Oxidative damage blocks thymineless death and trimethoprim poisoning in Escherichia coli

Cells that cannot synthesize one of the DNA precursors, dTTP, due to thyA mutation or metabolic poisoning, undergo thymineless death (TLD), — a chromosome-based phenomenon of unclear mechanisms. In E. coli , thymineless death is caused either by denying thyA mutants thymidine supplementation or by treating wild type cells with trimethoprim. Two recent reports promised a potential breakthrough in TLD understanding, suggesting significant oxidative damage during thymine starvation. Oxidative damage in vivo comes from Fenton's reaction, when hydrogen peroxide meets ferrous iron to produce hydroxyl radical. Therefore, TLD could kill via irreparable double-strand breaks behind replication forks, when starvation-caused single-strand DNA gaps are attacked by hydroxyl radicals. We tested the proposed Fenton-TLD connection, in both thyA mutants denied thymidine, as well as in trimethoprim-treated WT cells, under three conditions: 1) intracellular iron chelation; 2) mutational inactivation of hydrogen peroxide (HP) scavenging; 3) acute treatment with sublethal HP concentrations. We found that TLD kinetics are affected by neither iron chelation, nor HP stabilization in cultures, indicating no induction of oxidative damage during thymine starvation. Moreover, acute exogenous HP treatments completely block TLD, apparently by blocking cell division — which may be a novel TLD prerequisite. Separately, the acute trimethoprim sensitivity of the rffC and recBCD mutants demonstrates how bactericidal power of this antibiotic could be amplified by inhibiting the corresponding enzymes. Importance Mysterious thymineless death strikes cells that are starved for thymine and therefore replicating their chromosomal DNA without dTTP. After 67 years of experiments testing various obvious and not so obvious explanations, thymineless death is still without a mechanism. Recently, oxidative damage via in vivo Fenton's reaction was proposed as a critical contributor to the irreparable chromosome damage during thymine starvation. We have tested this idea by either blocking in vivo Fenton's reaction (expecting no thymineless death) or by amplifying oxidative damage (expecting hyper thymineless death). Instead, we found that blocking Fenton's reaction has no influence on thymineless death, while amplifying oxidative damage prevents thymineless death altogether. Thus, oxidative damage does not contribute to thymineless death, while the latter remains enigmatic.

In situ bioremediation of Fenton’s reaction–treated oil spill site, with a soil inoculum, slow release additives, and methyl-β-cyclodextrin

A residential lot impacted by spills from a leaking light heating oil tank was treated with a combination of chemical oxidation and bioremediation to avoid technically challenging excavation. The tank left emptied in the ground was used for slow infiltration of the remediation additives to the low permeability, clayey soil. First, hydrogen peroxide and citrate chelate was added for Fenton’s reaction–based chemical oxidation, resulting in a ca. 50% reduction from the initial 25,000 mg/kg average oil concentration in the soil below the tank. Part of this was likely achieved through mobilization of oily soil into the tank, which was beneficial in regards to the following biological treatment. By first adding live bacteria in a soil inoculum, and then oxygen and nutrients in different forms, an approximately 90% average reduction was achieved. To further enhance the effect, methyl-β-cyclodextrin surfactant (CD) was added, resulting finally in a 98% reduction from the initial average level. The applicability of the surfactant was based on laboratory-scale tests demonstrating that CD promoted oil degradation and, unlike pine soap, was not utilized by the bacteria as a carbon source, and thus inhibiting degradation of oils regardless of the positive effect on biological activity. The effect of CD on water solubility for different hydrocarbon fractions was tested to serve as the basis for risk assessment requirements for authorizing the use of the surfactant at the site.

Phytosynthesis of Palladium Nanoclusters: An Efficient Nanozyme for Ultrasensitive and Selective Detection of Reactive Oxygen Species

Hydrogen peroxide is a low-reactivity reactive oxygen species (ROS); however, it can easily penetrate cell membranes and produce highly reactive hydroxyl radical species through Fenton’s reaction. Its presence in abnormal amounts can lead to serious diseases in humans. Although the development of a simple, ultrasensitive, and selective method for H2O2 detection is crucial, this remains a strategic challenge. The peroxidase mimetic activity of palladium nanoclusters (PdNCs) has not previously been evaluated. In this study, we developed an ultrasensitive and selective colorimetric detection method for H2O2 using PdNCs. An unprecedented eco-friendly, cost-effective, and facile biological method was developed for the synthesis of PdNCs. This is the first report of the biosynthesis of PdNCs. The synthesized nanoclusters had a significantly narrow size distribution profile and high stability. The nanoclusters were demonstrated to possess a peroxidase mimetic activity that could oxidize peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB). Various interfering substances in serum (100 μM phenylalanine, cysteine, tryptophan, arginine, glucose, urea, Na+, Fe2+, PO43−, Mn+2, Ca2+, Mg2+, Zn2+, NH4+, and K+) were included to evaluate the selectivity of the assay, and oxidation of TMB occurred only in the presence of H2O2. Therefore, PdNCs show an efficient nanozyme for the peroxidase mimetic activity. The assay produced a sufficient signal at the ultralow concentration of 0.0625 µM H2O2. This colorimetric assay provides a real-time, rapid, and easy-to-use platform for the detection of H2O2 for clinical purposes.

Fenton’s reaction-based chemical oxidation in suboptimal conditions can lead to mobilization of oil hydrocarbons but also contribute to the total removal of volatile compounds

Fenton’s reaction-based chemical oxidation is in principle a method that can be utilized for all organic fuel residues thus making it a potential all-purpose, multi-contaminant, in situ application for cases in which storage and distribution of different types of fuels have resulted in contamination of soil or groundwater. Since peroxide breakdown reactions are also expected to lead to a physical transport of the target compound, this secondary physical removal, or rebound concentrations related to it, is prone to be affected by the chemical properties of the target compound. Also, since soil conditions are seldom optimal for Fenton’s reaction, the balance between chemical oxidation and transport may vary. In this study, it was found that, with a high enough hydrogen peroxide concentration (5 M), methyl tert-butyl ether–spiked groundwater could be treated even under suboptimal conditions for chemical mineralization. In these cases, volatilization was not only contributing to the total removal but also leading to rebound effects similar to those associated with air sparging techniques. Likewise for diesel, temporal transport from soil to the aqueous phase was found to lead to false positives that outweighed the actual remediation effect through chemical mineralization.

An Original HPLC Method with Coulometric Detection to Monitor Hydroxyl Radical Generation via Fenton Chemistry

Hydroxyl radicals (•OH) can be generated via Fenton chemistry catalyzed by transition metals. An in vitro Fenton system was developed to test both the inhibition and stimulation of •OH formation, by monitoring salicylate aromatic hydroxylation derivatives as markers of •OH production. The reaction was optimized with either iron or copper, and target analytes were determined by means of an original HPLC method coupled to coulometric detection. The method granted good sensitivity and precision, while method applicability was tested on antioxidant compounds with and without chelating properties in different substance to metal ratios. This analytical approach shows how Fenton’s reaction can be monitored by HPLC coupled to coulometric detection, as a powerful tool for studying molecules′ redox behavior.