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.

Optical determination of deoxyribonucleic acid (DNA) -DNA reassociation kinetics was applied to the classification of 32 selected strains of hydrogen-oxidizing bacteria belonging to the genera Pseudomonas, Alcaligenes, and Paracoccus. The renaturation studies revealed a high intraspecies DNA homology for some strains of the species Pseudomonas palleronii, Pseudomonas pseudoflava, and Alcaligenes paradoxus, supporting former taxonomic concepts of different authors. The 12 denitrifying strains belonging to the genus Paracoccus were shown to be interrelated at various levels of percent degree of binding, and at least two clusters of very high genome-DNA relatedness have been found. DNA-DNA reassociation kinetics were also used for the calculation of the average molecular weight of genome DNA. The genome molecular weight of the hydrogen-oxidizing bacteria investigated in this study ranged from 3 X lo9 to 5 x log.Defined as a physiological group, the "hydrogen bacteria" are aerobic chemolithotrophs that can use the oxidation of molecular hydrogen as an energy source. To accommodate these organisms, the genus Hydrogenomonas was proposed by Orla-Jensen in 1909. Davis et d. (5) rejected the genus Hydrogenomonas and proposed the assignment of all gram-negative strains of hydrogen-oxidizing bacteria to Pseudomonas, Alcaligenes, or Paracoccus. By this and later investigations of gram-negative, POlarly flagellated, hydrogen-oxidizing bacteria and related strains (1,2,5,6), the following nine hydrogen-oxidizing species of the genera Pseudomonas, Alcaligenes, and Paracoccus were established: Pseudomonas facilis, P. flava, P. pseudojlava, P. palleronii, P. saccharophila, Alcaligenes eutrophus, A. paradoxus, A. ruhlandii, and Paracoccus denitrificans. Serological methods have been used to show relatedness between strains of these species (2, 8). In spite of the intensive application of nucleic acid hybridization techniques for the classification of the pseudomonads (14, 16, 17), only very few deoxyribonucleic acid (DNA) -DNA reassociations have been reported for hydrogen-oxidizing strains of the genera Pseudomonas and Al5ali-genes (2, 18).The present investigation was undertaken to confirm, by DNA-DNA reassociation studies, the establishment of new genera and species for Nine new denitrifying strains, isolated by T.Nokhal from our institute, were included in this study. Their characterization and comparison with known strains of Paracoccus denitrificans will be described in a separate paper (Nokhal and Schlegel, manuscript in preparation). MATERIALS AND METHODSBacterial strains and culture conditions. The bacterial strains used in this study are listed in Table 1. In addition, Xanthomonas peiargonii ICPB P121 was used as a reference in molecular weight determinations and was grown in nutrient broth (Difco). The culture conditions and the media for growth and maintenance of the hydrogen-oxidizing strains have been described elsewhere (2). The purity of each strain was checked by microscopic observation and by plating on nutrien...

Paracoccus denitrificans strains Stanier 381 (DSM 65), Morris (DSM 413), and Vogt 11 (DSM 415) and eleven newly isolated strains were compared with respect to the localization of hydrogenase and its regulation. In all strains hydrogenase was found to be membrane-bound and not able to reduce pyridine nucleotides. The enzyme was inducible in strain 381 and was found only in cells grown with hydrogen as the sole hydrogen donor; in cells grown under mixotrophic or heterotrophic conditions the hydrogenase activity was zero. In all other strains hydrogenase was constitutive and was present in cells grown under autotrophic, mixotrophic and heterotrophic conditions. Under the latter conditions the specific hydrogenase activity was even higher than under mixotrophic conditions.

No abstract

Two out of eleven newly isolated strains of Paracoccus denitrificans were investigated by light and electron microscopic methods and compared with two strains of P. denitrificans already kept in culture collections. Samples were taken from different growth phases revealing short rods and nearly spherical cells in the exponential growth phase, and an increasing ratio of nearly spherical cells in the stationary growth phase. Cell division followed the binary fission mode; higher cell aggregates were not observed. Fine structural analysis revealed extracellular surface material stainable with Ruthenium red, a gram-negative cell wall and different storage material inclusions. Structural properties and variations within the four strains under investigation are discussed and compared with those of related bacteria.