Specific Action of 4-Nitropyridine 1-Oxide on <i>Escherichia coli</i> K-12 Pro ⁺ Strains Leading to the Isolation of Proline-Requiring Mutants: Mechanism of Action of 4-Nitropyridine 1-Oxide

<jats:p> Possible mechanisms involved in the action of 4-nitropyridine 1-oxide (4NPO) on <jats:italic>Escherichia coli</jats:italic> K-12 <jats:italic>pro</jats:italic> <jats:sup>+</jats:sup> cells in Penassay broth leading to the selective isolation of <jats:italic>proA</jats:italic> <jats:sup>−</jats:sup> and/or <jats:italic>proB</jats:italic> <jats:sup>−</jats:sup> mutants but not <jats:italic>proC</jats:italic> <jats:sup>−</jats:sup> mutant were studied. Reconstruction experiments between <jats:italic>pro</jats:italic> <jats:sup>+</jats:sup> and <jats:italic>pro</jats:italic> <jats:sup>−</jats:sup> cells, together with experiments on the bactericidal action of 4NPO on <jats:italic>pro</jats:italic> <jats:sup>+</jats:sup> and <jats:italic>pro</jats:italic> <jats:sup>−</jats:sup> cells, indicated that 4NPO is more toxic for <jats:italic>pro</jats:italic> <jats:sup>+</jats:sup> and <jats:italic>proC</jats:italic> <jats:sup>−</jats:sup> cells than for <jats:italic>proA</jats:italic> <jats:sup>−</jats:sup> and <jats:italic>proB</jats:italic> <jats:sup>−</jats:sup> cells. These results, coupled with data indicating little mutagenicity of 4NPO on <jats:italic>E. coli</jats:italic> cells, led us to conclude that the selection of <jats:italic>proA</jats:italic> <jats:sup>−</jats:sup> and/or <jats:italic>proB</jats:italic> <jats:sup>−</jats:sup> cells that arose spontaneously in the <jats:italic>pro</jats:italic> <jats:sup>+</jats:sup> culture is a possible mechanism for the action of 4NPO. Examination of 4NPO sensitivity of <jats:italic>pro</jats:italic> <jats:sup>+</jats:sup> transductants derived from <jats:italic>proA</jats:italic> <jats:sup>−</jats:sup> and <jats:italic>proB</jats:italic> <jats:sup>−</jats:sup> cells with P1 <jats:italic>vir</jats:italic> phage and <jats:italic>pro</jats:italic> <jats:sup>+</jats:sup> cells as donor and of <jats:italic>pro</jats:italic> <jats:sup>+</jats:sup> spontaneous revertants derived from those <jats:italic>pro</jats:italic> <jats:sup>−</jats:sup> cells suggested that 4NPO-sensitive gene(s) should be on, or very close to, the <jats:italic>proA</jats:italic> and <jats:italic>proB</jats:italic> loci and that both products of <jats:italic>proA</jats:italic> and <jats:italic>proB</jats:italic> genes may be involved in the sensitivity of bacteria to 4NPO. The fact that the 4NPO-sensitive allele is dominant over the 4NPO-resistant allele further indicated the possible correlation between gene products of <jats:italic>proA</jats:italic> and <jats:italic>proB</jats:italic> and the 4NPO sensitivity of bacteria. Experiments on metabolic conversion of 4NPO with bacterial cells proved that the major metabolic pathway of the agent is reduction to (possibly via 4-nitroso-) 4-hydroxylamino- and 4-amino-pyridine 1-oxides, and then to 4-aminopyridine. Investigation of the effect of structural modification of 4NPO on the elective selection of Pro <jats:sup>−</jats:sup> mutants in Pro <jats:sup>+</jats:sup> culture further suggested that the structural feature indispensable for the action of the agent is the hydroxyl-amino or its more oxidized state at the 4 position and the <jats:italic>N</jats:italic> -oxide moiety at the 1 position on the pyridine skeleton. Action of 4NPO in minimal medium was found to be bacteriostatic on <jats:italic>pro</jats:italic> <jats:sup>+</jats:sup> cells but not on <jats:italic>pro</jats:italic> <jats:sup>−</jats:sup> cells, leading to the formation of long nonseptate multinucleate filament cells on <jats:italic>pro</jats:italic> <jats:sup>+</jats:sup> cells. Possible biochemical mechanisms of the selective toxicity of 4NPO for <jats:italic>pro</jats:italic> <jats:sup>+</jats:sup> and <jats:italic>pro</jats:italic> <jats:sup>−</jats:sup> cells a ...