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
There are two distinct nickel resistance loci on plasmid pTOM9 from Achromobacter xylosoxidans 31A, ncc and nre. Expression of the nreB gene was specifically induced by nickel and conferred nickel resistance on both A. xylosoxidans 31A and Escherichia coli. E. coli cells expressing nreB showed reduced accumulation of Ni 2؉ , suggesting that NreB mediated nickel efflux. The histidine-rich C-terminal region of NreB was not essential but contributed to maximal Ni 2؉ resistance.Nickel is the 24th most abundant element in the earth's crust and has been detected in different media in all parts of the biosphere. Nickel is classified as a borderline metal ion because it has both soft and hard metal properties and can bind to sulfur, nitrogen, and oxygen groups (3). In many bacteria, nickel is required for enzymes such as urease, CO dehydrogenase, and hydrogenase (5, 10). However, excess nickel is toxic. Nickel binds to proteins and nucleic acids and frequently inhibits enzymatic activity, DNA replication, transcription, and translation (1). Several nickel-resistant bacteria have been isolated from heavy-metal-contaminated sites. Well-studied examples include Ralstonia metallidurans CH34 and Achromobacter xylosoxidans 31A (8, 24). The determinant responsible for nickel resistance in R. metallidurans CH34, cnr (cobalt-nickel resistance), encodes three regulatory genes (cnrY, cnrX, and cnrH) and three structural genes encoding the subunits of the Co-Ni efflux pump (cnrC, cnrB, and cnrA) (8,26). The cnr determinant is similar to the ncc determinant (nickel-cobalt-cadmium resistance) of A. xylosoxidans 31A. The proposed gene products for the efflux system CnrCBA and NccCBA are largely homologous to the gene products for the three subunits of the better-characterized CzcCBA cation-proton antiporter and probably have a similar function (16,17,27). In addition to the ncc locus, A. xylosoxidans 31A contains another distinct nickel resistance locus, nre, located on plasmid pTOM9. The nre locus confers low-level nickel resistance on both Ralstonia and Escherichia coli strains (24). The closest homologue of the deduced nreB gene product is NrsD from Synechocystis sp. strain PCC 6803 (6). Both NreB and NrsD belong to the major facilitator superfamily (MFS), and computer analysis indicates 12 putative transmembrane helices in each (11,20). Additionally, both proteins possess histidine-rich C termini possibly implicated in metal binding (6).In this study, we characterized the nre locus of A. xylosoxidans 31A and showed that only nreB is required for nickel resistance. In A. xylosoxidans, nreB was specifically induced by nickel but not by cobalt or zinc. The histidine-rich C terminus was not essential for NreB function but was necessary for maximum nickel resistance. E. coli cells harboring nreB showed reduced uptake of nickel compared to that of wild-type cells. The data support our hypothesis that NreB is a Ni 2ϩ transporter responsible for Ni 2ϩ efflux and resistance in A. xylosoxidans 31A and E. coli. MATERIALS AND METHODSBacterial...
The nucleotide sequence of the gene that encodes the fermentative, multifunctional alcohol dehydrogenase (ADH) in Alcaligenes eutrophus, and of adjacent regions on a 1.8-kilobase-pair PstI fragment was determined. From the deduced amino acid sequence, a molecular weight of 38,549 was calculated for the ADH subunit. The amino acid sequence reveals homologies from 22.3 to 26.3% with zinc-containing alcohol dehydrogenases from eucaryotic organisms (Schizosaccharomyces pombe, Zea mays, mouse, horse liver, and human liver). Most of the 22 amino acid residues, which are strictly conserved in this group of ADHs (H. Jornvall, B. Persson, and J. Jeffery, Eur. J. Biochem. 167:195-201, 1987), either were present in the A. eutrophus enzyme or had been substituted by related amino acids. The A. eutrophus adh gene was transcribed in Escherichia coli only under the control of the lac promoter, but was not expressed by its own promoter. A sequence resembling the E. coli consensus promoter DNA sequence did not contain the invariant T, but a G, in the potential -10 region. In the transposon-induced mutants HC1409 and HC1421, which form ADH constitutively, the insertions of Tn5::mob were localized 56 and 66 base pairs, respectively, upstream of the presumptive translation initiation codon. In contrast to the promoter, the A. eutrophus ribosome-binding site with a GGAG Shine-Dalgarno sequence 6 base pairs upstream of the translation initiation codon was accepted by the E. coli translation apparatus. A stable hairpin structure, which may provide a transcription termination signal, is predicted to occur in the mRNA, with its starting point 21 base pairs downstream from the translation termination codon.Recently, we described the cloning of an 11.5-kilobasepair (kbp) EcoRI fragment which encodes the gene for fermentative alcohol dehydrogenase (ADH) from the strict aerobe Alcaligenes eutrophus (44). This enzyme is a tetramer of relative molecular weight 156,000 and consists of four subunits of equal size. The ADH catalyzes the NAD(P)-dependent oxidation of ethanol, 2,3-butanediol, and acetaldehyde and the reduction of acetaldehyde, acetoin, and diacetyl (66). The wild type synthesizes this multifunctional ADH, together with an NAD-linked lactate dehydrogenase, only when the cells are cultivated under conditions of restricted oxygen supply (58). In addition to the ability to evolve molecular hydrogen (45, 71) and to synthesize poly-,-hydroxybutyric acid (71), both enzymes appear to provide a safety valve for the release of excess reducing power in the absence of exogenous hydrogen acceptors such as oxygen or nitrate.Although many primary structures of eucaryotic ADHs have been elaborated (12,40,54), relatively little information exists on the primary structures of procaryotic ADHs (13, 51) and their genes.
In genetic studies on the catabolism of acetoin in Alcaligenes eutrophus, we used TnS::mob-induced mutants which were impaired in the utilization of acetoin as the sole carbon source for growth. The transposonharboring EcoRI restriction fragments from 17 acetoin-negative and slow-growing mutants (class 2a) and from six pleiotropic mutants of A. eutrophus, which were acetoin-negative and did not grow chemolithoautotrophically (class 2b), were cloned from pHC79 gene banks. The insertions of TnS were mapped on four different chromosomal EcoRI restriction fragments (A, C, D, and E) in class 2a mutants. The native DNA fragments were cloned from a XL47 or from a cosmid gene bank. Evidence is provided that fragments A (21 kilobase pairs [kb]) and C (7.7 kb) are closely linked in the genome; the insertions of TnS covered a region of approximately 5 kb. Physiological experiments revealed that this region encodes for acetoin:dichlorophenol-indophenol oxidoreductase, a fast-migrating protein, and probably for one additional protein that is as yet unknown. In mutants which were not completely impaired in growth on acetoin but which grew much slower and after a prolonged lag phase, fragments D (7.2 kb) and E (8.1 kb) were inactivated by insertion of TnS::mob. No structural gene could be assigned to the D or E fragments. In class 2b mutants, insertions of Tn5 were mapped on fragment B (11.3 kb). This fragment complemented pleiotropic hno mutants in trans; these mutants were impaired in the formation of a rpoN-like protein. The expression of the gene cluster on fragments A and C seemed to be rpoN dependent.
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