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 facilis, Pseudomonas delaJieldii, E. Falsen (EF) Group 13, EF Group 16, and Several Clinical Isolates, with the Species facilis comb. nov. , Acidovorax delaJieldii comb. nov., and 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 ). 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, (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 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), 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. previously (27). Most strains were grown at 28°C; the single exception was Numerical analysis of phenotypic features. The scoring Xylophilus ampelinus NCPPB 2217T (T = type strain), which used for the auxanographic and biochemical tests has been was grown at 24°C. described previously (43). Of 251 features tested, 195 were Preparation of high-molecular-weight DNA. Cells were used in the numerical analysis. The API 20NE tests (21 grown in Roux flasks for 2 to 3 days. DNA was isolated by features) were not included in the computer analysis as they using the method of Marmur (31). duplicated other tests, and 35 other features were omitted DNA base composition. The average guanine-plus-cytosine because all 70 strains tested were either negative or had the (G+C) value was determined by using the thermal denatur- same quantitative positive value. Hydrogen oxidation was ation method (11) and was calculated by using the equation not included because not all reference strains were tested for of Marmur and Doty (32), as modified by De Ley (7). this character. The interstrain similarities were calculated by DNA:rRNA hybridization. DNA was further purified by using the Gower similarity coefficient (S,) (47). Cluster CsCl gradient centrifugation (10). Fixation of thermally analysis by the unweighted average pair group method (43) denatured DNA on cellulose nitrate filters and chemical was performed by using the CLUSTAN 2.1 program of estimation of the amount of filter-fixed DNA were performed Wishart (55) and the Siemens model 7570-C computer of the as described previously (10,51). [3H]rRNA from Acidovorax Centraal Digitaal Rekencentrum, Rijksuniversiteit, Ghent, facilis ATCC 1122tIT was isolated and purified as described Belgium. The reproducibility of the clustering was estimated by De Ley and De Smedt (10). Purified 23s [3H]rRNAs from by including duplicate tests for 19 strains. The centrotype Comamonas acidovorans Stanier 14T, Comamonas terri- strains were calculated as described previously (49). gena NCIB 8193T, and Hydrogenophaga palleronii Stanier Polyacrylamide gel electrophoresis of proteins. All strains 362tlT and 16s [14C]rRNA from [Alcaligenes] paradoxus were grown on nutrient agar at 28°C for 40 h in Roux flasks. ATCC 17713tlT were available from members of our re- Whole-cell protein extracts were prepared, and sodium search group (13, 14, 53). Hybridizations between labeled dodecyl sulfate-polyacrylamide gel electrophoresis was per- 16s or 23s rRNA and filter-fixed DNA were carried out as formed by using small modifications of the procedure of described previously (10). Each hybrid was characterized by Laemmli (30), as described previously (28). The normalized 386 WILLEMS ET AL. HT. J. SYST. BACTERIOL.

TABLE 1. Strainsused Name as received Strain“ Other designation(s) Source, place, and/or year of isolation Strains assigned to Acidovorax facilis [Pseudomonas]facilis ATCC 1122gT LMG 2193T, CCUG 2113T Lawn soil, United States [Pseudomonas]facilis ATCC 15376 LMG 2194, CCUG 14278 Unknown [Pseudomonas]facilis DSM 550 LMG 6598, CCUG 15919, ATCC 17695 Lawn soil [Pseudomonas]facilis DSM 620 LMG 6599, CCUG 15920 Soil Strains assigned to Acidovorax delqfieldii [Pseudomonas]delqfieldii ATCC 1750ST LMG 5943T, CCUG 1779T Soil enriched with PHB as a sole carbon source [Pseudomonas] delafieldii ATCC 17506tl’ LMG 1792t1’, CCUG 14277 Soil enriched with PHB as a sole carbon source [Pseudomonas]delqfieldii ATCC 17506t2’ LMG 1792t2’, CCUG 14277 Soil enriched with PHB as a sole carbon source [Pseudomonas] delqfieldii ATCC 17508 LMG 5944, CCUG 14478 Soil enriched with PHB as a sole carbon source [Pseudomonas]delafieldii CCUG 16516 LMG 8905 Wound secretion, open fracture, foot, Goteborg, Sweden, 1984 [Pseudomonas]delqfieldii CCUG 16566 LMG 8906 Secretion, Goteborg, Sweden, 1984 [Pseudomonas]delafieldii CCUG 20074 LMG 7679 Nasopharynx, Skovde, Sweden, 1987 [Pseudomonas]delqfieldii CCUG 23830B LMG 8909 Central venous catheder, 54-yr-old male, Gote- borg, Sweden, 1989 [Pseudomonas]delqfieldii CCUG 24594 LMG 9090 Tibia puncture, Halmstadt, Sweden, 1989 EF group 16 CCUG 3746A LMG 7164 Dialysis equipment from artificial kidney, Gote- borg, Sweden, 1974 EF group 13 CCUG 11056tl’ LMG 7166tlb Pus from cemented femur, 76-yr-old female, Goteborg, Sweden, 1981 EF group 13 CCUG 11056t2’ LMG 7166t2’ Pus from cemented femur, 76-yr-old female, Goteborg, Sweden, 1981 EF group 13 CCUG 11062 LMG 7168 Trachea, 3-mo-old baby, Goteborg, Sweden, 1981 EF group 13 CCUG 12929 LMG 7167 Joint aspiration, Goteborg, Sweden, 1982 EF group 13 CCUG 18251 LMG 7186 Blood, Goteborg, Sweden, 1985 Unidentified CCUG 21358 LMG 8916 Human sperm, Skovde, Sweden, 1987 Strains assigned to Acidovorax temperans [Pseudomonas delqfieldii] CCUG 18750 LMG 7379 Goteborg, Sweden, 1986 [Pseudomonas pseudoalcaligenes] CUETM 85-21 LMG 9370, CCUG 24957, G-4767 Laboratory sink drain, 1983 [Pseudomonas pseudoalcaligenes] CUETM 85-23 LMG 9083, CCUG 24954, G-4713 Well water, 1983 [Pseudomonas pseudoalcaligenes] CUETM 85-25 LMG 9084, CCUG 24955, G-4522 Cerebrospinal fluid, 1982 [Pseudomonas] species CCUG 22192 LMG 8451 Wound, Gavle, Sweden, 1988 [Pseudomonas] species CCUG 22215 LMG8452 Wound secretion, Gavle, Sweden, 1988 [Alcaligenes denitrificans] CIP 239.74 LMG 3332, CCUG 21717 Blood culture, France [Alcaligenes denitrificans] CIP 471.74 LMG 3334, CCUG 21716 Unknown EF group 13 CCUG 11779T LMG 716ST Urine, 68-yr-old male, Goteborg, Sweden, 1981 EF group 16 CCUG 2168B LMG 6435 Dialysis equipment, Goteborg, Sweden, 1973 EF group 16 CCUG 3688A LMG 7163 Active sludge from a wastewater purification plant, Goteborg, Sweden, 1974 EF group 16 CCUG 9443Aa LMG 6436 Human liver (pathological), Goteborg, Sweden, 1980 EF group 16 CCUG 10556 LMG 6437 Tap water, hospital, Goteborg, Sweden, 1981 EF group 16 CCUG 16573 LMG 6438 Cervix, Goteborg, Sweden, 1984 EF group 16 CCUG 17140 LMG 6439 Drain, joint puncture, 2-yr-old child, brebro, Sweden, 1984 Unidentified CCUG 17727A LMG 6730 Urine, Grenoble, France, 1981 Unidentified CCUG 18350 LMG 8915 Hospital laborabory, Goteborg, Sweden Unidentified CCUG 23689 LMG 8768 Bronchial secretion, Stockholm, Sweden, 1988 Strains not assigned to the genus Acidovorax [Pseudomonas delqfieldifl CCUG 23155 LMG 8907 Water, Castres, France [Pseudomonas delafieldii] CCUG 23156 LMG 8908 Water, Castres, France EF group 16 CCUG 3182 LMG 7162, LMG 9291 Nonchlorinated water, Goteborg, Sweden, 1974 Unidentified CCUG 21294 LMG 8440 Roots of Brassica, Lund, Sweden Unidentified CCUG 23153 LMG 8767 Water, France Reference strains Comamonas acidovorans Stanier 14T LMG 1226T, ATCC 15SaST, CCUG 14481T Soil enriched with acetamide, Delft, The Nether- lands, 1926 Comamonas acidovorans ATCC 17406 LMG 1790, CCUG 15340 Soil enriched with p-hydroxybenzoate Comamonas acidovorans ATCC 17476 LMG 1791, CCUG 15337 Great Britain Comamonas acidovorans CCUG 274B LMG 7098 Urine, male, Goteborg, Sweden, 1968 Comamonas terrigena NICB 8193T LMG 1253T CCUG 21UT (= LMG 59293, Hay infusion filtrate, United States CCUG lj327T, ATCC 8461T Comamonas terrigena NICB 2581 LMG 1249, CCUG 2474 Soil Comamonas terrigena CCUG 12940 LMG 5520 Blood, 1982 Comamonas terrigena NCIB 2582 LMG 1251, CCUG 2475, ATCC 14636 Soil Comamonas testosteroni NCTC 1069gT LMG 1786T, CCUG 142aT, ATCC 11996T Soil, Berkeley, Calif. Comamonas testosteroni ATCC 17407 LMG 1787, CUG 15341 Soil enriched with anthranilate Comamonas testosteroni ATCC 17409 LMG 1788, CUG 15339 Soil enriched with kynurenate, Berkeley, Calif., 1963 Hydrogenophaga flava DSM 619T LMG 2MT, CCUG 165gT, ATCC 33667T Mud from ditch, 1942 Hydrogenophaga palleronii Stanier 362tlT6 LMG 2366tlTb (= CCUG 203349, ATCC Water enriched for hydrogen bacteria in an atmo- 17724T (= CCUG 1780T) sphere containing 6% O2 Hydrogenophaga palleronii Stanier 366 LMG 6346, CCUG 17387, CCUG 20336, Water enriched for hydrogen bacteria in an atmo- ATCC 17728 sphere containing 6% O2 Hydrogenophaga palleronii RH2 LMG 6348, CCUG 20338 Gottingen, Federal Republic of Germany Hydrogenophaga pseudoflava GA3T LMG 5945T, CCUG 13799T,ATCC 3366gT Water, River Weende, Federal Republic of Ger- many Continued on following page VOL.40, 1990 ACIDOVORAX GEN. NOV. 387

TABLE 1-Continued Name as received Strain“ Other designation($ Source, place, andor year of isolation Hydrogenophaga pseudofava GA2 LMG 6351, CCUG 17389, Unknown CCUG 20339 Hydrogenophaga pseudofava DSM 1084 LMG 7584, CCUG 20741, Z- Mud and soil, River Moskwa, USSR 1107 (= LMG 8355 = CCUG 22764) Hydrogenophaga taeniospiralis DSM 2082T LMG 7170T, CCUG 15921T Soil, Spain [Pseudomonas] avenue NCPPB lollT LMG 2117T CCUG 15838T, Zea mays, United States, 1958 ATCC 19660T [Pseudomonas] cattleyae NCPPB 961T LMG 5286T ATCC 33619T, Unknown CCUG 2<97ST [Pseudomonas pseudoalcaligenes] PDDCC 750OT LMG 5376T, CCUG 17393T Citrullus lanatus, United States, 1977 subsp. citrulli [Pseudomonas pseudoalcaligenes] PDDCC 7733T LMG 5691T, CCUG 17394T Amorphophallus rivieri cv. Konjac, Shi- subsp. konjaci zuoka Prefecture, Japan, 1977 [Pseudomonas] rubrilineans NCPPB 920T LMG 228IT, CCUG 15837T, Saccharum oficinarum cv. R445, ATCC 19307T Reunion, 1960 [Pseudomonas] saccharophila ATCC lS946T LMG 22SST, LMG 7831T Mud from a stagnant pool “[Pseudomonas] setariae” NCPPB 1392 LMG 1806, CCUG 15836, Oryza sativa, Japan, 1955 ATCC 19882 [Alcaligenes]latus Palleroni H-4T LMG 3321T, CCUG 10983T, Soil, California ATCC 29712T [Alcaligenes]paradoxus ATCC 17713tlTb LMG 1797tlTb,CCUG 1777T Soil in mineral medium under an atmo- sphere containing 91% H2, 4% 02,and 5% coz [Alcaligenes]paradoxus ATCC 17712 LMG 3572, CCUG 15916 Soil in an atmosphere containing 8% H2, 6% 02,and 5% C02 [Alcaligenesl paradoxus ATCC 17549tlb LMG 1796tlb, CCUG 1778 Soil enriched with panthotenate [Rhodocyclus]gelatinosus NCIB 8290T LMG 4311T, ATCC 17011T, Acetate enrichment, pH 6.6 CCUG 15841T, CCUG 21977T Xylophilus ampelinus NCPPB 2217T LMG 5856T, ATCC 33914T, Vitis vinifera var. sultana, Crete, 1966 CCUG 21976T

a Strain number as received. ATCC, American Type Culture Collection, Rockville, Md.; CCUG, Culture Collection of the University of Goteborg Department of Clinical Bacteriology, University of Goteborg, Goteborg, Sweden; CIP, Collection de 1’Institut Pasteur, Paris, France; CUETM, Collection Unit6 Ecotoxicologie Microbienne, Villeneuve d’Ascq, France; DSM, Deutsche Sammlung von Mikroorganismen, Braunschweig, Federal Republic of Germany; G, G. L. Gilardi, Hospital for Joint Diseases and Medical Center, New York, N.Y.; LMG, Culture Collection, Laboratorium voor Microbiologie, State University of Ghent, Ghent, Belgium; NCIB, National Collection of Industrial Bacteria, Aberdeen, Scotland; NCPPB, National Collection of Plant Pathogenic Bacteria, Hatching Green, England; NCTC, National Collection of Type Cultures, Central Public Health Laboratory, London, England; Z, G. A. Zavarzin, Institute of Microbiology, MOSCOW,USSR. We isolated two stable colony types from the original culture and labeled them colony types tl and t2. Since both types had almost identical protein electrophoretic patterns, we only used colony type tl in some tests. densitometric traces of the protein electrophoretic patterns 3688A, CCUG 9443Aa, CCUG 10556, CCUG 16573, CCUG were grouped by numerical analysis, using the Pearson 17140, and CCUG 17727A. These strains were grown on product moment correlation coefficient (r) as described by Columbia blood agar as described above, harvested, Pot et al. (39). Points (units of distance travelled) 4 to 179 and washed, freeze dried, and methanolyzed (both alkaline and 236 to 300 of the interpolated traces were used in the acidic) as described previously (22, 23). The resulting fatty numerical analysis. The first three points were omitted to acid methyl esters were analyzed on a gas chromatograph exclude the stacking gel-separation gel interface; points 180 equipped with a flame ionization detector (43). The hydrox- to 235 were omitted because for some strains this zone ylated fatty acids were separated from the nonhydroxylated contained one or a few high-density protein bands, the fatty acids on a silica BOND ELUT extraction column position of which varied slightly from one strain to another, (Analytichem International, Harbor City, Calif.), analyzed, while in the rest of the trace the strains produced very and identified separately by gas chromatography-mass spec- similar protein banding patterns. Taking into account these trometry (Ion-trap 700 instrument; Finnigan, San Jose, high-density protein bands when the Pearson product mo- Calif.) after trifluoroacetylation and trimethylsilylation (33). ment correlation coefficients were calculated would have Numerical analysis of gas chromatographic data. Principal- caused strains to group far apart in the numerical analysis, component analysis was carried out by using the soft inde- when in fact they are highly related. A similar approach was pendent modeling of class analogy method (57), as previ- also used in the study of Campylobacter and Klebsiella ously described (24). The software program (in a PC-DOS species (18, 35). Points 301 to 400, corresponding to the version) was obtained from Pattern Recognition Systems, bottom one-fourth of the traces in Fig. 4, were not included Bergen, Norway. because under our experimental conditions this zone con- tained very few protein bands. RESULTS Gas chromatography of cellular fatty acids. The following strains were examined for cellular fatty acid composition: DNA base composition. The average G+C values of the Acidovorax facilis ATCC 11228T, ATCC 15376, DSM 550, strains which we studied are shown in Table 2. and DSM 620; ATCC 17505=, ATCC DNA:rRNA hybridizations. The specific activities of the 17506t1, ATCC 17508, CCUG 20074, CCUG 3746A, CCUG 23s [3H]rRNAs from Acidovorax facilis ATCC 1122gT, 11056t1, CCUG 11062, CCUG 12929, and CCUG 18251; and Hydrogenophaga palleronii Stanier 362tlT, Cornamonas ac- Acidovorax temperans CCUG 18750, CCUG 22192, CCUG idovorans Stanier 14T, and Cornamonas terrigena NCIB 22215, CIP 239.74, CCUG 11779T, CCUG 2168B, CCUG 8193= and of the 16s [14C]rRNA from [Alcaligenes] para- 388 WILLEMS ET AL. INT. J. SYST.BACTERIOL.

TABLE 2. DNA base compositions of strains and parameters of hybrids of DNAs with labeled rRNAs from type strains of organisms belonging to the acidovorans rRNA complex Hybridized with:

r3H1rRNA [3H]rRNA from [3H]rRNA from [3H]rRNA from "4C1rRNA from G+ Acif;oovmorax Hydrogenophaga Cornamonas Cornamonas acidovorans terrigena NCIB [Afcu'igenesl DNA from: value facilis ATCC St~~~~~&TStanier 14T paradoxus 8193T (mol%) 11228T ATCC 17713t17'

Acidovorax facilis ATCC 1122gT 64.7" 79.8 0.12 76.3' 0.06' 77.0" 0.09" 75. 0' 0.08' 77.5' 0.07' Acidovorax facilis ATCC 15376 63.7" 79.3 0.05 76.0' 0.06' 76.5' 0.09' 78.0' 0.08' Acidovorax facilis DSM 550 64.2 80.4 0.10 Acidovorax facilis DSM 620 63.9 80.6 0.11 Acidovorax delafieldii ATCC 17505T 65.6 78.0 0.10 75.6' 0.06' 77.2' 0.11" 75.1' 0.05' 76.6" 0.02" Acidovorax delafieldii ATCC 17506t1 65.3 79.4 0.12 75.3' 0.09' 77.1' 0.09' Acidovorax delafieldii ATCC 17508 66.3 77.2 0.13 Acidovorax delafieldii CCUG 3746A 65.3 78.6 0.13 Acidovorax delafieldii CCUG 11056tl 65.4 79.8 0.09 Acidovorax delafieldii CCUG 11062 65.4 80.1 0.08 Acidovorax delafieldii CCUG 12929 66.2 77.5 0.09 Acidovorax delafieldii CCUG 18251 66.0 78.3 0.10 Acidovorax delafieldii CCUG 20074 66.0 80.1 0.08 Acidovorax temperans CCUG 2168B 62.9 77.9 0.13 75.6 0.08 76.2 0.24 76.3 0.12 Acidovorax temperans CCUG 3688A 64.9 78.9 0.12 Acidovorax temperans CCUG 9443Aa 66.1 77.2 0.10 Acidovorax temperans CCUG 10556 64.0 79.8 0.12 Acidovorax temperans CCUG 11779T 62.4 80.3 0.09 74.6 0.12 76.4 0.09 Acidovorax temperans CCUG 16573 63.9 78.5 0.07 Acidovorax temperans CCUG 17140 63.6 78.8 0.15 Acidovorax temperans CCUG 17727A 63.6 80.2 0.11 Acidovorax temperans CCUG 22192 64.6 79.1 0.10 Acidovorax temperans CCUG 22215 63.5 79.1 0.12 Acidovorax temperans CCUG 23689 63.5 78.8 0.07 Acidovorax temperans CIP 239.74 63 .O 78.4 0.10 76.6 0.03 76.9 0.11 77.7 0.12 75.8 0.07 Acidovorax ternperans CIP 471.74 63.3 79.3 0.15 76.8 0.10 77.7 0.15 75.9 0.07 EF group 16 strain CCUG 3182 66.9 78.1 0.09 Unidentified strain CCUG 23153 65.5 78.2 0.06 Unidentified strain CCUG 21294 61.0 76.1 0.08 Cornamonas acidovorans Stanier 14T 66.6" 76.6 0.11 73.6' 0.07' 80.6" 0.12" 75.0b 0.15b 77 .Od 0.10d Comamonas terrigena NCIB 8193T 64.0d 75.5 0.20 73.9' 0.14' 759 0.19d 81.1' 0.32' 75.5" 0.16" Comamonas testosteroni NCTC 10698T 62.5" 75.9 0.19 75.6' 0.17' 76.5" 0.17" 76.7' 0.13' 76.5" 0.18" Hydrogenophaga Java DSM 619T 66.7" 75.1 0.06 78.2' 0.07' 75.5" 0.02" 76.0" 0.04' Hydrogenophaga palleronii Stanier 362t lT 67.3' 75.2 0.05 80.4' 0.07' 76.0" 0.05" 73 .4' 0.06' 77.5" 0.06' Hydrogenophaga pseudofava GA3T 66.4' 75.0 0.08 77.4' 0.09' 76.0' 0.08' 75.2' 0.09' Hydrogenophaga taeniospiralis DSM 2082T 64.8' 75.2 0.04 79.0' 0.05' 75.0' 0.04' [Pseudomonas] avenue NCPPB lollT 69.8' 76.6 0.08 75.6' 0.07' 76.6" 0.12" 75.7' 0.09' 76.5" 0.09' [Pseudomonas] saccharophila ATCC 15946T 69.1' 74.2 0.05 73.4' 0.05' 72.6" 0.05" 71.8' 0.05' 73 .4' 0.04h [Alcaligenes] latus Palleroni H-4T 70.0" 73.0 0.14 73 .4' 0. 10' 74.5" 0.12" 71.6' 0.15' 75 .Or 0.08' [Alcaligenes] paradoxus ATCC 17713tlT 67.0" 75.4 0.04 76.0' 0.03' 76.5" 0.03" 74.8' 0.05' 81.Od 0. 06" [Rhodocyclus] gelatinosus NCIB 8290T 71.9 73.4 0.04 71.9' 0.01' 74.5' 0.05" 73.5' 0.06' 71.0' 0.02' Xylophilus ampelinus NCPPB 2217= 68.5 76.8 0.09 75.8' 0.06' 76.0" 0.09" 75.6' 0. 10' 76.2' 0. 10' " Data from reference 12. Data from reference 53. Data from reference 54. Data from reference 14. Data from reference 26.

doxus ATCC 17713tlT were 63,000, 413,000, 47,000, values of 77.2"C or more versus rRNA from Acidovorax 107,000, and 7,000 dpm/pg of rRNA, respectively. The facilis ATCC 11228T. TmCe,values obtained for repre- results of the DNA:rRNA hybridization experiments are sentative strains belonging to the other rRNA subbranches shown in Table 2 and are presented as a dendrogram based in the acidovorans rRNA complex varied from 73.0 to on Tmce)values in Fig. 1. Our results allowed the delineation 76.8"C. The mean Tm(e)level at which the Acidovorax facilis of a new rRNA subbranch within the acidovorans rRNA rRNA subbranch is linked to the other rRNA subbranches of complex. This subbranch consists of 4 Acidovorax facilis the acidovorans rRNA complex is 76.0 +- 1.1"C (Fig. 1). strains, 9 Acidovorax delajieldii strains, 13 Acidovorax tem- DNA:DNA hybridizations. The results of DNA:DNA hy- perans strains, EF group 16 strain CCUG 3182, and uniden- bridization experiments are shown in Fig. 2. Three DNA tified strain CCUG 23153. All of these strains yielded Tm(e) homology groups were delineated. The first one was formed VOL. 40, 1990 ACZDOVORAX GEN. NOV. 389

ACIDOVORANS rRNA COMPLEX h I

I a I v) 0 3 < z X v) a a 3 U % g m U -Iz v a a: W a F Z a z -1 CE 2 ‘E 7 5 FE f zv) U W 80 5: AC. DElAFlELDll W U AC. TEMPERANS 0

3 3LL 8 d “I 1 C. TESTOSTERONI, [P.] AVENAE [R.] GELATINOSUS, [A.] LATUS [P.] SACCHAROPHllA

70 III

-1

60

FIG. 1. rRNA cistron similarity dendrogram of the acidovorans rRNA complex. Trn(=)values are from Table 2 and from references 12,53, and 54. The bars represent TrnCe,ranges within the individual rRNA branches. The roman numerals indicate the roots of the rRNA superfamilies sensu De Ley (8); I+II, 111, and IV correspond to the gamma, beta, and alpha subclasses, respectively, of the class Proteobacteria (48). A., Alcaligenes; AC., Acidovorax; C., Comamonas; H., Hydrogenophaga; P., Pseudomonas; R., Rhodocyclus; X., Xylophilus. Brackets indicate presently misnamed taxa.

ATCC 11 228T ATCC 15376 DSM 550 DSM 620 ATCC 1750611 ATCC 17506t2 ATCC 1750!jT ATCC 17508 CCUG 3746A AC. DELAFIELDII CCUG 1105611 CCUG 11062 CCUG 12929 CCUG 18251 CCUG 20074 CCUG 18750 CCUG 1177ST CCUG 10556 CCUG 21 688 CCUG 9443Aa CCUG 3688A CCUG 16573 C. TEMPERANS CCUG 17140 CCUG 22192 CCUG 22215 CCUG 17727A CCUG 23689 CIP 239.74 CIP 471.74 CCUG 3182 CCUG 23153 CCUG 21294 FIG. 2. Average degrees of binding obtained from DNA:DNA hybridizations between representative Acidovorax strains and possibly related strains. 390 WILLEMS ET AL. INT. J. SYST. BACTERIOL.

by the four Acidovorax facilis strains; the second one vos, 100 90 80 70 consisted of 10 Acidovorax delafieldii strains; and the third I I I 1 group comprised 14 Acidovorax temperans strains. Strains ATCC 17408 CCUG 3182, CCUG 21294, and CCUG 23153 did not belong C. ACIDOVORANS [ A~~~~ =- to any of these three groups, nor did they exhibit any CCUG 2748 r Nciearw - significant DNA binding with each other (Fig. 2). C. TESTOSTERONI 1 /Ei:tg Numerical analysis of phenotypic features. A total of 70 CCUG 12840 , NCTC 106W 3 strains, including 29 reference strains belonging to the aci- C. TERRIGENA dovorans rRNA complex, were compared by performing a CCUGl8750 CCUG 21 88B numerical analysis of auxanographic and biochemical fea- CIP 239.74 CIP 471.74 tures. For practical reasons explained previously (53), [Al- CCUG 11779' CCUG 10556 caligenes] latus Palleroni H-4T and Xylophilus ampelinus CCUG 18513 CCUG 17140 NCPPB 2217T were not included in the numerical analysis. AC. TEMPERANS CCUG 3888A Strains tested in duplicate clustered at an average S, of 95% CCUG 17727A (25 sets of comparisons). All strains clustered at S, values above 68.4%. The following phena were delineated at S, values above 83% (Fig. 3): Comamonas acidovorans; Co- mamonas terrigena and Comamonas testosteroni; Acidovo- CUETM 85-21 rax temperans and one phenotypically abberant Acidovorax delafieldii strain (strain CCUG 3746A); Hydrogenophaga palleronii; Acidovorax facilis and EF group 16 strain CCUG 3182; Acidovorax delafieldii and unidentified or misnamed strains CCUG 21294, CCUG 23153, CCUG 23155, and EF GROUP 16 - CCW 3182 CCUG 23156; [Alcaligenes] paradoxus; Hydrogenophaga pseudoJava; and a phenon formed by representative strains ATCC 175oBtl ATCC 175057 - of [Pseudomonas] avenue, [Pseudomonas] rubrilineans, CCUG 20074 LLIPseudomonas]setariae," [Pseudomonas] cattleyae, and CCUG 11062 1 CCUG 16518 [Pseudomonaspseudoalcaligenes] subsp. konjaci. [Pseudo- CCUG 16568 AC. DELAFlELDll CCUG m3oB CCUG 1 1056tl monas pseudoalcaligenes] subsp. citrulli PDDCC 7500T, # CCUG 24594 I another phytopathogenic [Pseudomonas] strain, is linked to CCUG 12828 CCUG 18251 l-- the latter phenon at an S, of 80.6%. The following strains CCUGzl35B - ATCC 17508 occupy separate positions in the dendrogram: [Rhodocyclus] - Ncmioiv NCPPB 8207 gelatinosus NCIB 8290T, HydrogenophagaJlavaDSM 619T, PHYTOPATHOGENIC NCPPB 13w [PSEUDOMONAS] NCPPB 881' and Hydrogenophaga taeniospiralis DSM 2082T. Pwcc rn' Protein gel electrophoresis. Thirty-one Acidovorax strains - wDcc7KIo' and five unidentified or misnamed strains were compared by [A.] PARADOXUS [ A$&?$G?ATCC 17!j4(kl '-9 using protein gel electrophoresis (Fig. 4). In a previous paper (53) we showed that the electrophoretic protein patterns of H. TAENlOSPlRALlS - [Pseudomonas] facilis and [Pseudomonas] delafieldii are H. PSEUDOFLAVA clearly different from those of other representative taxa belonging to the acidovorans rRNA complex. Therefore, we 100 90 70 did not include any reference strains from the acidovorans 8o %SG rRNA complex in this protein electrophoretic study. Repro- FIG. 3. Dendrogram obtained by unweighted average linkage ducibility was checked in the following ways: different runs cluster analysis of SG values, expressed as percentages. Each of the of the reference profile of Psychrobacter immobilis LMG 70 strains was characterized by 195 phenotypic features. For abbre- 1125 (not shown in Fig. 4) clustered at r > 0.95; repeated viations see the legend to Fig. 1. runs of any protein extract clustered at r 1 0.93; and duplicate protein extracts, prepared independently from five strains, clustered at r > 0.91. The protein patterns for the erence taxa). The common pattern was characterized by Acidovorax facilis and Acidovorax delajieldii strains were unbranched fatty acids with chain lengths ranging from 12 to visually similar, although not identical; the patterns for 14 18 carbon atoms; palmitoleic acid (16:1), palmitic acid (16:0), Acidovorax temperans strains also visually resembled each and cis-vaccenic acid (All-18:l) were the most abundant other, but they showed more variation. In the numerical constituents. The two hydroxylated acids, 3-hydroxyoc- analysis the four Acidovorax facilis strains clustered at r = tanoic acid (3-OH-8:O) and 3-hydroxydecanoic acid (3-OH- 0.93 and grouped together with two clusters of the 13 10:0), were present in significant amounts in all of the strains Acidovorax delafieldii strains tested at r = 0.90. The 14 of the proposed genus Acidovorax which we examined. Acidovorax temperans strains clustered at r = 0.87. The The fatty acid data for the Acidovorax strains were ana- following five strains occupied separate positions on the lyzed by using soft independent modeling of class analogy dendrogram: strains CCUG 21294, CCUG 3182, CCUG principal-component analysis. This technique transforms 23153, CCUG 23155, and CCUG 23156. similarities and dissimilarities among the individual strains Numerical analysis of gas chromatographic cellular fatty into distances in a two-dimensional plot (Fig. 5). The sepa- acid patterns. Table 3 shows the cellular fatty acid compo- ration along the first and second principal components sitions of the species examined, including the compositions accounted for 66 and 13% of the variation among the strains, of several reference taxa belonging to the acidovorans rRNA respectively, whereas the third principal component (data complex. We found only small and mostly quantitative not shown) accounted for 8%. The strains of Acidovorax differences among the individual species (including the ref- facilis and Acidovorax delafieldii formed a group on the left I L’O 8’0 6’0 O’t 0 n

L

S1113Vtl ‘3V

t U L’O 8’0 6.0 O’t 0 3 392 WILLEMS ET AL. INT. J. SYST.BACTERIOL.

TABLE 3. Cellular fatty acid compositions of Acidovorax facilis, Acidovorax delafieldii, Acidovorax temperans, Comamonas spp., Hydrogenophaga spp., [Alcaligenes]paradoxus, and [Pseudomonas] avenae

% of total in: Fatty Acidovorax Acidovorax Acidovorax Hydrogenophaga Comamonas [Alcaligenes] [Pseudomonas] acid facilis delafieldii temperans SPP. SPP. paradoxus avenae (n = 4)" (n = 9) (n = 12) (n = 13)b (n = (n = 3)b (n = 12:O" 2 .9-3 .4d 2.4-3.0 3.4-5.4 0-1.4 2.6-6.5 2.7-3.0 2.3 13:O ND 0-0.1 0.1-0.3 ND ND ND ND 14:O 2.8-3.6 2.9-3.9 0.6-1.9 0.2-4.7 0.5-3.9 0.6-1.0 2.4 15:1 0.1 0.1-0.2 0.1-0.5 tr-3.1 0-tr 0.1-0.7 ND 15:o 0.2-0.3 0.2-1.1 0.2-0.8 0.2-5.1 0.2-1.1 0.1-1.9 0.3 16:1 39.4-45.7 35.144.1 45.149.3 25.3-5 1.7 22.241.5 32.2-3 3.4 41.6 16:O 19 3-25.6 21.6-32.8 19.6-25.2 16.3-27.2 14.428.3 20.7-26.5 29.0 17:l ND 0-0.2 04.1 0-0.7 0-tr tr ND 17:O 0.1-0.2 0.1-0.3 0.1-0.4 . 0.2-7.1 tr-6.8 0.2-0.9 0.2 A9-18: 1" 0.2-0.6 0.1-0.9 0.1-0.3 0.1-0.7 ' 0-0.8 0.6-0.8 0.5 A1 1- 18 :lf 18.0-22.9 13.9-22.6 15.6-22.6 7.2-23.3 11.6-16.6 12.3-20.1 15.9 18:O 0.2-0.3 0.2-0.5 0.2-0.3 0.2-1.5 tr-0.8 0.3-0.5 0.3 17:cyc 0.1-0.9 0.1-1.3 0-0.1 tr-21.1 1.2-15.2 3.7-6.0 0.2 19:cyc ND ND ND 0-tr 0-tr ND ND 3-OH-8:O 1.9-2.6 1.42.5 1.0-2.3 1.0-5.6 0-2.3 1.9-2.3 ND 3-OH-lO:O 4.9-6.0 3.6-5.4 3.14.8 0-6.3 4.9-17.3 4.8-5.6 5.8 3-OH-18:O ND ND ND 0-tr ND ND ND 2-OH-12:O ND ND ND ND ND ND ND 2-OH-14:O ND ND ND ND 0-0.8 3.0 ND 2-OH-16:O ND ND ND 0-tr 0-6.9 ND ND 2-OH-18:O ND ND ND ND 0-1.7 ND ND

a n is the number of strains investigated; the type strain was included for each species. Data from reference 53. Number of carbon atoms:number of double bonds. Range among strains. tr, Less than 0.1%; ND, not detected. Oleic acid. cis-Vaccenic acid. differentiated phenotypically from the other taxa in the tained with the S1 nuclease method, our results are for the acidovorans rRNA complex (Table 4). most part slightly lower. It is our experience that strains with The separate position of the genus Acidovorax within the a high level of rRNA cistron similarity and no significant acidovorans rRNA complex was also confirmed by the DNA binding (initial renaturation rate method) can represent results of studies on the regulation of aromatic amino acid at least separate species (41, 53). Therefore, our results biosynthesis (3) and by the results of DNA:DNA hybridiza- indicate that Acidovorax facilis, Acidovorax delafeldii, and tion experiments (1, 25, 40). Acidovorax temperans are separate genotypic entities at the The division of the genus Acidovorax into three species, species level. Acidovorax facilis, Acidovorax delajieldii, and Acidovorax Numerical analysis of 195 phenotypic features (Fig. 3) temperans (Table l), is based on the results of DNA:DNA confirmed that the Acidovorax strains constitute three dis- hybridization experiments and phenotypic tests. We delin- tinct phena, which are clearly different from the reference eated three separate DNA groups, which showed no signif- taxa and correspond to the three DNA homology groups. icant levels of DNA binding with each other (Fig. 2). The Phenotypically, Acidovorax facilis and Acidovorax delajiel- first DNA group, corresponding to Acidovorax facilis, con- dii are more similar to each other (SG, 83%) than to Acidovo- tains only four former [Pseudomonas] facilis strains. The rax temperans (SG, 78%). When creating [Pseudomonas] second DNA group, corresponding to Acidovorax delajiel- delafeldii, Davis et al. (5) described this species as very dii, contains most of the former [Pseudomonas] delajieldii similar to [Pseudomonas]facilis and recommended 12 fea- strains, most of the EF group 13 strains, and one EF group tures for the differentiation of these two species. Nine of 16 strain. The third DNA group, corresponding to Acidovo- these features were also included in our phenotypic analysis, rax temperans, contains most of the EF group 16 strains, one but we found that only three of them (growth on 2-ketoglu- EF group 13 strain, and various presently misnamed or conate, adipate, and citraconate) are valid for discriminating unnamed strains. The levels of DNA homology between between Acidovorax facilis and Acidovorax delajieldii. Dif- [Pseudomonas] delafieldii and [Pseudomonas]facilis have ferentiating features for the three Acidovorax species are been reported to be 49% (S1 nuclease method [25]), 83% shown in Table 5. (direct binding method [40]), and 42 and 100% (competition The close relationship between Acidovorax facilis and method [40]); all of these values were determined at a Acidovorax delafieldii was also apparent from the results of temperature which was 25°C below the thermal denaturation protein gel electrophoresis and fatty acid analysis. In the temperature. Using the initial renaturation rate method at dendrogram in Fig. 4, Acidovorax facilis forms a separate the optimal renaturation temperature (16°C below the ther- group within the Acidovorax delafieldii cluster at r = 0.90 mal denaturation temperature) we found no significant DNA (Fig. 4). The Acidovorax temperans strains form a separate binding between representative strains of these two species. and more diffuse cluster at r = 0.87. In the score plot Although we do not yet know the exact correlation between obtained by principal-component analysis of the fatty acid the results obtained with our method and the results ob- data, the Acidovorax facilis and Acidovorax delafeldii VOL.40. 1990 ACIDOVORAX GEN. NOV. 393

A

0

0 0. 0 A 0 0 8 A 0 0

0

0

0 0

A A

SCORES ON PC 1 FIG. 5. Score plot of Acidovorax strains, based on cellular fatty acid data. PC, Principal component. Symbols: 0,Acidovoraxfacilis; A, Acidovorax delafieldii; e, Acidovorax temperans. strains form a single group on the left side, while the tested does correlate with the general pattern of Acidovorax Acidovorax temperans strains form a group on the right side delafieldii. Furthermore, the protein gel electrophoresis re- (Fig. 5). sults (Fig. 4) and the fatty acid analysis results (Fig. 5) Strain CCUG 3746A belongs to the Acidovorax delafieldii exclude strain CCUG 3746A from Acidovorax temperans. DNA group (Fig. 2). In the dendrogram based on the Therefore, we assigned strain CCUG 3746A to Acidovorax phenotypic analysis, however, it is in the Acidovorax tem- delafieldii as a slightly aberrant strain. perans cluster (Fig. 3). This aberration was caused by the Strains CCUG 3182, CCUG 21294, and CCUG 23153 have inability of this strain to use L-arabinose, D-galactose, D- T,(,, values of 78.1, 76.1, and 78.2"C, respectively, versus mannose, and D-ribose as sole carbon sources. These four rRNA from Acidovorax fucilis ATCC 11228T. In the dendro- sugars are normally used by Acidovorax delafieldii (and gram of the phenotypic analysis (Fig. 3) strain CCUG 3182 Acidovorax facilis), but not by Acidovorax temperans (Table groups with the Acidovorax facilis phenon at an SG of 87%, 5). We repeated the auxanographic tests for this strain three while strains CCUG 21294 and CCUG 23153 are located in times and consistently obtained the same results. The assim- the Acidovorax delafieldii phenon (Fig. 3). However, these ilation pattern of strain CCUG 3746A for the other substrates strains could not be assigned to any of the three DNA groups

TABLE 4. Differentiating characteristics for the genus Acidovorax and related taxa belonging to the acidovorans rRNA complex Growth on: Nondiffusible G+C vs rRNA from D-Melibiose, L-Arabitol, yellow value Acidovorax facilis Taxon D- D- M'toseq D-ranose, xylitol, pigment (mol%)a ATCC 11228T ("C) Glucose Xylose sucrose glucosamine 5-ketogluconate

Acidovorax +b - - - 62-66 77.2-80.6 Hydrogenophaga + D D - 6569 76.0 k 1.0 Comamonas - - - - 60-69 76.0 * 1.0 [Alcaligenes]paradoxus + + - - 6769 76.0 2 1.0 - - [Pseudomonas] avenue + + 67-70 76.0 _+ 1.0 [Pseudomonas] saccharophila + + + + 69 73.1 2 1.4

a As determined by the thermal denaturation method. +, Present in 90% or more of the strains; -, present in ~WGor less of the strains; D, different reactions occur in different species. 394 WILLEMS ET AL. INT. J. SYST.BACTERIOL.

TABLE 5. Differentiating characteristics for the three Acidovorax species

Nitrite Autotrophic growth Species 2-Ketog1uconatey -_>.- __ Gelatinase L-Arabinose, -nlnnr-n- -.. with hydrogen D-ribose, r

~ Acidovorax facilis +a - - - + + Acidovorax delafieldii + + + d d d Acidovorax temperans - + d + - - +, Present in 90% or more of the strains; -, present in 10% or less of the strains; d, present in 11 to 89% of the strains.

(Fig. 2), and protein electrophoretically they are different voracious; M.L. masc. n. Acidovorax, acid devouring [bac- from the Acidovorax species (Fig. 4). They may belong to teria]). Cells are straight to slightly curved rods, 0.2 to 0.7 by the genus Acidovorax, but they cannot be assigned to any of 1.0 to 5.0 pm. They occur singly or in short chains and are the species described here. motile by means of a single polar . Gram negative. Two strains isolated from water in France, [Pseudomonas Oxidase positive. Urease activity varies among strains. delafieldii] CCUG 23155 and CCUG 23156, also occupy Some strains grow on Christensen urea agar, but lack urease separate positions in the dendrogram obtained by numerical according to API 20NE tests. No pigment is produced on analysis of the electrophoretic data (Fig. 4). Linked to all nutrient agar. Aerobic. Chemoorganotrophic. Acidovorax other strains at r values of 0.80 and 0.70, respectively, they facilis and several Acidovorax delafieldii strains are capable are the two most aberrant strains which were included in the of lithoautotrophic growth by using hydrogen as an energy analysis. In the dendrogram of the phenotypic analysis these source. Oxidative carbohydrate metabolism occurs with strains group in the Acidovorax delafieldii phenon (Fig. 3). oxygen as the terminal electron acceptor; alternatively, The protein patterns of both strain CCUG 23155 and strain some strains of Acidovorax delafieldii and Acidovorax tem- CCUG 23156 have a heavy protein band in the upper part of perans are capable of heterotrophic denitrification of nitrate. the gel, but their overall pattern visually resembles that of Good growth is obtained on media containing organic acids, Acidovorax delajieldii (Fig. 4). Both strains probably belong amino acids, or peptone, but only a limited number of sugars to the genus Acidovorax, but our present data do not allow are used for growth; but 54% of the acids are consumed, assignment to any species. DNA:DNA hybridization data compared with about 22% of the carbohydrates (Table 6). will be required to provide decisive evidence. Two hydroxylated fatty acids, 3-hydroxyoctanoic acid (3- Rode and Giffhorn (42) isolated D-tartrate-utilizing strain OH-8:O) and 3-hydroxydecanoic acid (3-OH-10:0), are al- G2 from water samples from a pond near Gottingen, Federal Republic of Germany, and identified it as a Pseudomonas ways present, 2-hydroxylated fatty acids are absent, and a strain. Later, Auling et al. (2) positively identified this strain cyclopropane-substituted fatty acid (17:cyc) is present in as [Pseudomonas] delafieldii by means of DNA:DNA hy- most of the strains. The mean G+C values of the DNAs bridization (degree of binding with [Pseudomonas] range from 62 to 66 mol%. In DNA-rRNA hybridizations all delafieldii ATCC 17505T, 57% [optical renaturation rate strains belonging to this new genus group in a Tm(e)range method]). We did not include strain G2 in our study, but it is from 77.2 to 80.6”C versus rRNA from Acidovorax facilis evident that this organism belongs in Acidovorax delafieldii. ATCC 11228T. The genus Acidovorax belongs to the aci- Its phenotypic features, mean G+C value (63.9 mol%), and dovorans rRNA complex and is equidistantly related to the fatty acid composition (2) match those of Acidovorax other taxa in this complex (e.g., Hydrogenophaga, Corna- delafieldii, except that strain G2 does not use adipate. monas, Xylophilus, [Alcaligenes]paradoxus, and [Pseudo- Hydrogen oxidation, one of the main differentiating fea- monas] avenue). The type species is Acidovorax facilis tures between [Pseudomonas]facilis and [Pseudomonas] (Schatz and Bovell 1952) comb. nov. delafieldii, is effected also by several Acidovorax delafieldii The descriptions of the morphological, biochemical, and strains, but not by Acidovorax temperans. In the past the nutritional features of the former species [Pseudomonas] taxonomic value of this feature was sometimes overesti- facilis and [Pseudomonas]delajieldii given by Davis et al. (5) mated. Hydrogen oxidation has been the subject of exten- and by Palleroni (36) were verified and were found to sive research. Schneider and Schlegel (45) reported that correlate fairly well with our own findings. However, be- [Pseudomonas]facilis contains only a membrane-associated cause Acidovorax delafieldii contains considerably more hydrogenase and no soluble hydrogenase. Although it was strains than [Pseudomonas]delajieldii, its description needs first assumed that hydrogen oxidation is plasmid encoded in to be updated. To facilitate comparison with the other two [Pseudomonas]facilis (20, 38), an analysis of plasmid DNA Acidovorax species described here, we also provide a de- restriction patterns showed that this is probably not so (52). tailed description of Acidovorax facilis below. Of the three Several Acidovorax delafieldii and Acidovorax temperans species which we included in the genus Acidovorax, Aci- strains have been isolated from clinical specimens. We have dovorax delafieldii is metabolically the most versatile, and no evidence for any pathogenicity in healthy humans, nor Acidovorax facilis is the least versatile. has a role for these organisms as opportunistic pathogens Description of Acidovorux fucilis (Schatz and Bovell 1952) been proven or rejected. The high level of relationship comb. nov. The description of Acidovorax facilis (fa’ci.lis. L. between soil and water isolates and isolates from various adj. facilis, ready, quick, referring to the ease of cultivation samples from clinical environments in the genus Acidovorax of the organism) is the same as that for the genus. Additional is striking. Comparable isolation sources have been reported biochemical and nutritional features are shown in Table 6. for Comamonas strains (14, 17, 21). Acidovorax facilis was isolated from lawn soil in the United Description of Acidovorax gen. nov. Acidovorax States (44). The mean G+C contents of the DNAs range (A.ci.do’vo.rax. L. neut. n. acidum, acid; L. adj. vorax, from 64 to 65 mol%. The type strain is strain ATCC 11228 VOL. 40, 1990 ACIDOVORAX GEN. NOV. 395

TABLE 6. Physiological, biochemical, and nutritional characteristics of Acidovorax species Acidovorax facilis Acidovorax delafieldii Acidovorax temperans Characteristic” No. of strains Result for No. of strains Result for No. of strains Result for positiveho. of type strain positiveho. of type strain positiveho. of type strain strains tested ATCC 11228 strains tested ATCC 17505 strains tested CCUG 11779 Growth at 37°C 314 14/14 + 18/18 Growth at 42°C 014 3/14 + 3/18 Growth in the presence of 1.5% NaCl 213 8/14 - 9/18 Growth on Drigalski-Conradiagar 014 12/14 + 15118 Catalase 414 7/13 + 12/18 Hydrolysis of Tween 80 314 14/14 + 17/17 Gelatinase 414 7/14 - 0/18 Nitrate reduction 414 14/14 + 17/18 Nitrite reduction 014 3/13 - 16/17 Denitrification 014 3/14 - 12/18 Autotrophic growth with hydrogen 414 9/14 - 0118 Resistance to penicillin (10 pg) 314 11/14 + 15/18 Growth on: D-Mannose 414 14/14 + 0118 L-Arabinose, D-ribose, D-galactose 414 13/14 + 0118 D-Mannitol, D-arabitol 414 13/14 + 10118 Sorbitol 414 13114 + 9118 D-Fucose 014 13/14’ + 0118 D-Fructose 414 14/14 + 17/18 Gluconate 414 14/14 + 11/18 2-Ketogluconate 014 14/14 + 2/18 D-Lactate, D-lactate + methionine 314 14/14 + 17/17 D-a- Alanhe 214 9/14 - 5/18 L-a-Alanine, L-threonine 414 13/14 + 13/18 L-Leucine 414 13/14 + 15118 L-Isoleucine 414 13/14 + 12/18 L-Norleucine 214 10114 - 12/18 DL-Norleucine 014 8/14 + 10118 L-Valine 414 8/14 + 10118 DL-Norvaline 014 0114 - 6/18 DL-2-Aminobutyrate 014 0114 - 4118 L-Serine 414 13/14 + 8/18 L-Methionine 114’ 0114 - 2/18 L-Phenylalanine 414 14/14 + 16/18 L-Tyrosine 414 14/14 + 14/18 L-Histidine 414 14/14 + 11/18 L-Tryptophan 414’ 2/14 - 0118 L-Aspartate 414 13/14 + 14/18 L-Ornithine 114‘ 8/14 + 6/18 L-Citrulline 014 4/14 + 10118 P-Alanine 414 14/14 + 7/18 DL-4-Aminobut yrate 414 14/14 + 11/18 Acetate 214 11/14 - 17/18 Propionate 414’ 10114 - 14/18 Isobutyrate 014 9/14 - 13/18 n-Valerate 414 13/14 + 17/18 Isovalerate 414’ 14/14 + 17118 n-Caproate 014 11/14 - 15/18 Heptanoate 014 0114 - 5/18 Maleate 014 9/14 - 5/18 Fumarate, L-malate 414 14/14 + 17/18 Glutarate 314 14/14 + 14/18 Adipate 014 14/14 + 18/18 Pimelate 314 14/14 + 18/18 DL-Glycerate 414 13/14 + 16/18 D-Tartrate 014 5/14 + 0118 meso-Tartrate 014 13/14 + 7/18 Pyruvate 414 12/14 + 14/18 Levulinate 114 6/14 - 15/18 2-Ketoglutarate 014 14/14’ + 4/18 Citraconate 014 14/14 + 6/18 Aconitate 014 11/14 - 0118 Citrate 014 2/14 - 0118 m-H ydrox ybenzoate 014 12/14 - 0118 p-H ydrox ybenzoate 214 14/14 + 3/18 Continued on following page 396 WILLEMS ET AL. INT.J. SYST.BACTERIOL.

TABLE &Continued

~~ ~~ ~~ ~ Acidovorax facilis Acidovorax delafieldii Acidovorux temperans Characteris tic” No. of strains Result for No. of strains Result for No. of strains Result for positiveho. of type strain positiveho. of type strain positiveho. of type strain strains tested ATCC 11228 strains tested ATCC 17505 strains tested CCUG 11779 Phthalate 014 - 8/14 - 0118 - Hydrolysis of 2-Naphthylphosphate (pH 8.5) 314 + 8/14 + 13/18 - 2-Naphthylphosphate (pH 5.4) 114 + 0114 - 0118 - 2-Naphthylmyristate 214 - 4/14 - 6/18 - ~-Cystyl-2-naphthylamide 014 - 4/14 - 4/18 -

a All Acidovorax strains are positive for the following characteristics: growth at 30°C; growth in the presence of 0.5% NaCl; growth on D-glucose, glycerol, butyrate, succinate, suberate, azelate, sebacate, m-lactate, D-malate, ~~-3-hydroxybutyrate,L-proline, and L-glutamate; hydrolysis of 2-naphthylbutyrate, 2-naphthylcaprylate, and L-leucyl-2-naphthylamide. All Acidovorax strains are negative for the following characteristics: growth in the presence of 3,4.5, or 6.5% NaCl; hemolysis; acid production in 10% lactose, in triple sugar iron medium, and in oxidative-fermentative medium containing D-glucose, D-fructose, D-xylose, maltose, or adonitol; production of H,S in triple sugar iron medium; amylase (not tested for strain CCUG 22192); hydrolysis of esculin, DNA (not tested for strain ATCC 15376), and acetamide (not tested for strains ATCC 15376 and CCUG 3746A); lysine and ornithine decarboxylases; arginine dihydrolase; p-galactosidase; fluorescein production; growth on cetrimide; indole production; growth on erythritol, D-arabinose, D-xylose, L-xylose, L-sorbose, L-rhamnose, adonitol, dulcitol, inositol, xylitol, L-arabitol, methyl-D-xyloside, methyl-D-mannoside, methyl-D-glucoside, N-acetylglucosamine, D-cellobiose, maltose, lactose, D-melibiose, sucrose, trehalose, p-gentiobiose, D-rnelezitose, D-raffinose, D-turanose, D-lyXOSe, D-tagatose, L-fucose, amygdalin, esculin, salicin, arbutin, inulin, starch, glycogen, 5-ketogluconate, glycolate, caprylate, pelargonate, caprate, oxalate, malonate, L-tartrate, itaconate, mesaconate, phenylacetate, benzoate, o- hydroxybenzoate, D-mandelate, L-mandelate, isophthalate, terephthalate, glycine, L-cysteine, D-tryptophan, L-lysine, L-arginine, trigonelline, DL-kynurenine, betaine, creatine, ethylamine, butylamine, amylamine, ethanolamine, benzylamine, diaminobutane, ~~-3-aminobutyrate,~~-S-aminovalerate, 2-aminobenzoate, 3-aminobenzoate, 4-aminobenzoate, urea, acetamide, sarcosine, spermine, histamine, tryptamine, and glucosamine; hydrolysis of ~-valyl-2-naphthylamide, N-benzoyl-~~-arginine-2-naphthylamide,N-glutaryl-phenylalanine-2-naphthylamide, 6-bromo-2-phosphodiamide-3-naphthoic acid-2-methoxyanilide (= naph- thol-AS-BI-phosphodiamide),6-bromo-2-naphthyl-a-~-galactopyranoside, 2-naphthyl-P-~-galactopyranoside, 6-bromo-2-hydroxy-3-naphthoic acid-2-methoxy- anilide-p-D-glucuronate (= naphthol-AS-BI-P-D-glucuronate),2-naphthyl-a-~-glucopyranoside, 6-bromo-2-naphthyl-P-~-glucopyranoside, 1-naphthyl-N-acetyl- P-D-glucosaminide, 6-bromo-2-naphthyl-a-~-mannopyranoside,and 2-naphthyl-a-~-fucopyranoside. Late reaction (positive after 3 to 5 days).

(= LMG 2193 = CCUG 2113); the mean G+C value of its ACKNOWLEDGMENTS DNA is 65 mol%. We thank API System S. A., Montalieu-Vercieu, France, for Description of Acidovorax deZaBeZdii (Davis, Stanier, Dou- supplying API galleries. E.F. gratefully acknowledges the qualified doroff, and Mandel 1970) comb. nov. The description of work at the bench of Lars Nehls, Kaety Plos, Ann Borjesson, Marie Acidovorax delafieldii (de.1a.fiel’di.i. M.L. gen. n. delufiel- Blomqvist, Eva Gunnarsson, and Gun Svensson. We thank all dii, of Delafield, named after F. P. Delafield, who first microbiologists who submitted interesting strains for identification. isolated these bacteria) is the same as that for the genus. J. De Ley and K.K. are indebted to the Fonds voor Geneeskundig Additional biochemical and nutritional features are shown in Wetenschappelijk Onderzoek, Belgium, for research and personnel Table 6. Acidovorax delafieldii strains were isolated from grants. A.W. is indebted to the Nationaal Fonds voor Wetenschap- soil, water, and various samples from clinical environments. pelijk Onderzoek, Belgium, for a position as Research Assistant. B.P. is indebted to the Instituut tot Aanmoediging van het Weten- The mean G+C contents of the DNAs are 65 to 66 mol%. schappelijk Onderzoek in Nijverheid en Landbouw, Belgium, for a Strain ATCC 17505 (= LMG 5943 = CCUG 1779), the type scholarship. Part of this research was carried out within the frame- strain of the former species [Pseudomonas]delajieldii, is the work of contract BAP-0138-B with the Biotechnology Action Pro- type strain of Acidovorax delafieldii. It was isolated from soil gram of the Commission of European Communities. with PHB as the sole carbon source (6). The mean G+C value of its DNA is 66 mol%. Since strain ATCC 17505T is LITERATURE CITED not the most representative strain of the newly described 1. Auling, G., M. Dittbrenner, M. Maarzahl, T. Nokhal, and M. species, the centrotype strain of the Acidovorax delajieldii Reh. 1980. Deoxyribonucleic acid relationships among hydro- cluster from our phenotypic analysis, strain CCUG 23830B gen-oxidizing strains of the genera Pseudomonas, Alcaligenes, (= LMG 8909), is a more suitable reference strain for this and Paracoccus. Int. J. Syst. Bacteriol. 30:123-128. species. 2. Auling, G., A. Probst, and R. M. Kroppenstedt. 1986. Chemo- Description of Acidovorax temperans sp. nov. The descrip- and molecular of D( -)-tartrate-utilizing pseudomo- nads. Syst. Appl. Microbiol. 8:114-120. tion of Acidovorax temperans (tem’pe.rans. L. v. temper- 3. Byng, G. S., J. L. Johnson, R. J. Whitaker, R. L. Gherna, and are, to moderate; M.L. pres. part. temperans, moderate, R. A. Jensen. 1983. The evolutionary pattern of aromatic amino referring to the moderate metabolic versatility of the species) acid biosynthesis and the emerging phylogeny of pseudomonad is the same as that for the genus. Biochemical and nutritional bacteria. J. Mol. Evol. 19:272-282. features are shown in Table 6. Acidovorax temperans strains 4. Davis, D. H., M. Doudoroff, R. Y. Stanier, and M. Mandel. 1969. were isolated from various samples from clinical environ- Proposal to reject the genus Hydrogenomonas: taxonomic im- ments; one strain was isolated from active sludge from a plications. Int. J. Syst. Bacteriol. 19:375-390. wastewater purification plant in Sweden. The mean G+C 5. Davis, D. H., R. Y. Stanier, M. Doudoroff, and M. Mandel. 1970. contents of the DNAs are 62 to 66 mol%. The centrotype Taxonomic studies on some Gram negative polarly flagellated “hydrogen bacteria” and related species. Arch. Mikrobiol. strain from the Acidovorax temperans cluster in our pheno- 70:1-13. typic analysis, strain CCUG 11779 (= LMG 7169), is the 6. Delafield, F. P., M. Doudoroff, N. J. Palleroni, C. J. Lusty, and type strain. It was isolated in 1981 from a urine sample of a R. Contopoulos. 1965. Decomposition of poly-p-hydroxybutyr- 68-year-old male patient in Goteborg, Sweden. The mean ate by pseudomonads. J. Bacteriol. 90:1455-1466. G+C value of its DNA is 62 mol%. 7. De Ley, J. 1970. Reexamination of the association between VOL. 40, 1990 ACIDOVORAX GEN. NOV. 397

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