Akaligenes eutrophus strain CH34, which was isolated as a bacterium resistant to cobalt, zinc, and cadmium ions, shares with A. eutrophus strain H16 the ability to grow lithoautotrophically on molecular hydrogen, to form a cytoplasmic NAD-reducing and a membrane-bound hydrogenase, and most metabolic attributes; however, it does not grow on fructose. Strain CH34 contains two plasmids, pMOL28 (163 kilobases) specifying nickel, mercury, and cobalt resistance and pMOL30 (238 kilobases) specifying zinc, cadmium, mercury, and cobalt resistance. The plasmids are self-transmissible in homologous matings, but at low frequencies. The transfer frequency was strongly increased with IncPl plasmids RP4 and pUZ8 as helper plasmids. The phenotypes of the wild type, cured strains, and transconjugants are characterized by the following MICs (Micromolar) in strains with the indicated phenotypes: Nic+, 2.5; Nic-, 0.6; Cob+A, 5.0; Cob+B, 20.0; Cob-, < 9.07; Zin+, 12.0; Zin-, 0.6; Cad', 2.5; and Cad-, 0.6. Plasmid-free cells of strain CH34 are still able to grow lithoautotrophicaily and to form both hydrogenases, indicating that the hydrogenase genes are located on the chromosome, in contrast to the Hox structural genes of strain H16, which are located on the megaplasmid pHG1 (450 kilobases).

Ralstonia metallidurans, formerly known as Alcaligenes eutrophus and thereafter as Ralstonia eutropha, is a beta-Proteobacterium colonizing industrial sediments, soils or wastes with a high content of heavy metals. The type strain CH34 carries two large plasmids (pMOL28 and pMOL30) bearing a variety of genes for metal resistance. A chronological overview describes the progress made in the knowledge of the plasmid-borne metal resistance mechanisms, the genetics of R. metallidurans CH34 and its taxonomy, and the applications of this strain in the fields of environmental remediation and microbial ecology. Recently, the sequence draft of the genome of R. metallidurans has become available. This allowed a comparison of these preliminary data with the published genome data of the plant pathogen Ralstonia solanacearum, which harbors a megaplasmid (of 2.1 Mb) carrying some metal resistance genes that are similar to those found in R. metallidurans CH34. In addition, a first inventory of metal resistance genes and operons across these two organisms could be made. This inventory, which partly relied on the use of proteomic approaches, revealed the presence of numerous loci not only on the large plasmids pMOL28 and pMOL30 but also on the chromosome. It suggests that metal-resistant Ralstonia, through evolution, are particularly well adapted to the harsh environments typically created by extreme anthropogenic situations or biotopes.

Many bacteria in the environment have adapted to the presence of toxic heavy metals. Over the last 30 years, this heavy metal tolerance was the subject of extensive research. The bacterium Cupriavidus metallidurans strain CH34, originally isolated by us in 1976 from a metal processing factory, is considered a major model organism in this field because it withstands milli-molar range concentrations of over 20 different heavy metal ions. This tolerance is mostly achieved by rapid ion efflux but also by metal-complexation and -reduction. We present here the full genome sequence of strain CH34 and the manual annotation of all its genes. The genome of C. metallidurans CH34 is composed of two large circular chromosomes CHR1 and CHR2 of, respectively, 3,928,089 bp and 2,580,084 bp, and two megaplasmids pMOL28 and pMOL30 of, respectively, 171,459 bp and 233,720 bp in size. At least 25 loci for heavy-metal resistance (HMR) are distributed over the four replicons. Approximately 67% of the 6,717 coding sequences (CDSs) present in the CH34 genome could be assigned a putative function, and 9.1% (611 genes) appear to be unique to this strain. One out of five proteins is associated with either transport or transcription while the relay of environmental stimuli is governed by more than 600 signal transduction systems. The CH34 genome is most similar to the genomes of other Cupriavidus strains by correspondence between the respective CHR1 replicons but also displays similarity to the genomes of more distantly related species as a result of gene transfer and through the presence of large genomic islands. The presence of at least 57 IS elements and 19 transposons and the ability to take in and express foreign genes indicates a very dynamic and complex genome shaped by evolutionary forces. The genome data show that C. metallidurans CH34 is particularly well equipped to live in extreme conditions and anthropogenic environments that are rich in metals.

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A 238-kilobase-pair plasmid, pMOL30, confers resistance to cadmium, zinc, and cobalt salts in Alcaligenes eutrophus CH34. After Tn5 mutagenesis, restriction nuclease analysis, and Southern DNA-DNA hybridization, a 9.1-kilobase-pair EcoRI fragment was found to harbor all of these resistance properties and was cloned into the broad-host-range hybrid plasmid pRK290. When transferred to a plasmid-free derivative of CH34, the hybrid plasmid conferred the same degree of resistance as the parent plasmid pMOL30. In two other Alcaligenes strains, the hybrid plasmid was expressed, but to a lower degree than in CH34 derivatives.Alcaligenes eutrophus CH34, initially isolated from a zinc decantation tank (13), is an aerobic gram-negative bacterium that displays two remarkable properties: (i) the ability to grow autotrophically with molecular hydrogen and (ii) resistance to the salts of the heavy metals cobalt, zinc, nickel, cadmium, and mercury (3,6,14,15). Strain CH34 contains two large plasmids (7) designated pMOL28 (163 kilobase pairs [kbp]) and pMOL30 (238 kbp). The plasmids were found to be involved in the expression of heavy-metal resistance but not in chemolithoautotrophy. pMOL30 specifies resistance to zinc (Zinr) and cadmium (Cadr) and a high level of cobalt resistance (CobBr). pMOL28 encodes resistance to nickel (Nicr) and a low level of cobalt resistance (CobAr) (14).In this communication, we describe the molecular cloning of the resistance markers encoded by pMOL30, as well as their expression in a plasmid-free variant of CH34 and in metal-sensitive Alcaligenes strains.The bacterial strains and plasmids used in this study are listed in Table 1. Nutrient broth and Luria broth were used as complex media. For testing the degree of resistance to the heavy-metal salts, a mineral medium (5) was used which contained, per liter of distilled water, 2 g of sodium gluconate, 6.06 g of Tris hydrochloride (pH 7.0), 4.68 g of NaCl, 1.49 g of KCI, 1.07 g of NH4Cl, 0.43 g of Na2SO4, 0.20 g of MgCI2 6H20, 0.03 g of CaCl2 -2H20, 0.23 g of Na2HPO4 12H20, 0.005 g of ferric ammonium citrate, and 1 ml of trace element solution SL7 (1). Analytical-grade salts of CdCI2 * H20, CoC12 6H20, NiCl2. 6H20, and ZnC12 were used to prepare 1.0 M stock solutions, which were sterilized by autoclaving and added to the medium at final concentrations of 1 mM NiCl2, 1 mM CdCI2, 2.5 mM ZnCl2, and 5.0 mM CoCl2. Heavy-metal-containing medium was solidified with 20 g of agar per liter; other solid media contained 15 g of agar per liter. For conjugation, overnight cultures of donor and recipient strains grown at 30°C in nutrient broth were mixed and plated onto nutrient broth agar. After 12 to 20 h of growth, the bacteria were suspended in saline (9 g of sodium chloride per liter), diluted, and plated to selective media. For transposon mutagenesis, Escherichia