scholarly journals Dihydrolipoamide Dehydrogenase Mutation Alters the NADH Sensitivity of Pyruvate Dehydrogenase Complex of Escherichia coli K-12

2008 ◽  
Vol 190 (11) ◽  
pp. 3851-3858 ◽  
Author(s):  
Youngnyun Kim ◽  
L. O. Ingram ◽  
K. T. Shanmugam
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Double Mutant ◽  
Pyruvate Dehydrogenase Complex ◽  
Anaerobic Growth ◽  
Lower Sensitivity ◽  
Dihydrolipoamide Dehydrogenase ◽  
Fermentation Products ◽  
E Coli ◽  
K 12

ABSTRACT Under anaerobic growth conditions, an active pyruvate dehydrogenase (PDH) is expected to create a redox imbalance in wild-type Escherichia coli due to increased production of NADH (>2 NADH molecules/glucose molecule) that could lead to growth inhibition. However, the additional NADH produced by PDH can be used for conversion of acetyl coenzyme A into reduced fermentation products, like alcohols, during metabolic engineering of the bacterium. E. coli mutants that produced ethanol as the main fermentation product were recently isolated as derivatives of an ldhA pflB double mutant. In all six mutants tested, the mutation was in the lpd gene encoding dihydrolipoamide dehydrogenase (LPD), a component of PDH. Three of the LPD mutants carried an H322Y mutation (lpd102), while the other mutants carried an E354K mutation (lpd101). Genetic and physiological analysis revealed that the mutation in either allele supported anaerobic growth and homoethanol fermentation in an ldhA pflB double mutant. Enzyme kinetic studies revealed that the LPD(E354K) enzyme was significantly less sensitive to NADH inhibition than the native LPD. This reduced NADH sensitivity of the mutated LPD was translated into lower sensitivity of the appropriate PDH complex to NADH inhibition. The mutated forms of the PDH had a 10-fold-higher Ki for NADH than the native PDH. The lower sensitivity of PDH to NADH inhibition apparently increased PDH activity in anaerobic E. coli cultures and created the new ethanologenic fermentation pathway in this bacterium. Analogous mutations in the LPD of other bacteria may also significantly influence the growth and physiology of the organisms in a similar fashion.

scholarly journals Metabolic Flux Control at the Pyruvate Node in an Anaerobic Escherichia coli Strain with an Active Pyruvate Dehydrogenase

2010 ◽  
Vol 76 (7) ◽  
pp. 2107-2114 ◽  
Author(s):  
Qingzhao Wang ◽  
Mark S. Ou ◽  
Y. Kim ◽  
L. O. Ingram ◽  
K. T. Shanmugam
Keyword(s):  
Escherichia Coli ◽  
Mutant Strain ◽  
Pyruvate Dehydrogenase ◽  
Metabolic Flux ◽  
Anaerobic Growth ◽  
Kinetic Properties ◽  
Fermentation Products ◽  
E Coli ◽  
Pyruvate Node

ABSTRACT During anaerobic growth of Escherichia coli, pyruvate formate-lyase (PFL) and lactate dehydrogenase (LDH) channel pyruvate toward a mixture of fermentation products. We have introduced a third branch at the pyruvate node in a mutant of E. coli with a mutation in pyruvate dehydrogenase (PDH*) that renders the enzyme less sensitive to inhibition by NADH. The key starting enzymes of the three branches at the pyruvate node in such a mutant, PDH*, PFL, and LDH, have different metabolic potentials and kinetic properties. In such a mutant (strain QZ2), pyruvate flux through LDH was about 30%, with the remainder of the flux occurring through PFL, indicating that LDH is a preferred route of pyruvate conversion over PDH*. In a pfl mutant (strain YK167) with both PDH* and LDH activities, flux through PDH* was about 33% of the total, confirming the ability of LDH to outcompete the PDH pathway for pyruvate in vivo. Only in the absence of LDH (strain QZ3) was pyruvate carbon equally distributed between the PDH* and PFL pathways. A pfl mutant with LDH and PDH* activities, as well as a pfl ldh double mutant with PDH* activity, had a surprisingly low cell yield per mole of ATP (Y ATP) (about 7.0 g of cells per mol of ATP) compared to 10.9 g of cells per mol of ATP for the wild type. The lower Y ATP suggests the operation of a futile energy cycle in the absence of PFL in this strain. An understanding of the controls at the pyruvate node during anaerobic growth is expected to provide unique insights into rational metabolic engineering of E. coli and related bacteria for the production of various biobased products at high rates and yields.

scholarly journals L-Alanine Prototrophic Suppressors Emerge from L-Alanine Auxotroph through Stress-Induced Mutagenesis in Escherichia coli

2021 ◽  
Vol 9 (3) ◽  
pp. 472
Author(s):  
Harutaka Mishima ◽  
Hirokazu Watanabe ◽  
Kei Uchigasaki ◽  
So Shimoda ◽  
Shota Seki ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Spontaneous Mutation ◽  
Point Mutations ◽  
Whole Genome Analysis ◽  
E Coli ◽  
Clone Pair ◽  
Induced Mutagenesis ◽  
Isogenic Mutants

In Escherichia coli, L-alanine is synthesized by three isozymes: YfbQ, YfdZ, and AvtA. When an E. coli L-alanine auxotrophic isogenic mutant lacking the three isozymes was grown on L-alanine-deficient minimal agar medium, L-alanine prototrophic mutants emerged considerably more frequently than by spontaneous mutation; the emergence frequency increased over time, and, in an L-alanine-supplemented minimal medium, correlated inversely with L-alanine concentration, indicating that the mutants were derived through stress-induced mutagenesis. Whole-genome analysis of 40 independent L-alanine prototrophic mutants identified 16 and 18 clones harboring point mutation(s) in pyruvate dehydrogenase complex and phosphotransacetylase-acetate kinase pathway, which respectively produce acetyl coenzyme A and acetate from pyruvate. When two point mutations identified in L-alanine prototrophic mutants, in pta (D656A) and aceE (G147D), were individually introduced into the original L-alanine auxotroph, the isogenic mutants exhibited almost identical growth recovery as the respective cognate mutants. Each original- and isogenic-clone pair carrying the pta or aceE mutation showed extremely low phosphotransacetylase or pyruvate dehydrogenase activity, respectively. Lastly, extracellularly-added pyruvate, which dose-dependently supported L-alanine auxotroph growth, relieved the L-alanine starvation stress, preventing the emergence of L-alanine prototrophic mutants. Thus, L-alanine starvation-provoked stress-induced mutagenesis in the L-alanine auxotroph could lead to intracellular pyruvate increase, which eventually induces L-alanine prototrophy.

scholarly journals pH-Dependent Catabolic Protein Expression during Anaerobic Growth of Escherichia coli K-12

2004 ◽  
Vol 186 (1) ◽  
pp. 192-199 ◽  
Author(s):  
Elizabeth Yohannes ◽  
D. Michael Barnhart ◽  
Joan L. Slonczewski
Keyword(s):  
Escherichia Coli ◽  
Ph Regulation ◽  
Metabolic Enzymes ◽  
Anaerobic Growth ◽  
High Ph ◽  
E Coli ◽  
Catabolic Enzymes ◽  
Periplasmic Proteins ◽  
Ph Dependent ◽  
K 12

ABSTRACT During aerobic growth of Escherichia coli, expression of catabolic enzymes and envelope and periplasmic proteins is regulated by pH. Additional modes of pH regulation were revealed under anaerobiosis. E. coli K-12 strain W3110 was cultured anaerobically in broth medium buffered at pH 5.5 or 8.5 for protein identification on proteomic two-dimensional gels. A total of 32 proteins from anaerobic cultures show pH-dependent expression, and only four of these proteins (DsbA, TnaA, GatY, and HdeA) showed pH regulation in aerated cultures. The levels of 19 proteins were elevated at the high pH; these proteins included metabolic enzymes (DhaKLM, GapA, TnaA, HisC, and HisD), periplasmic proteins (ProX, OppA, DegQ, MalB, and MglB), and stress proteins (DsbA, Tig, and UspA). High-pH induction of the glycolytic enzymes DhaKLM and GapA suggested that there was increased fermentation to acids, which helped neutralize alkalinity. Reporter lac fusion constructs showed base induction of sdaA encoding serine deaminase under anaerobiosis; in addition, the glutamate decarboxylase genes gadA and gadB were induced at the high pH anaerobically but not with aeration. This result is consistent with the hypothesis that there is a connection between the gad system and GabT metabolism of 4-aminobutanoate. On the other hand, 13 other proteins were induced by acid; these proteins included metabolic enzymes (GatY and AckA), periplasmic proteins (TolC, HdeA, and OmpA), and redox enzymes (GuaB, HmpA, and Lpd). The acid induction of NikA (nickel transporter) is of interest because E. coli requires nickel for anaerobic fermentation. The position of the NikA spot coincided with the position of a small unidentified spot whose induction in aerobic cultures was reported previously; thus, NikA appeared to be induced slightly by acid during aeration but showed stronger induction under anaerobic conditions. Overall, anaerobic growth revealed several more pH-regulated proteins; in particular, anaerobiosis enabled induction of several additional catabolic enzymes and sugar transporters at the high pH, at which production of fermentation acids may be advantageous for the cell.

scholarly journals Expression and lipoylation in Escherichia coli of the inner lipoyl domain of the E2 component of the human pyruvate dehydrogenase complex

1993 ◽  
Vol 289 (1) ◽  
pp. 81-85 ◽  
Author(s):  
J Quinn ◽  
A G Diamond ◽  
A K Masters ◽  
D E Brookfield ◽  
N G Wallis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Structural Studies ◽  
Gene Encoding ◽  
E Coli ◽  
Inner Lipoyl Domain ◽  
Dehydrogenase Complex ◽  
Lipoyl Domain

The dihydrolipoamide acetyltransferase subunit (E2p) of mammalian pyruvate dehydrogenase complex has two highly conserved lipoyl domains each modified with a lipoyl cofactor bound in amide linkage to a specific lysine residue. A sub-gene encoding the inner lipoyl domain of human E2p has been over-expressed in Escherichia coli. Two forms of the domain have been purified, corresponding to lipoylated and non-lipoylated species. The apo-domain can be lipoylated in vitro with partially purified E. coli lipoate protein ligase, and the lipoylated domain can be reductively acetylated by human E1p (pyruvate dehydrogenase). Availability of the two forms will now allow detailed biochemical and structural studies of the human lipoyl domains.

scholarly journals Genome-Wide Expression Analysis Indicates that FNR of Escherichia coli K-12 Regulates a Large Number of Genes of Unknown Function

2005 ◽  
Vol 187 (3) ◽  
pp. 1135-1160 ◽  
Author(s):  
Yisheng Kang ◽  
K. Derek Weber ◽  
Yu Qiu ◽  
Patricia J. Kiley ◽  
Frederick R. Blattner
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Dna Binding ◽  
Growth Conditions ◽  
Anaerobic Growth ◽  
Computer Search ◽  
Anaerobic Conditions ◽  
Metabolic Potential ◽  
Fermentation Products ◽  
K 12

ABSTRACT The major regulator controlling the physiological switch between aerobic and anaerobic growth conditions in Escherichia coli is the DNA binding protein FNR. To identify genes controlled by FNR, we used Affymetrix Antisense GeneChips to compare global gene expression profiles from isogenic MG1655 wild-type and Δfnr strains grown in glucose minimal media under aerobic or anaerobic conditions. We found that 297 genes contained within 184 operons were regulated by FNR and/or by O2 levels. The expression of many genes known to be involved in anaerobic respiration and fermentation was increased under anaerobic growth conditions, while that of genes involved in aerobic respiration and the tricarboxylic acid cycle were repressed as expected. The expression of nine operons associated with acid resistance was also increased under anaerobic growth conditions, which may reflect the production of acidic fermentation products. Ninety-one genes with no presently defined function were also altered in expression, including seven of the most highly anaerobically induced genes, six of which we found to be directly regulated by FNR. Classification of the 297 genes into eight groups by k-means clustering analysis indicated that genes with common gene expression patterns also had a strong functional relationship, providing clues for studying the function of unknown genes in each group. Six of the eight groups showed regulation by FNR; while some expression groups represent genes that are simply activated or repressed by FNR, others, such as those encoding functions for chemotaxis and motility, showed a more complex pattern of regulation. A computer search for FNR DNA binding sites within predicted promoter regions identified 63 new sites for 54 genes. We suggest that E. coli MG1655 has a larger metabolic potential under anaerobic conditions than has been previously recognized.

scholarly journals Escherichia coli Pyruvate Dehydrogenase Complex Is an Important Component of CXCL10-Mediated Antimicrobial Activity

2015 ◽  
Vol 84 (1) ◽  
pp. 320-328 ◽  
Author(s):  
Kirsten M. Schutte ◽  
Debra J. Fisher ◽  
Marie D. Burdick ◽  
Borna Mehrad ◽  
Amy J. Mathers ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Cell Surface ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Gene Encoding ◽  
Content Type ◽  
E Coli ◽  
Novel Therapeutic Strategies ◽  
Dehydrogenase Complex

Chemokines are best recognized for their role within the innate immune system as chemotactic cytokines, signaling and recruiting host immune cells to sites of infection. Certain chemokines, such as CXCL10, have been found to play an additional role in innate immunity, mediating CXCR3-independent killing of a diverse array of pathogenic microorganisms. While this is still not clearly understood, elucidating the mechanisms underlying chemokine-mediated antimicrobial activity may facilitate the development of novel therapeutic strategies effective against antibiotic-resistant Gram-negative pathogens. Here, we show that CXCL10 exerts antibacterial effects on clinical and laboratory strains ofEscherichia coliand report that disruption of pyruvate dehydrogenase complex (PDHc), which converts pyruvate to acetyl coenzyme A, enablesE. colito resist these antimicrobial effects. Through generation and screening of a transposon mutant library, we identified two mutants with increased resistance to CXCL10, both with unique disruptions of the gene encoding the E1 subunit of PDHc,aceE. Resistance to CXCL10 also occurred following deletion of eitheraceForlpdA, genes that encode the remaining two subunits of PDHc. Although PDHc resides within the bacterial cytosol, electron microscopy revealed localization of immunogold-labeled CXCL10 to the bacterial cell surface in both theE. coliparent andaceEdeletion mutant strains. Taken together, our findings suggest that while CXCL10 interacts with an as-yet-unidentified component on the cell surface, PDHc is an important mediator of killing by CXCL10. To our knowledge, this is the first description of PDHc as a key bacterial component involved in the antibacterial effect of a chemokine.

Sign in / Sign up

Export Citation Format

Share Document