Summary
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.
When E. coli encounters environmental cystine, it rapidly imports it, reduces it to cysteine and then exports most of the cysteine. This odd behavior is ultimately an awkward way to minimize the disulfide stress that results from the import of cystine.
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.
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.
Summary
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.
Toxic levels of intracellular sulfide arise either from the degradation of excess cysteine or from an influx of hydrogen sulfide itself. Sulfide inhibits the primary respiratory cytochrome bo oxidase, and continued respiration depends upon the presence of the secondary cytochrome bd oxidase. This scenario is likely common to sulfidic environments, such as the mammalian gut.
Rapid identification of both species and even specific strains of human pathogenic bacteria grown on standard agar has been achieved from the volatiles they produce using a disposable colorimetric ...sensor array in a Petri dish imaged with an inexpensive scanner. All 10 strains of bacteria tested, including Enterococcus faecalis and Staphylococcus aureus and their antibiotic-resistant forms, were identified with 98.8% accuracy within 10 h, a clinically important time frame. Furthermore, the colorimetric sensor arrays also proved useful as a simple research tool for the study of bacterial metabolism and as an easy method for the optimization of bacterial production of fine chemicals or other fermentation processes.
In aerobic environments, mutants of Escherichia coli that lack peroxidase and catalase activities (Hpx⁻) accumulate submicromolar concentrations of intracellular H₂O₂. We observed that in defined ...medium these strains constitutively expressed members of the Fur regulon. Iron-import proteins, which Fur normally represses, were fully induced. H₂O₂ may antagonize Fur function by oxidizing the Fur:Fe²⁺ complex and inactivating its repressor function. This is a potential problem, as in iron-rich environments excessive iron uptake would endanger H₂O₂-stressed cells by accelerating hydroxyl-radical production through the Fenton reaction. However, the OxyR H₂O₂-response system restored Fur repression in iron-replete Luria-Bertani medium by upregulating the synthesis of Fur protein. Indeed, when the OxyR binding site upstream of fur was disrupted, Hpx⁻ mutants failed to repress transporter synthesis, and they exhibited high levels of intracellular free iron. Mutagenesis and bacteriostasis resulted. These defects were eliminated by mutations or chelators that slowed iron import, confirming that dysregulation of iron uptake was the root problem. Thus, aerobic organisms must grapple with a conundrum: how to monitor iron levels in oxidizing environments that might perturb the valence of the analyte. The induction of Fur synthesis by the OxyR response comprises one evolutionary solution to that problem.
We report the first measurement of monoenergetic muon neutrino charged current interactions. MiniBooNE has isolated 236 MeV muon neutrino events originating from charged kaon decay at rest ...(K^{+}→μ^{+}ν_{μ}) at the NuMI beamline absorber. These signal ν_{μ}-carbon events are distinguished from primarily pion decay in flight ν_{μ} and νover ¯_{μ} backgrounds produced at the target station and decay pipe using their arrival time and reconstructed muon energy. The significance of the signal observation is at the 3.9σ level. The muon kinetic energy, neutrino-nucleus energy transfer (ω=E_{ν}-E_{μ}), and total cross section for these events are extracted. This result is the first known-energy, weak-interaction-only probe of the nucleus to yield a measurement of ω using neutrinos, a quantity thus far only accessible through electron scattering.
The observation of neutrino oscillations is clear evidence for physics beyond the standard model. To make precise measurements of this phenomenon, neutrino oscillation experiments, including ...MiniBooNE, require an accurate description of neutrino charged current quasielastic (CCQE) cross sections to predict signal samples. Using a high-statistics sample of nu_(mu) CCQE events, MiniBooNE finds that a simple Fermi gas model, with appropriate adjustments, accurately characterizes the CCQE events observed in a carbon-based detector. The extracted parameters include an effective axial mass, M_(A)(eff)=1.23+/-0.20 GeV, that describes the four-momentum dependence of the axial-vector form factor of the nucleon, and a Pauli-suppression parameter, kappa=1.019+/-0.011. Such a modified Fermi gas model may also be used by future accelerator-based experiments measuring neutrino oscillations on nuclear targets.
Intrinsic positive end-expiratory pressure (PEEP) and gas trapping are recognised hazards during ventilation of patients with airflow obstruction. Demonstration of these phenomena on conventional ...lung models using realistic ventilation settings is difficult. We describe an Intrinsic PEEP Model that is able to demonstrate dynamic gas trapping and intrinsic PEEP at realistic ventilation settings, and demonstrate its ability to develop intrinsic PEEP in a timeframe useful for teaching. The model uses a Heimlich valve to permit a lower resistance on inspiration than expiration. The model was tested using a series of typical ventilation settings which, when applied in a clinical setting on patients with airflow obstruction issues, would result in prolonged low expiratory flow and the development of intrinsic PEEP of 10 to 20 cmH2O, and ultimately significant gas trapping. The IPM can be used to demonstrate this effect and the ventilator adjustments required to minimise these problems.
The MiniBooNE Collaboration reports a search for nu_{micro} and nuover_{micro} disappearance in the Deltam;{2} region of 0.5-40 eV;{2}. These measurements are important for constraining models with ...extra types of neutrinos, extra dimensions, and CPT violation. Fits to the shape of the nu_{micro} and nuover_{micro} energy spectra reveal no evidence for disappearance at the 90% confidence level (C.L.) in either mode. The test of nuover_{micro} disappearance probes a region below Deltam;{2} = 40 eV;{2} never explored before.
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