ENZYMOLOGICAL CHARACTERIZATION OF THE NUCLEASE DOMAIN FROM THE BACTERIAL TOXIN COLICIN E9 FROM ESCHERICHIA-COLI

The cytotoxicity of the bacterial toxin colicin E9 is due to a nonspec ific DNase that penetrates the cytoplasm of the infected organism and causes cell death. We report the first enzymological characterization of the overexpressed and purified 15 kDa DNase domain (E9 DNase) from this class of toxin. CD spectroscopy shows the E9 DNase to be structur ed in solution, and analytical ultracentrifugation data indicate that the enzyme is a monomer. The nuclease activity of the E9 DNase was com pared with the well-studied, non-specific DNase I by using a spectroph otometric assay with calf thymus DNA as the substrate. Both enzymes re quire divalent metal ions for activity but, unlike DNase I, the E9 DNa se is not activated by Ca2+ ions. Somewhat surprisingly, the E9 DNase shows optimal activity and linear kinetics in the presence of transiti on metals such as Ni2+ and Co2+ but displays non-linear kinetics with metals such as Mg2+ and Ca2+. Conversely, Ni2+ and other transition me tals showed poor activity in a plasmid-based nicking assay, yielding s ignificant amounts of linearized plasmid, whereas Mg2+ was very active , with the main intermediate being open-circle DNA. The results sugges t that, on entry into bacterial cells, the E9 DNase is likely to exhib it primarily Mg2+-dependent nicking activity against chromosomal DNA, although other metals could also be utilized to introduce both single- and double-strand cleavages.