Substituents at N6 and C-5' control selective uptake and toxicity of the adenine-nucleotide bacteriocin, agrocin 84, in Agrobacteria

Abstract

The inhibition of a sensitive strain of Agrobacterium radiobacter by the nucleotide bacteriocin agrocin 84 has been studied. A structure-function study of the agrocin 84 molecule was undertaken. Two agrocin 84 nucleotide fragments lacking either the N6 or 5'-phosphoramidate substituents were used in uptake studies of [32P2]agrocin 84. It was established that the plasmid-controlled, strain-specific uptake of agrocin 84 is determined by the N6-D-glucofuranosyloxyphosphoramidate substituent. This conclusion is further supported by the markedly reduced uptake of the 32P-labelled fragment lacking the N6 substituent. Equilibrium dialysis studies also indicate that the N6 substituent is 'recognised' by a binding protein involved in the uptake of agrocin 84 into sensitive strains. The nucleotide fragment bearing the N6 substituent is a competitive inhibitor for the uptake of agrocin 84 in vivo with a Ki = 1.0 x 10(-7) M and is itself selectively transported into a sensitive strain at a rate comparable with agrocin 84, but unlike agrocin 84 is non-toxic. By contrast, the fragment bearing the 5'-phosphoramidate is taken up by both sensitive and insensitive strains at a barely measurable rate and is toxic to both.