Dataset Information

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. 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.

SUBMITTER: Hwang S

PROVIDER: S-EPMC5847655 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Peptide Transporter CstA Imports Pyruvate in Escherichia coli K-12.

Hwang Soonkyu S Choe Donghui D Yoo Minseob M Cho Sanghyuk S Kim Sun Chang SC Cho Suhyung S Cho Byung-Kwan BK

Journal of bacteriology 20180312 7

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 ...[more]

PMID: 29358499

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Project description:Escherichia coli and many other bacterial species can enter into a viable but nonculturable (VBNC) state, which is a survival strategy adopted by cells exposed to adverse environmental conditions. Pyruvate is known to be one factor that promotes resuscitation of VBNC cells. Here we studied the role of a pyruvate-sensing network, composed of the histidine kinase-response regulator systems BtsS/BtsR and YpdA/YpdB and the target gene btsT, encoding the high-affinity pyruvate/H+ symporter BtsT, in the resuscitation of VBNC E. coli K-12 cells after exposure to cold for 120 days. Analysis of the proteome of VBNC cells revealed upregulation, relative to exponentially growing cells, of BtsT and other proteins involved in pyruvate metabolism. Provision of pyruvate stimulated protein and DNA biosynthesis, and thus resuscitation, in wild-type but not btsSR ypdAB mutant VBNC cells. This result was corroborated by time-dependent tracking of the resuscitation of individual VBNC E. coli cells observed in a microfluidic system. Finally, transport assays revealed that 14C-labeled pyruvate was rapidly taken up into VBNC cells by BtsT. These results provide the first evidence that pyruvate is taken up as a carbon source for the resuscitation of VBNC E. coli cells.IMPORTANCE Viable but nonculturable (VBNC) bacteria do not form colonies in standard medium but otherwise retain their metabolic activity and can express toxic proteins. Many bacterial genera, including Escherichia, Vibrio, and Listeria, have been shown to enter the VBNC state upon exposure to adverse conditions, such as low temperature, radiation, and starvation. Ultimately, these organisms pose a public health risk with potential implications for the pharmaceutical and food industries, as dormant organisms are especially difficult to selectively eliminate and VBNC bacteria can be resuscitated if placed in an environment with appropriate nutrition and temperature. Here we used a microfluidic system to monitor the resuscitation of single VBNC cells over time. We provide new molecular insights into the initiation of resuscitation by demonstrating that VBNC E. coli cells rapidly take up pyruvate with an inducible high-affinity transporter, whose expression is triggered by the BtsSR-YpdAB sensing network.

Dataset Information

Peptide Transporter CstA Imports Pyruvate in Escherichia coli K-12.

Publications

Peptide Transporter CstA Imports Pyruvate in Escherichia coli K-12.

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