In vivo processing of DNase colicins E2 and E7 is required for their import into the cytoplasm of target cells.
DNase colicins E2 and E7, both of which appropriate the BtuB/Tol translocation machinery to cross the outer membrane, undergo a processing step as they enter the cytoplasm. This endoproteolytic cleavage is essential for their killing action. A processed form of the same size, 18.5 kDa, which corresponds to the C-terminal catalytic domain, was detected in the cytoplasm of bacteria treated with either of the two DNase colicins. The inner-membrane protease FtsH is necessary for the processing that allows the translocation of the colicin DNase domain into the cytoplasm. The processing occurs near residue D420, at the same position as the FtsH-dependent cleavage in RNase colicins E3 and D. The cleavage site is located 30 amino acids upstream of the DNase domain. In contrast, the previously reported periplasm-dependent colicin cleavage, located at R452 in colicin E2, was shown to be generated by the outer-membrane protease OmpT and we show that this cleavage is not physiologically relevant for colicin import. Residue R452, whose mutated derivatives led to toxicity defect, was shown to have no role in colicin processing and translocation, but it plays a key role in the catalytic activity, as previously reported for other DNase colicins. Membrane associated forms of colicins E2 and E7 were detected on target cells as proteinase K resistant peptides, which include both the receptor-binding and DNase domains. A similar, but much less proteinase K-resistant form was also detected with RNase colicin E3. These colicin forms are not relevant for colicin import, but their detection on the cell surface indicates that whole nuclease-colicin molecules are found in a stable association with the outer-membrane receptor BtuB of the target cells.
Institut de Biologie Physico-Chimique, CNRS, UPR 9073, Paris, France.
Liliana
L
Mora
Institut de Biologie Physico-Chimique, CNRS, UPR 9073, Paris, France.
Miklos
M
de Zamaroczy
D003087:Colicins / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology*
D003087:Colicins; D029968:Escherichia coli Proteins; D003851:Deoxyribonucleases