Expression of the PitA phosphate/metal transporter of Escherichia coli is responsive to zinc and inorganic phosphate levels.

Escherichia coli possesses two major systems for inorganic phosphate (P(i)) uptake. The Pst system (pstSCAB) is inducible by low phosphate concentrations whereas the low-affinity transporter (pitA) has been described as constitutively expressed. PitA catalyses transport of metal [Mg(II), Ca(II)]-phosphate complexes, and mutations in pitA confer Zn(II) resistance. Here we report that pitA transcription is not constitutive; activity of a single-copy pitA-lacZ transcriptional fusion (monolysogen) was maximal at high extracellular Zn(II) (150 microM), in the absence of added P(i), and in a well-defined pitA mutant strain. Intracellular zinc levels were unaffected by adding Zn(II) to the medium for both the wild-type and mutant strains. However, in the wild-type strain, Mg levels (per gram of dry biomass) fell by eightfold in cells grown with added Zn(II) and by 20-fold when Zn(II) and P(i) were added to cultures. Mutation of pitA reduced the effects of external Zn(II) and phosphate levels on Mg pools, consistent with competition or inhibition by Zn(II) of PitA. The mechanism of pitA regulation by extracellular Zn(II) and P(i) is unknown but appears not to involve Fur or other well-characterized regulators.

Approaches to assess the oral bioaccessibility of persistent organic pollutants: a critical review.

Oral bioaccessibility, also known as in vitro gastrointestinal extraction or the physiologically based extraction test (PBET), is an important tool when assessing the risk to humans from persistent organic pollutants (POPs) (and metals). The approach seeks to mimic the processes of human food digestion and thereby assess the bioavailability of POPs (and metals) from materials consumed either accidentally or intentionally in the diet. In vitro conditions are created to simulate various actions in the stomach and intestines (although some methods also include the mouth compartment). This paper reviews the current status of oral bioaccessibility with respect to the release of POPs from soil and related samples of environmental importance. Particular emphasis is placed on the parameters that influence gastrointestinal extraction including gastric and intestinal pH, enzymes, bile salts, food constituents and residence time. In addition, important developments in the use of in vitro gastrointestinal extraction are highlighted. These developments include the use of epithelial Caco-2 cells to mimic the intestinal cell lining, the potential for biotransformation of POPs into estrogenic metabolites as a result of colon microbiota, and the use of in vivo studies to validate existing approaches.

Determination of (234)U/ (238)U isotope ratios in environmental waters by quadrupole ICP-MS after U stripping from alpha-spectrometry counting sources.

The 234U/238U isotope ratio has been widely used as a tracer for geochemical processes in underground aquifers. Quadrupole-based inductively coupled plasma mass spectrometry (ICP-MS) equipped with a high-efficiency nebulizer and a membrane desolvator was employed for the determination of 234U/238U isotope ratios in natural water samples. The instrumental limit of detection for 234U was at the low pg L(-1) level with very low sample consumption. Measurement precision (234U/238U) was 3-5% for bottled mineral water with elevated uranium concentration (>1 microg L(-1)). For the analysis of groundwater samples from the Almonte-Marisma underground aquifer (Huelva, Spain), uranium was stripped from stainless steel planchets that had previously been used as radiometric counting sources for alpha-particle spectrometry. Potential spectral interferences from other metals introduced during the dissolution were investigated. Matrix-matched blank solutions were needed to subtract the background on 234U due to the formation of platinum argides, and to allow for mass bias correction and background correction. The Pt appears to be an impurity present in the stainless steel, either as a minor component by itself or after extraction from the anode and a subsequent uranium electrodeposition. The 234U/238U isotope ratio data were in very good agreement with those of alpha spectrometry, while precision was improved by a factor of up to 10 and counting time was reduced down to approximately 20 min (10 replicate measurements).

Speciation of protein-bound trace elements by gel electrophoresis and atomic spectrometry.

The metabolism of trace elements, in particular their binding to proteins in biological systems is of great importance in biochemical, toxicological, and pharmacological studies. As a result there has been a sustained interest over the last two decades in the speciation of protein-bound metals. Various analytical approaches have been employed, combining efficient separation of metalloproteins by liquid chromatography or electrophoresis with high-sensitivity elemental detection. Slab-gel electrophoresis (GE) is a key platform for high-resolution protein separation, and has been combined with autoradiography and various atomic spectrometric techniques for in-gel determination of protein-bound metals. Recently, the combination of GE with state-of-the-art inductively coupled plasma-mass spectrometry (ICP-MS), particularly when linked to laser ablation (LA) for direct gel interrogation, has opened up new opportunities for rapid characterization of metalloproteins. The use of GE and atomic spectrometry for the speciation of protein-bound trace elements is reviewed in this paper. Technical requirements for gel electrophoresis/atomic spectrometric measurement are considered in terms of method compatibilities, detection capability and potential usefulness. The literature is also surveyed to illustrate current status and future trends.

Detection and characterization of zinc- and cadmium-binding proteins in Escherichia coli by gel electrophoresis and laser ablation-inductively coupled plasma-mass spectrometry.

Metals bound to proteins play key roles in structure stabilization, catalysis, and metal transport in cells, but metals may also be toxic. As a consequence, cells have developed mechanisms to control metal concentrations through binding to proteins. We have used a hyphenated strategy linking gel electrophoresis with laser ablation-inductively coupled plasma-mass spectrometry in order to detect, map, and quantify metal-binding proteins synthesized in Escherichia coli under zinc- and cadmium-stress conditions. We report the development of a powerful analytical method suitable for detection and characterization of metalloproteins in complex, unfractionated bacterial cell extracts. The approach was validated by using an E. coli strain overexpressing the cyanobacterial metallothionein protein SmtA. We observed induction of SmtA synthesis by zinc and binding of both zinc and cadmium cations by this protein. A profile of zinc- and cadmium-binding proteins was obtained from E. coli cytoplasmic fractions. Analysis of induction patterns and metal contents demonstrated the presence of proteins with high metal content which, on further study, should lead to the identification of novel metal-binding proteins.