Uncoupling of Substrate-Level Phosphorylation in Escherichia coli during Glucose-Limited Growth

ABSTRACTThe respiratory chain ofEscherichia colicontains three different cytochrome oxidases. Whereas the cytochromebooxidase and the cytochromebd-I oxidase are well characterized and have been shown to contribute to proton translocation, physiological data suggested a nonelectrogenic functioning of the cytochromebd-II oxidase. Recently, however, this view was challenged by anin vitrobiochemical analysis that showed that the activity of cytochromebd-II oxidase does contribute to proton translocation with an H+/e−stoichiometry of 1. Here, we propose that this apparent discrepancy is due to the activities of two alternative catabolic pathways: the pyruvate oxidase pathway for acetate production and a pathway with methylglyoxal as an intermediate for the production of lactate. The ATP yields of these pathways are lower than those of the pathways that have so far always been assumed to catalyze the main catabolic flux under energy-limited growth conditions (i.e., pyruvate dehydrogenase and lactate dehydrogenase). Inclusion of these alternative pathways in the flux analysis of growingE. colistrains for the calculation of the catabolic ATP synthesis rate indicates an electrogenic function of the cytochromebd-II oxidase, compatible with an H+/e−ratio of 1. This analysis shows for the first time the extent of bypassing of substrate-level phosphorylation inE. coliunder energy-limited growth conditions.

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A Rhodobacter sphaeroides Protein Mechanistically Similar to Escherichia coli DksA Regulates Photosynthetic Growth

mBio ◽

10.1128/mbio.01105-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 7

Author(s):

Christopher W. Lennon ◽

Kimberly C. Lemmer ◽

Jessica L. Irons ◽

Max I. Sellman ◽

Timothy J. Donohue ◽

...

Keyword(s):

Escherichia Coli ◽

Structure Function ◽

Rhodobacter Sphaeroides ◽

Fatty Acid Content ◽

Regulatory Protein ◽

Growth Conditions ◽

Globular Domain ◽

Content Type ◽

E Coli

ABSTRACTDksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the “stringent response” to nutrient starvation in the gammaproteobacteriumEscherichia coliand for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly relatedRhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length toE. coliDksA but lacks the Zn finger motif of theE. coliDksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of anE. colistrain lacking thedksAgene and modulates transcriptionin vitrowithE. coliRNA polymerase (RNAP) similarly toE. coliDksA. RSP2654 reduces RNAP-promoter complex stabilityin vitrowith RNAPs fromE. coliorR. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity toE. coliDksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsphas distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp.IMPORTANCEThe role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis inRhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

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Global Lysine Acetylation in Escherichia coli Results from Growth Conditions That Favor Acetate Fermentation

Journal of Bacteriology ◽

10.1128/jb.00768-18 ◽

2019 ◽

Vol 201 (9) ◽

Cited By ~ 6

Author(s):

Birgit Schilling ◽

Nathan Basisty ◽

David G. Christensen ◽

Dylan Sorensen ◽

James S. Orr ◽

...

Keyword(s):

Escherichia Coli ◽

Enzyme Activity ◽

Regulatory Mechanism ◽

Growth Conditions ◽

Lysine Acetylation ◽

Label Free ◽

Content Type ◽

E Coli ◽

Regulatory Mode ◽

Specific Sugar

ABSTRACT Lysine acetylation is thought to provide a mechanism for regulating metabolism in diverse bacteria. Indeed, many studies have shown that the majority of enzymes involved in central metabolism are acetylated and that acetylation can alter enzyme activity. However, the details regarding this regulatory mechanism are still unclear, specifically with regard to the signals that induce lysine acetylation. To better understand this global regulatory mechanism, we profiled changes in lysine acetylation during growth of Escherichia coli on the hexose glucose or the pentose xylose at both high and low sugar concentrations using label-free mass spectrometry. The goal was to see whether lysine acetylation differed during growth on these two different sugars. No significant differences, however, were observed. Rather, the initial sugar concentration was the principal factor governing changes in lysine acetylation, with higher sugar concentrations causing more acetylation. These results suggest that acetylation does not target specific metabolic pathways but rather simply targets accessible lysines, which may or may not alter enzyme activity. They further suggest that lysine acetylation principally results from conditions that favor accumulation of acetyl phosphate, the principal acetate donor in E. coli. IMPORTANCE Bacteria alter their metabolism in response to nutrient availability, growth conditions, and environmental stresses using a number of different mechanisms. One is lysine acetylation, a posttranslational modification known to target many metabolic enzymes. However, little is known about this regulatory mode. We investigated the factors inducing changes in lysine acetylation by comparing growth on glucose and xylose. We found that the specific sugar used for growth did not alter the pattern of acetylation; rather, the amount of sugar did, with more sugar causing more acetylation. These results imply that lysine acetylation is a global regulatory mechanism that is responsive not to the specific carbon source per se but rather to the accumulation of downstream metabolites.

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Stress-Induced MazF-Mediated Proteins in Escherichia coli

mBio ◽

10.1128/mbio.00340-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 8

Author(s):

Akanksha Nigam ◽

Tamar Ziv ◽

Adi Oron-Gottesman ◽

Hanna Engelberg-Kulka

Keyword(s):

Escherichia Coli ◽

Proteomic Analysis ◽

Normal Growth ◽

Growth Conditions ◽

Translation Machinery ◽

Content Type ◽

E Coli ◽

Adverse Conditions ◽

Almost All ◽

Induced Proteins

ABSTRACT Escherichia coli mazEF is an extensively studied stress-induced toxin-antitoxin (TA) system. The toxin MazF is an endoribonuclease that cleaves RNAs at ACA sites. By that means, under stress, the induced MazF generates a stress-induced translation machinery (STM) composed of MazF-processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Here, we performed a proteomic analysis of all the E. coli stress-induced proteins that are mediated through the chromosomally borne mazF gene. We show that the mRNAs of almost all of them are characterized by the presence of an ACA site up to 100 nucleotides upstream of the AUG initiator. Therefore, under stressful conditions, induced MazF processes mRNAs that are translated by STM. Furthermore, the presence of the ACA sites far upstream (up to 100 nucleotides) of the AUG initiator may still permit translation by the canonical translation machinery. Thus, such dual-translation mechanisms enable the bacterium under stress also to prepare proteins for immediate functions while coming back to normal growth conditions. IMPORTANCE The stress response, the strategy that bacteria have developed in order to cope up with all kinds of adverse conditions, is so far understood at the level of transcription. Our previous findings of a uniquely modified stress-induced translation machinery (STM) generated in E. coli under stress by the endoribonucleolytic activity of the toxin MazF opens a new chapter in understanding microbial physiology under stress at the translational level. Here, we performed a proteomic analysis of all the E. coli stress-induced proteins that are mediated by chromosomally borne MazF through STM.

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Inactivation of Escherichia coli by Polychromatic Simulated Sunlight: Evidence for and Implications of a Fenton Mechanism Involving Iron, Hydrogen Peroxide, and Superoxide

Applied and Environmental Microbiology ◽

10.1128/aem.02419-13 ◽

2013 ◽

Vol 80 (3) ◽

pp. 935-942 ◽

Cited By ~ 17

Author(s):

Michael B. Fisher ◽

Kara L. Nelson

Keyword(s):

Escherichia Coli ◽

Hydrogen Peroxide ◽

Enteric Bacteria ◽

Growth Conditions ◽

Simulated Sunlight ◽

Wild Type ◽

Intracellular Iron ◽

Content Type ◽

E Coli ◽

Degree Of Sensitization

ABSTRACTSunlight inactivation ofEscherichia colihas previously been shown to accelerate in the presence of oxygen, exogenously added hydrogen peroxide, and bioavailable forms of exogenously added iron. In this study, mutants unable to effectively scavenge hydrogen peroxide or superoxide were found to be more sensitive to polychromatic simulated sunlight (without UVB wavelengths) than wild-type cells, while wild-type cells grown under low-iron conditions were less sensitive than cells grown in the presence of abundant iron. Furthermore, prior exposure to simulated sunlight was found to sensitize cells to subsequent hydrogen peroxide exposure in the dark, but this effect was attenuated for cells grown with low iron. Mutants deficient in recombination DNA repair were sensitized to simulated sunlight (without UVB wavelengths), but growth in the presence of iron chelators reduced the degree of sensitization conferred by this mutation. These findings support the hypothesis that hydrogen peroxide, superoxide, and intracellular iron all participate in the photoinactivation ofE. coliand further suggest that the inactivation rate of enteric bacteria in the environment may be strongly dependent on iron availability and growth conditions.

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O-Antigen-Dependent Colicin Insensitivity of UropathogenicEscherichia coli

Journal of Bacteriology ◽

10.1128/jb.00545-18 ◽

2018 ◽

Vol 201 (4) ◽

Cited By ~ 12

Author(s):

Connor Sharp ◽

Christine Boinett ◽

Amy Cain ◽

Nicholas G. Housden ◽

Sandip Kumar ◽

...

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Growth Conditions ◽

Membrane Receptors ◽

Cell Surface Receptor ◽

Dominant Factor ◽

Content Type ◽

E Coli ◽

O Antigen ◽

K 12

ABSTRACTThe outer membrane of Gram-negative bacteria presents a significant barrier for molecules entering the cell. Nevertheless, colicins, which are antimicrobial proteins secreted byEscherichia coli, can target otherE. colicells by binding to cell surface receptor proteins and activating their import, resulting in cell death. Previous studies have documented high rates of nonspecific resistance (insensitivity) of variousE. colistrains toward colicins that is independent of colicin-specific immunity and is instead associated with lipopolysaccharide (LPS) in the outer membrane. This observation poses a contradiction: why doE. colistrains have colicin-expressing plasmids, which are energetically costly to retain, if cells around them are likely to be naturally insensitive to the colicin they produce? Here, using a combination of transposon sequencing and phenotypic microarrays, we show that colicin insensitivity of uropathogenicE. colisequence type 131 (ST131) is dependent on the production of its O-antigen but that minor changes in growth conditions render the organism sensitive toward colicins. The reintroduction of O-antigen intoE. coliK-12 demonstrated that it is the density of O-antigen that is the dominant factor governing colicin insensitivity. We also show, by microscopy of fluorescently labelled colicins, that growth conditions affect the degree of occlusion by O-antigen of outer membrane receptors but not the clustered organization of receptors. The result of our study demonstrate that environmental conditions play a critical role in sensitizingE. colitoward colicins and that O-antigen in LPS is central to this role.IMPORTANCEEscherichia coliinfections can be a major health burden, especially with the organism becoming increasingly resistant to “last-resort” antibiotics such as carbapenems. Although colicins are potent narrow-spectrum antimicrobials with potential as future antibiotics, high levels of naturally occurring colicin insensitivity have been documented which could limit their efficacy. We identify O-antigen-dependent colicin insensitivity in a clinically relevant uropathogenicE. colistrain and show that this insensitivity can be circumvented by minor changes to growth conditions. The results of our study suggest that colicin insensitivity amongE. coliorganisms has been greatly overestimated, and as a consequence, colicins could in fact be effective species-specific antimicrobials targeting pathogenicE. colisuch as uropathogenicE. coli(UPEC).

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Effect of Intracellular Expression of Antimicrobial Peptide LL-37 on Growth of Escherichia coli Strain TOP10 under Aerobic and Anaerobic Conditions

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00825-13 ◽

2013 ◽

Vol 57 (10) ◽

pp. 4707-4716 ◽

Cited By ~ 9

Author(s):

Wei Liu ◽

Shi Lei Dong ◽

Fei Xu ◽

Xue Qin Wang ◽

T. Ryan Withers ◽

...

Keyword(s):

Escherichia Coli ◽

Growth Conditions ◽

Anaerobic Growth ◽

Anaerobic Conditions ◽

Content Type ◽

E Coli ◽

Intracellular Expression ◽

Aerobic And Anaerobic Growth ◽

Aerobic And Anaerobic ◽

Aerobic And Anaerobic Conditions

ABSTRACTAntimicrobial peptides (AMPs) can cause lysis of target bacteria by directly inserting themselves into the lipid bilayer. This killing mechanism confounds the identification of the intracellular targets of AMPs. To circumvent this, we used a shuttle vector containing the inducible expression of a human cathelicidin-related AMP, LL-37, to examine its effect onEscherichia coliTOP10 under aerobic and anaerobic growth conditions. Induction of LL-37 caused growth inhibition and alteration in cell morphology to a filamentous phenotype. Further examination of theE. colicell division protein FtsZ revealed that LL-37 did not interact with FtsZ. Moreover, intracellular expression of LL-37 results in the enhanced production of reactive oxygen species (ROS), causing lethal membrane depolarization under aerobic conditions. Additionally, the membrane permeability was increased after intracellular expression of LL37 under both aerobic and anaerobic conditions. Transcriptomic analysis revealed that intracellular LL-37 mainly affected the expression of genes related to energy production and carbohydrate metabolism. More specifically, genes related to oxidative phosphorylation under both aerobic and anaerobic growth conditions were affected. Collectively, our current study demonstrates that intracellular expression of LL-37 inE. colican inhibit growth under aerobic and anaerobic conditions. While we confirmed that the generation of ROS is a bactericidal mechanism for LL-37 under aerobic growth conditions, we also found that the intracellular accumulation of cationic LL-37 influences the redox and ion status of the cells under both growth conditions. These data suggest that there is a new AMP-mediated bacterial killing mechanism that targets energy metabolism.

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Distinct Acid Resistance and Survival Fitness Displayed by Curli Variants of Enterohemorrhagic Escherichia coli O157:H7

Applied and Environmental Microbiology ◽

10.1128/aem.02315-10 ◽

2011 ◽

Vol 77 (11) ◽

pp. 3685-3695 ◽

Cited By ~ 48

Author(s):

Michelle Q. Carter ◽

Maria T. Brandl ◽

Jacqueline W. Louie ◽

Jennifer L. Kyle ◽

Diana K. Carychao ◽

...

Keyword(s):

Escherichia Coli ◽

Acid Resistance ◽

Cell Aggregation ◽

Oxygen Depletion ◽

Growth Conditions ◽

Enterohemorrhagic Escherichia Coli ◽

Surface Attachment ◽

Content Type ◽

E Coli ◽

Physiological Properties

ABSTRACTCurli are adhesive fimbriae ofEnterobacteriaceaeand are involved in surface attachment, cell aggregation, and biofilm formation. Here, we report that both inter- and intrastrain variations in curli production are widespread in enterohemorrhagicEscherichia coliO157:H7. The relative proportions of curli-producing variants (C+) and curli-deficient variants (C−) in anE. coliO157:H7 cell population varied depending on the growth conditions. In variants derived from the 2006 U.S. spinach outbreak strains, the shift between the C+and C−subpopulations occurred mostly in response to starvation and was unidirectional from C−to C+; in variants derived from the 1993 hamburger outbreak strains, the shift occurred primarily in response to oxygen depletion and was bidirectional. Furthermore, curli variants derived from the same strain displayed marked differences in survival fitness: C+variants grew to higher concentrations in nutrient-limited conditions than C−variants, whereas C−variants were significantly more acid resistant than C+variants. This difference in acid resistance does not appear to be linked to the curli fimbriaeper se, since acsgAdeletion mutant in either a C+or a C−variant exhibited an acid resistance similar to that of its parental strain. Our data suggest that natural curli variants ofE. coliO157:H7 carry several distinct physiological properties that are important for their environmental survival. Maintenance of curli variants in anE. coliO157:H7 population may provide a survival strategy in which C+variants are selected in a nutrient-limited environment, whereas C−variants are selected in an acidic environment, such as the stomach of an animal host, including that of a human.

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PmrD Is Required for Modifications to Escherichia coli Endotoxin That Promote Antimicrobial Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05052-14 ◽

2015 ◽

Vol 59 (4) ◽

pp. 2051-2061 ◽

Cited By ~ 35

Author(s):

Erica J. Rubin ◽

Carmen M. Herrera ◽

Alexander A. Crofts ◽

M. Stephen Trent

Keyword(s):

Escherichia Coli ◽

Protein Interactions ◽

Lipid A ◽

Bacterial Resistance ◽

Functional Divergence ◽

Polymyxin B ◽

Growth Conditions ◽

Wild Type ◽

Content Type ◽

E Coli

ABSTRACTInSalmonella enterica, PmrD is a connector protein that links the two-component systems PhoP-PhoQ and PmrA-PmrB. WhileEscherichia coliencodes a PmrD homolog, it is thought to be incapable of connecting PhoPQ and PmrAB in this organism due to functional divergence from theS. entericaprotein. However, our laboratory previously observed that low concentrations of Mg2+, a PhoPQ-activating signal, leads to the induction of PmrAB-dependent lipid A modifications in wild-typeE. coli(C. M. Herrera, J. V. Hankins, and M. S. Trent, Mol Microbiol 76:1444–1460, 2010,http://dx.doi.org/10.1111/j.1365-2958.2010.07150.x). These modifications include phosphoethanolamine (pEtN) and 4-amino-4-deoxy-l-arabinose (l-Ara4N), which promote bacterial resistance to cationic antimicrobial peptides (CAMPs) when affixed to lipid A. Here, we demonstrate thatpmrDis required for modification of the lipid A domain ofE. colilipopolysaccharide (LPS) under low-Mg2+growth conditions. Further, RNA sequencing shows thatE. colipmrDinfluences the expression ofpmrAand its downstream targets, including genes coding for the modification enzymes that transfer pEtN andl-Ara4N to the lipid A molecule. In line with these findings, apmrDmutant is dramatically impaired in survival compared with the wild-type strain when exposed to the CAMP polymyxin B. Notably, we also reveal the presence of an unknown factor or system capable of activatingpmrDto promote lipid A modification in the absence of the PhoPQ system. These results illuminate a more complex network of protein interactions surrounding activation of PhoPQ and PmrAB inE. colithan previously understood.

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Pathways of Energy Metabolism Required for Phenotypic Expression of Nif+Kp Genes in Escherichia Coli

Australian Journal of Biological Sciences ◽

10.1071/bi9790637 ◽

1979 ◽

Vol 32 (6) ◽

pp. 637 ◽

Cited By ~ 3

Author(s):

Mary L Skotnicki ◽

Barry G Rolfe

Keyword(s):

Escherichia Coli ◽

Nitrogen Fixation ◽

Energy Metabolism ◽

Electron Transport ◽

Nitrogenase Activity ◽

Phenotypic Expression ◽

E Coli ◽

Substrate Level Phosphorylation ◽

Fumarate Reduction ◽

Substrate Level

In E. coli K12 (F'nif+ KP) hybrids, electron-transport-dependent phosphorylation is not necessary for anaerobic nitrogen fixation, and substrate level phosphorylation can provide sufficient ATP from glucose for nitrogenase activity, The fumarate-reduction system, however, is essential in these hybrids for the transfer of electrons to nitrogenase, This system is probably also involved in maintaining the membrane in the energized state, thereby allowing nitrogen fixation to occur

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Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.00781-12 ◽

2012 ◽

Vol 78 (15) ◽

pp. 5238-5246 ◽

Cited By ~ 9

Author(s):

Dongfei Han ◽

Ji-Young Ryu ◽

Robert A. Kanaly ◽

Hor-Gil Hur

Keyword(s):

Escherichia Coli ◽

Pseudomonas Putida ◽

Hypothetical Protein ◽

Aldehyde Group ◽

Agrobacterium Vitis ◽

T7 Promoter ◽

Content Type ◽

E Coli ◽

Cell Extracts ◽

Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

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