Double-Viewing-Position Single-Particle Inductively Coupled Plasma-Atomic Emission Spectrometry for the Selection of ICP Sampling Position in SP-ICP Measurements.

Double-viewing-position single-particle inductively coupled plasma-atomic emission spectrometry (DVP-SP-ICP-AES) measures emission intensity at two ICP vertical positions simultaneously using a single photomultiplier tube. A particle travelling up the ICP gives two consecutive temporal emission peaks. The Yb II 328.937-nm emission intensity of the two peaks for single Yb2O3 particles of diameter of 200 - 2000 nm are plotted against each other in a correlation plot. The correlation is poor when the gas temperature at the lower observation position is approximately the boiling point of the particles. Poor particle vaporization at the center of the central channel occurs because the gas temperature is 400 K lower than the temperature at the rim. The correlation is improved by shifting the observation positions up or using helium-argon mixed carrier gas to increase the gas temperature. Gas temperature is an important parameter for precise single particle-ICP measurements. DVP-SP-ICP-AES can be used to identify poor particle vaporization without the need of temperature measurement.

Copper efflux is induced during anaerobic amino acid limitation in Escherichia coli to protect iron-sulfur cluster enzymes and biogenesis.

Adaptation to changing environments is essential to bacterial physiology. Here we report a unique role of the copper homeostasis system in adapting Escherichia coli to its host-relevant environment of anaerobiosis coupled with amino acid limitation. We found that expression of the copper/silver efflux pump CusCFBA was significantly upregulated during anaerobic amino acid limitation in E. coli without the supplement of exogenous copper. Inductively coupled plasma mass spectrometry analysis of the total intracellular copper content combined with transcriptional assay of the P(cusC)-lacZ reporter in the presence of specific Cu(I) chelators indicated that anaerobic amino acid limitation led to the accumulation of free Cu(I) in the periplasmic space of E. coli, resulting in Cu(I) toxicity. Cells lacking cusCFBA and another copper transporter, copA, under this condition displayed growth defects and reduced ATP production during fumarate respiration. Ectopic expression of the Fe-S cluster enzyme fumarate reductase (Frd), or supplementation with amino acids whose biosynthesis involves Fe-S cluster enzymes, rescued the poor growth of ΔcusC cells. Yet, Cu(I) treatment did not impair the Frd activity in vitro. Further studies revealed that the alternative Fe-S cluster biogenesis system Suf was induced during the anaerobic amino acid limitation, and ΔcusC enhanced this upregulation, indicating the impairment of the Fe-S cluster assembly machinery and the increased Fe-S cluster demands under this condition. Taken together, we conclude that the copper efflux system CusCFBA is induced during anaerobic amino acid limitation to protect Fe-S cluster enzymes and biogenesis from the endogenously originated Cu(I) toxicity, thus facilitating the physiological adaptation of E. coli.

Tracking bismuth antiulcer drug uptake in single Helicobacter pylori cells.

Bismuth-based drugs have long been used for the treatment of Helicobacter pylori infection. In this work, the metal content in H. pylori was monitored at the single-cell level by time-resolved inductively coupled plasma mass spectrometry, and ∼2.9 × 10(7) Mg atoms/cell was determined for the wild-type. Bacteria treated with a Bi antiulcer drug deposited nearly 1.0 × 10(6) Bi atoms/cell, whereas the uptake process took ∼3 h to reach the half-maximum. Interference of ferric ions on bismuth uptake was demonstrated, suggesting that the metallodrug can utilize certain iron-transport pathways in the pathogen. The approach provides a general strategy for monitoring metals in single cells, facilitating exploration of metal-relevant bioprocesses.

Purple acid phosphatase-like sequences in prokaryotic genomes and the characterization of an atypical purple alkaline phosphatase from Burkholderia cenocepacia J2315.

Purple acid phosphatases (PAP) are a group of dimetallic phosphohydrolase first identified in eukaryotes. Bioinformatics analysis revealed 57 prokaryotic PAP-like sequences in the genomes of 43 bacteria and 4 cyanobacteria species. A putative PAP gene (BcPAP) from the bacteria Burkholderia cenocepacia J2315 was chosen for further studies. Synteny analysis showed that this gene is present as an independent gene in most of the members of the genus Burkholderia. The predicted 561 a.a. polypeptide of BcPAP was found to harbour all the conserved motifs of the eukaryotic PAPs and an N-terminal twin-arginine translocation signal. Expression and biochemical characterization of BcPAP in Escherichia coli revealed that this enzyme has a relatively narrow substrate spectrum, preferably towards phosphotyrosine, phosphoserine and phosphoenolpyruvate. Interestingly, this enzyme was found to have a pH optimum at 8.5, rather than an acidic optima exhibited by eukaryotic PAPs. BcPAP contains a dimetallic ion centre composed of Fe and Zn, and site-directed mutagenesis confirmed that BcPAP utilizes the invariant residues for metal-ligation and catalysis. The enzyme is secreted by the wild type bacteria and its expression is regulated by the availability of orthophosphate. Our findings suggest that not all members in the PAP family have acidic pH optimum and broad substrate specificity.

Microwave plasma treatment of polymer surface for irreversible sealing of microfluidic devices.

Microwave plasma was generated in a glass bottle containing 2-3 Torr of oxygen for plasma treatment of a polymer surface. A "kitchen microwave oven" and a dedicated microwave digestion oven were used as the power source. Poly(dimethylsiloxane)(PDMS) slabs treated by a 30 W plasma for 30-60 s sealed irreversibly to form microfluidic devices that can sustain solution flow of an applied pressure of 42 psi without leaking. Experimental set up and conditions for the production of a homogeneous plasma to activate the PDMS surface for irreversible sealing are described in detail. The surface of a microwave plasma-treated PDMS slab was characterized using atomic force microscopy (AFM) and attenuated total reflection-Fourier Transform infrared spectroscopy (ATR-FTIR). The plasma-treated surface bears silica characteristics.

[Speciation of iron using capillary electrophoresis inductively coupled plasma atomic emission spectrometry].

The separation of iron species, e. g. Fe2+ -Fe3+, Fe2+ -Fe(phen)3(2+), and Fe2+ -Fe3+, by mixing with complexing reagents of o-phenanthroline and EDTA, and Fe2+ -Fe3+ with o-phenanthroline was developed by capillary electrophoresis inductively coupled plasma atomic emission spectronetry (CE-ICP-AES). The effects of electrophoresis conditions such as voltage, buffer solution pH, and complex concentration on iron species are discussed. Satisfactory separation conditions for iron speciation have been acquired. Cations and anions, containing complexing agents, do not interfere in the separation of iron species. The method has advantage to CE-UV.