Visualising the concept of metabolic regulation in bacteria: a simple laboratory experiment on polyhydroxybutyrate storage

Regulation is a key concept for understanding the dynamics of metabolism in bacteria. This report outlines a simple laboratory experiment aimed at studying a key form of regulation in bacterial metabolism, exemplifying how microbes switch the flux of carbon towards alternative metabolic fates as a function of nutrient availability and according to their physiological status. The experiment involves the use of a Sinorhizobium meliloti wild-type strain and its isogenic derivative carrying a null mutation in the gene encoding the synthase that generates the storage polymer polyhydroxybutyrate (PHB). These strains serve to characterise the effects of growth phase and carbon availability at the end of balanced growth on the extent of PHB accumulation by rhizobia. The experimental set-up can be completed in a main 4-h laboratory session, followed by an additional 1-h session after an initial incubation for subsequent bacterial-colony counting (48 h), and requires equipment usually available at any university. The protocol described here is performed by the undergraduates of our biochemistry course. We consider this exercise to be a useful complementary tool for spotlighting the concept of metabolic regulation in bacteria and for promoting scientific thinking along with the development of students' wet-bench laboratory skills.