The archaebacterium Methanobacterium thermoautotrophicum was grown at 65 degrees C in H2- and Pi-limited chemostat cultures at dilution rates corresponding to 3- and 4-h doubling times, respectively. Under these conditions the steady state concentration of cyclic 2,3-diphosphoglycerate was 44 mM in the H2-limited cells and 13 mM in the cells grown under Pi limitation. Flux of Pi into the cyclic pyrophosphate pool was estimated by two 32P-labeling procedures: approach to isotopic equilibrium and replacement of prelabeled cyclic diphosphoglycerate with unlabeled compound. The results unequivocally demonstrate turnover of the phosphoryl groups; either both phosphoryl groups of the cyclic pyrophosphate leave together or the second leaves at a faster rate. The half-life of the rate-determining step for loss of the phosphoryl groups was approximately equal to the culture doubling time. The Pi flowing into the cyclic diphosphoglycerate pool accounted for 19% of the total Pi flux into Pi-limited cells and 43% of the total for H2-limited cells. The high phosphate flux through the large cyclic diphosphoglycerate pool suggests that this molecule plays an important role in the phosphorus metabolism of this methanogen.
