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 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 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.

Aerobic purification of N5,N10-methylenetetrahydromethanopterin dehydrogenase, separated from N5,N10-methenyltetrahydromethanopterin cyclohydrolase, from Methanobacterium thermoautotrophicum strain Marburg

1989 ◽  
Vol 35 (4) ◽  
pp. 499-507 ◽  
Author(s):  
Biswarup Mukhopadhyay ◽  
Lacy Daniels
Keyword(s):  
Enzyme Activity ◽  
Ammonium Sulfate ◽  
Dehydrogenase Activity ◽  
Specific Activity ◽  
Methanobacterium Thermoautotrophicum ◽  
Anaerobic Conditions ◽  
Cell Extracts ◽  
First Time ◽  
Aerobic And Anaerobic ◽  
Aerobic And Anaerobic Conditions

The N5,N10-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum strain Marburg has been purified with reasonable yield and much higher specific activity than previously reported. For the first time it has been shown that both N5,N10-methylenetetrahydromethanopterin dehydrogenase and N5,N10-methenyltetrahydromethanopterin cyclohydrolase activities were stable under air and could be purified using aerobic operations. The dehydrogenase activity from Methanobacterium thermoautotrophicum Marburg was stable in phosphate buffer with or without glycerol or ammonium sulfate under both aerobic and anaerobic conditions. However, the presence of either 2-mercaptoethanol or dithiothreitol in the enzyme solution destroyed the enzyme activity during both aerobic and anaerobic incubations. Dehydrogenase was purified 62-fold using Phenyl-Sepharose and DEAE-Sephadex chromatography in succession under air. Both of these chromatographic methods separated dehydrogenase activity from N5,N10-methenyltetrahydromethanopterin cyclohydrolase; DEAE-Sephadex provided the best separation. Phenyl-Sepharose chromatography of the supernatant of cell extracts containing ammonium sulfate at 60% of saturation provided a 4.7-fold purification and 98% recovery of cyclohydrolase; this result established the air stability of N5,N10-methenyltetrahydromethanopterin cyclohydrolase from Methanobacterium thermoautotrophicum Marburg.Key words: methylenetetrahydromethanopterin dehydrogenase, methenyltetrahydromethanopterin cyclohydrolase, Methanobacterium, aerobic purification, oxygen stability.

scholarly journals Growth of cell wall-defective variants of Escherichia coli: comparison of aerobic and anaerobic induction frequencies

1977 ◽  
Vol 6 (2) ◽  
pp. 166-171
Author(s):  
T W Huber ◽  
A W Brinkley
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
High Frequency ◽  
Anaerobic Incubation ◽  
Anaerobic Conditions ◽  
Group Iii ◽  
E Coli ◽  
Induction Frequency ◽  
Group I ◽  
Aerobic And Anaerobic

A method for quantitating the conversion of Escherichia coli to colony-forming, cell wall-defective (CWD) bacteria has been developed. The induction frequency, i.e., the percentage of the population recovered as CWD colonies was determined for 20 randomly selected clinical isolates of E. coli under aerobic and anaerobic incubation conditions. Penicillin (1,000 U/ML) was the inducing agent. The 20 strains segregated into three groups. Group I organisms produced CWD colonies with high frequency both aerobically and anaerobically. Grout II organisms showed a much higher induction frequency anaerobically than aerobically. Group III organisms were poor inducers. Thirty percent of the strains were group I, 50% were group II, and 20% were group III organisms. These data indicate that anaerobic conditions enhance the induction and growth of CWD E. coli in the research laboratory and suggest that anaerobic incubation may be important in recovery of medically significant CWD bacteria.

scholarly journals Diverse effects of nitric oxide reductase NorV on Aeromonas hydrophila virulence-associated traits under aerobic and anaerobic conditions

2019 ◽  
Vol 50 (1) ◽  
Author(s):  
Jin Liu ◽  
Yuhao Dong ◽  
Nannan Wang ◽  
Shuiyan Ma ◽  
Chengping Lu ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Aeromonas Hydrophila ◽  
Dependent Manner ◽  
Nitric Oxide Reductase ◽  
Wild Type ◽  
Anaerobic Conditions ◽  
Aerobic Conditions ◽  
Significant Difference ◽  
Aerobic And Anaerobic ◽  
Aerobic And Anaerobic Conditions

Abstract NorV has been known to be an anaerobic nitric oxide reductase associated with nitric oxide (NO) detoxification. Recently, we showed that the norV gene of Aeromonas hydrophila was highly upregulated after co-culturing with Tetrahymena thermophila. Here, we demonstrated that the transcription and expression levels of norV were upregulated in a dose-dependent manner after exposure to NO under aerobic and anaerobic conditions. To investigate the roles of norV in resisting predatory protists and virulence of A. hydrophila, we constructed the norV gene-deletion mutant (ΔnorV). Compared to the wild type, the ΔnorV mutant showed no significant difference in growth at various NO concentrations under aerobic conditions but significantly stronger NO-mediated growth inhibition under anaerobic conditions. The deletion of norV exhibited markedly decreased cytotoxicity, hemolytic and protease activities under aerobic and anaerobic conditions. Also, the hemolysin co-regulated protein (Hcp) in the ΔnorV mutant showed increased secretion under aerobic conditions but decreased secretion under anaerobic conditions as compared to the wild-type. Moreover, the inactivation of norV led to reduced resistance to predation by T. thermophila, decreased survival within macrophages and highly attenuated virulence in zebrafish. Our data indicate a diverse role for norV in the expression of A. hydrophila virulence-associated traits that is not completely dependent on its function as a nitric oxide reductase. This study provides insights into an unexplored area of NorV, which will contribute to our understanding of bacterial pathogenesis and the development of new control strategies for A. hydrophila infection.

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