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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.

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The Zymomonas mobilis genes encoding alcohol dehydrogenase I (adhA), alcohol dehydrogenase H (adhB), and pyruvate decarboxylase (pdc) were overexpressed in Escherichia coli and Z. mobUis by using a broad-host-range vector containing the tac promoter and the lacP repressor gene. Maximal IPIG (isopropyl-1%-D-thiogalactopyranoside) induction of these plasmid-borne genes in Z. mobilis resulted in a 35-fold increase in alcohol dehydrogenase I activity, a 16.7-fold increase in alcohol dehydrogenase H activity, and a 6.3-fold increase in pyruvate decarboxylase activity. Small changes in the activities of these enzymes did not affect glycolytic flux in cells which are at maximal metabolic activity, indicating that flux under these conditions is controlled at some other point in metabolism. Expression ofadfA, adhB, orpdc at high specific activities (above 8 lU/mg of cell protein) resulted in a decrease in glycolytic flux (negative flux control coefficients), which was most pronounced for pyruvate decarboxylase. Growth rate and flux are imperfectly coupled in this organism. Neither a twofold increase in flux nor a 50%o decline from maximal flux caused any immediate change in growth rate. Thus, the rates of biosynthesis and growth in this organism are not limited by energy generation in rich medium.Zymomonas mobilis represents an excellent model system for metabolic flux control analysis (8,33). This organism is an obligately fermentative gram-negative bacterium that utilizes the Entner-Douderoff (ED) pathway for glycolysis. More than 95% of the glucose metabolized is converted into ethanol and carbon dioxide with a low ATP yield (29). The glycolytic and ethanologenic enzymes in Z. mobilis represent the sole route for energy generation, and together they constitute approximately 50% of soluble cellular protein (2,3,34). The biochemistry and kinetics of the enzymes involved have been characterized (6,21,30,31,34,38,41), and some have recently been proposed to form complexes in vivo (1). In contrast to Embden-Meyerhof glycolysis, which exhibits considerable allosteric control, the ED pathway lacks two key allosteric enzymes, namely, phosphofructokinase and an allosteric hexokinase (2, 29, 41). On the basis of biochemical characterizations and the small metabolite pools in Z. mobilis, it has been proposed that little allosteric regulation operates within the Z. mobilis ED glycolytic pathway (2, 5). Consequently, carbon flux may be limited by the specific activities of pathway enzymes.Most of the Z. mobilis genes encoding ED enzymes and ethanologenic enzymes have been cloned and sequenced (4,(8)(9)(10)(11)(12)24). To facilitate the study of flux control by using these genes, a regulated expression vector is needed for Z. mobilis and none have been previously reported. In this article, we describe the modification and use of a broad-hostrange vector (18) that allows partial control of plasmid-borne genes. This vector was used to investigate the effects of the ethanologenic genes (adhA, adhB, and pdc) on metabolic activity and g...

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...

Cell extracts of methanogens and the thermoacidophile Sulfolobus solfataricus contained little or no folic acid (pteroylglutamate) or pteroylpolyglutamate activity (less than 0.1 nmol/g [dry weight]). However, the halophile Halobacterium salinarum contained pteroylmono- or pteroyldiglutamates, and Halobacterium volcanii and Halobacterium halobium contained pteroyltriglutamates at levels equivalent to those in eubacteria (greater than 1 nmol/g [dry weight]).

The enzymes involved in the purine interconversion pathway of wild-type and purine analog-resistant strains of Methanobacterium thermoautotrophicum Marburg were assayed by radiometric and spectrophotometric methods. Wild-type cells incorporated labeled adenine, guanine, and hypoxanthine, whereas mutant strains varied in their ability to incorporate these bases. Adenine, guanine, hypoxanthine, and xanthine were activated by phosphoribosyltransferase activities present in wild-type cell extracts. Some mutant strains simultaneously lost the ability to convert both guanine and hypoxanthine to the respective nucleotide, suggesting that the same enzyme activates both bases. Adenosine, guanosine, and inosine phosphorylase activities were detected for the conversion of base to nucleoside. Adenine deaminase activity was detected at low levels. Guanine deaniinase activity was not detected. Nucleoside kinase activities for the conversion of adenosine, guanosine, and inosine to the respective nucleotides were detected by a new assay. The nucleotide-interconverting enzymes AMP deaminase, succinyl-AMP synthetase, succinyl-AMP lyase, IMP dehydrogenase, and GMP synthetase were present in extracts; GMP reductase was not detected. The results indicate that this autotrophic methanogen has a complex system for the utilization of exogenous purines.As the terminal organisms in many anaerobic food chains, the methanogens play a key role in biodegradation reactions. These organisms are phylogenetically diverse and metabolically rather limited, being capable of methanogenesis from only a few simple substrates (20, 21). They utilize novel coenzymes, lipids, cell walls, and metabolic reactions (3,20,21,37). Molecular biology studies have revealed aspects of gene organization and regulation (6, 21) that support the inclusion of methanogens in the kingdom Archaebacteria and show their similarity to both eubacteria and eucaryotes. The genetic approach to the study of these organisms has yielded mutants and physiological information, but genetic exchange mechanisms are in the very early stages of development (4, 32, 45).Although great strides have been made in understanding methanogens, little is known about the biosynthesis of nucleotides in these organisms. Purine nucleotides can be synthesized from simple precursors by de novo pathways; however, salvage of preformed bases and nucleosides can provide an energy-efficient alternative. Salvage pathways have been found in eucaryotes (1, 13, 16) and eubacteria (12, 43) and appear to be present in archaebacteria (5,23,33,42). Knowledge of purine salvage pathways in methanogens is important because it increases our knowledge of methanogen metabolism, which can be applied to the selection and use of radioactive compounds for labeling nucleic acids and their precursors. It provides insight and understanding of the mechanism of action of purine analogs and allows for the isolation of purine analog-resistant mutants blocked in various steps of the salvage pathways. Because methanogens are resistant to...