Characterization of the specific pyruvate transport system in Escherichia coli K-12

Abstract

A mutant of Escherichia coli K-12 Bacterial cells are capable of taking up a variety of carboxylic acids from the surrounding medium. The translocation of carboxylic acids across the bacterial membrane is mediated by transport mechanisms such as facilitative diffusion, active transport, and vectorial acylation (for a review, see reference 11). Of all the carboxylate transport systems of bacteria studied, the tricarboxylic acid cycle dicarboxylates (1,9,13,16,17) and tricarboxylates (6,12,28) have received the most attention. The transport or uptake of a number of monocarboxylates by bacteria has also been reported, but the molecular mechanisms involved are less well established. Thus, specific transport systems have been suggested for the glycolytic intermediates D-and L-lactate (3, 19), pyruvate (4, 14, 19), glycolate (23), and phosphoenolpyruvate and 2-and 3-phosphoglycerate (26), as well as for monocarboxylates such as hexuronates (5), hexonates (24), acetate (29), and propionate (10).Preliminary studies by Kornberg and Smith (14) on the genetic control of intermediary metabolism revealed that a phosphoenolpyruvate synthase (pps)-deficient mutant with restored enzymatic activity still could not grow on pyruvate as the sole carbon source. Kornberg and Smith concluded that the cells must also have a pyruvate transport system and a defect in this system prevented growth on pyruvate. This purported pyruvate transport (usp) mutant could still take up lactate from the growth medium, indicating that pyruvate and lactate do not share the same transport system. Matin and Konings (19), on the basis of studies on the uptake of lactate and succinate in membrane vesicles of Escherichia coli K-12, concluded from inhibition studies that pyruvate and lactate may share the same transport system. In none of these studies was pyruvate transport thoroughly examined. Pyruvate has also been shown to be taken up by a system with high affinity in Rhodopseudomonas spheroides (4). This system has a wide substrate specificity, being competitively inhibited by a variety of short-chain fatty acids, as well as by lactate. * Corresponding author.To characterize the pyruvate transport system in E. coli K-12 whole cells, we chose a mutant strain deficient in pyruvate dehydrogenase and phosphoenolpyruvate synthase. Although these two major routes of pyruvate metabolism were blocked, pyruvate could still be metabolized in cells by a variety of enzymes including lactate dehydrogenase, pyruvate oxidase, and amino acid transaminases. To minimize this problem, pyruvate uptake was also studied in membrane vesicles devoid of cytoplasmic contents. The whole cell and vesicle results indicate that uptake of pyruvate in E. coli is due to a specific active transport system. MATERIALS AND METHODSBacterial strains and growth conditions. Whole cell studies were done with a mutant strain of E. coli K-12 [pps relAl thyA56 metBI azi-54 ton-54 tsx-97 A(aroP-ace)F73] kindly provided by the E. coli Genetic Stock Center (CGSC 5688). This mutant strain, designa...

The formate channel FocA exports the products of mixed-acid fermentation

“…A single anion channel, FocA, that can keep the intracellular concentrations of these molecules low must constitute a major evolutionary advantage. However, the capacity of FocA to export the central metabolic intermediate pyruvate seems contradictory, in particular because the uptake of pyruvate is an active process (31,32). Under conditions of mixed-acid fermentation, extracellular pyruvate was detected when glucose was supplied (22), suggesting that intracellular levels of pyruvate may rise to a point where FocA again can serve as an overflow.…”

Reexamination of the Physiological Role of PykA in Escherichia coli Revealed that It Negatively Regulates the Intracellular ATP Levels under Anaerobic Conditions

“…One of the important characteristics of the EB216-ΔpykA strain is its reutilization of the secreted pyruvate. Interestingly, from examination of the transcriptomic analysis data (see Table S1 in the supplemental material), we found that the yhjX gene, which encodes a putative pyruvate transporter (22)(23)(24), was 15-fold upregulated compared with the control strain EB216 at 48 h. Figure 2A shows that the expression strength of the yhjX gene is highly associated with the fermentation process (from 216 to 5,703 fragments per kilobase per million reads [FPKM], which was 10 to 15 times higher than that measured in strain EB216 during the n-butanol-producing period). To investigate whether yhjX is associated with pyruvate reassimilation, the yhjX gene was knocked out in the EB216 and EB216-ΔpykA strains.…”

Overcoming Fluctuation and Leakage Problems in the Quantification of Intracellular 2-Oxoglutarate Levels in Escherichia coli

“…The quantification of their internal concentrations would depend on the extent of help provided by their respective uptake mechanisms, similar to what KgtP is to 2OG. Such transporters have been documented in the literature, including a pyruvate transporter (26,29) and the fumarate/malate/succinate Dct uptake systems (11,27,33). Even with a recycle mechanism, however, metabolite leakage presents a challenge for accurate quantification of cellular metabolites.…”