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...