DOMAINS OF COLICIN-M INVOLVED IN UPTAKE AND ACTIVITY

Colicin M inhibits murein biosynthesis by interfering with bactoprenyl phosphate carrier regeneration. It belongs to the group B colicins th e 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 c olicin M, cma, resulting in derivatives that no longer killed cells. M utations in the tonB gene suppressed, in an allele-specific manner, so me of the cma mutations, suggesting that interaction of colicin M with TonB may be required for colicin M uptake. Among the hydroxylamine-ge nerated colicin M-inactive cma mutants was one which carried cysteine in place of arginine at position 115. This colicin derivative still bo und 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-g enerated 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 impor tant 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 Ton B across the outer membrane and that the C-terminal portion of the mol ecule is likely to contain the activity domain.