pH-Dependent Catabolic Protein Expression during Anaerobic Growth of Escherichia coli 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.

Download Full-text

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

Journal of Bacteriology ◽

10.1128/jb.00104-08 ◽

2008 ◽

Vol 190 (11) ◽

pp. 3851-3858 ◽

Cited By ~ 76

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.

Download Full-text

Regulation of Aerobic and Anaerobic d-Malate Metabolism of Escherichia coli by the LysR-Type Regulator DmlR (YeaT)

Journal of Bacteriology ◽

10.1128/jb.01665-09 ◽

2010 ◽

Vol 192 (10) ◽

pp. 2503-2511 ◽

Cited By ~ 12

Author(s):

Hanna Lukas ◽

Julia Reimann ◽

Ok Bin Kim ◽

Jan Grimpo ◽

Gottfried Unden

Keyword(s):

Escherichia Coli ◽

Malate Dehydrogenase ◽

Major Effect ◽

Anaerobic Growth ◽

Two Component System ◽

E Coli ◽

High Level ◽

K 12 ◽

Aerobic And Anaerobic ◽

Malate Metabolism

ABSTRACT Escherichia coli K-12 is able to grow under aerobic conditions on d-malate using DctA for d-malate uptake and the d-malate dehydrogenase DmlA (formerly YeaU) for converting d-malate to pyruvate. Induction of dmlA encoding DmlA required an intact dmlR (formerly yeaT) gene, which encodes DmlR, a LysR-type transcriptional regulator. Induction of dmlA by DmlR required the presence of d-malate or l- or meso-tartrate, but only d-malate supported aerobic growth. The regulator of general C4-dicarboxylate metabolism (DcuS-DcuR two-component system) had some effect on dmlA expression. The anaerobic l-tartrate regulator TtdR or the oxygen sensors ArcB-ArcA and FNR did not have a major effect on dmlA expression. DmlR has a high level of sequence identity (49%) with TtdR, the l- and meso-tartrate-specific regulator of l-tartrate fermentation in E. coli. dmlA was also expressed at high levels under anaerobic conditions, and the bacteria had d-malate dehydrogenase activity. These bacteria, however, were not able to grow on d-malate since the anaerobic pathway for d-malate degradation has a predicted yield of ≤0 ATP/mol d-malate. Slow anaerobic growth on d-malate was observed when glycerol was also provided as an electron donor, and d-malate was used in fumarate respiration. The expression of dmlR is subject to negative autoregulation. The network for regulation and coordination of the central and peripheral pathways for C4-dicarboxylate metabolism by the regulators DcuS-DcuR, DmlR, and TtdR is discussed.

Download Full-text

Faculty Opinions recommendation of Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1032868.374523 ◽

2006 ◽

Author(s):

David Cane

Keyword(s):

Escherichia Coli ◽

Biological Research ◽

E Coli ◽

Complete Set ◽

K 12

Download Full-text

Structural genes for nitrate-inducible formate dehydrogenase in Escherichia coli K-12.

Genetics ◽

10.1093/genetics/125.4.691 ◽

1990 ◽

Vol 125 (4) ◽

pp. 691-702 ◽

Cited By ~ 6

Author(s):

B L Berg ◽

V Stewart

Keyword(s):

Escherichia Coli ◽

Nitrate Reductase ◽

Formate Dehydrogenase ◽

Recombinant Dna ◽

Structural Genes ◽

Respiratory Pathway ◽

E Coli ◽

Formate Oxidation ◽

Operon Expression ◽

K 12

Abstract Formate oxidation coupled to nitrate reduction constitutes a major anaerobic respiratory pathway in Escherichia coli. This respiratory chain consists of formate dehydrogenase-N, quinone, and nitrate reductase. We have isolated a recombinant DNA clone that likely contains the structural genes, fdnGHI, for the three subunits of formate dehydrogenase-N. The fdnGHI clone produced proteins of 110, 32 and 20 kDa which correspond to the subunit sizes of purified formate dehydrogenase-N. Our analysis indicates that fdnGHI is organized as an operon. We mapped the fdn operon to 32 min on the E. coli genetic map, close to the genes for cryptic nitrate reductase (encoded by the narZ operon). Expression of phi(fdnG-lacZ) operon fusions was induced by anaerobiosis and nitrate. This induction required fnr+ and narL+, two regulatory genes whose products are also required for the anaerobic, nitrate-inducible activation of the nitrate reductase structural gene operon, narGHJI. We conclude that regulation of fdnGHI and narGHJI expression is mediated through common pathways.

Download Full-text

Potentiation by L-cysteine of the bactericidal effect of hydrogen peroxide in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.152.1.81-88.1982 ◽

1982 ◽

Vol 152 (1) ◽

pp. 81-88

Author(s):

E H Berglin ◽

M B Edlund ◽

G K Nyberg ◽

J Carlsson

Keyword(s):

Escherichia Coli ◽

Hydrogen Peroxide ◽

Metal Ion ◽

Chelating Agent ◽

Fold Increase ◽

Bactericidal Effect ◽

Anaerobic Conditions ◽

Dna Breakage ◽

E Coli ◽

K 12

Under anaerobic conditions an exponentially growing culture of Escherichia coli K-12 was exposed to hydrogen peroxide in the presence of various compounds. Hydrogen peroxide (0.1 mM) together with 0.1 mM L-cysteine or L-cystine killed the organisms more rapidly than 10 mM hydrogen peroxide alone. The exposure of E. coli to hydrogen peroxide in the presence of L-cysteine inhibited some of the catalase. This inhibition, however, could not fully explain the 100-fold increase in hydrogen peroxide sensitivity of the organism in the presence of L-cysteine. Of other compounds tested only some thiols potentiated the bactericidal effect of hydrogen peroxide. These thiols were effective, however, only at concentrations significantly higher than 0.1 mM. The effect of L-cysteine and L-cystine could be annihilated by the metal ion chelating agent 2,2'-bipyridyl. DNA breakage in E. coli K-12 was demonstrated under conditions where the organisms were killed by hydrogen peroxide.

Download Full-text

The Small Noncoding DsrA RNA Is an Acid Resistance Regulator in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.186.18.6179-6185.2004 ◽

2004 ◽

Vol 186 (18) ◽

pp. 6179-6185 ◽

Cited By ~ 65

Author(s):

Richard A. Lease ◽

Dorie Smith ◽

Kathleen McDonough ◽

Marlene Belfort

Keyword(s):

Escherichia Coli ◽

Stress Response ◽

Resistance Genes ◽

Acid Resistance ◽

Dna Arrays ◽

E Coli ◽

Specific Mrnas ◽

Steady State Levels ◽

Control Translation ◽

K 12

ABSTRACT DsrA RNA is a small (87-nucleotide) regulatory RNA of Escherichia coli that acts by RNA-RNA interactions to control translation and turnover of specific mRNAs. Two targets of DsrA regulation are RpoS, the stationary-phase and stress response sigma factor (σs), and H-NS, a histone-like nucleoid protein and global transcription repressor. Genes regulated globally by RpoS and H-NS include stress response proteins and virulence factors for pathogenic E. coli. Here, by using transcription profiling via DNA arrays, we have identified genes induced by DsrA. Steady-state levels of mRNAs from many genes increased with DsrA overproduction, including multiple acid resistance genes of E. coli. Quantitative primer extension analysis verified the induction of individual acid resistance genes in the hdeAB, gadAX, and gadBC operons. E. coli K-12 strains, as well as pathogenic E. coli O157:H7, exhibited compromised acid resistance in dsrA mutants. Conversely, overproduction of DsrA from a plasmid rendered the acid-sensitive dsrA mutant extremely acid resistant. Thus, DsrA RNA plays a regulatory role in acid resistance. Whether DsrA targets acid resistance genes directly by base pairing or indirectly via perturbation of RpoS and/or H-NS is not known, but in either event, our results suggest that DsrA RNA may enhance the virulence of pathogenic E. coli.

Download Full-text

afa-8 Gene Cluster Is Carried by a Pathogenicity Island Inserted into the tRNAPhe of Human and Bovine Pathogenic Escherichia coli Isolates

Infection and Immunity ◽

10.1128/iai.69.2.937-948.2001 ◽

2001 ◽

Vol 69 (2) ◽

pp. 937-948 ◽

Cited By ~ 48

Author(s):

Lila Lalioui ◽

Chantal Le Bouguénec

Keyword(s):

Escherichia Coli ◽

Direct Repeat ◽

Pathogenicity Island ◽

Genomic Region ◽

Open Reading Frames ◽

Blood Isolate ◽

Distinctive Features ◽

E Coli ◽

Pathogenic Escherichia Coli ◽

K 12

ABSTRACT We recently described a new afimbrial adhesin, AfaE-VIII, produced by animal strains associated with diarrhea and septicemia and by human isolates associated with extraintestinal infections. Here, we report that the afa-8 operon, encoding AfaE-VIII adhesin, from the human blood isolate Escherichia coli AL862 is carried by a 61-kb genomic region with characteristics typical of a pathogenicity island (PAI), including a size larger than 10 kb, the presence of an integrase-encoding gene, the insertion into a tRNA locus (pheR), and the presence of a small direct repeat at each extremity. Moreover, the G+C content of the afa-8 operon (46.4%) is lower than that of the E. coli K-12/MG1655 chromosome (50.8%). Within this PAI, designated PAI IAL862, we identified open reading frames able to code for products similar to proteins involved in sugar utilization. Four probes spanning these sequences hybridized with 74.3% of pathogenicafa-8-positive E. coli strains isolated from humans and animals, 25% of human pathogenic afa-8-negativeE. coli strains, and only 8% of fecal strains (P = 0.05), indicating that these sequences are strongly associated with the afa-8 operon and that this genetic association may define a PAI widely distributed among human and animal afa-8-positive strains. One of the distinctive features of this study is that E. coli AL862 also carries another afa-8-containing PAI (PAI IIAL862), which appeared to be similar in size and genetic organization to PAI IAL862 and was inserted into the pheV gene. We investigated the insertion sites of afa-8-containing PAI in human and bovine pathogenic E. coli strains and found that this PAI preferentially inserted into the pheV gene.

Download Full-text

Identification and Function of the pdxY Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5′-Phosphate Biosynthesis in Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.180.7.1814-1821.1998 ◽

1998 ◽

Vol 180 (7) ◽

pp. 1814-1821 ◽

Cited By ~ 52

Author(s):

Yong Yang ◽

Ho-Ching Tiffany Tsui ◽

Tsz-Kwong Man ◽

Malcolm E. Winkler

Keyword(s):

Escherichia Coli ◽

De Novo ◽

Specific Activity ◽

Salvage Pathway ◽

Pyridoxal Kinase ◽

Triple Mutant ◽

Reading Frame ◽

E Coli ◽

Specific Activities ◽

K 12

ABSTRACT pdxK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5′-phosphate (PLP) coenzyme biosynthesis. The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase. Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete E. coli genome. Minimal clones of pdxY + overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette intopdxY and crossed the resultingpdxY::ΩKanr mutation into the bacterial chromosome of a pdxB mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts ofpdxK and pdxY single and double mutants. Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6vitamer kinases in E. coli and that their function is confined to the PLP salvage pathway. Last, we show thatpdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNATyr synthetase) in a multifunctional operon.pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in thetyrS-pdxY intercistronic region.

Download Full-text

Reclassification of Shigella species as later heterotypic synonyms of Escherichia coli in the Genome Taxonomy Database

10.1101/2021.09.22.461432 ◽

2021 ◽

Author(s):

Donovan H Parks ◽

Maria Chuvochina ◽

Peter R Reeves ◽

Scott A Beatson ◽

Philip Hugenholtz

Keyword(s):

Escherichia Coli ◽

Type Strain ◽

Shigella Flexneri ◽

Laboratory Strain ◽

Single Species ◽

Species Definition ◽

E Coli ◽

Shigella Species ◽

K 12 ◽

Common Laboratory

Members of the genus Shigella have high genomic similarity to Escherichia coli and are often considered to be atypical members of this species. In an attempt to retain Shigella species as recognizable entities, they were reclassified as Escherichia species in the Genome Taxonomy Database (GTDB) using an operational average nucleotide identity (ANI)-based approach nucleated around type strains. This resulted in nearly 80% of E. coli genomes being reclassified to new species including the common laboratory strain E. coli K-12 (to 'E. flexneri') because it is more closely related to the type strain of Shigella flexneri than it is to the type strain of E. coli. Here we resolve this conundrum by treating Shigella species as later heterotypic synonyms of E. coli, present evidence supporting this reclassification, and show that assigning E. coli/Shigella strains to a single species is congruent with the GTDB-adopted genomic species definition.

Download Full-text