Domains of colicin M involved in uptake and activity

Abstract

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.

The Crystal Structure of the Lipid II-degrading Bacteriocin Syringacin M Suggests Unexpected Evolutionary Relationships between Colicin M-like Bacteriocins

“…The translocation domain of colicin M spans the N-terminal 37 amino acids, with binding to TonB mediated by the first 7 amino acids, which constitute the TonB box. Mutation or deletion of the TonB box results in a protein that does not possess cytotoxicity but is not deficient in receptor binding or catalytic activity (46). A similar loss of cytotoxicity is also observed for ⌬10 syringacin M, so it is reasonable to postulate that this region of syringacin M contains a binding box that is equivalent in binding the TonB orthologue of P. syringae.…”

Crystal Structure of Colicin M, a Novel Phosphatase Specifically Imported by Escherichia coli>

“…Mutations in the TonB box (L4N, L4P, V6R, V6G, and V6E) inactivate colicin M. Sensitivity to colicin M is restored in cells in which the mutation V6R is combined with the chromosomal TonB mutation Q160L. Suppression of the V6R mutation by the Q160L mutation suggests interaction of colicin M with TonB through regions in which these mutations are localized (6). In FhuA, the TonB box is not visible (32,33), but cocrystals of FhuA with a C-proximal fragment of TonB (residues 158 -235) reveal a FhuA N-proximal ␤-strand (TonB box) bound to a three-stranded ␤-sheet of TonB (8).…”

“…2C), a small ␤-sheet (␤1, ␤2, ␤6, and ␤7) connects the two domains by hydrogen bridges and may contribute to the overall stability of the protein. A mutant in this region, R115C, is defective in colicin M uptake (6).…”

“…These functional domains have been assigned based on the following findings. Mutations in the TonB box (residues 2-8) abolish colicin M uptake (6). Colicin M lacking a 5-kDa N-terminal fragment binds to cells via the receptor FhuA but does not enter cells (23), and this fragment kills cells when it is translocated into the periplasm via osmotic shock, which bypasses the uptake system (24).…”

“…Removal of the C-terminal Lys-Arg residues by carboxypeptidase B inactivates colicin M (23). Finally, randomly generated inactive point mutants are located in the C-domain (6).…”