Function and Regulation of the Pyruvate Transporter CstA in .

Pyruvate is a central metabolite that connects many metabolic pathways in living organisms. To meet the cellular pyruvate requirements, the enterobacterium has at least three pyruvate uptake systems-the H/pyruvate symporter BtsT, and two thus far less well-characterized transporters, YhjX and CstA. BtsT and CstA belong to the putative carbon starvation (CstA) family (transporter classification TC# 2.A.114). We have created an mutant that cannot grow on pyruvate as the sole carbon source and used it to characterize CstA as a pyruvate transporter. Transport studies in intact cells confirmed that CstA is a highly specific pyruvate transporter with moderate affinity and is energized by a proton gradient. When cells of a reporter strain were cultured in complex medium, expression was maximal only in stationary phase. A DNA affinity-capture assay combined with mass spectrometry and an in-vivo reporter assay identified Fis as a repressor of expression, in addition to the known activator cAMP-CRP. The functional characterization and regulation of this second pyruvate uptake system provides valuable information for understanding the complexity of pyruvate sensing and uptake in .