A Vector Library for Silencing Central Carbon Metabolism Genes with Antisense RNAs in Escherichia coli

ABSTRACTWe describe here the construction of a series of 71 vectors to silence central carbon metabolism genes inEscherichia coli. The vectors inducibly express antisense RNAs called paired-terminus antisense RNAs, which have a higher silencing efficacy than ordinary antisense RNAs. By measuring mRNA amounts, measuring activities of target proteins, or observing specific phenotypes, it was confirmed that all the vectors were able to silence the expression of target genes efficiently. Using this vector set, each of the central carbon metabolism genes was silenced individually, and the accumulation of metabolites was investigated. We were able to obtain accurate information on ways to increase the production of pyruvate, an industrially valuable compound, from the silencing results. Furthermore, the experimental results of pyruvate accumulation were compared toin silicopredictions, and both sets of results were consistent. Compared to the gene disruption approach, the silencing approach has an advantage in that anyE. colistrain can be used and multiple gene silencing is easily possible in any combination.

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The Role of Metabolites in the Link between DNA Replication and Central Carbon Metabolism in Escherichia coli

Genes ◽

10.3390/genes11040447 ◽

2020 ◽

Vol 11 (4) ◽

pp. 447

Author(s):

Klaudyna Krause ◽

Monika Maciąg-Dorszyńska ◽

Anna Wosinski ◽

Lidia Gaffke ◽

Joanna Morcinek-Orłowska ◽

...

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Protein Interactions ◽

Carbon Metabolism ◽

Molecular Mechanisms ◽

Central Carbon Metabolism ◽

Replication Initiation ◽

Cell Physiology ◽

E Coli ◽

Central Carbon

A direct link between DNA replication regulation and central carbon metabolism (CCM) has been previously demonstrated in Bacillus subtilis and Escherichia coli, as effects of certain mutations in genes coding for replication proteins could be specifically suppressed by particular mutations in genes encoding CCM enzymes. However, specific molecular mechanism(s) of this link remained unknown. In this report, we demonstrate that various CCM metabolites can suppress the effects of mutations in different replication genes of E. coli on bacterial growth, cell morphology, and nucleoid localization. This provides evidence that the CCM-replication link is mediated by metabolites rather than direct protein-protein interactions. On the other hand, action of metabolites on DNA replication appears indirect rather than based on direct influence on the replication machinery, as rate of DNA synthesis could not be corrected by metabolites in short-term experiments. This corroborates the recent discovery that in B. subtilis, there are multiple links connecting CCM to DNA replication initiation and elongation. Therefore, one may suggest that although different in detail, the molecular mechanisms of CCM-dependent regulation of DNA replication are similar in E. coli and B. subtilis, making this regulation an important and common constituent of the control of cell physiology in bacteria.

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Metabolic Flux Ratio Analysis of Genetic and Environmental Modulations of Escherichia coli Central Carbon Metabolism

Journal of Bacteriology ◽

10.1128/jb.181.21.6679-6688.1999 ◽

1999 ◽

Vol 181 (21) ◽

pp. 6679-6688 ◽

Cited By ~ 263

Author(s):

Uwe Sauer ◽

Daniel R. Lasko ◽

Jocelyne Fiaux ◽

Michel Hochuli ◽

Ralf Glaser ◽

...

Keyword(s):

Escherichia Coli ◽

Carbon Metabolism ◽

Metabolic Flux ◽

Flux Ratio ◽

Tca Cycle ◽

Central Carbon Metabolism ◽

E Coli ◽

Chemostat Cultures ◽

Central Carbon ◽

Pep Carboxykinase

ABSTRACT The response of Escherichia coli central carbon metabolism to genetic and environmental manipulation has been studied by use of a recently developed methodology for metabolic flux ratio (METAFoR) analysis; this methodology can also directly reveal active metabolic pathways. Generation of fluxome data arrays by use of the METAFoR approach is based on two-dimensional13C-1H correlation nuclear magnetic resonance spectroscopy with fractionally labeled biomass and, in contrast to metabolic flux analysis, does not require measurements of extracellular substrate and metabolite concentrations. METAFoR analyses of E. coli strains that moderately overexpress phosphofructokinase, pyruvate kinase, pyruvate decarboxylase, or alcohol dehydrogenase revealed that only a few flux ratios change in concert with the overexpression of these enzymes. Disruption of both pyruvate kinase isoenzymes resulted in altered flux ratios for reactions connecting the phosphoenolpyruvate (PEP) and pyruvate pools but did not significantly alter central metabolism. These data indicate remarkable robustness and rigidity in central carbon metabolism in the presence of genetic variation. More significant physiological changes and flux ratio differences were seen in response to altered environmental conditions. For example, in ammonia-limited chemostat cultures, compared to glucose-limited chemostat cultures, a reduced fraction of PEP molecules was derived through at least one transketolase reaction, and there was a higher relative contribution of anaplerotic PEP carboxylation than of the tricarboxylic acid (TCA) cycle for oxaloacetate synthesis. These two parameters also showed significant variation between aerobic and anaerobic batch cultures. Finally, two reactions catalyzed by PEP carboxykinase and malic enzyme were identified by METAFoR analysis; these had previously been considered absent in E. colicells grown in glucose-containing media. Backward flux from the TCA cycle to glycolysis, as indicated by significant activity of PEP carboxykinase, was found only in glucose-limited chemostat culture, demonstrating that control of this futile cycle activity is relaxed under severe glucose limitation.

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Flux redistribution of central carbon metabolism for efficient production of L‐tryptophan in Escherichia coli

Biotechnology and Bioengineering ◽

10.1002/bit.27665 ◽

2021 ◽

Author(s):

Bo Xiong ◽

Yongduo Zhu ◽

Daoguang Tian ◽

Shuai Jiang ◽

Xiaoguang Fan ◽

...

Keyword(s):

Escherichia Coli ◽

Carbon Metabolism ◽

Central Carbon Metabolism ◽

Efficient Production ◽

Central Carbon ◽

Flux Redistribution

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HexR Controls Glucose-Responsive Genes and Central Carbon Metabolism in Neisseria meningitidis

Journal of Bacteriology ◽

10.1128/jb.00659-15 ◽

2015 ◽

Vol 198 (4) ◽

pp. 644-654 ◽

Cited By ~ 6

Author(s):

Ana Antunes ◽

Giacomo Golfieri ◽

Francesca Ferlicca ◽

Marzia M. Giuliani ◽

Vincenzo Scarlato ◽

...

Keyword(s):

Energy Metabolism ◽

Neisseria Meningitidis ◽

Microarray Analysis ◽

Carbon Metabolism ◽

Tricarboxylic Acid Cycle ◽

Transcriptional Regulator ◽

Central Carbon Metabolism ◽

Binding Motif ◽

Content Type ◽

Central Carbon

ABSTRACTNeisseria meningitidis, an exclusively human pathogen and the leading cause of bacterial meningitis, must adapt to different host niches during human infection.N. meningitidiscan utilize a restricted range of carbon sources, including lactate, glucose, and pyruvate, whose concentrations vary in host niches. Microarray analysis ofN. meningitidisgrown in a chemically defined medium in the presence or absence of glucose allowed us to identify genes regulated by carbon source availability. Most such genes are implicated in energy metabolism and transport, and some are implicated in virulence. In particular, genes involved in glucose catabolism were upregulated, whereas genes involved in the tricarboxylic acid cycle were downregulated. Several genes encoding surface-exposed proteins, including the MafA adhesins andNeisseriasurface protein A, were upregulated in the presence of glucose. Our microarray analysis led to the identification of a glucose-responsivehexR-like transcriptional regulator that controls genes of the central carbon metabolism ofN. meningitidisin response to glucose. We characterized the HexR regulon and showed that thehexRgene is accountable for some of the glucose-responsive regulation;in vitroassays with the purified protein showed that HexR binds to the promoters of the central metabolic operons of the bacterium. Based on DNA sequence alignment of the target sites, we propose a 17-bp pseudopalindromic consensus HexR binding motif. Furthermore,N. meningitidisstrains lackinghexRexpression were deficient in establishing successful bacteremia in an infant rat model of infection, indicating the importance of this regulator for the survival of this pathogenin vivo.IMPORTANCENeisseria meningitidisgrows on a limited range of nutrients during infection. We analyzed the gene expression ofN. meningitidisin response to glucose, the main energy source available in human blood, and we found that glucose regulates many genes implicated in energy metabolism and nutrient transport, as well as some implicated in virulence. We identified and characterized a transcriptional regulator (HexR) that controls metabolic genes ofN. meningitidisin response to glucose. We generated a mutant lacking HexR and found that the mutant was impaired in causing systemic infection in animal models. SinceN. meningitidislacks known bacterial regulators of energy metabolism, our findings suggest that HexR plays a major role in its biology by regulating metabolism in response to environmental signals.

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Elucidation of Regulatory Modes for Five Two-Component Systems in Escherichia coli Reveals Novel Relationships

mSystems ◽

10.1128/msystems.00980-20 ◽

2020 ◽

Vol 5 (6) ◽

Author(s):

Kumari Sonal Choudhary ◽

Julia A. Kleinmanns ◽

Katherine Decker ◽

Anand V. Sastry ◽

Ye Gao ◽

...

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Target Gene ◽

Target Genes ◽

Rna Seq ◽

Content Type ◽

E Coli ◽

Component Systems ◽

Two Component ◽

Two Component Systems

ABSTRACT Escherichia coli uses two-component systems (TCSs) to respond to environmental signals. TCSs affect gene expression and are parts of E. coli’s global transcriptional regulatory network (TRN). Here, we identified the regulons of five TCSs in E. coli MG1655: BaeSR and CpxAR, which were stimulated by ethanol stress; KdpDE and PhoRB, induced by limiting potassium and phosphate, respectively; and ZraSR, stimulated by zinc. We analyzed RNA-seq data using independent component analysis (ICA). ChIP-exo data were used to validate condition-specific target gene binding sites. Based on these data, we do the following: (i) identify the target genes for each TCS; (ii) show how the target genes are transcribed in response to stimulus; and (iii) reveal novel relationships between TCSs, which indicate noncognate inducers for various response regulators, such as BaeR to iron starvation, CpxR to phosphate limitation, and PhoB and ZraR to cell envelope stress. Our understanding of the TRN in E. coli is thus notably expanded. IMPORTANCE E. coli is a common commensal microbe found in the human gut microenvironment; however, some strains cause diseases like diarrhea, urinary tract infections, and meningitis. E. coli’s two-component systems (TCSs) modulate target gene expression, especially related to virulence, pathogenesis, and antimicrobial peptides, in response to environmental stimuli. Thus, it is of utmost importance to understand the transcriptional regulation of TCSs to infer bacterial environmental adaptation and disease pathogenicity. Utilizing a combinatorial approach integrating RNA sequencing (RNA-seq), independent component analysis, chromatin immunoprecipitation coupled with exonuclease treatment (ChIP-exo), and data mining, we suggest five different modes of TCS transcriptional regulation. Our data further highlight noncognate inducers of TCSs, which emphasizes the cross-regulatory nature of TCSs in E. coli and suggests that TCSs may have a role beyond their cognate functionalities. In summary, these results can lead to an understanding of the metabolic capabilities of bacteria and correctly predict complex phenotype under diverse conditions, especially when further incorporated with genome-scale metabolic models.

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Dynamic Metabolic Rewiring Enables Efficient Acetyl Coenzyme A Assimilation in Paracoccus denitrificans

mBio ◽

10.1128/mbio.00805-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 4

Author(s):

Katharina Kremer ◽

Muriel C. F. van Teeseling ◽

Lennart Schada von Borzyskowski ◽

Iria Bernhardsgrütter ◽

Rob J. M. van Spanning ◽

...

Keyword(s):

Growth Rates ◽

Carbon Metabolism ◽

Paracoccus Denitrificans ◽

High Growth ◽

Central Carbon Metabolism ◽

High Biomass ◽

Acetyl Coa ◽

Acetyl Coenzyme A ◽

Content Type ◽

Central Carbon

ABSTRACT During growth, microorganisms have to balance metabolic flux between energy and biosynthesis. One of the key intermediates in central carbon metabolism is acetyl coenzyme A (acetyl-CoA), which can be either oxidized in the citric acid cycle or assimilated into biomass through dedicated pathways. Two acetyl-CoA assimilation strategies in bacteria have been described so far, the ethylmalonyl-CoA pathway (EMCP) and the glyoxylate cycle (GC). Here, we show that Paracoccus denitrificans uses both strategies for acetyl-CoA assimilation during different growth stages, revealing an unexpected metabolic complexity in the organism’s central carbon metabolism. The EMCP is constitutively expressed on various substrates and leads to high biomass yields on substrates requiring acetyl-CoA assimilation, such as acetate, while the GC is specifically induced on these substrates, enabling high growth rates. Even though each acetyl-CoA assimilation strategy alone confers a distinct growth advantage, P. denitrificans recruits both to adapt to changing environmental conditions, such as a switch from succinate to acetate. Time-resolved single-cell experiments show that during this switch, expression of the EMCP and GC is highly coordinated, indicating fine-tuned genetic programming. The dynamic metabolic rewiring of acetyl-CoA assimilation is an evolutionary innovation by P. denitrificans that allows this organism to respond in a highly flexible manner to changes in the nature and availability of the carbon source to meet the physiological needs of the cell, representing a new phenomenon in central carbon metabolism. IMPORTANCE Central carbon metabolism provides organisms with energy and cellular building blocks during growth and is considered the invariable “operating system” of the cell. Here, we describe a new phenomenon in bacterial central carbon metabolism. In contrast to many other bacteria that employ only one pathway for the conversion of the central metabolite acetyl-CoA, Paracoccus denitrificans possesses two different acetyl-CoA assimilation pathways. These two pathways are dynamically recruited during different stages of growth, which allows P. denitrificans to achieve both high biomass yield and high growth rates under changing environmental conditions. Overall, this dynamic rewiring of central carbon metabolism in P. denitrificans represents a new strategy compared to those of other organisms employing only one acetyl-CoA assimilation pathway.

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