Abstract
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.
Highlights
Under stress conditions the production of colicins is a response that E. coli strains have developed to facilitate the invasion of environmental populations competing for limited nutrients [1]
Colicin Forms Detected in DNase Colicin Treated Bacteria In the case of RNase colicins an endoproteolytic cleavage step, occurring either before or during translocation across the inner membrane, was shown to be necessary to promote the release of the C-terminal toxic domain into the cytoplasm [10]
We detected in crude cell extracts a small processed form (PF) of about 18 kDa (Figure 1A, lane 3) that, as shown below, corresponds to the DNase domain of colicin E2
Summary
Under stress conditions the production of colicins is a response that E. coli strains have developed to facilitate the invasion of environmental populations competing for limited nutrients [1]. Colicinogenic strains are protected against both endogenous and exogenous toxin molecules by the constitutive expression of an immunity (Imm) protein, which in the case of nuclease type colicins forms a tight heterodimer complex with the colicins [3,4]. Nuclease colicins are one of the two main classes of colicins Both require colicin translocation across the outer membrane. While killing by one class is based on a pore-forming activity at the inner membrane that results in depolarization of the target cell, the second utilizes nucleolytic activities targeting chromosomal DNA (colicins E2, E7 and E9), ribosomal (colicin E3) or transfer RNAs (colicins E5 and D). The nuclease type of colicins requires their translocation across both the outer and inner membranes. During early steps of invasion the first two domains are required for receptor binding and translocation across the outer membrane.
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call