A 76-residue polypeptide of colicin E9 confers receptor specificity and inhibits the growth of vitamin B-12-dependent Escherichia coli 113/3 cells

The mechanism by which E colicins recognize and then bind to BtuB receptors in the outer membrane of Escherichia coli cells is a poorly understood fir st step in the process that results in cell killing. Using N- and C-termina l deletions of the N-terminal 448 residues of colicin E9, we demonstrated t hat the smallest polypeptide encoded by one of these constructs that retain ed receptor-binding activity consisted of residues 343-418. The results of the in vivo receptor-binding assay were supported by an alternative competi tion assay that we developed using a fusion protein consisting of residues 1-497 of colicin E9 fused to the green fluorescent protein as a fluorescent probe of binding to BtuB in E. coli cells. Using this improved assay, we d emonstrated competitive inhibition of the binding of the fluorescent fusion protein by the minimal receptor-binding domain of colicin E9 and by vitami n B-12. Mutations located in the minimum R domain that abolished or reduced the biological activity of colicin E9 similarly affected the competitive b inding of the mutant colicin protein to BtuB. The sequence of the 76-residu e R domain in colicin E9 is identical to that found in colicin E3, an RNase type E colicin. Comparative sequence analysis of colicin E3 and cloacin DF 13, which is also an RNase-type colicin but uses the IutA receptor to bind to E. coli cells, revealed significant sequence homology throughout the two proteins, with the exception of a region of 92 residues that included the minimum R domain. We constructed two chimeras between cloacin DF13 and coli cin E9 in which (i) the DNase domain of colicin E9 was fused onto the T+R d omains of cloacin DF13; and (ii) the R domain and DNase domain of colicin E 9 were fused onto the T domain of cloacin DF13. The killing activities of t hese two chimeric colicins against indicator strains expressing BtuB or Iut A receptors support the conclusion that the 76 residues of colicin E9 confe r receptor specificity. The minimum receptor-binding domain polypeptide inh ibited the growth of the vitamin B-12-dependent E. coli 113/3 mutant cells, demonstrating that vitamin B-12 and colicin E9 binding is mutually exclusi ve.