The linked resistance to nickel and cobalt of Ralstonia eutropha-like strain CH34 (Alcaligenes eutrophus CH34) is encoded by the cnr operon, which is localized on the megaplasmid pMOL28. The regulatory genes cnrYXH have been cloned, overexpressed, and purified in Escherichia coli. CnrY fractionated as a 10.7-kDa protein in in vitro translation assays. CnrX, a periplasmic protein of 16.5 kDa, was overproduced and purified as a histidine-tagged fusion protein in E. coli. His-CnrX was found to posses a secondary structure content rich in alpha-helical and beta-sheet structures. CnrH, a sigma factor of the extracytoplasmic function family, was purified as an N-terminally histidine-tagged fusion. In gel shift mobility assays, His-CnrH, in the presence of E. coli core RNA polymerase enzyme, could retard at least two different promoter DNA targets, cnrYp and cnrHp, localized within the cnrYXH locus. These promoters and their transcription start sites were confirmed by primer extension. Purified His-CnrX did not inhibit the DNA-binding activity of His-CnrH and is therefore unlikely to be an anti-sigma factor, as previously hypothesized (EMBL M91650 description entry). To study the transcriptional response of the regulatory locus to metals and to probe promoter regions, transcriptional fusions were constructed between fragments of cnrYXH and the luxCDABE, luciferase reporter genes. Nickel and cobalt specifically induced the cnrYXH-luxCDABE fusion at optimal concentrations of 0.3 mM Ni 2؉ and 2.0 mM Co 2؉ in a noncomplexing medium for metals. The two promoter regions P Y (upstream cnrY) and P H (upstream cnrH) were probed and characterized using this vector and were found to control the nickelinducible regulatory response of the cnr operon. The cnrHp promoter was responsible for full transcription of the cnrCBA structural resistance genes, while the cnrYp promoter was necessary to obtain metal-inducible transcription from the cnrHp promoter. The zinc resistance phenotype (ZinB) of a spontaneous cnr mutant strain, AE963, was investigated and could be attributed to an insertion of IS1087, a member of the IS2 family of insertion elements, within the cnrY gene.The resistance of Ralstonia eutropha-like strain CH34 (Alcaligenes eutrophus CH34) against multiple heavy metals can be regarded as a phenomenon in its own right (15) and has found increasing applications within the field of environmental technology (29). The metal resistance determinants, which are localized predominantly on either of the two indigenous megaplasmids pMOL28 (15, 28) and pMOL30 (15), have been recently reviewed by Taghavi et al. (27). One important application has been the development of metal-specific biosensors based on in vivo and in vitro gene fusions of CH34 heavy metal resistance determinants. This was possible only through a detailed knowledge of the regulation of the metal resistances at the molecular level (2).The cnr operon, located on pMOL28 (13, 28), encodes a phenotype of inducible resistance to 5 mM Co 2ϩ and 3 mM Ni 2ϩ in minimal medium. R...

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An environmental assessment of hydrotropes was conducted under the Organisation for Economic Co-operation and Development (OECD) Screening Information Data Sets (SIDS) for High Production Volume (HPV) Program via the Global International Council of Chemical Associations (ICCA) Hydrotropes Consortium. The assessment and its conclusions were presented at a meeting of the OECD member countries in Washington, DC in 2005. The SIDS Initial Assessment Report (SIAR) was accepted by the membership. Their conclusion was “The chemicals in this [hydrotropes] category are of low priority for further work because of their low hazard profile.” Hydrotropes are used to solubilize the water-insoluble ingredients of cleaning and personal care products including, for example, powder and liquid laundry detergents, hard-surface cleaners, machine dishwashing rinse aids, hand dishwashing liquids, body washes, shampoos, hair conditioners, and liquid hand and face soaps. Global production equals approximately 46 500 metric tons, a little more than half of which is used in the United States. The 8 chemicals accounted for in the “hydrotropes category” include ammonium, Ca, K, and Na salts that are described by 10 Chemical Abstract Service (CAS) registration numbers. The 8 chemical entities are generally comparable and predictable in their chemical behavior and that measured and/or modeled data for members from one subgroup can be applied to other subgroups and to the hydrotropes category as a whole. The assessment is based on a search for and evaluation of available data on physical–chemical properties, biodegradability, removal by wastewater treatment, and aquatic toxicity. Reliable ecotoxicity and environmental fate data were found for selected members of the category. Partitioning, once released into the environment, and exposure in surface waters were modeled for consumer use and manufacturing scenarios relevant to the United States, Europe, and Australia. The models indicate 99+% of the hydrotropes will partition to water. Furthermore, given the low potential for hydrotropes reaching the terrestrial environment and their lack of persistence or bioaccumulation, the focus of the assessment is on the aquatic environment, specifically the water compartment. Aquatic risks were assessed in each scenario using what is referred to as the PEC/PNEC ratio. The modeled predicted environmental concentration (PEC), accounting for volume released and wastewater treatment, is divided by the predicted no effect concentration (PNEC) derived from the aquatic toxicity tests. The closest a predicted environmental concentration came to the toxicity threshold is 0.125 (or 12.5% of the no effect concentration) and that is for a hypothetical manufacturing facility that produces the entire annual volume of hydrotropes and discharges to a small (10%ile) stream under low flow (7Q10) conditions. PEC/PNEC ratios were considerably smaller for consumer use scenarios. The ratios were 0.0002 for a low flow (7Q10) stream scenario in the United States, 0.026 to 0.089 for...