1887

Abstract

Summary: E colicins are plasmid-coded, protein antibiotics which bind to the BtuB outer membrane receptor of cells and are then translocated either to the outer surface of the cytoplasmic membrane in the case of the pore-forming colicin E1, or to the cytoplasm in the case of the enzymic colicins E2-E9. Translocation has been proposed to be dependent on a putative TolA box; a pentapeptide (DGSGW) located in the N-terminal 39 residues of several Toldependent colicins. In this study, site-directed mutagenesis was used to change each of the residues of the putative TolA box of colicin E9 to alanines. In the case of the two glycine residues, the resulting mutant proteins were indistinguishable from the native colicin E9 protein in a biological assay; whereas the other three residues when mutated to alanines resulted in a complete loss of biological activity. Mutagenesis of two serine residues flanking the putative TolA box, Ser34 and Ser40, to alanines did not abolish the biological activity of the mutant colicin E9, although the zones of growth inhibition were hazy and slow to form. The size of the zone of inhibition was significantly smaller than the control in the case of the Ser40Ala mutant. The ColE9/lm9 complex was isolated from the three biologically inactive TolA box alanine mutants. In competition assays all three mutant protein complexes were capable of protecting sensitive cells against killing by the native ColE9/lm9 complex. On removal of the Im9 protein from the three mutant ColE9/lm9 complexes, all three mutant colicins exhibited DNase activity. These results confirm the importance of the putative TolA box in the biological activity of colicin E9, and suggest that the TolA box has a role in the translocation of colicin E9.

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1997-09-01
2021-10-19
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