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Analysis of the nucleotide sequence of an Escherichia coli colicin S4 determinant revealed 76% identity to the pore-forming domain of the colicin A protein, 77% identity to the colicin A immunity protein, and 82% identity to the colicin A lysis protein. The N-terminal region, which is responsible for the Tol-dependent uptake of colicin S4, has 94% identity to the N-terminal region of colicin K. By contrast, the predicted receptor binding domain shows no sequence similarities to other colicins. Mutants that lacked the OmpW protein were resistant to colicin S4.

The hydrophobic C terminus of pore-forming colicins associates with and inserts into the cytoplasmic membrane and is the target of the respective immunity protein. The hydrophobic region of colicin U of Shigella boydii was mutated to identify determinants responsible for recognition of colicin U by the colicin U immunity protein. Deletion of the tip of the hydrophobic hairpin of colicin U resulted in a fully active colicin that was no longer inactivated by the colicin U immunity protein. Replacement of eight amino acids at the tip of the colicin U hairpin by the corresponding amino acids of the related colicin B resulted in colicin U(575-582ColB), which was inactivated by the colicin U immunity protein to 10% of the level of inactivation of the wild-type colicin U. The colicin B immunity protein inactivated colicin U(575-582ColB) to the same degree. These results indicate that the tip of the hydrophobic hairpin of colicin U and of colicin B mainly determines the interaction with the corresponding immunity proteins and is not required for colicin activity. Comparison of these results with published data suggests that interhelical loops and not membrane helices of pore-forming colicins mainly interact with the cognate immunity proteins and that the loops are located in different regions of the A-type and E1-type colicins. The colicin U immunity protein forms four transmembrane segments in the cytoplasmic membrane, and the N and C termini face the cytoplasm.

The uptake of colicin U into sensitive cells is dependent on the Tol system. Like colicin A and the E-type colicins, colicin U contains the consensus pentapeptide DG(T/S)G(S/W) in the N-terminal 36 amino acids, which have been proposed to be involved in the interaction of colicin U with the Tol proteins. The role of the DGTGW motif in colicin U was studied by converting it into the TonB box motif DTMVV of colicin B. Uptake of the mutated colicin U (DTMVV) depended on TonB and ExbB and still remained TolA- and TolQ-dependent, but no longer required TolB. The Ton system could obviously replace the TolB function in the uptake of the mutated colicin U. The colicin U derivative is the first colicin whose uptake depends on the Ton and the Tol systems.

Uptake of a new colicin, colicin 10 (Col10), into cells of Escherichia coli required TonB, ExbBD (Ton system), but its cognate receptor, Tsx, functioned independently of Ton and TolQRAB (Tol system). Uptake of Col10 also required TolC which is unique for a Ton-coupled translocation through the outer membrane. A 2470 bp DNA fragment from the natural plasmid pCol10 encoding the Col10 activity (cta), immunity (cti) and lysis (ctl) genes was sequenced. The Cta, Cti and Ctl proteins, as deduced from the nucleotide sequences, consisted of 490 (M(r) 53,342), 96 (M(r) 11,586) and 43 (M(r) 4484) amino acid residues, respectively. Col10 (Cta) was highly homologous to colicin E1 in two regions which determined the common TolC requirement for uptake and the pore-forming activity. Col10 and E1 differed entirely in the regions which are predicted to determine the Ton dependence of Col10 and the Tol dependence of E1, and binding to the receptors Tsx and BtuB, respectively. The region responsible for the Ton-dependent uptake of Col10 was localized in the sequence ranging from residues 1 to 43 (Ton region), and the region responsible for the Tol-dependent uptake of colicin E1 extended from residues 1 to 34 (Tol region). Each Tol-dependent colicin contained a pentapeptide homologous to the sequence DGSGS in the Tol region of E1 which is proposed to be implicated in Tol-dependent uptake (TolA box). After the exchange of the Ton and the Tol regions between Col10 and E1, the Col10-E1 fusion protein was carried into cells via the Ton system and BtuB, whereas the E1-Col10 fusion protein was imported via the Tol system and Tsx. Although the immunity proteins of Col10 and E1 displayed a low homology, Cti conferred full immunity to E1, in contrast to the immunity protein of E1 which did not protect cells against Col10. It is proposed that Col10 belongs to the colicin E1, Ia, Ib group as opposed to the colicin A, B, N group of pore-forming colicins. Col10 consists of 4 domains of which two are very similar and two are very different to E1, supporting our previous proposal that colicins evolved by recombination of DNA fragments which encode uptake and activity domains.

This article intends to inform a broader audience on a fascinating class of protein toxins (bacteriocins) which usually kill only cells of the same species. Those who gained a deeper interest in bacteriocins can find a comprehensive description of the field in a recent book based on a conference (James et al. 1992), and in more specialized review articles dealing with certain aspects (Pugsley 1984a, b), or certain colicins (De Graaf and Oudega 1986; Harkness and Olschläger 1991; Lazdunski et al. 1988). The older literature has been reviewed by Brandis and Smarda (1971), Reeves (1972), Hardy (1975) and Konisky (1982).

Colicin M inhibits murein biosynthesis by interfering with bactoprenyl phosphate carrier regeneration. It belongs to the group B colicins the uptake of which through the outer membrane depends on the TonB, ExbB and ExbD proteins. These colicins contain a sequence, called the TonB box, which has been implicated in transport via TonB. Point mutations were introduced by PCR into the TonB box of the structural gene for colicin M, cma, resulting in derivatives that no longer killed cells. Mutations in the tonB gene suppressed, in an allele-specific manner, some of the cma mutations, suggesting that interaction of colicin M with TonB may be required for colicin M uptake. Among the hydroxylamine-generated colicin M-inactive cma mutants was one which carried cysteine in place of arginine at position 115. This colicin derivative still bound to the FhuA receptor and killed cells when translocated across the outer membrane by osmotic shock treatment. It apparently represents a new type of transport-deficient colicin M. Additional hydroxylamine-generated inactive derivatives of colicin M carried mutations centered on residues 193-197 and 223-252. Since these did not kill osmotically shocked cells the mutations must be located in a region which is important for colicin M activity. It is concluded that the TonB box at the N-terminal end of colicin M must be involved in colicin uptake via TonB across the outer membrane and that the C-terminal portion of the molecule is likely to contain the activity domain.