Kinetics of phosphate uptake, growth, and accumulation of cyclic diphosphoglycerate in a phosphate-limited continuous culture of Methanobacterium thermoautotrophicum

Abstract

The archaebacterium Methanobacterium thermoautotrophicum was grown in continuous culture at 65 degrees C in a phosphate-limited medium at specific growth rates from 0.06 to 0.28 h-1 (maximum growth rate [mu max] = 0.36 h-1). Cyclic-2,3-diphosphoglycerate (cyclic DPG) levels ranged from 2 to 20 mM in Pi-limited cells, compared with about 30 mM in batch-grown cells. The Monod constant for Pi-limited growth was 5 nM. Pi uptake rates were determined by following the disappearance of 32Pi from the medium. Interrupting the H2 supply stopped the uptake of Pi and the release of organic phosphates. Little or no efflux of Pi occurred in the presence or absence of H2. Pi uptake of cells adapted to nanomolar Pi concentrations could be accounted for by the operation of one uptake system with an apparent Km of about 25 nM and a Vmax of 58 nmol of Pi per min per g (dry weight). Uptake curves at 30 microM Pi or above were biphasic due to a sevenfold decrease in Vmax after an initial phase of rapid movement of Pi into the cell. Under these conditions the growth rate slowed to zero and the cyclic DPG pool expanded before growth resumed. Thus, three properties of M. thermoautotrophicum make it well adapted to live in a low-P environment: the presence of a low-Km, high-Vmax uptake system for Pi; the ability to accumulate cyclic DPG rapidly; and a growth strategy in which accumulation of Pi and cyclic DPG takes precedence over a shift-up in growth rate when excess Pi becomes available.