In Escherichia coli Ammonia Inhibits Cytochrome bo3 But Activates Cytochrome bd-I

Interaction of two redox enzymes of Escherichia coli, cytochrome bo3 and cytochrome bd-I, with ammonium sulfate/ammonia at pH 7.0 and 8.3 was studied using high-resolution respirometry and absorption spectroscopy. At pH 7.0, the oxygen reductase activity of none of the enzymes is affected by the ligand. At pH 8.3, cytochrome bo3 is inhibited by the ligand, with 40% maximum inhibition at 100 mM (NH4)2SO4. In contrast, the activity of cytochrome bd-I at pH 8.3 increases with increasing the ligand concentration, the largest increase (140%) is observed at 100 mM (NH4)2SO4. In both cases, the effector molecule is apparently not NH4+ but NH3. The ligand induces changes in absorption spectra of both oxidized cytochromes at pH 8.3. The magnitude of these changes increases as ammonia concentration is increased, yielding apparent dissociation constants Kdapp of 24.3 ± 2.7 mM (NH4)2SO4 (4.9 ± 0.5 mM NH3) for the Soret region in cytochrome bo3, and 35.9 ± 7.1 and 24.6 ± 12.4 mM (NH4)2SO4 (7.2 ± 1.4 and 4.9 ± 2.5 mM NH3) for the Soret and visible regions, respectively, in cytochrome bd-I. Consistently, addition of (NH4)2SO4 to cells of the E. coli mutant containing cytochrome bd-I as the only terminal oxidase at pH 8.3 accelerates the O2 consumption rate, the highest one (140%) being at 27 mM (NH4)2SO4. We discuss possible molecular mechanisms and physiological significance of modulation of the enzymatic activities by ammonia present at high concentration in the intestines, a niche occupied by E. coli.

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Cardiolipin enhances the enzymatic activity of cytochrome bd and cytochrome bo3 solubilized in dodecyl-maltoside

Scientific Reports ◽

10.1038/s41598-021-87354-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Amer H. Asseri ◽

Albert Godoy-Hernandez ◽

Hojjat Ghasemi Goojani ◽

Holger Lill ◽

Junshi Sakamoto ◽

...

Keyword(s):

Escherichia Coli ◽

Oxygen Consumption ◽

Integral Membrane Proteins ◽

Terminal Oxidase ◽

Geobacillus Thermodenitrificans ◽

E Coli ◽

Cytochrome Bd ◽

Cytochrome Bo3 ◽

Dodecyl Maltoside ◽

Consumption Activity

AbstractCardiolipin (CL) is a lipid that is found in the membranes of bacteria and the inner membranes of mitochondria. CL can increase the activity of integral membrane proteins, in particular components of respiratory pathways. We here report that CL activated detergent-solubilized cytochrome bd, a terminal oxidase from Escherichia coli. CL enhanced the oxygen consumption activity ~ twofold and decreased the apparent KM value for ubiquinol-1 as substrate from 95 µM to 35 µM. Activation by CL was also observed for cytochrome bd from two Gram-positive species, Geobacillus thermodenitrificans and Corynebacterium glutamicum, and for cytochrome bo3 from E. coli. Taken together, CL can enhance the activity of detergent-solubilized cytochrome bd and cytochrome bo3.

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Short-chain aurachin D derivatives are selective inhibitors of E. coli cytochrome bd-I and bd-II oxidases

Scientific Reports ◽

10.1038/s41598-021-03288-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Melanie Radloff ◽

Isam Elamri ◽

Tamara N. Grund ◽

Luca F. Witte ◽

Katharina F. Hohmann ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Reductase Activity ◽

Mitochondrial Respiratory Chain ◽

Short Chain ◽

Inhibitory Effects ◽

Selective Inhibitors ◽

E Coli ◽

Terminal Oxidases ◽

Cytochrome Bd ◽

Oxygen Reductase

AbstractCytochrome bd-type oxidases play a crucial role for survival of pathogenic bacteria during infection and proliferation. This role and the fact that there are no homologues in the mitochondrial respiratory chain qualify cytochrome bd as a potential antimicrobial target. However, few bd oxidase selective inhibitors have been described so far. In this report, inhibitory effects of Aurachin C (AurC-type) and new Aurachin D (AurD-type) derivatives on oxygen reductase activity of isolated terminal bd-I, bd-II and bo3 oxidases from Escherichia coli were potentiometrically measured using a Clark-type electrode. We synthesized long- (C10, decyl or longer) and short-chain (C4, butyl to C8, octyl) AurD-type compounds and tested this set of molecules towards their selectivity and potency. We confirmed strong inhibition of all three terminal oxidases for AurC-type compounds, whereas the 4(1H)-quinolone scaffold of AurD-type compounds mainly inhibits bd-type oxidases. We assessed a direct effect of chain length on inhibition activity with highest potency and selectivity observed for heptyl AurD-type derivatives. While Aurachin C and Aurachin D are widely considered as selective inhibitors for terminal oxidases, their structure–activity relationship is incompletely understood. This work fills this gap and illustrates how structural differences of Aurachin derivatives determine inhibitory potency and selectivity for bd-type oxidases of E. coli.

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Are Escherichia coli OXPHOS complexes concentrated in specialized zones within the plasma membrane?

Biochemical Society Transactions ◽

10.1042/bst0361032 ◽

2008 ◽

Vol 36 (5) ◽

pp. 1032-1036 ◽

Cited By ~ 34

Author(s):

Tchern Lenn ◽

Mark C. Leake ◽

Conrad W. Mullineaux

Keyword(s):

Escherichia Coli ◽

Plasma Membrane ◽

Respiratory Function ◽

Mitochondrial Membrane ◽

Terminal Oxidase ◽

Inner Mitochondrial Membrane ◽

High Concentration ◽

E Coli ◽

Cytochrome Bd ◽

Biochemical Level

Most organisms are able to synthesize ATP by OXPHOS (oxidative phosphorylation). Mitochondria in eukaryotes perform OXPHOS in the inner mitochondrial membrane, whereas the plasma membrane is used by prokaryotes. However, whereas OXPHOS is a well-understood process at the biochemical level, relatively little is known about its operation at the level of the whole-organelle/cell. We observed that a fluorescently labelled terminal oxidase, the cytochrome bd complex, is heterogeneously distributed in the Escherichia coli plasma membrane. This observation forms the basis of a working hypothesis that patches of the E. coli plasma membrane (‘respirazones’) are dedicated to respiratory function by the high concentration of OXPHOS components in these zones relative to the adjacent membrane. The formulation and physiological significance of this hypothesis are discussed in this paper.

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The Amino Acids Motif -32GSSYN36- in the Catalytic Domain of E. coli Flavorubredoxin NO Reductase Is Essential for Its Activity

Catalysts ◽

10.3390/catal11080926 ◽

2021 ◽

Vol 11 (8) ◽

pp. 926

Author(s):

Maria C. Martins ◽

Susana F. Fernandes ◽

Bruno A. Salgueiro ◽

Jéssica C. Soares ◽

Célia V. Romão ◽

...

Keyword(s):

Amino Acids ◽

Catalytic Domain ◽

Reductase Activity ◽

Conserved Residues ◽

Mutant Proteins ◽

E Coli ◽

C Terminus ◽

Bona Fide ◽

Oxygen Reductase ◽

Soluble Enzymes

Flavodiiron proteins (FDPs) are a family of modular and soluble enzymes endowed with nitric oxide and/or oxygen reductase activities, producing N2O or H2O, respectively. The FDP from Escherichia coli, which, apart from the two core domains, possesses a rubredoxin-like domain at the C-terminus (therefore named flavorubredoxin (FlRd)), is a bona fide NO reductase, exhibiting O2 reducing activity that is approximately ten times lower than that for NO. Among the flavorubredoxins, there is a strictly conserved amino acids motif, -G[S,T]SYN-, close to the catalytic diiron center. To assess its role in FlRd’s activity, we designed several site-directed mutants, replacing the conserved residues with hydrophobic or anionic ones. The mutants, which maintained the general characteristics of the wild type enzyme, including cofactor content and integrity of the diiron center, revealed a decrease of their oxygen reductase activity, while the NO reductase activity—specifically, its physiological function—was almost completely abolished in some of the mutants. Molecular modeling of the mutant proteins pointed to subtle changes in the predicted structures that resulted in the reduction of the hydration of the regions around the conserved residues, as well as in the elimination of hydrogen bonds, which may affect proton transfer and/or product release.

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Inhibition of Escherichia coli Trimethylamine-N-oxide Reductase by Food Preservatives1

Journal of Food Protection ◽

10.4315/0362-028x-45.3.241 ◽

1982 ◽

Vol 45 (3) ◽

pp. 241-243 ◽

Cited By ~ 2

Author(s):

M. KRUK ◽

J. S. LEE

Keyword(s):

Escherichia Coli ◽

Benzoic Acid ◽

Sorbic Acid ◽

Reductase Activity ◽

Resting Cells ◽

E Coli

Trimethylamine-N-oxide (TMA-O) reductase activity of resting cells of Escherichia coli was inhibited by tetrasodium ethylenediaminetetraacetate (Na4EDTA), benzoic acid (BA and methylparaben (MP). The 50% inhibitory concentrations of Na4EDTA, BA and MP were 20.2, 1.2 and 32.4 mM, respectively. BA at pH 6.5 or below most effectively inhibited the TMA-O reductase. Sorbic acid (SA), up to 0.70 mM, had no effect on TMA-O reductase activity, but SA inhibited the growth and subsequent TMA production in E. coli at or above 0.3S mM.

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A factor produced by Escherichia coli K-12 inhibits the growth of E. coli mutants defective in the cytochrome bd quinol oxidase complex: enterochelin rediscovered

Microbiology ◽

10.1099/00221287-144-12-3297 ◽

1998 ◽

Vol 144 (12) ◽

pp. 3297-3308 ◽

Cited By ~ 14

Author(s):

G. M. Cook ◽

C. Loder ◽

B. Soballe ◽

G. P. Stafford ◽

J. Membrillo-Hernandez ◽

...

Keyword(s):

Escherichia Coli ◽

Quinol Oxidase ◽

E Coli ◽

Cytochrome Bd ◽

K 12

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Mechanistic and structural diversity between cytochrome bd isoforms of Escherichia coli

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2114013118 ◽

2021 ◽

Vol 118 (50) ◽

pp. e2114013118

Author(s):

Tamara N. Grund ◽

Melanie Radloff ◽

Di Wu ◽

Hojjat G. Goojani ◽

Luca F. Witte ◽

...

Keyword(s):

Escherichia Coli ◽

Pathogenic Bacteria ◽

Reduction Rate ◽

Structural Diversity ◽

Multidrug Resistant ◽

E Coli ◽

Structural And Functional Properties ◽

Oxygen Reductase ◽

General Architecture ◽

Structural Insights

The treatment of infectious diseases caused by multidrug-resistant pathogens is a major clinical challenge of the 21st century. The membrane-embedded respiratory cytochrome bd-type oxygen reductase is a critical survival factor utilized by pathogenic bacteria during infection, proliferation and the transition from acute to chronic states. Escherichia coli encodes for two cytochrome bd isoforms that are both involved in respiration under oxygen limited conditions. Mechanistic and structural differences between cydABX (Ecbd-I) and appCBX (Ecbd-II) operon encoded cytochrome bd variants have remained elusive in the past. Here, we demonstrate that cytochrome bd-II catalyzes oxidation of benzoquinols while possessing additional specificity for naphthoquinones. Our data show that although menaquinol-1 (MK1) is not able to directly transfer electrons onto cytochrome bd-II from E. coli, it has a stimulatory effect on its oxygen reduction rate in the presence of ubiquinol-1. We further determined cryo-EM structures of cytochrome bd-II to high resolution of 2.1 Å. Our structural insights confirm that the general architecture and substrate accessible pathways are conserved between the two bd oxidase isoforms, but two notable differences are apparent upon inspection: (i) Ecbd-II does not contain a CydH-like subunit, thereby exposing heme b595 to the membrane environment and (ii) the AppB subunit harbors a structural demethylmenaquinone-8 molecule instead of ubiquinone-8 as found in CydB of Ecbd-I. Our work completes the structural landscape of terminal respiratory oxygen reductases of E. coli and suggests that structural and functional properties of the respective oxidases are linked to quinol-pool dependent metabolic adaptations in E. coli.

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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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Characterization of Multidrug-Resistant Escherichia coli Isolates from Animals Presenting at a University Veterinary Hospital

Applied and Environmental Microbiology ◽

10.1128/aem.00599-11 ◽

2011 ◽

Vol 77 (20) ◽

pp. 7104-7112 ◽

Cited By ~ 59

Author(s):

Maria Karczmarczyk ◽

Yvonne Abbott ◽

Ciara Walsh ◽

Nola Leonard ◽

Séamus Fanning

Keyword(s):

Escherichia Coli ◽

Molecular Mechanisms ◽

Gene Array ◽

Multidrug Resistant ◽

Genetic Determinants ◽

Gene Encoding ◽

Content Type ◽

E Coli ◽

Class 1

ABSTRACTIn this study, we examined molecular mechanisms associated with multidrug resistance (MDR) in a collection ofEscherichia coliisolates recovered from hospitalized animals in Ireland. PCR and DNA sequencing were used to identify genes associated with resistance. Class 1 integrons were prevalent (94.6%) and contained gene cassettes recognized previously and implicated mainly in resistance to aminoglycosides, β-lactams, and trimethoprim (aadA1,dfrA1-aadA1,dfrA17-aadA5,dfrA12-orfF-aadA2,blaOXA-30-aadA1,aacC1-orf1-orf2-aadA1,dfr7). Class 2 integrons (13.5%) contained thedfrA1-sat1-aadA1gene array. The most frequently occurring phenotypes included resistance to ampicillin (97.3%), chloramphenicol (75.4%), florfenicol (40.5%), gentamicin (54%), neomycin (43.2%), streptomycin (97.3%), sulfonamide (98.6%), and tetracycline (100%). The associated resistance determinants detected includedblaTEM,cat,floR,aadB,aphA1,strA-strB,sul2, andtet(B), respectively. TheblaCTX-M-2gene, encoding an extended-spectrum β-lactamase (ESβL), andblaCMY-2, encoding an AmpC-like enzyme, were identified in 8 and 18 isolates, respectively. The mobility of the resistance genes was demonstrated using conjugation assays with a representative selection of isolates. High-molecular-weight plasmids were found to be responsible for resistance to multiple antimicrobial compounds. The study demonstrated that animal-associated commensalE. coliisolates possess a diverse repertoire of transferable genetic determinants. Emergence of ESβLs and AmpC-like enzymes is particularly significant. To our knowledge, theblaCTX-M-2gene has not previously been reported in Ireland.

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Transcriptional Responses of Escherichia coli K-12 and O157:H7 Associated with Lettuce Leaves

Applied and Environmental Microbiology ◽

10.1128/aem.07454-11 ◽

2012 ◽

Vol 78 (6) ◽

pp. 1752-1764 ◽

Cited By ~ 64

Author(s):

Ryan C. Fink ◽

Elaine P. Black ◽

Zhe Hou ◽

Masayuki Sugawara ◽

Michael J. Sadowsky ◽

...

Keyword(s):

Escherichia Coli ◽

Molecular Mechanisms ◽

Leaf Surface ◽

Short Term ◽

Transcriptional Responses ◽

Content Type ◽

E Coli ◽

Leaf Surfaces ◽

K 12

ABSTRACTAn increasing number of outbreaks of gastroenteritis recently caused byEscherichia coliO157:H7 have been linked to the consumption of leafy green vegetables. Although it is known thatE. colisurvives and grows in the phyllosphere of lettuce plants, the molecular mechanisms by which this bacterium associates with plants are largely unknown. The goal of this study was to identifyE. coligenes relevant to its interaction, survival, or attachment to lettuce leaf surfaces, comparingE. coliK-12, a model system, andE. coliO157:H7, a pathogen associated with a large number of outbreaks. Using microarrays, we found that upon interaction with intact leaves, 10.1% and 8.7% of the 3,798 shared genes were differentially expressed in K-12 and O157:H7, respectively, whereas 3.1% changed transcript levels in both. The largest group of genes downregulated consisted of those involved in energy metabolism, includingtnaA(33-fold change), encoding a tryptophanase that converts tryptophan into indole. Genes involved in biofilm modulation (bhsAandybiM) and curli production (csgAandcsgB) were significantly upregulated inE. coliK-12 and O157:H7. BothcsgAandbhsA(ycfR) mutants were impaired in the long-term colonization of the leaf surface, but onlycsgAmutants had diminished ability in short-term attachment experiments. Our data suggested that the interaction ofE. coliK-12 and O157:H7 with undamaged lettuce leaves likely is initiated via attachment to the leaf surface using curli fibers, a downward shift in their metabolism, and the suppression of biofilm formation.

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