Phosphohydrolases of a Bacillus subtilis Mutant Accumulating Inosine and Hypoxanthine

Abstract

Although adenine-requiring auxotrophs of Bacillus subtilis accumulate large quantities of inosine or hypoxanthine, or of both, they do not accumulate inosine-5′-monophosphate (IMP). Experiments directed at understanding this phenomenon were conducted with an adenineless auxotroph and with a mutant derived from it which lacked alkaline phosphohydrolase. It was found that B. subtilis contains four different phosphohydrolases. Only one is an extracellular enzyme; it is a 5′-nucleotide phosphohydrolase which can be inhibited by addition of CuSO 4 to the medium. Of the three cellular enzymes, only one, an acid phosphohydrolase, cannot attack 5′-nucleotides; this enzyme is not repressed by inorganic phosphate. One of the two remaining surface-bound enzymes is a nonspecific alkaline phosphohydrolase which attacks both 5′-nucleotides and p -nitrophenyl phosphate; this is the only phosphohydrolase that is markedly repressed by inorganic phosphate. The other surface-bound enzyme is a nonrepressible 5′-nucleotide phosphohydrolase with double p H optima: one at neutrality and the other near p H 9.0. The experiments indicate that the absence of IMP in the extracellular broth is due to degradation of internally accumulated IMP to inosine by the cellular 5′-nucleotide phosphohydrolase.