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

Anzai, Takumi; Imamura, Sousuke; Ishihama, Akira; Shimada, Tomohiro

doi:10.1099/mgen.0.000442

Cited by 16 publications

(20 citation statements)

References 63 publications

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“…The aceE and aceF genes constitute an operon together with lpd and the transcriptional repressor gene pdhR as pdhR ‐ aceE ‐ aceF ‐ lpd in this order [37]. Transcriptional repression of this operon by PdhR is derepressed by pyruvate [37,38]; thus, overexpression of the PDHc subunits suggested the intracellular accumulation of pyruvate. The aceA and aceB genes constitute an operon with the regulatory kinase gene aceK as aceB ‐ aceA ‐ aceK in this order, which is negatively regulated by the repressor IclR [39].…”

Section: Resultsmentioning

confidence: 99%

Acetate overflow metabolism regulates a major metabolic shift after glucose depletion in Escherichia coli

et al. 2021

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Acetate overflow refers to the metabolism by which a large part of carbon incorporated as glucose into Escherichia coli cells is catabolized and excreted as acetate into the medium. We previously found that mutants for the acetate overflow pathway enzymes phosphoacetyltransferase (Pta) and acetate kinase (AckA) showed significant diauxic growth after glucose depletion in E. coli. Here, we analyzed the underlying mechanism in the pta mutant. Proteomic and other analyses revealed an increase in pyruvate dehydrogenase complex subunits and a decrease in glyoxylate shunt enzymes, which resulted from pyruvate accumulation. Since restoration of these enzyme levels by overexpressing PdhR (pyruvate‐sensing transcription factor) or deleting iclR (gene encoding a pyruvate‐ and glyoxylate‐sensing transcription factor) alleviated the growth lag of the pta mutant after glucose depletion, these changes were considered as the reason for the phenotype. Given the evidence for decreased coenzyme A (HS‐CoA) levels in the pta mutant, the growth inhibition after glucose depletion was partly explained by limited availability of HS‐CoA in the cell. The findings provide insights into the role of acetate overflow in metabolic regulation, which may be useful for biotechnological applications.

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Section: Resultsmentioning

confidence: 99%

Acetate overflow metabolism regulates a major metabolic shift after glucose depletion in Escherichia coli

et al. 2021

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“…PdhR represses the entire operon and autoregulates its own synthesis by binding to the pdhR promoter region. Repression by PdhR is relieved by pyruvate and PdhR controls an additional small regulon of about 20 genes [ 47 ]. CRISPRi can inhibit transcription by blocking initiation or elongation.…”

Section: Resultsmentioning

confidence: 99%

“…For example, deletions of pdhR lead to overexpression of the pyruvate dehydrogenase complex and reduced maximum specific growth rates [ 54 ]. In wild‐type E. coli PdhR autoregulates its own synthesis and serves as a regulator to 16‐23 other genes [ 47 ]. Its central function is to relieve the repression of pdhRp at high pyruvate concentrations, thereby enhancing the expression of pdhR , aceE , and aceF which accelerates pyruvate degradation to acetyl‐CoA.…”

Section: Discussionmentioning

confidence: 99%

CRISPRi enables fast growth followed by stable aerobic pyruvate formation in Escherichia coli without auxotrophy

Ziegler

Hägele

Gäbele

2021

Engineering in Life Sciences

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CRISPR interference (CRISPRi) was applied to enable the aerobic production of pyruvate in Escherichia coli MG1655 under glucose excess conditions by targeting the promoter regions of aceE or pdhR. Knockdown strains were cultivated in aerobic shaking flasks and the influence of inducer concentration and different sgRNA binding sites on the production of pyruvate was measured. Targeting the promoter regions of aceE or pdhR triggered pyruvate production during the exponential phase and reduced expression of aceE. In lab‐scale bioreactor fermentations, an aceE silenced strain successfully produced pyruvate under fully aerobic conditions during the exponential phase, but loss of productivity occurred during a subsequent nitrogen‐limited phase. Targeting the promoter region of pdhR enabled pyruvate production during the growth phase of cultivations, and a continued low‐level accumulation during the nitrogen‐limited production phase. Combinatorial targeting of the promoter regions of both aceE and pdhR in E. coli MG1655 pdCas9 psgRNA_aceE_234_pdhR_329 resulted in the stable aerobic production of pyruvate with non‐growing cells at YP/S = 0.36 ± 0.029 gPyruvate/gGlucose in lab‐scale bioreactors throughout an extended nitrogen‐limited production phase.

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“…RT-qPCR analysis was performed according to a standard procedure [ 40 ]. The nitrogen replete and deplete conditions were made according to Brown et al with some modifications [ 22 ].…”

Section: Methodsmentioning

confidence: 99%

Whole set of constitutive promoters for RpoN sigma factor and the regulatory role of its enhancer protein NtrC in Escherichia coli K-12

2021

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The promoter selectivity of Escherichia coli RNA polymerase (RNAP) is determined by its promoter-recognition sigma subunit. The model prokaryote E. coli K-12 contains seven species of the sigma subunit, each recognizing a specific set of promoters. Using genomic SELEX (gSELEX) screening in vitro, we identified the whole set of ‘constitutive’ promoters recognized by the reconstituted RNAP holoenzyme alone, containing RpoD (σ70), RpoS (σ38), RpoH (σ32), RpoF (σ28) or RpoE (σ24), in the absence of other supporting regulatory factors. In contrast, RpoN sigma (σ54), involved in expression of nitrogen-related genes and also other cellular functions, requires an enhancer (or activator) protein, such as NtrC, for transcription initiation. In this study, a series of gSELEX screenings were performed to search for promoters recognized by the RpoN RNAP holoenzyme in the presence and absence of the major nitrogen response enhancer NtrC, the best-characterized enhancer. Based on the RpoN holoenzyme-binding sites, a total of 44 to 61 putative promoters were identified, which were recognized by the RpoN holoenzyme alone. In the presence of the enhancer NtrC, the recognition target increased to 61–81 promoters. Consensus sequences of promoters recognized by RpoN holoenzyme in the absence and presence of NtrC were determined. The promoter activity of a set of NtrC-dependent and -independent RpoN promoters was verified in vivo under nitrogen starvation, in the presence and absence of RpoN and/or NtrC. The promoter activity of some RpoN-recognized promoters increased in the absence of RpoN or NtrC, supporting the concept that the promoter-bound NtrC-enhanced RpoN holoenzyme functions as a repressor against RpoD holoenzyme. Based on our findings, we propose a model in which the RpoN holoenzyme fulfils the dual role of repressor and transcriptase for the same set of genes. We also propose that the promoter recognized by RpoN holoenzyme in the absence of enhancers is the ‘repressive’ promoter. The presence of high-level RpoN sigma in growing E. coli K-12 in rich medium may be related to the repression role of a set of genes needed for the utilization of ammonia as a nitrogen source in poor media. The list of newly identified regulatory targets of RpoN provides insight into E. coli survival under nitrogen-depleted conditions in nature.

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

Cited by 16 publications

References 63 publications

Acetate overflow metabolism regulates a major metabolic shift after glucose depletion in Escherichia coli

Acetate overflow metabolism regulates a major metabolic shift after glucose depletion in Escherichia coli

CRISPRi enables fast growth followed by stable aerobic pyruvate formation in Escherichia coli without auxotrophy

Whole set of constitutive promoters for RpoN sigma factor and the regulatory role of its enhancer protein NtrC in Escherichia coli K-12

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