Release of Immunity Protein Requires Functional Endonuclease Colicin Import Machinery

Abstract

Bacteria producing endonuclease colicins are protected against the cytotoxic activity by a small immunity protein that binds with high affinity and specificity to inactivate the endonuclease. This complex is released into the extracellular medium, and the immunity protein is jettisoned upon binding of the complex to susceptible cells. However, it is not known how and at what stage during infection the immunity protein release occurs. Here, we constructed a hybrid immunity protein composed of the enhanced green fluorescent protein (EGFP) fused to the colicin E2 immunity protein (Im2) to enhance its detection. The EGFP-Im2 protein binds the free colicin E2 with a 1:1 stoichiometry and specifically inhibits its DNase activity. The addition of this hybrid complex to susceptible cells reveals that the release of the hybrid immunity protein is a time-dependent process. This process is achieved 20 min after the addition of the complex to the cells. We showed that complex dissociation requires a functional translocon formed by the BtuB protein and one porin (either OmpF or OmpC) and a functional import machinery formed by the Tol proteins. Cell fractionation and protease susceptibility experiments indicate that the immunity protein does not cross the cell envelope during colicin import. These observations suggest that dissociation of the immunity protein occurs at the outer membrane surface and requires full translocation of the colicin E2 N-terminal domain.

Complete Genome Sequence of a Yersinia enterocolitica “Old World” (3/O:9) Strain and Comparison with the “New World” (1B/O:8) Strain

“…Colicin E2 is known to cause DNA breakdown. Moreover, the release of colicin E2 protein requires the presence of the periplasmic and inner membrane Toll components (9). The colicin E2 gene cluster encodes three components of this operon: colicin, immunity protein, and lysis signal peptide.…”

Colicin E2 Is Still in Contact with Its Receptor and Import Machinery When Its Nuclease Domain Enters the Cytoplasm

“…Thus, ColE2 remained associated with its receptor and the Tol machinery for at least 30 min. Although the exact amount of time required for complete import of nuclease colicins is unknown, previous reports suggest that nuclease colicins enter the cytoplasm from 3.5 to 20 min after their addition to cells (15,21,34). These findings demonstrate that ColE2 releases neither its receptor nor its import machinery when its nuclease domain enters the cytoplasm.…”

“…Colicins A, B, E1, and E2 and ColE2(R544A H575A) were purified from E. coli W3110 as previously described (13,15,26,29 (5). Colicins were added at the multiplicities (numbers of molecules per cell) indicated below, and the initial rate of K ϩ efflux was determined in the linear part of the K ϩ efflux curve as previously described (6).…”

FtsH-dependent Processing of RNase Colicins D and E3 Means That Only the Cytotoxic Domains Are Imported into the Cytoplasm

“…The binding of the complex per se is however not enough to liberate the Imm protein. The dissociation of colicins E9-and E2-Imm complexes requires the unfolding of the colicin, as it contacts the energy-transducing Tol system in the periplasm (12,13).To transfer the cytotoxic domain of the colicin molecule across the inner membrane into the cytoplasm, nuclease colicins need to parasitize more of the cell functions than the pore-forming colicins. The DNase domains of colicins E9 and E2 exhibit nonvoltage-gated channel forming activity in planar lipid bilayers, which involves changes in their conformation (14).…”

“…The binding of the complex per se is however not enough to liberate the Imm protein. The dissociation of colicins E9-and E2-Imm complexes requires the unfolding of the colicin, as it contacts the energy-transducing Tol system in the periplasm (12,13).…”