Antioxidants are ineffective at quenching reactive oxygen species inside bacteria and should not be used to diagnose oxidative stress.

A wide variety of stresses have been proposed to exert killing effects upon bacteria by stimulating the intracellular formation of reactive oxygen species (ROS). A key part of the supporting evidence has often been the ability of antioxidant compounds to protect the cells. In this study, some of the most-used antioxidants-thiourea, glutathione, N-acetylcysteine, and ascorbate-have been examined. Their ability to quench superoxide and hydrogen peroxide was verified in vitro, but the rate constants were orders of magnitude too slow for them to have an impact upon superoxide and peroxide concentrations in vivo, where these species are already scavenged by highly active enzymes. Indeed, the antioxidants were unable to protect the growth and ROS-sensitive enzymes of E. coli strains experiencing authentic oxidative stress. Similar logic posits that antioxidants cannot substantially quench hydroxyl radicals inside cells, which contain abundant biomolecules that react with them at diffusion-limited rates. Indeed, antioxidants were able to protect cells from DNA damage only if they were applied at concentrations that slow metabolism and growth. This protective effect was apparent even under anoxic conditions, when ROS could not possibly be involved, and it was replicated when growth was similarly slowed by other means. Experimenters should discard the use of antioxidants as a way of detecting intracellular oxidative stress and should revisit conclusions that have been based upon such experiments. The notable exception is that these compounds can effectively degrade hydrogen peroxide from environmental sources before it enters cells.

Cystine import is a valuable but risky process whose hazards Escherichia coli minimizes by inducing a cysteine exporter.

The structure of free cysteine makes it vulnerable to oxidation by molecular oxygen; consequently, organisms that live in oxic habitats have acquired the ability to import cystine as a sulfur source. We show that cystine imported into Escherichia coli can transfer disulfide bonds to cytoplasmic proteins. To minimize this problem, the imported cystine is rapidly reduced. However, this conversion of cystine to cysteine precludes product inhibition of the importer, so cystine import continues into cells that are already sated with cysteine. The burgeoning cysteine pool is itself hazardous, as cysteine promotes the formation of reactive oxygen species, triggers sulfide production and competitively inhibits a key enzyme in the isoleucine biosynthetic pathway. The Lrp transcription factor senses the excess cysteine and induces AlaE, an export protein that pumps cysteine back out of the cell until transcriptional controls succeed in lowering the amount of the importer. While it lasts, the overall phenomenon roughly doubles the NADPH demand of the cell. It comprises another example of the incompatibility of the reduced cytoplasms of microbes with the oxic world in which they dwell. It also reveals one natural source of cytoplasmic disulfide stress and sheds light on a role for broad-spectrum amino acid exporters.

The cytochrome bd oxidase of Escherichia coli prevents respiratory inhibition by endogenous and exogenous hydrogen sulfide.

When sulfur compounds are scarce or difficult to process, Escherichia coli adapts by inducing the high-level expression of sulfur-compound importers. If cystine then becomes available, the cystine is rapidly overimported and reduced, leading to a burgeoning pool of intracellular cysteine. Most of the excess cysteine is exported, but some is adventitiously degraded, with the consequent release of sulfide. Sulfide is a potent ligand of copper and heme moieties, raising the prospect that it interferes with enzymes. We observed that when cystine was provided and sulfide levels rose, E. coli became strictly dependent upon cytochrome bd oxidase for continued respiration. Inspection revealed that low-micromolar levels of sulfide inhibited the proton-pumping cytochrome bo oxidase that is regarded as the primary respiratory oxidase. In the absence of the back-up cytochrome bd oxidase, growth failed. Exogenous sulfide elicited the same effect. The potency of sulfide was enhanced when oxygen concentrations were low. Natural oxic-anoxic interfaces are often sulfidic, including the intestinal environment where E. coli dwells. We propose that the sulfide resistance of the cytochrome bd oxidase is a key trait that permits respiration in such habitats.

Topological Aspects of Symmetry Breaking in Triangular-Lattice Ising Antiferromagnets.

Using a specially designed Monte Carlo algorithm with directed loops, we investigate the triangular lattice Ising antiferromagnet with coupling beyond the nearest neighbors. We show that the first-order transition from the stripe state to the paramagnet can be split, giving rise to an intermediate nematic phase in which algebraic correlations coexist with a broken symmetry. Furthermore, we demonstrate the emergence of several properties of a more topological nature such as fractional edge excitations in the stripe state, the proliferation of double domain walls in the nematic phase, and the Kasteleyn transition between them. Experimental implications are briefly discussed.

Physiological Roles and Adverse Effects of the Two Cystine Importers of Escherichia coli.

UNLABELLED: When cystine is added to Escherichia coli, the bacterium becomes remarkably sensitive to hydrogen peroxide. This effect is due to enlarged intracellular pools of cysteine, which can drive Fenton chemistry. Genetic analysis linked the sensitivity to YdjN, a secondary transporter that along with the FliY-YecSC ABC system is responsible for cystine uptake. FliY-YecSC has a nanomolar Km and is essential for import of trace cystine, whereas YdjN has a micromolar Km and is the predominant importer when cystine is more abundant. Oddly, both systems are strongly induced by the CysB response to sulfur scarcity. The FliY-YecSC system can import a variety of biomolecules, including diaminopimelate; it is therefore vulnerable to competitive inhibition, presumably warranting YdjN induction under low-sulfur conditions. But the consequence is that if micromolar cystine then becomes available, the abundant YdjN massively overimports it, at >30 times the total sulfur demand of the cell. The imported cystine is rapidly reduced to cysteine in a glutathione-dependent process. This action avoids the hazard of disulfide stress, but it precludes feedback inhibition of YdjN by cystine. We conjecture that YdjN possesses no cysteine allosteric site because the isostructural amino acid serine might inappropriately bind in its place. Instead, the cell partially resolves the overaccumulation of cysteine by immediately excreting it, completing a futile import/reduction/export cycle that consumes a large amount of cellular energy. These unique, wasteful, and dangerous features of cystine metabolism are reproduced by other bacteria. We propose to rename ydjN as tcyP and fliY-yecSC as tcyJLN. IMPORTANCE: In general, intracellular metabolite pools are kept at steady, nontoxic levels by a sophisticated combination of transcriptional and allosteric controls. Surprisingly, in E. coli allosteric control is utterly absent from the primary importer of cystine. This flaw allows massive overimport of cystine, which causes acute vulnerability to oxidative stress and is remedied only by wasteful cysteine efflux. The lack of import control may be rationalized by the unusual properties of cysteine itself. This phenomenon justifies the existence of countervailing cysteine export systems, whose purpose is otherwise hard to understand. It also highlights an unexpected link between sulfur metabolism and oxidative damage. Although this investigation focused upon E. coli, experiments confirmed that similar phenomena occur in other species.

Evidence for columnar order in the fully frustrated transverse field Ising model on the square lattice.

Using extensive classical and quantum Monte Carlo simulations, we investigate the ground-state phase diagram of the fully frustrated transverse field Ising model on the square lattice. We show that pure columnar order develops in the low-field phase above a surprisingly large length scale, below which an effective U(1) symmetry is present. The same conclusion applies to the quantum dimer model with purely kinetic energy, to which the model reduces in the zero-field limit, as well as to the stacked classical version of the model. By contrast, the 2D classical version of the model is shown to develop plaquette order. Semiclassical arguments show that the transition from plaquette to columnar order is a consequence of quantum fluctuations.

Quantification of Hydrogen Sulfide and Cysteine Excreted by Bacterial Cells.

Bacteria release cysteine to moderate the size of their intracellular pools. They can also evolve hydrogen sulfide, either through dissimilatory reduction of oxidized forms of sulfur or through the deliberate or inadvertent degradation of intracellular cysteine. These processes can have important consequences upon microbial communities, because excreted cysteine autoxidizes to generate hydrogen peroxide, and hydrogen sulfide is a potentially toxic species that can block aerobic respiration by inhibiting cytochrome oxidases. Lead acetate strips can be used to obtain semiquantitative data of sulfide evolution (Oguri et al., 2012). Here we describe methods that allow more-quantitative and discriminatory measures of cysteine and hydrogen sulfide release from bacterial cells. An illustrative example is provided in which Escherichia coli rapidly evolves both cysteine and sulfide upon exposure to exogenous cystine (Chonoles Imlay et al., 2015; Korshunov et al., 2016).