Structural and functional properties of colicin M

Abstract

Colicin M of Escherichia coli C1139 was isolated in pure form. It consisted of a single polypeptide with a molecular weight of 27,000 ± 2,000. Colicin M lysed sensitive cells of E. coli but had to act continuously up to the point when lysis commenced (after 20 min). Colicin M was largely resistant to hydrolysis by trypsin except when adsorbed to cells. Within 4 to 5 min after addition of colicin M, cells could be rescued by trypsin or sodium dodecyl sulfate. Later, colicin M was apparently inaccessible to these inactivating agents. Killing of cells by colicin M required Ca2+ ions. Cells could be rescued with ethylene glycol-bis(f-aminoethyl ether)-N,N'-tetraacetate (EGTA) immediately before the onset of lysis. Under these conditions, colicin M remained bound to the cells, and it became again sensitive to trypsin. We conclude that under the influence of EGTA colicin M is removed from its site of action and becomes again accessible to trypsin at the cell surface. Colicin M was originally defined on the basis that resistant cells were cross-resistant to phages Ti and T5 (6). Later it was shown that colicin M causes lysis of sensitive cells (2). Both observations were of interest since the uptake of the toxic protein required the function coded by the

Monoclonal antibodies against the FhuA (TonA) protein of the Escherichia coli outer membrane

“…Phage T1, T5, 4~80, BF23, and TuIb were from our stock. Colicins B and M were isolated in pure form according to published procedures [4,5]. Lipopolysaccharide was obtained from K. Hantke and was prepared as described by Galanos et al [6].…”

The biology of colicin M

“…This is ur, usuai as the synthesis of most colicins can be induced by mitomycin C or other activators of the host cells SOS response [2]. However, when colicin M is found together with colicin B on the same plasmid (e.g., pColBM-Cl139) [16], its synthesis is mitomycin C inducible [17]. E. coil strains bearing the ColBM plasmid produce up to 103 times more colic'in following mitomycin C induction.…”

Colicin B consists of a single polypeptide chain

“…The amino acid composition of colicin B was determined after hydrolysis for 18 h at ll0°C in 6 N HCI under nitrogen with the Biotronic amino acid analyzer LC6000E. Freeze-dried samples were oxidized with performic acid [1] to determine cy-steine as cysteic acid and methionine as methione sulfone.…”

“…Colicin B activity was determined with strain P8, a fhuA (tonA) mutant of E. coil AB2847 aroB thi tsx malT [1]. Protein concentration was assayed with Fluram [9,10] using bovine serum albumin as standard.…”

“…We have characterized the molecule of colicin M of Escherichia coli [1], its uptake into sensitive cells [1][2][3], and its mode of action [4,5]. The same plasmid of strain Cl139 that encodes colicin M also determines colicin B synthesis and immunity [1]. Cloning the genes for colicin B and M into multicopy vectors indicates spatial linkage between the two (T. Olschl~iger and E. Schramm, this institute).…”