Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34.

The lead resistance operon, pbr, of Ralstonia metallidurans (formerly Alcaligenes eutrophus) strain CH34 is unique, as it combines functions involved in uptake, efflux, and accumulation of Pb(II). The pbr lead resistance locus contains the following structural resistance genes: (i) pbrT, which encodes a Pb(II) uptake protein; (ii) pbrA, which encodes a P-type Pb(II) efflux ATPase; (iii) pbrB, which encodes a predicted integral membrane protein of unknown function; and (iv) pbrC, which encodes a predicted prolipoprotein signal peptidase. Downstream of pbrC, the pbrD gene, encoding a Pb(II)-binding protein, was identified in a region of DNA, which was essential for functional lead sequestration. Pb(II)-dependent inducible transcription of pbrABCD from the PpbrA promoter is regulated by PbrR, which belongs to the MerR family of metal ion-sensing regulatory proteins. This is the first report of a mechanism for specific lead resistance in any bacterial genus.

A microbial biosensor to predict bioavailable nickel in soil and its transfer to plants.

Ralstonia eutropha strain AE2515 was constructed and optimised to serve as a whole-cell biosensor for the detection of bioavailable concentrations of Ni2+ and Co2+ in soil samples. Strain AE2515 is a Ralstonia eutropha CH34 derivative containing pMOL1550, in which the cnrYXH regulatory genes are transcriptionally fused to the bioluminescent luxCDABE reporter system. Strain AE2515 was standardised for its specific responses to Co2+ and Ni2+. The detection limits for AE2515 were 0.1 microM Ni2+ and 9 microM Co2+, respectively. The signal to noise (S/N) bioluminescence response and the metal cation concentration could be linearly correlated: for Ni2+ this was applicable within the range 0.1-60 microM, and between 9 and 400 microM for Co2+. The AE2515 biosensor strain was found to be highly selective for nickel and cobalt: no induction was observed with Zn(II), Cd(II), Mn(II), Cu(III) and Cr(VI). In mixed metal solutions, the bioluminescent response always corresponded to the nickel concentrations. Only in the presence of high concentrations of Co2+ (2 mM), the sensitivity to nickel was reduced due to metal toxicity. AE2515 was used to quantify the metal bioavailability in various nickel-enriched soils, which had been treated with additives for in situ metal immobilisation. The data obtained with strain AE2515 confirmed that the bioavailability of nickel was greatly reduced following the treatment of the soils with the additives beringite and steel shots. Furthermore, the data were found to correlate linearly with those on the biological accumulation of Ni2+ in specific parts of important agricultural crops, such as maize and potato. Therefore, the test can be used to assess the potential transfer of nickel to organisms of higher trophic levels, in this case maize and potato plants grown on nickel-enriched soils, and the potential risk of transfer of these elements to the food chain.

Regulation of the cnr cobalt and nickel resistance determinant of Ralstonia eutropha (Alcaligenes eutrophus) CH34.

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 possess 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 nickel-inducible 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.

Identification of a gene cluster, czr, involved in cadmium and zinc resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa CMG103 was isolated from a metal-polluted river in Pakistan and displayed a high level of Zn and Cd resistance. An omega-Km transposon mutant of strain CMG103, which showed a substantial decrease in resistance to Zn and Cd, was obtained. A 12.8 kb region determining Zn and Cd resistance in strain CM103 was cloned by complementing the mutant strain, and its nt sequence was determined. Five genes, czrSRCBA, involved in Zn and Cd resistance, were identified. The predicted gene products of czrCBA show a significant similarity with the proteins encoded by the plasmid borne metal resistant determinants czc, cnr and ncc of Ralstonia strains, which determine a chemiosmotic cation-antiporter efflux system. The predicted CzrS and CzrR proteins show a significant similarity to the sensor and regulatory protein, respectively, of two component regulatory systems, such as CopS/CopR and PcoS/PcoR involved in the regulation of plasmid-borne Cu-resistant determinants, and CzcS/CzcR involved in the regulation of czc. The cloned czr region contained downstream of czrCBA additional ORFs whose predicted gene products are similar to proteins involved in catabolism of aromatic compounds. DNA-DNA hybridization indicated strong conservation of czr in other environmental P. aeruginosa isolates and in the P. aeruginosa type strain PAO1, a clinical isolate. This was confirmed by a comparison of the sequence of the CMG103 czr region with the currently available genome sequence of strain PAO1. A high sequence identity (till 99% at the nt level) and organizatory conservation of the czr region of CMG103 was found in PAO1 as well regarding coding sequences as intervening sequences between ORFs. The czr locus was localized between coordinates 2400 and 2550 kb on the physical map of the chromosome of PAO1.

Siderophore-mediated iron uptake in Alcaligenes eutrophus CH34 and identification of aleB encoding the ferric iron-alcaligin E receptor.

Siderophore production in response to iron limitation was observed in Alcaligenes eutrophus CH34, and the corresponding siderophore was named alcaligin E. Alcaligin E was characterized as a phenolate-type siderophore containing neither catecholate nor hydroxamate groups. Alcaligin E promoted the growth of siderophore-deficient A. eutrophus mutants under iron-restricted conditions and promoted 59Fe uptake by iron-limited cells. However, the growth of the Sid- mutant AE1152, which was obtained from CH34 by Tn5-Tc mutagenesis, was completely inhibited by the addition of alcaligin E. AE1152 also showed strongly reduced 59Fe uptake in the presence of alcaligin E. This indicates that a gene, designated aleB, which is involved in transport of ferric iron-alcaligin E across the membrane is inactivated. The aleB gene was cloned, and its putative amino acid sequence showed strong similarity to those of ferric iron-siderophore receptor proteins. Both wild-type strain CH34 and aleB mutant AE1152 were able to use the same heterologous siderophores, indicating that AleB is involved only in ferric iron-alcaligin E uptake. Interestingly, no utilization of pyochelin, which is also a phenolate-type siderophore, was observed for A. eutrophus CH34. Genetic studies of different Sid- mutants, obtained after transposon mutagenesis, showed that the genes involved in alcaligin E and ferric iron-alcaligin E receptor biosynthesis are clustered in a 20-kb region on the A. eutrophus CH34 chromosome in the proximity of the cys-232 locus.

A straightforward approach to isolate DNA sequences with potential linkage to the retinoblastoma locus.

From a human-Chinese hamster somatic cell hybrid a clone was derived containing chromosome 13 in duplicate as its only human material. This clone was used to construct a human chromosome 13-specific recombinant DNA-library. Overlapping Sau3AI DNA sequences (11.9-17.2 kb) from the cell hybrid were inserted into the lambda phage vector EMBL4. From eleven recombinants having a human insert thirteen putative unique DNA sequences were isolated and cloned into the plasmid vector pBR329. A human-mouse hybrid containing a human chromosome 13 with a deletion of 13q14 and lacking its undeleted homologue was constructed to be used in a selection procedure for DNA sequences belonging to band q14. Three probes originating from two different phages were assigned to 13q14 because they did not hybridise to DNA from this cell hybrid. One of these 13q14 probes detects a low frequency (2/44) MspI restriction fragment length polymorphism. The probes are now being used for screening a cosmid library to find adjacent polymorphic sequences with a RFLP information content suitable for application in the diagnosis of hereditary retinoblastoma.