A cadmium resistance gene, designated cadD, has been identified in and cloned from the Staphylococcus aureusplasmid pRW001. The gene is part of a two-component operon which contains the resistance gene cadD and an inactive regulatory gene, cadX*. A high degree of sequence similarity was observed between cadD and thecadB-like gene from S. lugdunensis, but no significant similarity was found with either cadA orcadB from the S. aureus plasmids pI258 and pII147. The positive regulatory gene cadX* is identical tocadX from pLUG10 over a stretch of 78 codons beginning at the N terminus, but it is truncated at this point and inactive. Sequence analysis showed that the cadmium resistance operon resides on a 3,972-bp element that is flanked by direct repeats of IS257. The expression of cadD in S. aureus and Bacillus subtilis resulted in low-level resistance to cadmium; in contrast, cadA andcadB from S. aureus induced higher level resistance. However, when the truncated version ofcadX contained in pRW001 is complemented intrans with cadX from plasmid pLUG10, resistance increased approximately 10-fold suggesting that the cadmium resistance operons from pRW001 and pLUG10 are evolutionarily related. Moreover, the truncated version ofcadX contained in pRW001 is nonfunctional and may have been generated by deletion during recombination to acquire the cadmium resistance element.
A wild-type strain of Methanobacterium thermoautotrophicum Marburg was transformed by DNA from strains resistant to 5-fluorouracil. Recipient cells were grown without selection on gellan gum (GELRITE) plates with DNA. Drug-resistant cells were recovered by replica plating the resulting colonies onto drug plates. Transformation required high-molecular-weight DNA with appropriate markers and was not observed on agar or in liquid media under a variety of conditions. A significant amount of molecular information has been uncovered about methanogenic archaebacteria with the use of methanogen DNAs that have been cloned into eubacterial hosts (7, 9). A method for performing genetic experiments in methanogens is necessary so that the cloned genes can be used as starting points for meaningful physiological studies. Techniques for mutant selection and methods for introducing methanogen genes into their original backgrounds are the minimal requirements for a genetic system. Some progress has been made in this direction for several species of archaebacteria. Several drug-resistant and auxotrophic mutants have been reported in various methanogen species (3,8,10,11,18, 20, 22). Among the halophilic archaebacteria, transfer mechanisms based on conjugation and transfection with DNA from a lytic phage have been demonstrated (6,17). Genetic transformation of the mesophile Methanococcus voltae PS in liquid culture was reported recently (3).We present evidence for a natural transformation system in the thermophilic methanogen Methanobacterium thermoautotrophicum Marburg. A marker for 5-fluorouracil (FU) resistance was transformed into the wild-type strain via DNAs from drug-resistant mutants.
MATERIALS AND METHODSStrains and media. Table 1 lists the strains used in this study. Halobacterium volcanii was grown in medium 97 at 37°C (5). Escherichia coli was grown in LB broth at 37°C. M. thermoautotrophicum strains were grown on mineral medium 2 as described previously (1, 24). Mineral medium plates contained 0.8% GELRITE gellan gum (Kelco Division of Merck & Co., Inc., San Diego, Calif.) plus a cation mixture composed of 1 g each of MgSO4 * 7H20 and CaC12 *2H20 per liter. The amount of Na2S -9H20 was reduced 10-fold to a final concentration of 0.006%. Mineral medium agar plates contained 2% Noble agar (Difco Laboratories, Detroit, Mich.) with or without the cation mixture. All plating operations, including preparation of plates after sterilization of anoxic medium, were done in an anaerobic chamber under an atmosphere of 95% N2 and 5% H2 (2). Additions were made by spreading liquid supplements on the solidified medium with a bent glass rod or a sterile plastic loop. Plates were incubated in pressure vessels (modified from references 1 and 2) under an atmosphere of H2-C02- H2S (80:19:1 [vol/vol]) at 200 to 300 kPa for 2 to 4 days at 60°C. To score colonies, the gas phase was exchanged with * Corresponding author. 653 nitrogen, and the vessels were brought into the anaerobic chamber.DNA purification. M. thermoautotrophicum cells were...
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