Cloning of plasmid genes encoding resistance to cadmium, zinc, and cobalt in Alcaligenes eutrophus CH34

Abstract

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

Classification of metal-resistant bacteria from industrial biotopes as Ralstonia campinensis sp. nov., Ralstonia metallidurans sp. nov. and Ralstonia basilensis Steinle et al. 1998 emend.

“…Resistance to Cd# + , Zn# + and Co# + has been shown to be located on a czc operon of the plasmid pMOL30 (240 kb), while Co# + combined with Ni# + resistance is located on the cnr operon of the plasmid pMOL28 (163 kb) (Liesegang et al, 1993 ;Mergeay et al, 1985 ;Nies et al, 1987 ;Siddiqui et al, 1989). Other plasmid-borne resistances to metals include resistance to copper (cop), lead ( pbr) and mercury (mer) on pMOL30 and resistance to chromate (chr) and mercury on pMOL28 (for a review see Mergeay, 2000).…”

CzcR-CzcS, a Two-component System Involved in Heavy Metal and Carbapenem Resistance in Pseudomonas aeruginosa

“…Proton-driven RND type efflux pumps conferring heavy metal resistance have been described in Ralstonia metallidurans (for a recent review, see Ref. 10) and include the Cnr system (nickel/cobalt) (11), the Ncc system (nickel/cobalt/cadmium) (12), and the Czc system (cobalt/zinc/cadmium) (13,14). In P. aeruginosa, an RND type efflux pump called CzrCBA was recently described in an environmental isolate where it contributes to the intrinsic resistance to zinc and cadmium (15).…”

Two‐component regulatory system involved in transcriptional control of heavy‐metal homoeostasis in Alcaligenes eutrophus

“…Standard molecular genetic techniques were used (Nies et al, 1987;Sambrook et al, 1989). For conjugal gene transfer, overnight cultures of donor strain Escherichia coli S17/1 (Simon et al, 1983) and the A. eutrophus recipient strains (1:1) grown at 30ЊC in complex medium were mixed and plated onto nutrient agar (Difco).…”

“…For conjugal gene transfer, overnight cultures of donor strain Escherichia coli S17/1 (Simon et al, 1983) and the A. eutrophus recipient strains (1:1) grown at 30ЊC in complex medium were mixed and plated onto nutrient agar (Difco). After overnight growth, the bacteria were suspended in saline (9 g l ¹1 of NaCl), diluted, and plated onto selective media as previously described (Nies et al, 1987). PCGENE was used as the standard computer program for the analysis of DNA sequences.…”

“…One of these, the czc determinant of plasmid pMOL30, mediates inducible resistance to Co 2+ , Zn 2+ and Cd 2+ in A. eutrophus (Nies et al, 1987;Nies, 1992;Kunito et al, 1996). The products of the genes czcA, czcB, and czcC form a membrane-bound protein complex catalysing an energydependent efflux of these three metal cations (Nies et al, 1989b).…”