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BtsT, a Novel and Specific Pyruvate/H⁺ Symporter in Escherichia coli.

ABSTRACT: The peptide transporter carbon starvation (CstA) family (transporter classification [TC] 2.A.114) belongs to the second largest superfamily of secondary transporters, the amino acid/polyamine/organocation (APC) superfamily. No representative of the CstA family has previously been characterized either biochemically or structurally, but we have now identified the function of one of its members, the transport protein YjiY of Escherichia coli Expression of the yjiY gene is regulated by the LytS-like histidine kinase BtsS, a sensor of extracellular pyruvate, together with the LytTR-like response regulator BtsR. YjiY consists of 716 amino acids, which form 18 putative transmembrane helices. Transport studies with intact cells provided evidence that YjiY is a specific and high-affinity transporter for pyruvate (K_m , 16 ?M). Furthermore, reconstitution of the purified YjiY into proteoliposomes revealed that YjiY is a pyruvate/H⁺ symporter. It has long been assumed that E. coli possesses a transporter(s) for pyruvate, but the present study is the first to definitively identify such a protein. Based on its function, we propose to change the name of the uncharacterized gene yjiY to btsT for Brenztraubensäure (the German word for pyruvate) transporter.IMPORTANCE BtsT (formerly known as YjiY) is found in many commensal and pathogenic representatives of the Enterobacteriaceae This study for the first time characterizes a pyruvate transporter in E. coli, BtsT, as a specific pyruvate/H⁺ symporter. When nutrients are limiting, BtsT takes up pyruvate from the medium, thus enabling it to be used as a carbon source for the growth and survival of E. coli.

SUBMITTER: Kristoficova I

PROVIDER: S-EPMC5738728 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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BtsT, a Novel and Specific Pyruvate/H+ Symporter in Escherichia coli.

Kristoficova Ivica I Vilhena Cláudia C Behr Stefan S Jung Kirsten K

Journal of bacteriology 20171220 2

The peptide transporter carbon starvation (CstA) family (transporter classification [TC] 2.A.114) belongs to the second largest superfamily of secondary transporters, the amino acid/polyamine/organocation (APC) superfamily. No representative of the CstA family has previously been characterized either biochemically or structurally, but we have now identified the function of one of its members, the transport protein YjiY of Escherichia coli Expression of the yjiY gene is regulated by ...[more]

PMID: 29061664

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Project description: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. coliIMPORTANCE 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.

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BtsT, a Novel and Specific Pyruvate/H⁺ Symporter in Escherichia coli.

Publications

BtsT, a Novel and Specific Pyruvate/H<sup>+</sup> Symporter in Escherichia coli.

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