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From pMOL28, one of the two heavy metal resistance plasmids of Alcaligenes eutrophus strain CH34, we cloned an EcoRI-PstI fragment into plasmid pVDZ'2. This hybrid plasmid conferred inducible nickel and cobalt resistance (cnr) in two distinct plasmid-freeA. eutrophus hosts, strains AE104 and H16. Resistances were not expressed in Escherichia coli. The nucleotide sequence of the 8.5-kb EcoRI-PstI fragment (8,528 bp) revealed seven open reading frames; two of these, cnrB and cnrA, were assigned with respect to size and location to polypeptides expressed in E. coli under the control of the bacteriophage T7 promoter. sensitive. The mutations located upstream of cnrC resulted in various phenotypic changes: (i) each mutation in one of the gene loci cnrYRH caused constitutivity, (ii) a mutation in cnrH resulted in different expression of cobalt and nickel resistance in the hosts H16 and AE104, and (iii) mutations in cnrY resulted in two-to fivefold-increased nickel resistance in both hosts. These genes are considered to be involved in the regulation of cnr. Comparison of cnr of pMOL28 with czc of pMOL30, the other large plasmid of CH34, revealed that the structural genes are arranged in the same order and determine proteins of similar molecular weights. The largest protein CnrA shares 46% amino acid similarity with CzcA (the largest protein of the czc operon). The other putative gene products, CnrB and CnrC, share 28 and 30%o similarity, respectively, with the corresponding proteins of czc.Alcaligenes eutrophus CH34 is a metal-resistant bacterium that carries two plasmids; pMOL28 (163 kb) determines resistance to nickel, cobalt, mercury, and chromate, and pMOL30 (238 kb) determines resistance to cadmium, zinc, cobalt, mercury, and copper (7,9,16,18). Metal-resistant bacteria isolated from low-grade ore deposits in Belgium and Zaire (7, 14) have several traits in common with strain CH34 and thus indicate the predominance of this type among metal-resistant bacteria. Nickel and cobalt resistance (cnr) encoded by pMOL28 has been studied in some detail. The pMOL28-encoded nickel and cobalt resistance is inducible (31) and is due to an energy-dependent specific efflux system (20,29,38). In rare mutants, nickel and cobalt resistance is expressed constitutively (32). Plasmid pMOL28 has been transferred to other wild-type strains of A. eutrophus, such as H16, N9A, and G29, and confers the ability to tolerate 3 mM NiCl2 and 5 mM CoCl2 to the transconjugants when they are grown on solid or in liquid media (16 degree of nickel resistance also was observed in Alcaligenes hydrogenophilus, Pseudomonas putida, and P. oleovorans. In all transconjugants the resistance to nickel and cobalt was constitutively expressed. Resistance was not expressed in Escherichia coli (30).The genes coding for chromate and cobalt-nickel resistance were cloned (18) from plasmid pMOL28, and the nucleotide sequence of the chromate resistance gene was determined (19). The genes of pMOL30, which determine resistance to cadmium, zinc, and cobalt (czc), have a...

Isolates of Paracoccus denitrijicans were obtained from various habitats by enrichment in a mineral medium, using molecular hydrogen as the hydrogen donor and nitrate as the hydrogen acceptor. A total of 11 strains were compared with the following three reference strains: P . denitrijcans Stanier 381T (type strain) (= DSM 65T = ATCC 17741T), Morris (= DSM 413 = ATCC 19367), and Vogt (= DSM 415). A computer analysis based on 235 characters indicated that the strains clustered into subgroups. Deoxyribonucleic acid-deoxyribonucleic acid homology determinations confirmed this suggestion. A formal description of the species is presented, and the taxonomic position of P . denitrijcans is discussed. Paracoccus denitrijicans (formerly known asMicrococcus denitriJicans) was first isolated by Beijerinck and Minkman ( 5 ) and was reisolated as a bacterium that was capable of using molecular hydrogen in denitrification by Verhoeven et al. (56) and Vogt (57). Although this species has a guanine-plus-cytosine (G + C) content similar to the G+C contents of some members of Micrococcus, it differs from other species of Micrococcus as follows (40): (i) P. denitr$cans forms rod-shaped cells in young cultures (34, 39, 55, 57); (ii) it is gram negative (9, 34, 56, 57); (iii) it has a cell wall peptidoglycan which contains a wide range of amino acids characteristic of gram-negative genera, and the cell wall contains diaminopimelic acid in place of the lysine characteristic of micrococci (4); and (iv) it contains ubiquinone as an electron carrier (46), which is characteristic of gram-negative bacteria. For these reasons a new genus, Paracoccus was created (9). The electron transport chain of P . denitrifcans resembles the electron transport chain of the inner mitochondria1 membrane more closely than the electron transport chain of any other bacterium (21). The hydrogenase of P . denitrifcans strain Stanier 381T (type strain) has been isolated and characterized (49, 50), and the regulation of hydrogenase formation as a diagnostic character of the reference strains of P . denitrijicans and of newly isolated strains has been studied (35).Although P. denitr8can.s was isolated more than 70 years ago (5) and has been used in many investigations, its taxonomic position is still ill defined; the genus was placed with "genera of uncertain affiliation" (11) because only two strains were available for study and taxonomic data were lacking.The objectives of this study were to isolate many new strains of P . denitrijicans, to determine their main features, to compare them with the strains kept in culture collections, to evaluate their similarities and delineate them from other related bacteria, and to determine their taxonomic niche.

The nickel-cobalt-cadmium resistance genes carried by plasmid pTOM9 of Alcaligenes xylosoxidans 31A are located on a 14.5-kb BamHI fragment. By random Tn5 insertion mutagenesis, the fragment was shown to contain two distinct nickel resistance loci, ncc and nre. The ncc locus causes a high-level combined nickel, cobalt, and cadmium resistance in strain AE104, which is a cured derivative of the metal-resistant bacterium Alcaligenes eutrophus CH34. ncc is not expressed in Escherichia coli. Among the bacterial nickel resistance genes, those ofAlcaligenes eutrophus CH34, a hydrogen-oxidizing bacterium containing two metal resistance plasmids, are the most studied (17). One plasmid, pMOL28 (163 kb), determines resistance to nickel, cobalt, chromate, and mercury; the other plasmid, pMOL30 (238 kb), determines resistance to cobalt, zinc, and cadmium (9, 21, 26). The mechanism of pMOL28-encoded nickel and cobalt resistance was shown to involve an energydependent efflux system for the metal ions (38, 46). This Cnr system enables A. eutrophus CH34 to grow in the presence of 3 mM nickel and 5 mM cobalt ions. The 7.1-kb cnr sequence revealed six potential genes, cnrYXHCBA. The genes cnrYXH encode proteins involved in the regulation of the cnr operon (15). Recently CnrH was suggested to be an alternative sigma factor belonging to the new ECF subfamily of c70 factors (16). CnrH controls the transcription of the structural genes cnrCBA encoding the subunits of the efflux pump (15). Interestingly, mutations in the cnr operon affecting its regulation also resulted in zinc resistance (4). The structural protein CnrA shows similarities to a set of three proteins (NolG, NolH, and NolI) that play a role in the nodulation of legumes, as well as to the protein EnvD, which functions in cell division (30).Other metal-resistant bacteria have been isolated, some of which tolerate up to 40 mM nickel (14,34,35). Two of these high-level nickel-resistant bacteria, Alcaligenes xylosoxidans 31A and A. eutrophus KT02, turned out to be closely related. Strain 31A contains two plasmids, and strain KT02 contains three plasmids. Strain 31A, initially isolated from a copper galvanization tank, tolerates relatively high concentrations of nickel (40 mM), cobalt (20 mM), zinc (10 mM), cadmium (1 mM), and copper (1 mM). Its metal resistance genes are located on two large plasmids, pTOM8 (340 kb) and pTOM9 (200 kb), which are transferable to A. eutrophus strains. In A. eutrophus AE104, a metal-sensitive cured mutant of A. eutrophus CH34, each plasmid confers resistance to nickel, cobalt, and cadmium, whereas the presence of both plasmids causes in addition copper and zinc resistance (36). The other strain, A. eutrophus KT02, shows similar metal resistances. They are also encoded by megaplasmids. With respect to the restriction nuclease patterns, one of these plasmids, pGOE2, strongly resembles plasmid pTOM9 ofA. xylosoxidans 31A. The nickelcobalt-cadmium resistance genes of pTOM8, pTOM9, and pGOE2 are located on 14.5-kb BamHI fragments which are apparently...

SummaryThe Alcaligenes eutrophus genes for (i-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase and poly([i-hydroxybutyric acid) synthase (PHB synthase) which comprise the three-step PHB-biosynthetic pathway, were cloned. Molecular studies revealed that these genes are organized in a single operon. The A. eutrophus PHB-biosynthetic genes are readily expressed in other bacteria, and DNA fragments harbouring the operon can be used as a cartridge to confer to other bacteria the ability to synthesize PHB from acetyl-CoA. The biochemical and physiological capabilities of A. eutrophus for the synthesis of a wide variety of polyhydroxyalkanoates are discussed.