Dataset Information

Expanded roles of pyruvate-sensing PdhR in transcription regulation of the Escherichia coli K-12 genome: fatty acid catabolism and cell motility.

ABSTRACT: The transcription factor PdhR has been recognized as the master regulator of the pyruvate catabolism pathway in Escherichia coli, including both NAD-linked oxidative decarboxylation of pyruvate to acetyl-CoA by PDHc (pyruvate dehydrogenase complex) and respiratory electron transport of NADH to oxygen by Ndh-CyoABCD enzymes. To identify the whole set of regulatory targets under the control of pyruvate-sensing PdhR, we performed genomic SELEX (gSELEX) screening in vitro. A total of 35 PdhR-binding sites were identified along the E. coli K-12 genome, including previously identified targets. Possible involvement of PdhR in regulation of the newly identified target genes was analysed in detail by gel shift assay, RT-qPCR and Northern blot analysis. The results indicated the participation of PdhR in positive regulation of fatty acid degradation genes and negative regulation of cell mobility genes. In fact, GC analysis indicated an increase in free fatty acids in the mutant lacking PdhR. We propose that PdhR is a bifunctional global regulator for control of a total of 16-23 targets, including not only the genes involved in central carbon metabolism but also some genes for the surrounding pyruvate-sensing cellular pathways such as fatty acid degradation and flagella formation. The activity of PdhR is controlled by pyruvate, the key node between a wide variety of metabolic pathways, including generation of metabolic energy and cell building blocks.

SUBMITTER: Anzai T

PROVIDER: S-EPMC7660256 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Expanded roles of pyruvate-sensing PdhR in transcription regulation of the Escherichia coli K-12 genome: fatty acid catabolism and cell motility.

Anzai Takumi T Imamura Sousuke S Ishihama Akira A Shimada Tomohiro T

Microbial genomics 20201001 10

The transcription factor PdhR has been recognized as the master regulator of the pyruvate catabolism pathway in Escherichia coli, including both NAD-linked oxidative decarboxylation of pyruvate to acetyl-CoA by PDHc (pyruvate dehydrogenase complex) and respiratory electron transport of NADH to oxygen by Ndh-CyoABCD enzymes. To identify the whole set of regulatory targets under the control of pyruvate-sensing PdhR, we performed genomic SELEX (gSELEX) screening in vitro. A total of 3 ...[more]

PMID: 32975502

Similar Datasets

Project description:Gene expression profiles of Escherichia coli K-12 W3110 were compared as a function of steady-state external pH. Cultures were grown to an optical density at 600 nm of 0.3 in potassium-modified Luria-Bertani medium buffered at pH 5.0, 7.0, and 8.7. For each of the three pH conditions, cDNA from RNA of five independent cultures was hybridized to Affymetrix E. coli arrays. Analysis of variance with an alpha level of 0.001 resulted in 98% power to detect genes showing a twofold difference in expression. Normalized expression indices were calculated for each gene and intergenic region (IG). Differential expression among the three pH classes was observed for 763 genes and 353 IGs. Hierarchical clustering yielded six well-defined clusters of pH profiles, designated Acid High (highest expression at pH 5.0), Acid Low (lowest expression at pH 5.0), Base High (highest at pH 8.7), Base Low (lowest at pH 8.7), Neutral High (highest at pH 7.0, lower in acid or base), and Neutral Low (lowest at pH 7.0, higher at both pH extremes). Flagellar and chemotaxis genes were repressed at pH 8.7 (Base Low cluster), where the cell's transmembrane proton potential is diminished by the maintenance of an inverted pH gradient. High pH also repressed the proton pumps cytochrome o (cyo) and NADH dehydrogenases I and II. By contrast, the proton-importing ATP synthase F1Fo and the microaerophilic cytochrome d (cyd), which minimizes proton export, were induced at pH 8.7. These observations are consistent with a model in which high pH represses synthesis of flagella, which expend proton motive force, while stepping up electron transport and ATPase components that keep protons inside the cell. Acid-induced genes, on the other hand, were coinduced by conditions associated with increased metabolic rate, such as oxidative stress. All six pH-dependent clusters included envelope and periplasmic proteins, which directly experience external pH. Overall, this study showed that (i) low pH accelerates acid consumption and proton export, while coinducing oxidative stress and heat shock regulons; (ii) high pH accelerates proton import, while repressing the energy-expensive flagellar and chemotaxis regulons; and (iii) pH differentially regulates a large number of periplasmic and envelope proteins.

Project description:Gene expression profiles of Escherichia coli K-12 W3110 were compared as a function of steady-state external pH. Cultures were grown with aeration to an optical density at 600 nm of 0.3 in potassium-modified Luria-Bertani medium buffered at pH 5.0, 7.0, and 8.7. For each of the three pH conditions, cDNA from RNA of five independent cultures was hybridized to Affymetrix E. coli arrays. Analysis of variance with a significance level of 0.001 resulted in 98% power to detect genes showing a twofold difference in expression. Normalized expression indices were calculated for each gene and intergenic region (IG). Differential expression among the three pH classes was observed for 763 genes and 353 IGs. Hierarchical clustering yielded six well-defined clusters of pH profiles, designated Acid High (highest expression at pH 5.0), Acid Low (lowest expression at pH 5.0), Base High (highest at pH 8.7), Base Low (lowest at pH 8.7), Neutral High (highest at pH 7.0, lower in acid or base), and Neutral Low (lowest at pH 7.0, higher at both pH extremes). Flagellar and chemotaxis genes were repressed at pH 8.7 (Base Low cluster), where the cell's transmembrane proton potential is diminished by the maintenance of an inverted pH gradient. High pH also repressed the proton pumps cytochrome o (cyo) and NADH dehydrogenases I and II. By contrast, the proton-importing ATP synthase F1Fo and the microaerophilic cytochrome d (cyd), which minimizes proton export, were induced at pH 8.7. These observations are consistent with a model in which high pH represses synthesis of flagella, which expend proton motive force, while stepping up electron transport and ATPase components that keep protons inside the cell. Acid-induced genes, on the other hand, were coinduced by conditions associated with increased metabolic rate, such as oxidative stress. All six pH-dependent clusters included envelope and periplasmic proteins, which directly experience external pH. Overall, this study showed that (i) low pH accelerates acid consumption and proton export, while coinducing oxidative stress and heat shock regulons; (ii) high pH accelerates proton import, while repressing the energy-expensive flagellar and chemotaxis regulons; and (iii) pH differentially regulates a large number of periplasmic and envelope proteins. Keywords: Steady State

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.

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.

Project description:Acid-adapted strains of Escherichia coli K-12 W3110 were obtained by serial culture in medium buffered at pH 4.6 (M. M. Harden, A. He, K. Creamer, M. W. Clark, I. Hamdallah, K. A. Martinez, R. L. Kresslein, S. P. Bush, and J. L. Slonczewski, Appl Environ Microbiol 81:1932-1941, 2015, https://doi.org/10.1128/AEM.03494-14). Revised genomic analysis of these strains revealed insertion sequence (IS)-driven insertions and deletions that knocked out regulators CadC (acid induction of lysine decarboxylase), GadX (acid induction of glutamate decarboxylase), and FNR (anaerobic regulator). Each acid-evolved strain showed loss of one or more amino acid decarboxylase systems, which normally help neutralize external acid (pH 5 to 6) and increase survival in extreme acid (pH 2). Strains from populations B11, H9, and F11 had an IS5 insertion or IS-mediated deletion in cadC, while population B11 had a point mutation affecting the arginine activator adiY The cadC and adiY mutants failed to neutralize acid in the presence of exogenous lysine or arginine. In strain B11-1, reversion of an rpoC (RNA polymerase) mutation partly restored arginine-dependent neutralization. All eight strains showed deletion or downregulation of the Gad acid fitness island. Strains with the Gad deletion lost the ability to produce GABA (gamma-aminobutyric acid) and failed to survive extreme acid. Transcriptome sequencing (RNA-seq) of strain B11-1 showed upregulated genes for catabolism of diverse substrates but downregulated acid stress genes (the biofilm regulator ariR, yhiM, and Gad). Other strains showed downregulation of H2 consumption mediated by hydrogenases (hya and hyb) which release acid. Strains F9-2 and F9-3 had a deletion of fnr and showed downregulation of FNR-dependent genes (dmsABC, frdABCD, hybABO, nikABCDE, and nrfAC). Overall, strains that had evolved in buffered acid showed loss or downregulation of systems that neutralize unbuffered acid and showed altered regulation of catabolism.IMPORTANCE Experimental evolution of an enteric bacterium under a narrow buffered range of acid pH leads to loss of genes that enhance fitness above or below the buffered pH range, including loss of enzymes that may raise external pH in the absence of buffer. Prominent modes of evolutionary change involve IS-mediated insertions and deletions that knock out key regulators. Over generations of acid stress, catabolism undergoes reregulation in ways that differ for each evolving strain.

Dataset Information

Expanded roles of pyruvate-sensing PdhR in transcription regulation of the Escherichia coli K-12 genome: fatty acid catabolism and cell motility.

Publications

Expanded roles of pyruvate-sensing PdhR in transcription regulation of the <i>Escherichia coli</i> K-12 genome: fatty acid catabolism and cell motility.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets