INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, OCt. 1990, p. 384-398 Vol. 40, No. 4 OO20-7713/90/040384-15$02.OO/O Copyright 0 1990, International Union of Microbiological Societies Acidovorax, a New Genus for Pseudomonas facilis, Pseudomonas delaJieldii, E. Falsen (EF) Group 13, EF Group 16, and Several Clinical Isolates, with the Species Acidovorax facilis comb. nov. , Acidovorax delaJieldii comb. nov., and Acidovorax temperans sp. nov. A. WILLEMS,l E. FALSEN,2 B. POT,l E. JANTZEN,3 B. HOSTE,l P. VANDAMME,' M. GILLIS,l* K. KERSTERS,l AND J. DE LEY' Laboratorium voor Microbiologie en Microbiele Genetica, Rijksuniversiteit, B-9000 Ghent, Belgium'; Culture Collection, Department of Clinical Bacteriology, University of Goteborg, ,5413 46 Goteborg, Sweden2; and Department of Methodology, National Institute of Public Health, N-0462 Oslo, Norway3 Pseudomonas facilis and Pseudomonas delafeldii are inappropriately assigned to the genus Pseudomonas. They belong to the acidovorans rRNA complex in rRNA superfamily I11 (i.e., the beta subclass of the Proteobacteria). The taxonomic relationships of both of these species, two groups of clinical isolates (E. Falsen [EF] group 13 and EF group 16), and several unidentified or presently misnamed strains were examined by using DNA:rRNA hybridization, numerical analyses of biochemical and auxanographic features and of fatty acid patterns, polyacrylamide gel electrophoresis of cellular proteins, and DNA:DNA hybridization. These organisms form a separate group within the acidovorans rRNA complex, and we propose to transfer them to a new genus, Acidovorax. We describe the following three species in this genus: the type species, Acidovorax facilis (formerly Pseudomonas facilis), with type strain LMG 2193 (= CCUG 2113 = ATCC 11228); Acidovorax delafeldii (for the former Pseudomonas delufeldii and most of the EF group 13 strains), with type strain LMG 5943 (= CCUG 1779 = ATCC 17505); and Acidovorax temperans (for several former Pseudomonas and Alcaligenes strains and most of the EF group 16 strains), with type strain CCUG 11779 (= LMG 7169). It is generally accepted today that the genus Pseudomo- [Pseudomonas]facilis and [Pseudomonas]delajieldii be- nas, as described in Bergey's Manual of Systematic Bacte- long to the acidovorans rRNA complex (rRNA homology riology (36), is multigeneric and cannot be maintained as a group I11 sensu Palleroni [36]) in rRNA superfamily I11 sensu single genus (3, 12, 50, 53, 56). Using a limited number of De Ley (8), which corresponds to the beta subclass of the strains, Palleroni et al. delineated five Pseudomonas rRNA Proteobacteria (48). As explained previously (53), the aci- homology groups on the basis of DNA:rRNA hybridization dovorans rRNA complex is a heterogeneous group of organ- data (37). These groups were later considerably extended isms, many of which should be generically renamed. Our and were shown to be only very remotely related to each main tool to reveal the basic taxonomic structure of this other (12, IS). Therefore, the genus Pseudomonas sensu group at the generic and suprageneric levels is DNA:rRNA strict0 should be restricted to the Pseudomonas JEclorescens hybridization. The finer taxonomic relationships within the rRNA group, which contains the type species, Pseudomonas subgroups revealed by DNA:rRNA hybridization are then aeruginosa (12). All other Pseudomonas species should be studied by using appropriate techniques, such as phenotypic generically renamed (indicated below by brackets). analysis, protein gel electrophoresis, DNA:DNA hybridiza- [Pseudomonas]facilis was originally described as Hydro- tion, etc. Up to now this polyphasic approach has led to the genomonas facilis, a hydrogen-oxidizing isolate from lawn revival of the genus Comamonas (14) and the creation of two soil (44). At that time all gram-negative hydrogen-oxidizing new genera, Xylophilus (54) and Hydrogenophaga (53), bacteria were grouped in a single genus, Hydrogenomonas. which accommodate several generically misnamed species In a study of the poly-P-hydroxybutyrate (PHB) metabolism from the acidovorans rRNA complex. of pseudomonads, Delafield et al. (6) compared morpholog- As reported previously (14, 53, 54), labeled rRNAs have ical and physiological properties of numerous PHB-using been prepared from the type strains of six species. By Pseudomonas and Hydrogenomonas strains. Hydrogenomo- hybridizing these rRNAs with DNAs from representative nas facilis was placed in their group I, together with five unidentified isolates. The genus Hydrogenomonas was later strains of all of the taxa belonging to the acidovorans rRNA rejected (4), Hydrogenomonas facilis was transferred to the complex, we delineated the following five rRNA sub- genus Pseudomonas as Pseudomonas facilis, and a new branches, which were linked at a Tm(e)level [Tm(e)is the species, Pseudomonas delajieldii, was created for the five temperature, in degrees Celsius, at which one-half of a unnamed strains belonging to group I of Delafield et al. DNA:rRNA hybrid is denatured] of 76.0 -+ 1.1"C: the Pseudomonas facilis and Pseudomonas delajieldii were de- Comamonas acidovorans rRNA subbranch, the Comamo- scribed as phenotypically similar; an important difference nas terrigena rRNA subbranch, the [Alcaligenes]paradoxus was the inability of Pseudomonas delafieldii to oxidize rRNA subbranch, the Xylophilus ampelinus rRNA sub- hydrogen (5). branch, and the Hydrogenophaga rRNA subbranch (53). Most other species of the acidovorans rRNA complex (e.g., [Pseudomonas]facilis, [Pseudomonas]delafieldii, [Pseudo- * Corresponding author monas] avenue, and Comamonas testosteroni) are located at 384 VOL. 40, 1990 ACZDOVORAX GEN. NOV. 385 the branching level of these five rRNA subbranches (53). its T,n(elvalue and by the percentage of rRNA binding (the Several species (e.g., [Pseudomonas] saccharophila, [Al- amount of rRNA [in micrograms] bound to 100 pg of caligenes] latus, and [Rhodocyclus]gelatinosus) are located filter-fixed DNA after RNase treatment). somewhat lower, at a T,,,(,, level of 73.1 5 1.4"C (53). DNA:DNA hybridization. The degree of binding, ex- A number of clinical isolates that were received for pressed as a percentage was determined spectrophotometri- identification at the Culture Collection of the University of cally by using the initial renaturation rate method (9), as Goteborg, Goteborg, Sweden, were examined by using described previously (53). Renaturations were performed in immunological methods (16) and were provisionally sorted 2x SSC (IxSSC is 0.15 M NaCl plus 0.015 M sodium into two unnamed groups, E. Falsen (EF) group 13 and EF citrate, pH 7.0) at an optimal renaturation temperature of group 16, which were found to be related to [Pseudomonas] 80.3"C and with a total DNA concentration of 0.102 mM base delajieldii (17). A routine numerical analysis of auxano- pairs. Degrees of binding of 25% and less do not represent graphic test results for a number of strains with mistaken any significant DNA binding. genus assignments showed that the following strains are also Morphological and biochemical features. We used the related to [Pseudomonas] delajieldii: (i) [Pseudomonas methods described by De Vos et al. (14). Nitrite reduction pseudoalcaligenes] strains CUETM 85-21, CUETM 85-23, was tested as described by Rossau et al. (43). Autotrophic and CUETM 85-25, which Gavini et al. (19) detected as a growth with hydrogen was tested on the basal medium separate cluster (cluster A2) in their phenotypic analysis of described by Meyer and Schlegel (34) to which 0.01% several Pseudomonas species; (ii) [Alcaligenesdenitrijicans] (wtlvol) yeast extract was added. For heterotrophic growth, strains CIP 239.74 and CIP 471.74, which clustered together 0.2% (wt/vol) succinic acid was added as a carbon source to with [Alcaligenes]paradoxus in the phenotypic study of the the same basal medium. To obtain a suitable atmosphere, we genus Alcaligenes performed by Kiredjian et al. (28); and (iii) modified the procedure of Sly (46) as described below. six unnamed clinical isolates which were received at the Anaerobic jars (type HP11; Oxoid) and gas-generating kits Culture Collection of the University of Goteborg for identi- (type BR38; Oxoid) were used without a catalyst; 4 h after fication. We studied the relationships of all of these strains, the gas-generating process was started, the pressure, which [Pseudomonas]facilis, [Pseudomonas]delajieldii, and sev- had then stabilized at about 0.6 bar was decreased to 0.05 eral unidentified or misidentified strains, mostly of clinical bar. Gas chromatographic analyses of the atmosphere in the origin, by using genotypic, chemotaxonomic and phenotypic jar gave the following mean composition: 47.0% N,, 13.0% techniques. We propose a new genus, Acidovorax, for these O,, 5.0% CO,, and 35.0% H,. In each jar four or five strains, strains, with three species, Acidovorax facilis, Acidovorax including one well-known hydrogen-oxidizing reference delajieldii, and Acidovorax temperans. strain, were tested on one petri dish each. The jars were incubated at 28°C for at least 3 weeks. Controls for each MATERIALS AND METHODS strain on autotrophic and heterotrophic medium were incu- bated in air. Autotrophic growth was regarded as positive if Bacterial strains. The strains which we used are listed in growth on the autotrophic medium in the jar was clearly Table 1. Most of these organisms were grown on nutrient visible and more abundant than growth on the same medium agar (0.1% [wt/vol] beef extract, 0.2% [wt/vol] yeast extract, incubated in air. All strains were tested at least twice. 0.5% [wt/vol] NaC1, 0.5% [wt/vol] peptone, 2% [wt/vol] Carbon substrate assimilation tests. API galleries (types agar; pH 7.4); exceptions were [Rhodocyclus] gelatinosus API 50CH, API SOAO, and API 50AA; API System S.A., and Xyfophilus ampelinus, which were grown on the media Montalieu-Vercieu, France) were used to test the assimila- described previously (54). For fatty acid analysis, cells were tion of 147 organic compounds as sole carbon sources. The grown for 40 h on Columbia agar (Oxoid Ltd., Basingstoke, experimental procedure which we used has been described England) containing 5% [vol/vol] defibrinated horse blood.