Apparent dependence of sporulation on synthesis of highly phosphorylated nucleotides in Bacillus subtilis.

Bacillus subtilis contains an enzyme that synthesizes the highly phosphorylated nucleotide adenosine 5',3'(2')-bis(triphosphate), abbreviated p(3)Ap(3). This enzyme can be demonstrated to be present in the cytoplasmic membrane of B. subtilis at all stages of growth and development. During vegetative growth its enzymatic activity is inhibited by phosphorylated metabolites of energy-rich carbon sources, as was shown with the aid of conditional sporulation mutants. The finding that a temperature-sensitive sporulation mutant with a mutation in the spoOF gene, an early sporulation gene, shows a temperature-sensitive activity of p(3)Ap(3)-synthetase indicates that p(3)Ap(3) plays an important role in sporulation, probably initiation of sporulation.

Studies on the control of development: isolation of Bacillus subtilis mutants blocked early in sporulation and defective in synthesis of highly phosphorylated nucleotides.

To test our model on the mechanism of initiation of differentiation in Bacillus subtilis, we tested early blocked (stage 0) sporulation mutants for their ability to synthesize highly phosphorylated nucleotides. We also isolated early blocked asporogenous mutants with the aid of the intercalating drug tilorone. Among all mutants tested we found that the spo0F-bearing strain was unable to synthesize adenosine 3'(2')-triphosphate 5'-triphosphate, pppAppp. A revertant of this mutant regained the ability to both sporulate and synthesize pppAppp. Ribosomes of the asporogenous mutant isolated at T2 (2 hr after the end of logarithmic growth) of sporulation, in contrast to the wild type, do not synthesize adenosine 3'(2')-diphosphate 5'-diphosphate, ppApp, or adenosine 3'(2')-diphosphate 5'-triphosphate, pppApp, but synthesize guanosine 3'(2')-diphosphate 5'-diphosphate, ppGpp, and guanosine 3'(2')-diphosphate 5'-triphosphate, pppGpp. This behavior is characteristic of ribosomes from vegetative, not sporulating, cells. Ribosomes from the sporogenous revertant behave like those of the wild type. The results suggest that the spo0F mutation may be a mutation in the structural gene for pppAppp synthetase. The inability to synthesize pppAppp in this strain also prevents the formation of "sporulation-specific ribosomes," i.e., ribosomes that synthetize ppApp and pppApp. The present experiments suggest that the nucleotide pppAppp participates in the initiation of sporulation by triggering a sequencies of events required for the production of heat-resistant spores.

Control of development: role of regulatory nucleotides synthesized by membranes of Bacillus subtilis in initiation of sporulation.

A model explaining the mechanism of initiation of differentiation is presented. It is based on the finding that sporulation in B. subtilis can be induced by the same nutrient deficiencies that also induce the synthesis of highly phosphorylated nucleotides. Two of these nucleotides are synthesized by membrane vesicles. Synthesis can be inhibited by the same metabolites of glucose that also inhibit sporulation. It is concluded, therefore, that the plasma membrane synthesizes unusual nucleotides in response to nutrient deficiencies. By several as yet unknown steps, these nucleotides then cuase changes in the metabolism of the organism leading to the formation of spores. Both structure and mechanism of synthesis of adenosine hexaphosphate, pppAppp, have been elucidated by use of ATP analogues.