started at about 15 min after addition of colicin M and was completed after 40 to 60 min. At this time, one-third of the protein had been released from the cells. The number of viable cells dropped within 10 min to 0.01%. Colicin M induced formation of spheroplasts in the presence of 16% sucrose. The electron microscope examination revealed that at first bulges in the cell envelope appear, most frequently occurring equatorially but also occurring at sites all over the cell. In the process of spheroplast formation, the cytoplasmic membrane often retreats from one-half of the outer membrane so that the cytoplasm is confined to one hemisphere. Sucrose did not prevent cells from dying unless cells were pregrown in a sucrose containing medium for several generations before colicin M was added. With cells pregrown in the presence of sucrose, the number of survivors was 100 times higher than in the absence of sucrose.