RNA synthesis as a measure of microbial growth in aquatic environments. I. Evaluation, verification and optimization of methods

Abstract

Measurement of rates of microbial (bacteria and unicellular algae) ribonucleic acid (RNA) synthesis in environmental samples by radioactive tracers requires several assumptions that we test and evaluate herein. After addition of 3H-adenine to water samples from eutrophic freshwater or pelagic and oceanic ecosystems, microorganisms assimilate it without (1) any luxury uptake or (2) any expansion of intracellular adenine nucleotide pools. The calculated rate of stable RNA synthesis tends to be overestimated during the initial time periods of labeling (i.e., less than 30 s for cultures of Serratia marinorubra, and less than 20 min for most environmental samples) due to kinetic compartmentalization of the nucleic acid precursor (ATP) pool. Time-series analyses are recommended because the effects of compartmentalization decrease with increasing incubation. The kinetics of 3H-adenine uptake and aspects of its subsequent assimilation, intermediary metabolism and breakdown are described in bacteria and natural assemblages of microorganisms. Mass-balance radioactivity inventories greatly facilitate interpretation of nucleic acid precursor assimilation. Finally, we demonstrate that independent measurements of precursor (ATP) specific radioactivity are required for any elucidation of rates of stable RNA synthesis (and thus microbial growth) in nature.