Colicin E9

Colicin E9 is a type of Colicin, a bacteriocin made by E. Coli which acts against other nearby E. Coli to kill them with its DNase activity; it digests the host's genome at specific locations, ultimately leading to the death of the cell.

Synthesis and releaseSynthesis and release

Colicin E9 in solution, ie in the cytoplasm after synthesis, is monomeric, and forms a high affinity complex with its immunity protein, Im9. The immunity protein does not directly bind to the active site, but instead to an exosite. This is bound while in the producing cell to protect it from the activity.

Mechanism of uptakeMechanism of uptake

The primary receptor for colicin E9 is the vitamin B12 receptor, BtuB. It then requires the outer membrane porin OmpF - either the two form the functional receptor, or OmpF is recruited for subsequent translocation. The OmpF association with the BtuB-colicin complex is weak and transient. After the interaction with OmpF, colicin E9 requires the Tol system to pass across the periplasm. The interaction with TolB is governed by a pentapeptide region in the N terminus. These residues are unstructured and highly flexible, but the TolB box of 5 residues is organised within this disordered domain.

OmpF acts synergistically with BtuB to protect bacteria against the action of colicin E9. This could indicate that OmpF is a component of the receptor apparatus. Alternatively the role of OmpF could be more to do with translocation rather than receptor recognition .

Once bound to the BtuB receptor, it is suggested that the coiled-coil receptor binding domain of the colicin unfolds to lose the immunity protein, Im9, and trigger translocation. This flexibility is crucial for translocation, and therefore the cytotoxicity, as shown by the addition of disulphide bonds. This reduces the flexibility and lowers the activity.

The endonuclease domain of colicin E9 is able to form ion channels in planar lipid bilayers. The E9 DNase mediates its own translocation across the cytoplasmic membrane, and the formation of ion channels is essential to this process. The association of colicin E9 with negative phospholipids results in a destabilisation of the DNase. This is protected by the colE9 immunity protein, Im9, but not by the binding of zinc to the active site. Formation of this destabilising complex preempts channel formation by the DNase, and makes up the first step in the translocation of colE9 across the E. coli inner membrane. The channels are then assumed to reseal themselves once the cytotoxic domain of the colicin has entered the cytoplasm.

The destabilisation of the DNase domain upon interaction with negative phospholipids increases its susceptibility to proteolysis and to thermal and chemical denaturation. Once associated, there is a massive disruption of protein tertiary structure, and the secondary structure instead interacts with the lipid bilayer - similar to the interaction between domains involved in Pore Formation in other colicins and the membranes that they disrupt.

The formation of a disulphide bond at D20C/E66C abolishes its channel forming ability, and its cytotoxicity (as it cannot penetrate cells) but has no effect on its DNase activity. It is still able to bind to the phospholipids, but not translocate across the membrane.

Killing ActivitiesKilling Activities

The cytotoxic activity of colE9 is DNase activity, where it hydrolyses the DNA . However, it is also able to form ion channels in planar lipid bilayers, similar to the pore-forming colicins. These channels do not cause cell death, instead they are related to the ability of the E9 DNase domain to translocate across the inner membrane.

The catalytic centre of the DNase domain contains the HNH motif, a site for DNA and metal (zinc ion) binding. Binding zinc stabilises the protein.

In response to the DNA damage by colE9, the E. coli cell initiates an SOS response, prior to cell death.