Peptide Transporter CstA Imports Pyruvate in Escherichia coli K-12

Abstract

Pyruvate is an important intermediate of central carbon metabolism and connects a variety of metabolic pathways in Escherichia coli. Although the intracellular pyruvate concentration is dynamically altered and tightly balanced during cell growth, the pyruvate transport system remains unclear. Here, we identified a pyruvate transporter in E. coli using high-throughput transposon sequencing. The transposon mutant library (a total of 5 × 105 mutants) was serially grown with a toxic pyruvate analog (3-fluoropyruvate [3FP]) to enrich for transposon mutants lacking pyruvate transport function. A total of 52 candidates were selected on the basis of a stringent enrichment level of transposon insertion frequency in response to 3FP treatment. Subsequently, their pyruvate transporter function was examined by conventional functional assays, such as those measuring growth inhibition by the toxic pyruvate analog and pyruvate uptake activity. The pyruvate transporter system comprises CstA and YbdD, which are known as a peptide transporter and a conserved protein, respectively, whose functions are associated with carbon starvation conditions. In addition to the presence of more than one endogenous pyruvate importer, it has been suggested that the E. coli genome encodes constitutive and inducible pyruvate transporters. Our results demonstrated that CstA and YbdD comprise the constitutive pyruvate transporter system in E. coli, which is consistent with the tentative genomic locus previously suggested and the functional relationship with the extracellular pyruvate sensing system. The identification of this pyruvate transporter system provides valuable genetic information for understanding the complex process of pyruvate metabolism in E. coli.IMPORTANCE Pyruvate is an important metabolite as a central node in bacterial metabolism, and its intracellular levels are tightly regulated to maintain its functional roles in highly interconnected metabolic pathways. However, an understanding of the mechanism of how bacterial cells excrete and transport pyruvate remains elusive. Using high-throughput transposon sequencing followed by pyruvate uptake activity testing of the selected candidate genes, we found that a pyruvate transporter system comprising CstA and YbdD, currently annotated as a peptide transporter and a conserved protein, respectively, constitutively transports pyruvate. The identification of the physiological role of the pyruvate transporter system provides valuable genetic information for understanding the complex pyruvate metabolism in Escherichia coli.

Function and Regulation of the Pyruvate Transporter CstA in Escherichia coli

“…BtsS/BtsR is a high-affinity pyruvate sensing system, while pyruvate acts as a low-affinity stimulus for the PyrS/PyrR system. Both systems regulate the expression of the pyruvate/H + symporter BtsT [20], as well as YhjX, a putative low-affinity pyruvate transporter belonging to the major facilitator superfamily [14,21].…”

“…BtsT displays high sequence similarity (75.4%) and identity (61.1%) to CstA [20]. Recently, CstA was identified as constitutively expressed pyruvate transporter by transposon mutagenesis in E. coli [21]. Nevertheless, its mode of action and substrate specificity remain unknown.…”

Reprogramming of gene expression in Escherichia coli cultured on pyruvate versus glucose

“…In contrast, only few transporter-encoding genes were upregulated. The most prominent ones included two genes coding for the BtsSR pyruvate sensor system (i.e., btsS and btsR) as well as major pyruvate transporter genes (i.e., btsT, yhjX and cstA (Hwang et al 2018).…”

Biotransformation and chiral resolution of d,l‐alanine into pyruvate and d‐alanine with a whole‐cell biocatalyst expressing l‐amino acid deaminase

“…Therefore, cutting off the pathways of pyruvate transport into cells may be feasible to prevent the utilization of pyruvate by the biocatalyst. BtsT was first characterized as a pyruvate transporter, soon after this, CstA and YbdD were demonstrated to be comprised of the constitutive pyruvate transporter system in E. coli, and the two proteins constitute a single pyruvate transporter complex [38,39]. Considering that the ybdD single knockout strain showed similar level of pyruvate uptake as that of cstA single knockout strain and the triple knockout mutant cstA ybdD btsT cannot grow [39], we knocked out cstA and btsT genes simultaneously in E. coli BL21 (DE3) to reduce the pyruvate uptake ( Fig.…”