Abstract
Nuclease colicins bind their target receptor in the outer membrane of sensitive cells in the form of a high affinity complex with their cognate immunity proteins. Upon cell entry the immunity protein is lost from the complex by means that are poorly understood. We have developed a sensitive fluorescence assay that has enabled us to study the molecular requirements for immunity protein release. Nuclease colicins use members of the tol operon for their translocation across the outer membrane. We have demonstrated that the amino-terminal 80 residues of the colicin E9 molecule, which is the region that interacts with TolB, are essential for immunity protein release. Using tol deletion strains we analyzed the cellular components necessary for immunity protein release and found that in addition to a requirement for tolB, the tolA deletion strain was most affected. Complementation studies showed that the mutation H22A, within the transmembrane segment of TolA, abolishes immunity protein release. Investigation of the energy requirements demonstrated that the proton motive force of the cytoplasmic membrane is critical. Taken together these results demonstrate for the first time a clear energy requirement for the uptake of a nuclease colicin complex and suggest that energy transduced from the cytoplasmic membrane to the outer membrane by TolA could be the driving force for immunity protein release and concomitant translocation of the nuclease domain.
Highlights
Membrane translocation is a formidable challenge for folded proteins
In common with most colicins, the DNase-type colicin E9 consists of three functional domains: the killing activity is contained in its carboxyl-terminal DNase domain; the central section contains the receptor-binding domain, which binds the vitamin B12 receptor, BtuB, in the outer membrane; and the amino-terminal translocation domain is needed for the entry of the cytotoxic domain into the target cell
Validation of the Alexa Fluor-labeled Immunity Protein and Its Release—The native cysteine residue at position 23 in Im9 was replaced by a serine residue, and we introduced a serine to cysteine mutation at position 6. This location was chosen for labeling with Alexa Fluor 594-C5-maleimide because it is not involved in binding the DNase domain of colicin E9 [34]
Summary
Bacterial Strains, and Media—E. coli DH5␣ was used as the host strain for cloning and mutagenesis. Immunity Protein Release Assay—Wild-type E. coli and E. coli tol mutants from overnight cultures were grown to midlog (A600 nm ϳ 0.4 – 0.6) in LB broth or M9 minimal medium containing 1 mM MgCl2, 0.2% (w/v) glucose, and casamino acids. Alexa Fluor-labeled colicin complexes were formed by preincubation of oxidized free colE9S-S constructs with Im9AF at a molar ratio of 1:1.5 and were subsequently added to 1 ml of cells in duplicate (10 nM final concentration). The cells were spun for 1 min at 10,000 rpm, and the RFU in 100 l of supernatant of the control and DTT-treated cells were measured in 96-well plates (optical bottom, Nunc) in triplicate. In all immunity protein release assays, control cells preincubated with colE9S-S-Im9AF but not treated with DTT were included in order to account for possible nonspecific effects such as cell surface proteolysis or vesicle shedding. Global analysis using BIAevaluation software 3.1 was used to fit corrected surface plasmon resonance responses to the theoretical 1:1 Langmuir binding model
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