scholarly journals The Disulfide Bond Formation Pathway Is Essential for Anaerobic Growth of Escherichia coli

2017 ◽  
Vol 199 (16) ◽  
Author(s):  
Brian M. Meehan ◽  
Cristina Landeta ◽  
Dana Boyd ◽  
Jonathan Beckwith
Keyword(s):  
Escherichia Coli ◽  
Disulfide Bond ◽  
Bond Formation ◽  
Anaerobic Growth ◽  
Strictly Anaerobic ◽  
Disulfide Bond Formation ◽  
Anaerobic Conditions ◽  
Content Type ◽  
E Coli ◽  
Laboratory Conditions

ABSTRACT Disulfide bonds are critical to the stability and function of many bacterial proteins. In the periplasm of Escherichia coli, intramolecular disulfide bond formation is catalyzed by the two-component disulfide bond forming (DSB) system. Inactivation of the DSB pathway has been shown to lead to a number of pleotropic effects, although cells remain viable under standard laboratory conditions. However, we show here that dsb strains of E. coli reversibly filament under aerobic conditions and fail to grow anaerobically unless a strong oxidant is provided in the growth medium. These findings demonstrate that the background disulfide bond formation necessary to maintain the viability of dsb strains is oxygen dependent. LptD, a key component of the lipopolysaccharide transport system, fails to fold properly in dsb strains exposed to anaerobic conditions, suggesting that these mutants may have defects in outer membrane assembly. We also show that anaerobic growth of dsb mutants can be restored by suppressor mutations in the disulfide bond isomerization system. Overall, our results underscore the importance of proper disulfide bond formation to pathways critical to E. coli viability under conditions where oxygen is limited. IMPORTANCE While the disulfide bond formation (DSB) system of E. coli has been studied for decades and has been shown to play an important role in the proper folding of many proteins, including some associated with virulence, it was considered dispensable for growth under most laboratory conditions. This work represents the first attempt to study the effects of the DSB system under strictly anaerobic conditions, simulating the environment encountered by pathogenic E. coli strains in the human intestinal tract. By demonstrating that the DSB system is essential for growth under such conditions, this work suggests that compounds inhibiting Dsb enzymes might act not only as antivirulents but also as true antibiotics.

scholarly journals Redesigning Escherichia coli Metabolism for Anaerobic Production of Isobutanol

2011 ◽  
Vol 77 (14) ◽  
pp. 4894-4904 ◽  
Author(s):  
Cong T. Trinh ◽  
Johnny Li ◽  
Harvey W. Blanch ◽  
Douglas S. Clark
Keyword(s):  
Escherichia Coli ◽  
Anaerobic Growth ◽  
Mode Analysis ◽  
Glycolytic Flux ◽  
Strictly Anaerobic ◽  
Anaerobic Conditions ◽  
Content Type ◽  
E Coli ◽  
Model Predictions ◽  
Chromosomal Genes

ABSTRACTFermentation enables the production of reduced metabolites, such as the biofuels ethanol and butanol, from fermentable sugars. This work demonstrates a general approach for designing and constructing a production host that uses a heterologous pathway as an obligately fermentative pathway to produce reduced metabolites, specifically, the biofuel isobutanol. Elementary mode analysis was applied to design anEscherichia colistrain optimized for isobutanol production under strictly anaerobic conditions. The central metabolism ofE. coliwas decomposed into 38,219 functional, unique, and elementary modes (EMs). The model predictions revealed that during anaerobic growthE. colicannot produce isobutanol as the sole fermentative product. By deleting 7 chromosomal genes, the total 38,219 EMs were constrained to 12 EMs, 6 of which can produce high yields of isobutanol in a range from 0.29 to 0.41 g isobutanol/g glucose under anaerobic conditions. The remaining 6 EMs rely primarily on the pyruvate dehydrogenase enzyme complex (PDHC) and are typically inhibited under anaerobic conditions. The redesignedE. colistrain was constrained to employ the anaerobic isobutanol pathways through deletion of 7 chromosomal genes, addition of 2 heterologous genes, and overexpression of 5 genes. Here we present the design, construction, and characterization of an isobutanol-producingE. colistrain to illustrate the approach. The model predictions are evaluated in relation to experimental data and strategies proposed to improve anaerobic isobutanol production. We also show that the endogenous alcohol/aldehyde dehydrogenase AdhE is the key enzyme responsible for the production of isobutanol and ethanol under anaerobic conditions. The glycolytic flux can be controlled to regulate the ratio of isobutanol to ethanol production.

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

2013 ◽  
Vol 57 (10) ◽  
pp. 4707-4716 ◽  
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.

scholarly journals Identification of the Thioredoxin Partner of Vitamin K Epoxide Reductase in Mycobacterial Disulfide Bond Formation

2018 ◽  
Vol 200 (16) ◽  
Author(s):  
Na Ke ◽  
Cristina Landeta ◽  
Xiaoyun Wang ◽  
Dana Boyd ◽  
Markus Eser ◽  
...  
Keyword(s):  
Vitamin K ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Cell Envelope ◽  
Bond Formation ◽  
Disulfide Bond Formation ◽  
Vitamin K Epoxide Reductase ◽  
Content Type ◽  
E Coli ◽  
Epoxide Reductase

ABSTRACTDisulfide bonds influence the stability and activity of many proteins. InEscherichia coli, the DsbA and DsbB enzymes promote disulfide bond formation. Other bacteria, including theActinobacteria, use instead of DsbB the enzyme vitamin K epoxide reductase (VKOR), whose gene is found either fused to or in the same operon as adsbA-like gene.Mycobacterium tuberculosisand other Gram-positive actinobacteria secrete many proteins with even numbers of cysteines to the cell envelope. These organisms have predicted oxidoreductases and VKOR orthologs. These findings indicate that such bacteria likely form disulfide bonds in the cell envelope. TheM. tuberculosisvkorgene complements anE. colidsbBdeletion strain, restoring the oxidation ofE. coliDsbA. While we have suggested that thedsbAgene linked to thevkorgene may express VKOR's partner in mycobacteria, others have suggested that two other extracytoplasmic oxidoreductases (DsbE or DsbF) may be catalysts of protein disulfide bond formation. However, there is no direct evidence for interactions of VKOR with either DsbA, DsbE, or DsbF. To identify the actual substrate of VKOR, we identified two additional predicted extracytoplasmic DsbA-like proteins using bioinformatics analysis of theM. tuberculosisgenome. Using the five potential DsbAs, we attempted to reconstitute disulfide bond pathways inE. coliand inMycobacterium smegmatis, a close relative ofM. tuberculosis. Our results show that onlyM. tuberculosisDsbA is oxidized by VKOR. Comparison of the properties ofdsbA- andvkor-null mutants inM. smegmatisshows parallels to the properties ofdsbmutations inE. coli.IMPORTANCEDisulfide bond formation has a great impact on bacterial pathogenicity. Thus, disulfide-bond-forming proteins represent new targets for the development of antibacterials, since the inhibition of disulfide bond formation would result in the simultaneous loss of the activity of several classes of virulence factors. Here, we identified five candidate proteins encoded by theM. tuberculosisgenome as possible substrates of theM. tuberculosisVKOR protein involved in disulfide bond formation. We then reconstituted the mycobacterial disulfide bond formation pathway inE. coliand showed that of the five candidates, onlyM. tuberculosisDsbA is efficiently oxidized by VKOR inE. coli. We also present evidence for the involvement of VKOR in DsbA oxidation inM. smegmatis.

scholarly journals Elevated Expression of GlpT and UhpT via FNR Activation Contributes to Increased Fosfomycin Susceptibility in Escherichia coli under Anaerobic Conditions

2015 ◽  
Vol 59 (10) ◽  
pp. 6352-6360 ◽  
Author(s):  
Kumiko Kurabayashi ◽  
Koichi Tanimoto ◽  
Shinobu Fueki ◽  
Haruyoshi Tomita ◽  
Hidetada Hirakawa
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Agents ◽  
Critical Issue ◽  
Multidrug Resistant ◽  
Anaerobic Growth ◽  
Pcr Analysis ◽  
Anaerobic Conditions ◽  
Content Type ◽  
E Coli ◽  
Elevated Expression

ABSTRACTBecause a shortage of new antimicrobial agents is a critical issue at present, and with the spread of multidrug-resistant (MDR) pathogens, the use of fosfomycin to treat infections is being revisited as a “last-resort option.” This drug offers a particular benefit in that it is more effective against bacteria growing under oxygen-limited conditions, unlike other commonly used antimicrobials, such as fluoroquinolones and aminoglycosides. In this study, we showed thatEscherichia colistrains, including enterohemorrhagicE. coli(EHEC), were more susceptible to fosfomycin when grown anaerobically than when grown aerobically, and we investigated how the activity of this drug was enhanced during anaerobic growth ofE. coli. Our quantitative PCR analysis and a transport assay showed thatE. colicells grown under anaerobic conditions had higher levels of expression ofglpTanduhpT, encoding proteins that transport fosfomycin into cells with their native substrates, i.e., glycerol-3-phosphate and glucose-6-phosphate, and led to increased intracellular accumulation of the drug. Elevation of expression of these genes during anaerobic growth requires FNR, a global transcriptional regulator that is activated under anaerobic conditions. Purified FNR bound to DNA fragments from regions upstream ofglpTanduhpT, suggesting that it is an activator of expression ofglpTanduhpTduring anaerobic growth. We concluded that the increased antibacterial activity of fosfomycin towardE. coliunder anaerobic conditions can be attributed to elevated expression of GlpT and UhpT following activation of FNR, leading to increased uptake of the drug.

scholarly journals Disulfide Bond Formation and Activation of Escherichia coli β-Galactosidase under Oxidizing Conditions

2012 ◽  
Vol 78 (7) ◽  
pp. 2376-2385 ◽  
Author(s):  
Joaquin Seras-Franzoso ◽  
Roman Affentranger ◽  
Mario Ferrer-Navarro ◽  
Xavier Daura ◽  
Antonio Villaverde ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Disulfide Bond ◽  
Cell Biology ◽  
Bond Formation ◽  
Homology Model ◽  
Disulfide Bond Formation ◽  
Content Type ◽  
Large Size ◽  
Reporter Enzyme ◽  
Tetrameric Structure

ABSTRACTEscherichia coliβ-galactosidase is probably the most widely used reporter enzyme in molecular biology, cell biology, and biotechnology because of the easy detection of its activity. Its large size and tetrameric structure make this bacterial protein an interesting model for crystallographic studies and atomic mapping. In the present study, we investigate a version ofEscherichia coliβ-galactosidase produced under oxidizing conditions, in the cytoplasm of an Origami strain. Our data prove the activation of this microbial enzyme under oxidizing conditions and clearly show the occurrence of a disulfide bond in the β-galactosidase structure. Additionally, the formation of this disulfide bond is supported by the analysis of a homology model of the protein that indicates that two cysteines located in the vicinity of the catalytic center are sufficiently close for disulfide bond formation.

scholarly journals Pyruvate Formate Lyase and Acetate Kinase Are Essential for Anaerobic Growth of Escherichia coli on Xylose

2004 ◽  
Vol 186 (22) ◽  
pp. 7593-7600 ◽  
Author(s):  
Adnan Hasona ◽  
Youngnyun Kim ◽  
F. G. Healy ◽  
L. O. Ingram ◽  
K. T. Shanmugam
Keyword(s):  
Escherichia Coli ◽  
Minimal Medium ◽  
High Capacity ◽  
Redox Balance ◽  
Anaerobic Growth ◽  
Acetate Kinase ◽  
Anaerobic Conditions ◽  
Pyruvate Formate Lyase ◽  
E Coli ◽  
Xylose Transport

ABSTRACT During anaerobic growth of bacteria, organic intermediates of metabolism, such as pyruvate or its derivatives, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate-level phosphorylation. In Escherichia coli, conversion of glucose to pyruvate yields 2 net ATPs, while metabolism of a pentose, such as xylose, to pyruvate only yields 0.67 net ATP per xylose due to the need for one (each) ATP for xylose transport and xylulose phosphorylation. During fermentative growth, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP from two pyruvates (one hexose equivalent) while still maintaining the overall redox balance. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose. An E. coli pfl mutant lacking pyruvate formate lyase cannot convert pyruvate to acetyl coenzyme A, the required precursor for acetate and ethanol production, and could not produce this additional ATP. E. coli pfl mutants failed to grow under anaerobic conditions in xylose minimal medium without any negative effect on their survival or aerobic growth. An ackA mutant, lacking the ability to generate ATP from acetyl phosphate, also failed to grow in xylose minimal medium under anaerobic conditions, confirming the need for the ATP produced by acetate kinase for anaerobic growth on xylose. Since arabinose transport by AraE, the low-affinity, high-capacity, arabinose/H+ symport, conserves the ATP expended in pentose transport by the ABC transporter, both pfl and ackA mutants grew anaerobically with arabinose. AraE-based xylose transport, achieved after constitutively expressing araE, also supported the growth of the pfl mutant in xylose minimal medium. These results suggest that a net ATP yield of 0.67 per pentose is only enough to provide for maintenance energy but not enough to support growth of E. coli in minimal medium. Thus, pyruvate formate lyase and acetate kinase are essential for anaerobic growth of E. coli on xylose due to energetic constraints.

scholarly journals Time-Dependent Proteome Alterations under Osmotic Stress during Aerobic and Anaerobic Growth in Escherichia coli

2006 ◽  
Vol 188 (20) ◽  
pp. 7165-7175 ◽  
Author(s):  
Arnim Weber ◽  
Stephanie A. Kögl ◽  
Kirsten Jung
Keyword(s):  
Escherichia Coli ◽  
Osmotic Stress ◽  
Integration Host Factor ◽  
Anaerobic Growth ◽  
Time Dependent ◽  
Transcriptional Level ◽  
Anaerobic Conditions ◽  
Growth And Survival ◽  
E Coli ◽  
Protein Gels

ABSTRACT Escherichia coli lives in the mammalian gastrointestinal tract anaerobically at high osmolarity as well as in the soil aerobically at varying osmolarities. Adaptation to these varying environmental conditions is crucial for growth and survival of E. coli. Two-dimensional protein gels were used to visualize global time-dependent changes (10 to 60 min) in the proteome of cells responding to osmotic stress (0.4 M NaCl or 0.7 M sorbitol) under aerobic or anaerobic conditions. The protein profiles revealed an induction of 12 proteins (Dps, HchA, HdhA, InfB, OsmC, OsmY, ProX, KatE, PspA, TalA, TktB, and TreF) under osmotic stress in an aerobic milieu. Eleven additional proteins (OtsB, YceI, YciE, YciF, YgaU, YjbJ, AcnA, MetL, PoxB, Ssb, and YhbO) were induced by osmotic stress imposed by NaCl. Most of the accumulated proteins were cross-protecting proteins (e.g., OsmY, OsmC, Dps, and KatE) which are regulated at the transcriptional level predominantly by RpoS and other regulators (e.g., integration host factor, OxyR, H-NS, LRP, and FIS). Comparative analysis of the proteome of E. coli grown under aerobic or anaerobic conditions under osmotic stress (NaCl) revealed an overlap of the up-regulated proteins of more than 50%. Ten proteins (PoxB, AcnA, TalA, TktB, KatE, PspA, Ssb, TreF, MetL, and YhbO) were detectable only under aerobic, high-osmolality conditions. Time-dependent alterations of the proteome were monitored, allowing classification of the up-regulated proteins into early, middle, and long-term phases of adaptation. Only a few proteins were found to be down-regulated upon osmotic stress.

Sign in / Sign up

Export Citation Format

Share Document