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 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 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.

Fumarate dependent protein composition under aerobic and anaerobic growth conditions in Escherichia coli

2020 ◽  
Vol 212 ◽  
pp. 103583 ◽  
Author(s):  
Kristin Surmann ◽  
Marius Stopp ◽  
Sebastian Wörner ◽  
Vishnu M. Dhople ◽  
Uwe Völker ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Composition ◽  
Growth Conditions ◽  
Anaerobic Growth ◽  
Dependent Protein ◽  
Aerobic And Anaerobic Growth ◽  
Aerobic And Anaerobic

scholarly journals Reactivity of nitric oxide with the [4Fe–4S] cluster of dihydroxyacid dehydratase from Escherichia coli

2009 ◽  
Vol 417 (3) ◽  
pp. 783-789 ◽  
Author(s):  
Xuewu Duan ◽  
Juanjuan Yang ◽  
Binbin Ren ◽  
Guoqiang Tan ◽  
Huangen Ding
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Enzyme Activity ◽  
Anaerobic Conditions ◽  
Iron Sulfur Clusters ◽  
E Coli ◽  
Endonuclease Iii ◽  
Iron Sulfur ◽  
Aerobic And Anaerobic ◽  
Aerobic And Anaerobic Conditions

Although the NO (nitric oxide)-mediated modification of iron–sulfur proteins has been well-documented in bacteria and mammalian cells, specific reactivity of NO with iron–sulfur proteins still remains elusive. In the present study, we report the first kinetic characterization of the reaction between NO and iron–sulfur clusters in protein using the Escherichia coli IlvD (dihydroxyacid dehydratase) [4Fe–4S] cluster as an example. Combining a sensitive NO electrode with EPR (electron paramagnetic resonance) spectroscopy and an enzyme activity assay, we demonstrate that NO is rapidly consumed by the IlvD [4Fe–4S] cluster with the concomitant formation of the IlvD-bound DNIC (dinitrosyl–iron complex) and inactivation of the enzyme activity under anaerobic conditions. The rate constant for the initial reaction between NO and the IlvD [4Fe–4S] cluster is estimated to be (7.0±2.0)×106 M−2·s−1 at 25 °C, which is approx. 2–3 times faster than that of the NO autoxidation by O2 in aqueous solution. Addition of GSH failed to prevent the NO-mediated modification of the IlvD [4Fe–4S] cluster regardless of the presence of O2 in the medium, further suggesting that NO is more reactive with the IlvD [4Fe–4S] cluster than with GSH or O2. Purified aconitase B [4Fe–4S] cluster from E. coli has an almost identical NO reactivity as the IlvD [4Fe–4S] cluster. However, the reaction between NO and the endonuclease III [4Fe–4S] cluster is relatively slow, apparently because the [4Fe–4S] cluster in endonuclease III is less accessible to solvent than those in IlvD and aconitase B. When E. coli cells containing recombinant IlvD, aconitase B or endonuclease III are exposed to NO using the Silastic tubing NO delivery system under aerobic and anaerobic conditions, the [4Fe–4S] clusters in IlvD and aconitase B, but not in endonuclease III, are efficiently modified forming the protein-bound DNICs, confirming that NO has a higher reactivity with the [4Fe–4S] clusters in IlvD and aconitase B than with O2 or GSH. The results suggest that the iron–sulfur clusters in proteins such as IlvD and aconitase B may constitute the primary targets of the NO cytotoxicity under both aerobic and anaerobic conditions.

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.

Differential effect of YidC depletion on the membrane proteome of Escherichia coli under aerobic and anaerobic growth conditions

PROTEOMICS ◽  
2010 ◽  
Vol 10 (18) ◽  
pp. 3235-3247 ◽  
Author(s):  
Claire E. Price ◽  
Andreas Otto ◽  
Fabrizia Fusetti ◽  
Dörte Becher ◽  
Michael Hecker ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Differential Effect ◽  
Growth Conditions ◽  
Anaerobic Growth ◽  
Membrane Proteome ◽  
Aerobic And Anaerobic Growth ◽  
Aerobic And Anaerobic

scholarly journals Effect of Anaerobic Growth on Quinolone Lethality with Escherichia coli

2006 ◽  
Vol 51 (1) ◽  
pp. 28-34 ◽  
Author(s):  
Muhammad Malik ◽  
Syed Hussain ◽  
Karl Drlica
Keyword(s):  
Escherichia Coli ◽  
Nalidixic Acid ◽  
Sodium Dodecyl ◽  
Anaerobic Growth ◽  
Aerobic Growth ◽  
Anaerobic Conditions ◽  
Dna Complexes ◽  
Chromosome Fragmentation ◽  
Aerobic And Anaerobic Growth ◽  
Aerobic And Anaerobic

ABSTRACT Quinolone activity against Escherichia coli was examined during aerobic growth, aerobic treatment with chloramphenicol, and anaerobic growth. Nalidixic acid, norfloxacin, ciprofloxacin, and PD161144 were lethal for cultures growing aerobically, and the bacteriostatic activity of each quinolone was unaffected by anaerobic growth. However, lethal activity was distinct for each quinolone with cells treated aerobically with chloramphenicol or grown anaerobically. Nalidixic acid failed to kill cells under both conditions; norfloxacin killed cells when they were grown anaerobically but not when they were treated with chloramphenicol; ciprofloxacin killed cells under both conditions but required higher concentrations than those required with cells grown aerobically; and PD161144, a C-8-methoxy fluoroquinolone, was equally lethal under all conditions. Following pretreatment with nalidixic acid, a shift to anaerobic conditions or the addition of chloramphenicol rapidly blocked further cell death. Formation of quinolone-gyrase-DNA complexes, observed as a sodium dodecyl sulfate (SDS)-dependent drop in cell lysate viscosity, occurred during aerobic and anaerobic growth and in the presence and in the absence of chloramphenicol. However, lethal chromosome fragmentation, detected as a drop in viscosity in the absence of SDS, occurred with nalidixic acid treatment only under aerobic conditions in the absence of chloramphenicol. With PD161144, chromosome fragmentation was detected when the cells were grown aerobically and anaerobically and in the presence and in the absence of chloramphenicol. Thus, all quinolones tested appear to form reversible bacteriostatic complexes containing broken DNA during aerobic growth, during anaerobic growth, and when protein synthesis is blocked; however, the ability to fragment chromosomes and to rapidly kill cells under these conditions depends on quinolone structure.

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

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
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.

scholarly journals Glutathionylspermidine metabolism in Escherichia coli

1995 ◽  
Vol 312 (2) ◽  
pp. 465-469 ◽  
Author(s):  
K Smith ◽  
A Borges ◽  
M R Ariyanayagam ◽  
A H Fairlamb
Keyword(s):  
Escherichia Coli ◽  
Glutathione Reductase ◽  
Growth Conditions ◽  
Anaerobic Growth ◽  
Growth Phases ◽  
Total Glutathione ◽  
Apparent Lack ◽  
E Coli ◽  
Catalytic Constant ◽  
Glutathione Disulphide

Intracellular levels of glutathione and glutathionylspermidine conjugates have been measured throughout the growth phases of Escherichia coli. Glutathionylspermidine was present in mid-log-phase cells, and under stationary and anaerobic growth conditions accounted for 80% of the total glutathione content. N1,N8-bis(glutathionyl)spermidine (trypanothione) was undetectable under all growth conditions. The catalytic constant kcat/Km of recombinant E. coli glutathione reductase for glutathionylspermidine disulphide was approx. 11,000-fold lower than that for glutathione disulphide. The much higher catalytic constant for the mixed disulphide of glutathione and glutathionylspermidine (11% that of GSSG), suggests a possible explanation for the low turnover of trypanothione disulphide by E. coli glutathione reductase, given the apparent lack of a specific glutathionylspermidine disulphide reductase in E. coli.

scholarly journals In vivo cycling of the Escherichia coli transcription factor FNR between active and inactive states

Microbiology ◽  
2005 ◽  
Vol 151 (12) ◽  
pp. 4063-4070 ◽  
Author(s):  
David P. Dibden ◽  
Jeffrey Green
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Oxygen Availability ◽  
Anaerobic Conditions ◽  
Aerobic Conditions ◽  
Fnr Protein ◽  
Transcription Regulators ◽  
Aerobic And Anaerobic ◽  
Aerobic And Anaerobic Conditions

FNR proteins are transcription regulators that sense changes in oxygen availability via assembly–disassembly of [4Fe–4S] clusters. The Escherichia coli FNR protein is present in bacteria grown under aerobic and anaerobic conditions. Under aerobic conditions, FNR is isolated as an inactive monomeric apoprotein, whereas under anaerobic conditions, FNR is present as an active dimeric holoprotein containing one [4Fe–4S] cluster per subunit. It has been suggested that the active and inactive forms of FNR are interconverted in vivo, or that iron–sulphur clusters are mostly incorporated into newly synthesized FNR. Here, experiments using a thermo-inducible fnr expression plasmid showed that a model FNR-dependent promoter is activated under anaerobic conditions by FNR that was synthesized under aerobic conditions. Immunoblots suggested that FNR was more prone to degradation under aerobic compared with anaerobic conditions, and that the ClpXP protease contributes to this degradation. Nevertheless, FNR was sufficiently long lived (half-life under aerobic conditions, ∼45 min) to allow cycling between active and inactive forms. Measuring the abundance of the FNR-activated dms transcript when chloramphenicol-treated cultures were switched between aerobic and anaerobic conditions showed that it increased when cultures were switched to anaerobic conditions, and decreased when aerobic conditions were restored. In contrast, measurement of the abundance of the FNR-repressed ndh transcript under the same conditions showed that it decreased upon switching to anaerobic conditions, and then increased when aerobic conditions were restored. The abundance of the FNR- and oxygen-independent tatE transcript was unaffected by changes in oxygen availability. Thus, the simplest explanation for the observations reported here is that the FNR protein can be switched between inactive and active forms in vivo in the absence of de novo protein synthesis.

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