INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1991, p. 427-444 Vol. 41, No. 3 0020-7713/91/030427- 18$02 .OO/O Copyright 0 1991, International Union of Microbiological Societies

Polyphasic Taxonomic Study of the Emended Genus : Relationship to Aquaspirillum aquaticum, E. Falsen Group 10, and Other Clinical Isolates A. WILLEMS,l B. POT,l E. FALSEN,2 P. VANDAMME,' M. GILLIS,l* K. KERSTERS,l AND J. DE LEY' Laboratorium voor Microbiologie en Microbiele Genetica, Rijksuniversiteit, B-9000 Ghent, Belgium, and Culture Collection, Department of Clinical Bacteriology, University of Goteborg, S-413 46 Goteborg, Sweden2

We used DNA-rRNA hybridization, DNA base composition, polyacrylamide gel electrophoresis of whole-cell proteins, DNA-DNA hybridization, numerical analysis of phenotypic features, and immunotyping to study the of the genus Comamonas. The relationships of this genus to Aquaspirillum aquaticum and a group of clinical isolates (E. Falsen group 10 [EF lo]) were studied. Our DNA and rRNA hybridization results indicate that the genus Comamonas consists of at least the following five genotypic groups: (i) Comamonas acidovoruns, (ii) Comamonas fesfosferoni,(iii) Comamonas ferrigena, (iv) A. aquaticum and a number of EF 10 strains, and (v) other EF 10 strains, several unnamed clinical isolates, and some misnamed strains of alcaligenes and Pseudomonas pseudoalcaligenes subsp. pseudoalcaligenes. The existence of these five groups was confirmed by the results of immunotyping and protein gel electrophoresis. A numerical analysis of morpho- logical, auxanographic, and biochemical data for the same organisms revealed the existence of three large phena. Two of these phena (C. acidovorans and C. tesfosferoni)correspond to two of the genotypic groups. The third phenon contains strains belonging to the other three genotypic groups, including most EF 10 strains and the type strains of C. ferrigena and A. aquaticum. The strains belonging to the third phenon were all incorporated into C. ferrigena, and we propose that the use of the name Aquaspirillum aquaticum should be discontinued. Emended descriptions of the genus Comamonas and C. ferrigena are presented.

De Vos et al. (9) revived the genus Comamonas and its [Pseudomonas alcaligenes] CUETM 25-3, and a number of type species, Comamonas terrigena, in a study in which unidentified clinical isolates from the Culture Collection of they performed DNA-rRNA and DNA-DNA hybridization the University of Goteborg (CCUG), Goteborg Sweden; 33 experiments, immunotyping experiments, gel electrophore- of the unidentified clinical strains were previously grouped sis of total cellular proteins, and a numerical analysis of by using serological techniques (10) and are referred to as E. phenotypic features. C. terrigena was shown to form a Falsen group 10 (EF 10) (11). separate taxon within the acidovorans rRNA complex in rRNA superfamily I11 (i.e., the beta subclass of the Proteo- [37]). This species can be differentiated phenotypi- MATERIALS AND METHODS cally from its nearest neighbors, such as [Pseudomonas] acidovorans and [Pseudomonas]testosteroni (9); the latter Bacterial strains. The strains which we used are listed in two species are generically misnamed according to present Table 1. Most of these strains were grown on nutrient agar phylogenetic data (misnamed taxa are enclosed in brackets). (0.1% [wthol] beef extract, 0.2% [wt/vol] yeast extract, On the basis of the results of studies in which several 0.5% [wthol] NaCl, 0.5% [wt/vol] peptone, 2% [wt/vol] Aquaspirillum techniques were used, Tamaoka et al. (38) later transferred agar; pH 7.4); the exceptions were strains of Leptothrix cholodnii, Rubrivivax gelatinosus, Xy- [Pseudomonas]acidovorans and [Pseudomonas]testoster- spp., and lophilus ampelinus, oni to the genus Comamonas as which were grown on the media de- and , respectively. For a more scribed previously (44). Most strains were grown at 28°C; the detailed review of the history of the genus Cornamonas see exceptions were X. ampelinus NCPPB 2217T (T = type [Aquaspirillum] references 9 and 42. strain), which was grown at 24"C, and psychrophilum In this paper we describe the results of a study of the IF0 13611T, which was grown at 18°C. genus Comamonas in which we performed DNA-rRNA and Morphological and biochemical characteristics. We used DNA-DNA hybridization experiments, determined DNA the methods described by De Vos et al. (9) to determine base compositions, and conducted immunotyping experi- morphological and biochemical characteristics. Nitrite re- ments, a numerical analysis of morphological, physiological, duction was tested as described by Rossau et al. (34). and biochemical characteristics, and a numerical analysis of Carbon substrate assimilation tests. API galleries (API protein gel electrophoretic patterns. On the basis of serolog- 50CH, API 50A0, and API 50AA; API System S.A., Mon- ical results and results of routine phenotypic analyses, a talieu-Vercieu, France) were used to test the assimilation of number of strains seemed to be highly related to the genus 147 organic compounds as sole carbon sources. The exper- Comamonas. These strains included [Pseudomonas imental procedure which we used has been described previ- pseudoalcaligenes] CUETM 85-15 and CUETM 85-24, ously (19). Numerical analysis of phenotypic characteristics. The re- sults of auxanographic and biochemical tests for 79 Coma- monas or possible Comamonas strains and 23 reference * Corresponding author. strains belonging to the acidovorans rRNA complex were

427 428 WILLEMS ET AL. INT. J. SYST.BACTERIOL.

TABLE 1. Strains used

~~ Other Source, place, and year Name Strain" strain designation(s) of isolation Assigned to C. acidovorans Comamonas acidovorans Stanier 14T LMG 1226T, ATCC 15668T, Soil enriched with acetamide, Delft, The Nether- CCUG 14481T (= LMG lands. 1926 6031T = LMG 8911T), CCUG 12692T (= LMG 8910T) C. acidovorans ATCC 9355tlb LMG 1801tl,b CCUG 1822 Soil enriched with indole C. acidovorans ATCC 15005 LMG 1802, CCUG 15338 Soil C. acidovorans ATCC 17406 LMG 1790, CCUG 15340 Soil enriched with p-hydroxybenzoate C. acidovorans ATCC 17476 LMG 1791, CCUG 15337 Great Britain C. acidovorans CCUG 274B LMG 7098 Urine, 60-yr-old male, Goteborg, Sweden, 1968 C. acidovorans CCUG 536tlb LMG 5931tl,b NCTC 9991 Pharyngeal swab (received as Comamonas perco- lans) C. acidovorans CCUG 727 LMG 5932, CIP 60.78, Pharyngeal biopsy specimen ATCC 17439 C. acidovorans CCUG 1686 LMG 7099 Sludge, enrichment on testosterone, Goteborg, Sweden, 1972 C. acidovorans CCUG 1711 LMG 7100 Pus, Goteborg, Sweden, 1972 C. acidovorans CCUG 2861 LMG 8926, ATCC 11299a, Colony variant of strain ATCC 11299 NCIB 9289 C. acidovorans CCUG 10545 LMG 7101 Tap water, Goteborg, Sweden, 1981 C. acidovorans CCUG 10726B LMG 7102 Contact lens, Goteborg, Sweden, 1981 C. acidovorans CCUG 15835 LMG 7103 Eye of newborn boy, Goteborg, Sweden, 1984 C. acidovorans CCUG 18325 LMG 8912 Tap water, Goteborg, Sweden, 1986 C. acidovorans CCUG 18417 LMG 7185 Industry water C. acidovorans CCUG 21074 LMG 8914 Urine, Goteborg, Sweden, 1987 C. acidovorans CCUG 23474 LMG 9146 Urine, Goteborg, Sweden, 1988 [Bordetella bronchiseptica] LRA 147.04.76 LMG 2842 Unknown [Achromobacter cystinovorum] NCIB 4854 LMG 1795, CCUG 2485 Soil, Nottingham, Great Britain, before 1936 Assigned to C. testosteroni Comamonas testosteroni NCTC 10698= LMG 1786T, CCUG 1426T, Soil, Berkeley, Calif. ATCC 11996T C. testosteroni ATCC 17407 LMG 1787, CCUG 15341 Soil enriched with anthranilate C. testosteroni ATCC 17409 LMG 1788, CCUG 15339 Soil enriched with kynurenate, Berkeley, Calif., 1963 C. testosteroni ATCC 17510tlb LMG 1789tl,b CCUG 14480 Soil enriched with poly-P-hydroxybutyrate, Berkeley, Calif., 1961 C. testosteroni ATCC 17510t2' LMG 1789t2,' CCUG 14480 Soil enriched with poly-P-hydroxybutyrate, Berkeley, Calif., 1961 C. testosteroni CCUG 178A LMG 7104 Pus, Goteborg, Sweden, 1968 C. testosteroni CCUG 1135 LMG 5933 Kidney, 26-yr-old woman, Goteborg, Sweden, 1971 C. testosteroni CCUG 1689 LMG 9290 Mud enriched with testosterone, Goteborg, Swe- den, 1972 C. testosteroni CCUG 4381 LMG 7105 Urine, Heidelberg, Federal Republic of Germany, 1975 C. testosteroni CCUG 12941 LMG 9386 Bronchial aspirate, United States, 1982 C. testosteroni CCUG 14479 LMG 7106, ATCC 15667 Delft, The Netherlands EF 10 CCUG 13889 LMG 6140, LRA 57.3.76 Unknown (received as Pseudomonas testosteroni) Assigned to C. terrigena Group 1' Comamonas terrigena NCIB 8193T LMG 1253T, CCUG 2MT Hay infusion filtrate, United States (= LMG 5929T), CCUG 15327T, ATCC 8461T C. terrigena NCIB 2581 LMG 1249, CCUG 2474 Soil C. terrigena NCIB 2582 LMG 1251, CCUG 2475, Soil ATCC 14636 C. terrigena CCUG 12940 LMG 5520, G-4425 Blood, 1982 EF 10 CCUG 4470 LMG 6164 Buccal cavity, man, Heidelberg, Federal Republic of Germany, 1975 Unidentified CCUG 17736 LMG 6733 Horse blood, Sweden, 1985 Group 2 [Aquaspirillum aquaticum] ATCC 11330T LMG 2370T, CCUG 1739ST Fresh water EF 10 CCUG 1192 LMG 5937 Clinical laboratory, University of California, Los Angeles (received as P. alcaligenes) EF 10 CCUG 2632tlb LMG 6162tl' Metal-cutting fluid, Goteborg, Sweden, 1973 EF 10 CCUG 8404 LMG 6163 Human, Heidelberg, Federal Republic of Ger- many, 1979 Continued on following page VOL.41, 1991 COMAMONAS TAXONOMY 429

TABLE 1-Continued

~~ strain Name Strain“ Other Source, place, and year designation(s1 of isolation EF 10 CCUG 9672 LMG 6015, LMG 9569 Human, Heidelberg, Federal Republic of Germany, 1980 EF 10 CCUG 9673 LMG 6016 Human, Heidelberg, Federal Republic of Germany, 1980 EF 10 CCUG 10330 LMG 6017 Pharyngeal mucosa of sclerosis patient, Heidelberg, Federal Republic of Germany, 1980 EF 10 CCUG 10475 LMG 5930 Urine, 74-yr-old woman, Goteborg, Sweden, 1980 EF 10 CCUG 15504 LMG 6112 Urine, Nice, France, 1984 EF 10 CCUG 15960 LMG 6142 Archeological site, 17th century latrine, Goteborg, Sweden, 1984 EF 10 CCUG 15988B LMG 8917 Archeological site, 17th century latrine, Goteborg, Sweden, 1984 EF 10 LRA 178.2.84 LMG 5900, CCUG 15848, Environment, 1980 (received as Alcaligenes G-3778 fizecalis) EF 10 LRA 180.2.84 LMG 5902, CCUG 15849, Environment, 1980 (received as Alcaligenes G-3791 faecalis) EF 10 LRA 193.3.84 LMG 5995, CCUG 15850, Environment, 1980 (received as Alcaligenes (3-3777 faecalis) Group 3 EF 10 AF 61 LMG 3475, CCUG 15333 Human material (received as Alcaligenes species) EF 10 CCUG 883 LMG 5936 Newcastle, Great Britain, 1922 (received as “C. percolans”) EF 10 CCUG 2601 LMG 5323 Feces, Goteborg, Sweden, 1973 EF 10 CCUG 3279 LMG 6013. NCTC 2902 USSR, 1929 (received as Vibrio alcaligenes or Vibrio cholerae) EF 10 CCUG 5531 LMG 6014 Human, Heidelberg, Federal Republic of Germany, 1976 EF 10 CCUG 11225 LMG 5324 Burst appendix, Goteborg, Sweden, 1981 EF 10 CCUG 13888 LMG 6139 Unknown (received as Pseudomonas testosteroni) EF 10 CCUG 13893 LMG 6141 Unknown EF 10 CCUG 14415 LMG 6018 Feces, diarrhoea, Goteborg, Sweden, 1983 EF 10 CCUG 17001tlb LMG 6357tlb Feces, Goteborg, Sweden, 1984 EF 10 CCUG 17321tlb LMG 6597tlb Feces, Goteborg, Sweden, 1985 EF 10 CCUG 18232tlb LMG 7096tlb Feces, Goteborg, Sweden, 1985 EF 10 CCUG 18805 LMG 7380, (3-3977 United States EF 10 CCUG 23299 LMG 8608 Feces, Goteborg, Sweden, 1988 EF 10 CCUG 23692 LMG 8766 Feces, Goteborg, Sweden, 1988 EF 10 CCUG 24553 LMG 9089, G-5602 Water for pharmaceutical use, United States (received as C. terrigena) EF 10 CCUG 25530 LMG 9568 Pus, abdominal cavity, Goteborg, Sweden, 1989 [Pseudomonas CIP 55.111 LMG 5518, CCUG 15342A, Rabbit blood culture pseudoalcaligenes] ATCC 17663 [Pseudomonas alcaligenes] CUETM 85-3 LMG 9082, CCUG 24953, Blood G-4519 [Pseudomonas CUETM 85-15 LMG 9371, CCUG 24958, Rabbit blood culture pseudoalcaligenes] CIP 55.111 (= LMG 5518) Genotypically separate C. terrigena strains [Pseudomonas delajieldii] CCUG 15581 LMG 8904 Goteborg, Sweden, 1984 EF 10 CCUG 18806 LMG 7381, G-2463 United States Unidentified Comamonas species CCUG 17739 LMG 6732 Contaminated horse blood, Sweden, 1985 Comamonas [acidovorans] CCUG 178B LMG 7097 Pus, 66-yr-old woman, Goteborg, Sweden, 1968 Strains not belonging to the genus Comamonas [Pseudomonaspseudoalcaligenes] CUETM 85-24 LMG 9369, CCUG 24956, Water, hospital environment, Long Island, N.Y., (3-4570 1983 [Pseudomonas]species CCUG 23632 LMG 8746 Antony , France, 1988 EF 16 CCUG 5180A LMG 7165 Industrial metal-cutting fluid, Goteborg, Sweden, 1976 Reference strains Acidovorax delajieldii ATCC 17505T LMG 5943T, CCUG 1779T Soil with poly-p-hydroxybutyrate as the sole carbon source Acidovorax delajeldii CCUG 20074 LMG 7679 Nasopharynx, Skovde, Sweden, 1987 Acidovorax delajeldii CCUG 23830B LMG 8909 Central venous catheder, 54-yr-old man, Goteborg, Sweden, 1981 Acidovorax facilis ATCC 11228T LMG 2193T, CCUG 2113T Lawn soil, United States Acidovorax facilis ATCC 15376 LMG 2194, CCUG 14278 Unknown Continued on following page 430 WILLEMS ET AL. INT. J. SYST.BACTERIOL.

TABLE 1-Continued Other strain Source, place, and year Name Strain“ designation(s) of isolation Acidovorax facilis DSM 550 LMG 6598, CCUG 15919, Lawn soil ATCC 17695 Acidovorax ternperans CIP 239.74 LMG 3332, CCUG 21717 Blood culture, France Acidovorax ternperans CIP 471.74 LMG 3334, CCUG 21716 Unknown Acidovorax ternperans CCUG 2168B LMG 6435 Dialysis equipment, Goteborg, Sweden, 1973 Acidovorax temperans CCUG 22192 LMG 8451 Wound, Gavle, Sweden, 1988 Acidovorax temperans CCUG 11779T LMG 7169T Urine, 68-yr-old man, Goteborg, Sweden, 1973 [Alcaligenes] latus Palleroni H-4T LMG 3321T, CCUG 10983T, Soil, California ATCC 29712T [Alcaligenes]paradoxus ATCC 17713tlTb LMG 1797tlT,b CCUG 1777T Soil in mineral medium under an atmosphere containing 91% H,, 4% 02,and 5% COz [Alcaligenes]paradoxus ATCC 17712 LMG 3572. CCUG 15916 Soil under an atmosphere containing H,, 6% O,, and 5% CO, [Alcaligenes]paradoxus ATCC 17549tl’ LMG 1796t1,’ CCUG 1778 Soil enriched with panthotenate Hydrogenophaga flava DSM 619T LMG 2MT, CCUG 1658T, Mud from ditch, 1942 ATCC 33667T Hydrogenophaga palleronii Stanier 362tlTb LMG 2366tlTb (= CCUG Water enriched for hydrogen bacteria in an 20334T), ATCC 17724T atmosphere containing 6% 0, (= CCUG 1780T) H. palleronii Stanier 366 LMG 6346, CCUG 17387, Water enriched for hydrogen bacteria in an CCUG 20336, ATCC 17728 atmosphere containing 6% 0, H. palleronii RH2 LMG 6348, CCUG 20338 Gottingen, Federal Republic of Germany Hydrogenophaga pseudoflava GA3T LMG 5945T, CCUG 13799T, Water, River Weende, Germany ATCC 33668T H. pseudoflava GA2 LMG 6351, CCUG 17389, Unknown CCUG 20339 H. pseudoflava DSM 1084 LMG 7584, CCUG 20741, Mud and soil, River Moskwa, USSR 2-1107 (= LMG 8355 = CCUG 22764) Hydrogenophaga taeniospiralis DSM 2082T LMG 7170T, CCUG 15921T Soil, Spain Leptothrix cholodnii CCM 1827 LMG 9467 Active sludge [Aquaspirillurn] anulus NCIB 9012T LMG 5404* Pond water [Aquaspirillurn] delicaturn NCIB 9419T LMG 4327T, ATCC 14667T Distilled water (= LMG 4328T) [Aquaspirillurn] giesbergeri ATCC 11334T LMG 4332T, NCIB 9073T Pond water (= LMG 4331T) [Aquaspirillum] gracile ATCC 19624T LMG 8201T Pond water [Aquaspirillurn] gracile ATCC 19625 LMG 4334 Pond water [Aquaspirillurn] rnetamorphurn NCIB 9509T LMG 4338T, ATCC 15280T Putrid infusion of freshwater shellfish (= LMG 4339T) [Aquaspirillurn]psychrophilum IF0 13611T LMG 5048T Ceratodon purpureus, Antarctica [Aquaspirillurn] sinuosum ATCC 9786T LMG 4347T Freshwater [Pseudornonas]avenue NCPPB lollT LMG 2117T, CCUG 1583tIT, Zea mays, United States, 1958 ATCC 19860T [Pseudornonas pseudoalcaligenes] PDDCC 7733T LMG 5691T. ATCC 33996T Arnorphophallus rivieri cv. Konjac, Shizuoka subsp. konjaci Prefecture, Japan, 1977 [Pseudornonas]rubrilineans NCPPB 920T LMG 2281T, CCUG 15837T, Saccharurn oficinarurn cv. R445, Reunion, 1960 ATCC 19307T [Pseudornonas]saccharophila ATCC 15946T LMG 2256T,.LMG 7831T Mud from a stagnant pool “[Pseudornonas] setariae” NCPPB 1392 LMG 1806, CCUG 15836, Oryza sativa, Japan, 1955 ATCC 19882 Rubrivivax gelatinosus NCIB 8290T LMG 4311T, ATCC 17011T, Acetate enrichment, pH 6.6 CCUG 15841T, CCUG 21977T Xylophilus arnpelinus NCPPB 2217T LMG 5856T, ATCC 33914T, Vitis vinifera var. sultana, Crete, 1966 CCUG 21976T

a Strain designation 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; CCM, Czechoslovak Collection of Microorganisms, Brno, Czechoslovakia; CIP, Collection de 1’Institut Pasteur, Paris, France; DSM, Deutsche Sammlung von Mikroorganismen, Braunschweig, Federal Republic of Germany; LMG, Culture Collection, Laboratorium voor Microbiologie, State University Gent, Ghent, Belgium; LRA, Laboratoire de Recherche, API System S.A., Montalieu-Vercieu, France; IFO, Institute for Fermentation, Osaka, Japan; 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; PDDCC, Culture Collection of the Plant Disease Division, New Zealand Department of Scientific and Industrial Research, Auckland, New Zealand. We isolated two stable colony types from the original culture and labeled them tl and t2. Since the two types had almost identical protein electrophoretic patterns, we used only colony type tl. On the basis of the results of DNA-rRNA and DNA-DNA hybridizations, a numerical analysis of protein electrophoretic patterns, and immunotyping, at least three clusters were delineated within the phenotypically homogeneous species C. terrigena. VOL. 41, 1991 COMAMONAS TAXONOMY 431

scored as described previously (34). Of the 251 characteris- chemical analysis, as described previously (5, 40). 23s tics tested, 207 were used in the numerical analysis. The [3H]rRNAs from C. testosteroni NCTC 1069gT and results of the API 20NE tests (21 characteristics) were not [Aquaspirillum aquaticum] ATCC 11330T were isolated and included in the computer analysis as these tests duplicated purified as described by De Ley and De Smedt (5). Purified other tests, and 23 other characteristics were omitted be- 23s [3H]rRNAs from C. acidovorans Stanier 14T and C. cause all 102 strains tested reacted in the same way. The terrigena NCIB 8193T were available from members of our levels of interstrain similarity were calculated by using the research group (8, 41). Hybridizations between labeled 16s Gower similarity coefficient (S,) (36). Cluster analysis by the or 23s rRNA and filter-fixed DNA were carried out as unweighted average pair group method (36) was performed described previously (5, 35). Each hybrid was characterized by using the CLUSTAN 2.1 program of Wishart (46) and the by its T,,+) value [Tm(e)is the temperature, in degrees Siemens model 7570-CX computer of the Centraal Digitaal Celsius, at which one-half of a DNA-rRNA duplex is dena- Rekencentrum, Rijksuniversiteit, Ghent, Belgium. The re- tured] and by the percentage of rRNA binding (the amount of producibility of the clustering results was estimated by rRNA, in micrograms, bound to 100 pg of filter-fixed DNA including duplicate tests for 12 strains. The centrotype after RNase treatment). strains were determined as described previously (33). Polyacrylamide gel electrophoresis of proteins. All strains RESULTS were grown on nutrient agar at 28°C for 40 h in Roux flasks. Whole-cell protein extracts were prepared, and sodium Numerical analysis of morphological, physiological, and dodecyl sulfate-polyacrylamide gel electrophoresis was per- biochemical characteristics. All 102 strains tested clustered formed by using slight modifications of the procedure of above an S, value of 64%. The reproducibility of the tests Laemmli (22), as described previously (20). The normalized was good; the average S, value for strains tested in duplicate densitometric traces of the protein electrophoretic patterns was 95% (23 sets of comparison). Figure 1 shows a sorted of 46 C. terrigena strains or possible C. terrigena strains and differentially shaded similarity matrix obtained by using were grouped by numerical analysis, using the Pearson unweighted average pair group clustering of S, values. In product moment correlation coefficient as described by Pot this study three Comamonas phena, with strains grouping et al. (30). Points (units of distance travelled) 4 to 179 and 241 above an S, value of 87%, were delineated. The C. aci- to 300 of the interpolated traces were used in the numerical dovorans phenon contained 20 strains, including type strain analysis. The first three points were omitted to exclude the Stanier 14 and one misnamed Bordetella bronchiseptica stacking gel-separation gel interface. Points 180 to 240 were strain; the centrotype strain was strain ATCC 15005. The C. omitted because this zone contains one or a few high-density testosteroni phenon contained 11 strains, including type protein bands with slightly variable positions. Since with the strain NCTC 10698 and one EF 10 strain; the centrotype Pearson product moment correlation coefficient more weight strain was the type strain. The C. terrigena phenon com- is given to high-density protein bands, protein patterns with prised 41 strains, including type strain NCIB 8193 and 3 a high overall level of similarity and a few nonmatching other strains of C. terrigena, the [Aquaspirillum aquaticum] major protein bands may cluster farther apart than protein type strain, 31 EF 10 strains, and 5 unnamed or misnamed patterns with marked overall differences in addition to a few strains; the centrotype strain was strain CCUG 2601. The C. matching high-density protein bands. A similar approach has testosteroni phenon and the C. terrigena phenon exhibited a been used previously in several studies (12, 26, 43). Points relatively high degree of similarity. The strains belonging to 300 to 400 were omitted because under our experimental these phena, together with strains CCUG 15581, CCUG conditions this zone contained very few significant protein 5180A, and CCUG 9672, grouped at S, values of at least bands and therefore was of little discriminatory value. 85%. The strains belonging to the reference taxa formed Preparation of high-molecular-weight DNA. Cells were separate clusters (Fig. 1) that were linked to the Comamonas grown in Roux flasks for 2 to 3 days, and high-molecular- clusters at S, values less than 81%. [P. pseudoalcaligenes] weight DNA was isolated by using the method of Marmur CUETM 85-24, C. acidovorans NCIB 4854 and CCUG (24). 178B, and [Pseudomonas]sp. strain CCUG 23632 exhibited DNA-DNA hybridization. The degree of binding, ex- less than 78% similarity with all of the other phena and pressed as a percentage, was determined spectrophotomet- occupied separate positions in the matrix (Fig. 1). rically by using the initial renaturation rate method (4), as Sodium dodecyl sulfate-polyacrylamidegel electrophoresis. described previously (41). Renaturation experiments were All Comamonas or possible Comamonas strains were in- performed in 2~ SSC (1 x SSC is 0.15 M NaCl plus 0.015 M cluded in the electrophoretic study. The 22 C. acidovorans sodium citrate, pH 7.0) at the optimal renaturation temper- strains tested produced very similar protein patterns; strain ature (8O.O"C) and with a total DNA concentration of 0.102 CCUG 178B was the most aberrant strain. Characteristic for mM base pairs. Degree of binding values of 25% and less almost all C. acidovorans strains was the presence of at least were not significant. one high-molecular-weight protein band at the top of the gel Immunotyping. The preparation of antigen, immunization, (Fig. 2). The 11 C. testosteroni strains tested (including one and immunodifhsion were performed as described previ- EF 10 strain) had very similar protein patterns, except at ously (39). molecular weights ranging from approximately 32,000 to DNA base composition. Average G + C contents were de- 34,000, where the positions of a few heavy protein bands termined by the thermal denaturation method (6) and were varied from strain to strain (Fig. 2). While the protein calculated by using the equation of Marmur and Doty (25), as electropherograms of C. acidovorans and C. testosteroni modified by De Ley (2). could be recognized visually, this was not the case for the DNA-rRNA hybridization. DNA was further purified by more varied protein patterns produced by C. terrigena CsCl gradient centrifugation (5). Thermally denatured DNA strains, EF 10 strains, and several possibly related strains. was fixed on cellulose nitrate filters (type SM 11358; Sarto- Therefore, the protein patterns of 46 of these strains were rius, Gottingen, Federal Republic of Germany), and the subjected to a numerical analysis in which we used un- amount of filter-fixed DNA was estimated by performing a weighted average pair group clustering of Pearson product wNP

FIG. 1. Sorted and differentially shaded matrixof S, values that were calculated by performing a numericalanalysis of 251 phenotypic characteristicsof 79 Comamonas or possible Cornamonas strains and 23 strains belonging to various reference taxa.A ., Alcaligenes;Ac., Acidovorax; C., Cornamonas; H., Hydrogenophaga; P., Pseudomonas. VOL.41, 1991 COMAMONAS TAXONOMY 433

FIG. 2. Normalized protein electropherograms of representative C. acidovorans and C.testosteroni strains. The positions of the molecular weight markers are indicated in lane MWM; these markers were, from left to right, lysozyme (molecular weight, 14,200), trypsin inhibitor (21,100), trypsinogen (24,000), carbonic anhydrase (29,000), glyceraldehyde-3-phosphate dehydrogenase (36,000), egg albumin (45,000), bovine albumin (66,000), and P-galactosidase (116,000). moment correlation coefficients (Fig. 3). Reference strains group comprised representative C. testosteroni strains; the belonging to the other taxa of the acidovorans rRNA com- third DNA group consisted of C. terrigena strains which plex were not included, because representative strains of represented electropherovars la and lb; the fourth DNA these taxa have quite distinct protein patterns (41, 45). The group contained [Aquaspirillum aquaticum] ATCC 11330T reproducibility with this method was rather good; repeated and six EF 10 strains and corresponded to electropherovar 2; runs of any of our protein extracts clustered at Pearson and the fifth DNA group was formed by EF 10 strains and product moment correlation coefficients of 20.91, and du- corresponded to electropherovar 3. The last three DNA plicate, independently prepared protein extracts from each groups together corresponded to the C. terrigena phenon of seven strains clustered at Pearson product moment cor- (Fig. 1). Unnamed strain CCUG 17739 did not belong to any relation coefficients of 20.89. The separation of the strains of the Comamonas DNA groups (Fig. 4). into four electropherovars (an infrasubspecific group of Immunotyping. Table 2 shows the results obtained with strains that have identical or similar protein gel patterns) is antisera against representative strains belonging to each of shown in Fig. 3. Electropherovar la contained C. terrigena the Comamonas groups and several reference strains and NCIB 8193T, NCIB 2581, and NCIB 2582; electropherovar antigens from various Comamonas strains and possibly lb contained C. terrigena CCUG 12940, EF 10 strain CCUG related strains. An immunovar is an infrasubspecific group of 4470, and unnamed strain CCUG 17736; electropherovar 2 strains that exhibit high levels of immunological cross- contained [Aquaspirillum aquaticum] ATCC 11330T and 13 reaction. EF 10 strains (listed in Table 1 under C. terrigena group 2); DNA base composition. The average G+C content of the and electropherovar 3 contained [P. pseudoalcaligenes] CIP strains which we studied is shown in Table 3. 55.111 and CUETM 85-15, [P. alcaligenes] CUETM 85-3, DNA-rRNA hybridization. The specific activities of the and 17 EF 10 strains (listed in Table 1 under C. terrigena 23s [3H]rRNAs from [Aquaspirillum aquaticum] ATCC group 3). In addition to these four electropherovars, several 11330T, C. acidovorans Stanier 14T, C. terrigena NCIB strains occupied separate positions on the dendrogram (Fig. 8193T, and C. testosteroni NCTC 10698Twere 38 x lo3, 62 3). x lo3, 107 X lo3, and 180 x lo3 dpm/pg, respectively. The DNA-DNA hybridization. The results of DNA-DNA hy- results of hybridizations between these four rRNAs and bridization experiments are shown in Fig. 4. Only a carefully DNAs from strains belonging to the three Comamonas selected set of strains was included. Five DNA homology species, [Aquaspirillum aquaticum], several EF 10 strains, groups, which exhibited no significant DNA homology with some unnamed or misnamed clinical isolates, and reference each other, were obtained. The first DNA group contained strains belonging to the different taxa in the acidovorans representative C. acidovorans strains; the second DNA rRNA complex (Table 3) showed that the genus Comamonas 434 WILLEMS ET AL. INT. J. SYST.BACTERIOL.

FIG. 3. Normalized protein electropherograms of some C. terrigeaa strains and possibly related strains. The results of the numerical analysis are presented as a Pearson product moment correlation coefficient (r) dendrogram, which was calculated by using the unweighted average pair group method. The protein patterns of representative strains of each of the different clusters and of strains occupying separate positions on the dendrogram are shown at the left, facing the corresponding branch or triangle. For an explanation of lane MWM, see the legend to Fig. 2. 0, zones omitted in numerical analysis. The numbers in the triangles indicate the numbers of strains in the clusters.

consists of at least five rRNA branches (Fig. 5). Two of these aquaticum] ATCC 11330T and 75.1 to 76.8"C versus rRNA branches represent species of this genus; the C. acidovorans from either strain Stanier 14T, strain NCTC 1069ST, or strain rRNA branch contains strains with Tm(e)values of at least NCIB 8193T (Table 3). A slightly higher level of relationship 79.5"C versus rRNA from C. acidovorans Stanier 14T, and was found for the four rRNA branches representing C. the C. testosteroni rRNA branch contains strains having testosteroni and C. terrigena; these branches were linked to Tm(e)values of at least 793°C versus rRNA from C. test- each other at a mean Tm(e)value of 76.9 L 0.9"C (Fig. 5) and osteroni NCTC 1069ST. In this study the other three Coma- to the C. acidovorans rRNA branch at a mean Tm(e)value of monas rRNA branches were labeled C. terrigena rRNA 76.2 k 0.6"C. The difference between these Tm(e)values was branches 1, 2, and 3 (Fig. 5). C. terrigena rRNA branch 1 rather small, but it was significant since it was based on the consisted of strains with T,(,> values of 78.4 to 81.2"C versus results of 97 different experiments. Reciprocal hybridiza- rRNA from C. terrigena NCIB 8193T (Table 3). C. terrigena tions between DNAs or rRNAs from strains belonging to rRNA branch 2 contained strains with T,(e) values of 80.0 to each of the five Comamonas rRNA branches and rRNAs or 80.9"C versus rRNA from [Aquaspirillum aquaticum] ATCC DNAs from reference strains belonging to the acidovorans 11330T (Table 3). C. terrigena rRNA branch 2 contained rRNA complex showed that the five Comamonas rRNA strains with TmCe)values of 80.0 to 80.9"C versus rRNA from branches were linked to the other rRNA branches in the [Aquaspirillum aquaticum] ATCC 11330T (Table 3). C. ter- acidovorans rRNA complex (e.g., the genera Acidovorax, rigena rRNA branch 3 comprised strains that had Tm(e) Hydrogenophaga, Xylophilus, and [Aquaspirillum]) at a values of 77.4 to 77.8"C versus rRNA from [Aquaspirillum base T,(e) level of 76.0 -+ 1.1"C (Fig. 5). VOL. 41. 1991 COMAMONAS TAXONOMY 435

Stanier 14T ATCC 17476 NClB 4854 ATCC 17406 CCUG 13889 NCTC 10698T ATCC 17407 NClB 8193T NClB 2581 CCUG 12940 CCUG 15504 CCUG 10475 CCUG 15960 CCUG 9673 CCUG 9672 LRA 180.2.84 ATCC 11330 CCUG 17739 CIP 55.111 CCUG 2601 CCUG 883 TERRIGENA 3 CCUG 182321 AF 61 CCUG 11225 FIG. 4. Average degrees of DNA-DNA binding between representative Cornamonas strains and possibly related strains.

DISCUSSION only very remotely related to the acidovorans rRNA com- Five Cumamonas groups. The combined results from the plex (31). Again according to Rule 42 (23), the epithet techniques which we used indicate that the genus Comamo- terrigenus should be retained in the species name since nus consists of at least five groups. The first Comamonas Comamonas terrigena was established in 1962 (16), while group corresponds to C. terrigena rRNA branch 1 (Table 3 the epithet aquaticus was first used in Aquaspirillum aquat- and Fig. 5), electropherovars la and lb (Fig. 3), immunovar icum in 1973 (17). We propose that the use of the name 1(Table 2), and DNA group 1 (Fig. 4). The strains belonging Aquaspirillum aquaticum should be discontinued. There- to electropherovars la and lb are very closely related fore, in Table 1 the strains belonging to Comamonas groups according to the results of all of the techniques which we 1, 2, and 3 are listed as members of C. terrigena groups 1, 2, used except numerical analysis of protein patterns; they and 3, respectively. These groups can be recognized by have visually similar protein patterns, but group far apart using protein gel electrophoresis, immunotyping, DNA- when numerical analysis is used mainly because of the DNA hybridization, and DNA-rRNA hybridization. How- presence of one heavy protein band with a molecular weight ever, we do not describe them as subspecies since at present of approximately 55,000 in C. terrigena electropherovar la. they cannot be differentiated morphologically, physiologi- Comamonas group 2 corresponds to C. terrigena rRNA cally, and biochemically. branch 2 (Table 3 and Fig. 5), electropherovar 2 (Fig. 3), Comamonas group 4 represents C. acidovorans. The immunovar 2 (Table 2), and DNA group 2 (Fig. 4). Likewise, strains of this group form a separate and homogeneous Comamonas group 3 corresponds to C. terrigena rRNA rRNA branch (Fig. 5 and Table 3), DNA group (Fig. 4), branch 3 (Table 3 and Fig. 5), electropherovar 3 (Fig. 3), immunovar (Table 2), electropherovar (Fig. 2), and phenon immunovar 3 (Table 2), and DNA group 3 (Fig. 4). The (Fig. 1). Finally, group 5 represents C. testosteroni, and its strains belonging to Comamonas groups 1, 2, and 3 form one strains likewise form a separate and homogeneous rRNA large phenon (Fig. 1).The 251 characteristics which we used branch (Fig. 5 and Table 3), DNA group (Fig. 4), immunovar did not allow us to differentiate these strains from one (Table 2), electropherovar (Fig. 2), and phenon (Fig. 1). another phenotypically. Therefore, we propose that these Differentiating characteristics for the three Comamonas three groups should be regarded as a single species. Since species are shown in Table 4. this taxon contains the type strains of two previously named DNA-rRNA hybridization revealed that C. testosteroni is species, Comamonas terrigena and Aquaspirillum aquati- slightly more closely related to the three C. terrigena groups cum, several possibilities seemed available for its name. than to C. acidovorans (Fig. 5). This was confirmed by the According to Rules 42 and 44 of the International Code of S,, values (47) of these taxa. In addition, in some immuno- Nomenclature of Bacteria (23), the genus name Comamonas typing experiments we observed slightly greater reactions Davis and Park 1962 has precedence over the genus name between strains of C. testosteroni and C. terrigena groups 1, Aquaspirillum Hylemon, Wells, Krieg, and Jannasch 1973. 2, and 3 (Table 2). The higher values occurred only in some Furthermore, the latter genus name is not suitable since the tests, but they may also indicate a somewhat closer relation- type species, Aquaspirillum serpens, is phylogenetically ship between the two groups. The results of DNA-DNA 436 WILLEMS ET AL. TABLE 2. Results of immunodiffusion analyses INT. J. SYST.BACTERIOL. Reaction with antiserum against?

~ ~~ ~ ~ Coma- Coma- Comamonas terrigena monas [Pseudo- Acido- Aci- Hydro- monas test- Immu- Immunovar 2 Immu- monas] vorux dovorax genophaga Antigen from: aci- strains novar avenae delajieldii facilis palleronii dovorans OS- teroni 1 strain 3 strain NCPPB ATCC ATCC Stanier '!.::CCUG NCIB CCUG CCUG CCUG lollT 17505T 11228T 362tlT 1135 8193T 10475 10330 2601 Comamonas acidovorans Stanier 14T 8 3 4 4 6 CCUG 274B 8 1 1 1 1 CCUG 536t1 6 CCUG 727 6 LRA 147.04.76 8 6 5 4 3 NCIB 4854 5 3 4 2 2 Comamonas testosteroni CCUG 1135 1 3 CCUG 12941 4 2 LRA 148.2.84 5 CCUG 13889 5 NCTC 10698T 4 6 Comamonas terrigena immunovar 1 NCIB 8193T 3 3 2 NCIB 2581 4 3 NCIB 2582 4 3 CCUG 12940 3 2 3 CCUG 17736 5 4 Comamonas terrigena immunovar 2 ATCC 11330 3 4 4 8 7 6 5 4 2 3 CCUG 8404 3 2 3 7 6 3 2 2 2 2 CCUG 2632t1 5 3 4 7 4 4 1 2 2 2 CCUG 9672 1 3 2 7 6 4 2 CCUG 9673 3 2 2 7 5 4 2 CCUG 10330 2 2 4 8 7 6 2 CCUG 10475 1 1 3 8 7 6 2 CCUG 15504 2 2 3 5 5 4 1 CCUG 15960 4 2 4 5 5 3 1 CCUG 15988B 0 1 1 4 5 5 1 LRA 178.2.84 3 2 2 6 6 4 4 LRA 180.2.84 3 1 2 7 7 4 4 LRA 193.3.84 1 1 1 7 7 5 4 Comamonas terrigena immunovar 3 AF 61 3 3 5 0 5 8 4 5 4 3 CCUG 883 4 4 3 0 3 8 3 3 2 2 CCUG 2601 3 2 3 1 2 9 2 2 2 1 CCUG 3279 2 2 1 0 7 8 1 CCUG 5531 1 1 3 0 4 5 2 CCUG 11225 2 2 4 2 2 8 2 2 0 0 CCUG 13888 0 3 2 4 2 6 2 CCUG 13893 2 2 2 0 4 7 3 CCUG 14415 2 2 2 0 4 6 2 CCUG 17001tl 1 1 1 4 5 7 1 CCUG 17321t1 1 1 1 3 4 7 2 CCUG 18232t1 3 2 3 4 3 7 3 CCUG 18805 4 3 4 3 7 3 CCUG 23299 3 3 2 1 5 2 CCUG 23692 0 0 0 1 6 CIP 55.111 2 3 3 3 7 3 CUETM 85-3 3 4 4 4 9 3 CUETM 85-15 2 3 3 4 9 2 Strains not belonging to the genus Comamo- nus [Pseudomonas pseudoalcaligenes] CUETM 3 2 2 2 4 2 3 4 2 2 85-24 EF 16 strain CCUG 5180A 3 3 2 2 3 1 4 4 3 3 Reference strains [Pseudomonas] avenae NCPPB lollT 1 Acidovorax delafieldii ATCC 17505T 1 Acidovorax facilis ATCC 11228T 2 Hydrogenophaga palleronii Stanier 362tlT 1

a 0, no precipitate; 1 or 2, weak reaction with uncertain interpretation; 3, weak reaction, usually revealing relatedness when serum specificity was high; 4 or 5, moderate reaction, revealing relatedness or identity with unsatisfactory serum; 6, 7, 8, or 9, strong reaction observed only with closely related strains. Each value is the average of the results of at least two successful immunodiffusion analyses performed under similar conditions. VOL.41, 1991 COMAMONAS TAXONOMY 437

TABLE 3. DNA base compositions of strains and parameters of the hybrids of their DNAs with labeled rRNAs from the type strains of C. acidovorans, C. testosteroni, C. terrigena, and [Aquaspirillum aquaticum]

Hybridized with [3H]rRNA from:

G+C Comamonas Cornamonas Cornamonas [Aquaspirillum DNA from strain: content acidovorans testosteroni terrigena aquaticum] Imo,%, Stanier 14T NCTC 10698' NCIB 8193T ATCC 11330T <--- ~ -, T,(=) % of rRNA T+) % of rRNA Tm(=) % of rRNA T&) % of rRNA ("C) binding ("C) binding ("C) binding ("C) binding C. acidovorans rRNA branch Comamonas acidovorans Stanier 14T 66.6" 80.6" 0.12" 76.5 0.09 75.0' 0.15' 75.4 0.15 C. acidovorans ATCC 9355t1 67.9" 81.0" 0.10" C. acidovorans ATCC 15005 68.5" 81.0" 0.10" C. acidovorans ATCC 17406 68.4" 79.5" 0.12" 76.4 0.10 76.2' 0.16' 75.5 0.11 C. acidovorans ATCC 17476 68.4" 81 .O" 0.10" 76.5 0.09 75.8' 0.16' 75.5 0.10 [Achromobacter cystinovorum] NCIB 4854 68.7' 80.5' 0.11' 76.7 0.09 76.0 0.15 C. testosteroni rRNA branch Comamonas testosteroni NCTC 10698T 62.5" 76.5" 0.17" 80.9 0.20 76.7' 0.13' 76.8 0.22 C. testosteroni ATCC 17407 64.5" 77.5" 0.17" 79.6 0.12 75 .9' 0.14' 74.4 0.15 C. testosteroni ATCC 17409 63.0" 77.5" 0.15" 80.8 0.17 77.7' 0.24' 75.9 0.22 C. testosteroni ATCC 17510tl 62.8" 76.0" 0.13" C. testosteroni ATCC 17510t2 63.2" 76.5" 0.13" C. terrigena rRNA branch 1 Comamonas terrigena NCIB 8193T 64.0' 75.9 0.19 77.5 0.17 81. 1' 0.32' 76.4 0.22 C. terrigena NCIB 2581 65.8' 75.5' 0.16' 74.3 0.13 79.4' 0.27' 77.0 0.19 C. terrigena CCUG 12940 66.1' 76.9' 0.14' 77.9 0.14 80.5' 0.23' 77.1 0.17 C. terrigena NCIB 2582 66.1' 76.0' 0.19' 77.2 0.18 81.2' 0.30' Unidentified strain CCUG 17736 65.6 77.0 0.13 80.3 0.25 76.6 0.22 EF 10 strain CCUG 4470 66.6 75.8 0.14 78.4 0.29 75.7 0.20 C. terrigena rRNA branch 2 [Aquaspirillum aquaticum] ATCC 11330T 64.0d 76.3d 0.19d 77.0 0.20 76.6d 0.25d 80.0 0.24 EF 10 strain CCUG 10475 62.8 77.4 0.20 77.8 0.36 80.8 0.26 EF 10 strain CCUG 9672 66.6 76.6 0.22 78.0 0.38 80.7 0.29 EF 10 strain CCUG 9673 66.6 77.2 0.21 77.9 0.36 80.7 0.27 EF 10 strain CCUG 15504 66.7 76.5 0.11 77.7 0.22 80.9 0.15 EF 10 strain CCUG 15960 66.1 76.6 0.24 77.4 0.39 80.6 0.29 EF 10 strain LRA 180.2.84 66.6 76.7 0.20 77.5 0.31 80.7 0.21 C. terrigena rRNA branch 3 EF 10 strain CCUG 2601 60.3 75.1 0.26 76.1 0.14 76.2 0.23 77.5 0.25 EF 10 strain CCUG 883 61.6 76.8 0.13 76.6 0.20 77.5 0.15 EF 10 strain CCUG 11225 63.3 75.1 0.23 76.5 0.15 75.8 0.18 77.6 0.31 EF 10 strain AF 61 61.0 76.5 0.13 76.5 0.18 77.6 0.15 EF 10 strain CIP 55.111 76.1 0.09 76.3 0.17 77.8 0.13 EF 10 strain CCUG 18232t1 61.7 76.0 0.10 76.6 0.24 77.7 0.14 EF 10 strain CCUG 23299 59.7 75.3 0.18 76.8 0.29 77.4 0.25 EF 10 strain CCUG 23692 59.6 75.5 0.17 76.7 0.27 76.8 0.22 C. terrigena Unidentified strain CCUG 17739 60.7 75.0 0.08 75.2 0.11 Strain not belonging to the genus Comamonas EF 16 strain CCUG 5180A 68.5 76.1 0.08 76.2 0.14 75.5 0.12 Reference strains Acidovorax delafieldii ATCC 17505* 65.6' 77.2' 0.11' 75.1' 0.05' 77.4 0.14 Acidovorax facilis ATCC 11228T 64.7" 77 .O" 0.09" 76.6 0.08 75.0' 0.08' 76.8 0.10 Acidovorax temperans CCUG 11779T 62.4 76.N 0.09 75.6 0.14 Acidovorux temperans CCUG 2168B 62.9 76.2- 0.26 76.9 0.12- Acidovorux temperans CIP 239.74 63.6 76.9 0.llf 77 .P 0.12- 77.3 0.12 Acidovorax temperans CIP 471.74 63.9 76.g 0.16 77.7f 0.19- 76.8 0.24 [Alcaligenes] latus Palleroni H-4T 7O.Og 74.6' 0.12' 71.6' 0.15' 75.0 0.12 [Alcaligenes]paradoxus ATCC 17713tlT 67 .O" 76.5" 0.03" 75.8 0.03 74.8' 0.05' 75.4 0.04 Hydrogenophaga flava DSM 619T 66.7" 75.5" 0.02" 77.1 0.07 Hydrogenophaga palleronii Stanier 362tlT 67.3' 76.0" 0.05" 75.3 0.04 73 .4' 0.06' 76.0 0.04 Hydrogenophaga pseudoflava CCUG 13799= 66.4' 76.0' 0.08' 75.1 0.06 75.2' 0.09' 77.5 0.07 Hydrogenophaga taeniospiralis DSM 2098T 64.8' 75.0' 0.04' Leptothrix cholodnii CCM 1827 72.2h O.OSh 75.3h 0.07h 72.4 0.02 [Aquaspirillum] anulus NCIB 9012T 58.0d 77.6d 0.12d 76.6 0.12 75Sd 0.18 74.3 0.10 [Aquaspirillum] delicatum NCIB 9419T 64.0d 75.1d 0.lod 75.1 0.06 75.1d 0.05 75.5 0.04 [Aquaspirillum]giesbergeri ATCC 11334T 57.0d 77.5d 0.18d 74.4d 0.20 75.3 0.12 [Aquaspirillum]gracile ATCC 19624T 66.3d 75.8d 0. 13d 76.3 0.07 [Aquaspirillum] gracile ATCC 19625 62.3d 76.2d 0.08d 75.8d 0.10 76.2 0.08 [Aquaspirillum] metamorphum NCIB 9509T 63.0d 75.4d O.lld 76.5 0.05 76.2d 0.05 77.1 0.15 Continued on following page 438 WILLEMS ET AL. INT.J. SYST.BACTERIOL.

TABLE 3-Continued Hybridized with [3H]rRNA from:

G+C Cornamonas Cornamonas Comamonas [Aquaspirillum terrigena aquaticum] DNA from strain: content acidovorans testosteroni (mol%) Stanier 14= NCTC 1069ST NCIB 8193T ATCC 11330T Tm(e) % of rRNA Tmce) % of rRNA Tm(,,) % of rRNA Tm(=) % of rRNA (“C) binding (“C) binding (“C) binding (“C) binding [Aquaspirillum] psychrophilum IF0 13611T 63 .Od 76. 6d 0.12d 75.9 0.14 76.8d 0.27 76.2 0.14 [Aquaspirillum] sinosum ATCC 9786T 58.0d 77.2d 0.17d 76.7 0.14 75.2d 0.16 76.0 0.15 [Pseudomonas]avenue NCPPB lollT 69.8b 76.6‘ 0.12‘ 75.5 0.04 75.7’ 0.09b 75.4 0.11 [Pseudomonas pseudoalcaligenes] subsp. 68.4 76.3d 0. 09d 76.3 0.08 konjaci PDDCC 7733T [Pseudomonas] rubrilineans NCPPB 920T 69.0‘ 77.5‘ 0.06‘ [Pseudomonas] saccharophila ATCC 15946T 69.1’ 72.6‘ 0.05‘ 71.5 0.04 71.8b 0.05’ 72.4 0.05 “[Pseudomonas]setariae” NCPPB 1392 69.1‘ 78.0’ 0.08’ 76.8 0.09 Rubrivivax gelatinosus NCIB 8290T 71.9g 74.5‘ 0.05‘ 70.7 0.03 73Sb 0.06’ 73.0 0.04 Xylophilus ampelinus NCPPB 2217T 68.5‘ 76.0‘ 0.10‘ 74.7 0.08 75.6b O.lOb 75.6 0.13

a Data from reference 7. ’Data from reference 41. ‘ Data from reference 9. Data from reference 31. Data from reference 45. Data from reference 43. Data from reference 18. Data from reference 44. Data from reference 8. hybridization experiments (Fig. 4) did not reveal these closer homology with the type strain of P. pseudoalcaligenes, relationships among the four rRNA branches. which is a genuine Pseudomonas species. Therefore, the Aberrant strains. Some strains could not be assigned to subculture which we obtained from the culture collection of any of the Comamonas groups by one or several of the the Institut Pasteur (Paris) is obviously not identical to the methods which we used. Strain CCUG 178B was received as strain used by Ralston-Barrett et al. (32). C. acidovorans. Its protein pattern was similar to the pat- Cornamonas strains are phenotypically easily confused terns of both C. acidovorans and C. testosteroni (Fig. 2), and with either P. alcaligenes or P. pseudoalcaligenes subsp. phenotypically this strain was somewhat intermediate be- pseudoalcaligenes, which can be isolated from similar clin- tween the two species (Fig. 1). We prefer not to assign it to ical samples. The latter two taxa belong to the genuine a species now and provisionally name it Cornamonas spe- pseudomonads in rRNA superfamily I1 (7). Numerical pro- cies. EF 10 strain CCUG 9672 was phenotypically aberrant files obtained from API 20NE systems do not allow differ- although it was most similar to C. terrigena (Fig. 1). The entiation of C. testosteroni and C. terrigena from P. alcali- main difference between strain CCUG 9672 and C. terrigena genes or P. pseudoalcaligenes subsp. pseudoalcaligenes. was the inability of this strain to use several organic acids Apart from the difference in flagellation, which is rather (e.g., acetate, propionate, isobutyrate, n-valerate, isovaler- difficult to verify, none of the identification tests recom- ate, n-caproate, glutarate, pimelate, and levulinate); accord- mended in the Manual of Clinical Microbiology (14) allows ing to the results obtained by using the other methods differentiation of C. testosteroni from P. alcaligenes, and (Tables 2 and 3 and Fig. 3 and 4), it belongs to C. terrigena only one test (production of acid from fructose) allows group 2 and we therefore listed it as such in Table 1. Another differentiation of C. testosteroni from P. pseudoalcaligenes EF 10 strain, strain CCUG 23692, does not belong to any of subsp. pseudoalcaligenes. An example of the confusion the C. terrigena groups according to the DNA-rRNA hybrid- among these taxa is that 6 of the 13 strains used by Pickett ization results. However, we assigned this strain to C. and Greenwood (29) as reference strains for P. alcaligenes terrigena group 3 because it belongs to the C. terrigena were later shown, in a phenotypic study by Gavini et al. (13), phenon and to the corresponding taxa immunovar 3 and to belong to subcluster Al, which is only distantly related to electropherovar 3. The following strains belong to C. terri- the genuine P. alcaligenes phenon. Two strains belonging to gena on phenotypic grounds (Fig. 1) but could not be subcluster Al, strains CCUG 12941 and CUETM 85-3, were assigned to one of the three groups in this species: strains found in this study to belong to C. testosteroni and C. CCUG 18806 and CCUG 15581 (unique protein patterns terrigena, respectively. [Fig. 31) and strain CCUG 17739 (separate on the basis of A number of auxanographic features that can be used for DNA-DNA hybridization results [Fig. 41 and unique protein differentiating Comamonas species from P. alcaligenes and pattern [Fig. 31). According to the results of the different P. pseudoalcaligenes subsp. pseudoalcaligenes are shown in genotypic and phenotypic methods, strains CCUG 5180A, Table 5. The two phytopathogenic taxa [P. pseudoalcali- CCUG 23632, and CUETM 85-24 are not related to the genus genes] subsp. citrulli and [P. pseudoalcaligenes] subsp. Comamonas (Tables 2 and 3 and Fig. 1 and 3). konjaci are not related to P. pseudoalcaligenes subsp. Confusion among the genus Comamonas, P. alcaligenes, and pseudoalcaligenes. They belong to the acidovorans rRNA P. pseudoalcaligenes. [P. pseudoalcaligenes] CIP 55.111 (= complex and are related to [Pseudomonas]avenue (15). Stanier 297), which we assigned to the species C. terrigena, Emended description of Comamonas (ex Davis and Park was reported by Ralston-Barrett et al. (32) to have 79% DNA 1962) De Vos et al. 1985 emend. Tamaoka et al. 1987. The VOL. 41, 1991 COMAMONAS TAXONOMY 439

Comamonas 60 70 80 T m(e) (OC) TABLE 4. Differentiation of the three species I I I I I C. aci- C.teroni testos- C. terrigena Characteristic [P.] SOLANACEARUM dovorans - -C. ACIDOVORANS Growth on: C. TESTOSTERONI L-Histidine, glycolate D-Fructose, D-mannitol C. TERRIGENA 1 ~~-2-Aminobutyrate, C. TERRIGENA 2 ~~-3-arninobutyrate, C. TERRIGENA 3 - phenylacetate XYLOPHILUS L-Methionine m-Tartrate, glycerol, HY DROGENOPHAGA L-threonine 2-Aminobenzoate [A.] PARADOXUS Benzoate Citrate ACIDOVORAX Testosteroneb I, Hydrolysis of acetamide Phosphoamidase' [AQUASPIRILLUM] Occurrence of 2-hydroxy fatty acidsd G+C content (mol%) 66.6-68.7 62.5-64.5 59.7-66.7

z a +, present in all strains; (+), present in 90 to 99% of the strains; d, present [P.] AVENAE in 11 to 89% of the strains; (-), absent in 90 to 99% of the strains; -, absent in all strains. t R. GELATINOSUS Data from reference 38. As determined with API ZYM systems. L. DISCOPHORA Data from references 27 and 41.

ALCALIGENACEAE They have been isolated from various clinical samples, but no evidence for any pathogenic effect on healthy humans has AUTHENTIC PSEUDOMONAS been reported. The type species is C. terrigena. Emended description of Comamonas terrigena (ex Hugh XANTHOMONAS 1962) De Vos et al. 1985. The species description given by De Vos et al. (9) is emended on the basis of 38 additional strains. ~ Cells are slightly curved rods or spirilla that are 0.3 to 0.5 by 60 70 80 T m(e) W) 1.1 to 4.4 pm (occasionally up to 7 pm). Phenotypic char- FIG. 5. Simplified rRNA cistron similarity dendrogram of rRNA acteristics are shown in Table 7. The G+C values of the superfamily 111. T,(,, values are from Table 3 and from previous DNAs range from 59.7 to 66.7 mol% (as determined by the papers (7-9, 44). Solid bars represent Tm[,)ranges within individual thermal denaturation method). C. terrigena strains are iso- rRNA branches. The branch of the authentic genus Pseudomonas is lated from soil, water, various clinical samples, hospital the Pseudomonas Jluorescens rRNA branch, which contains the environments, horse blood, and rabbit blood. The type strain type species. The roman numerals indicate the roots of the rRNA is strain NCIB 8193; it was isolated from a hay infusion superfamilies sensu De Ley (3); I11 and IV correspond to the beta filtrate. The G+C value of strain NCIB 8193T DNA is 64.0 and alpha subclasses, respectively, and I + I1 constitutes a major part of the gamma subclass of the (37). A., Alca- mol% (as determined by the thermal denaturation method). ligenes; C., Comamonas; L., Leptothrix; P., Pseudomonas; R., C. terrigena contains at least three distinct groups. DNA Rubrivivax.

TABLE 5. Auxanographic differentiation of the genus Comamonas, P. alcaligenes, and P. pseudoalcaligenes previous descriptions of the genus Comamonas (9, 38) are subsp. pseudoalcaligenes" emended below. Comamonas cells are straight or curved Growth on: rods or spirilla that are 0.3 to 0.8 by 1.1 to 4.4 pm. Occasionally longer, irregularly curved cells or spirilla that are 5 to 7 pm long occur. Motile by means of polar or bipolar tufts of one to five flagella. No diffusible pigments are produced on nutrient agar. The average G+C values of the cate DNA range from 59.7 to 68.7 mol% (as determined by the Comamonas 74 - b + - + thermal denaturation method). The genus Comamonas be- - Pseudomonas alcaligenes '- longs to the acidovorans rRNA complex in rRNA superfam- 3 + d Pseudomonas pseudoal- 5 + - +- (= ily I11 the beta subclass of the Proteobacteria), where its caligenes subsp. nearest neighbors are the genera Acidovorax, Hydrogenoph- pseudoalcaligenes aga, and Xylophilus, as well as several [Aquaspirillum] species, the [P.]avenae group, and [Alcaligenes]paradoxus. a The results were obtained by using auxanographic API galleries and are in Features that differentiate the genus Comamonas from these agreement with data reported in Bergey 's Manual of Systematic Bacteriology '28,'. taxa are shown in Table 6. Comamonas strains are isolated +, present in all strains tested; -, absent in all strains tested: d, present from soil or water in natural or industrial environments. in 1 to 99% of the strains tested. 440 WILLEMS ET AL. INT.J. SYST.BACTERIOL.

TABLE 6. Characteristics of the genus Comamonas and other generic groups belonging to the acidovorans rRNA complex

[Pseudomonas] Characteristic Comamonas Acidovoraxa Hydrogenophagab Xylophilusd [Aqua~pirilluml~ avenue T:;iz$ rRNA brand Cell morphology Rods or spirilla Rods Rods Rods Spirilla Flagella Polar, monotrichous -&? + + - - Bipolar tufts + - - - + Peritrichous - - - + - Yellow nondiffusible pigment - - 4- + - Occurrenceh S, FW, CS S, FW, CS S, FW S, FW FW Growth at 30°C + + + + (+I Growth on nutrient agar + + + + - within 3 days Chemolithotrophic growth - d + d with H, Oxidase + + + + + Nitrate reduction d + D d d Growth on: L- Arabinose - D D + D D-Arabitol, sorbitol - d D + L- Arabitol - - - + D-Galactose - D D + D-Glucose - + + + D D-Mannose - D D + D-Xylose - - D + D Propionate (+I d d d - Isobutyrate (+I d - d D-Tartrate (-1 d D d -1 Citrate d (-1 (-) + - Citraconate d d - d Mesaconate d - - + L-Serine (-1 d D d P- Alanine d d - d L-Valine d d d - G+C content (mol%y 59.7-68.7 62.0-66.0 65.0-69.0 66.8-69.4 68.0-69.0 57.0-65.0 67 .O-70.0

a Data from reference 43. Data from reference 41. Data from reference 18. Data from reference 45. Data for [Aquaspirillum] anulus, [Aquaspirillum] delicatum, [Aquaspirillum] giesbergeri, [Aquaspirillum] gracile, [Aquaspirillum] metamorphum, [Aquaspirillum] psychrophilum, and [Aquaspirillum] sinuosum taken from reference 21. Data for [P.]avenue, [P.]cattleyae, [P.pseudoalcaligenes] subsp. citrulli, [P.pseuodalcaligenes] subsp. konjaci, [P.]rubrilineans, and "[P.]setariae" taken from references 15 and 28. +, present in all strains; (+), present in 90 to 99% of the strains; d, present in 11 to 89% of the strains; D, different reaction for different species; (-), absent in 90 to 99% of the strains; -, absent in all strains. S, soil; FW, freshwater; CS, clinical samples; IP, infected plants (phytopathogenic bacteria). Results not reported for [Aquaspirillum] gracile. j As determined by the thermal denaturation method.

hybridization, protein electrophoresis, and iminunotyping, Additional description of Comamonus acidovoruns (den but not morphological, auxanographic, and biochemical Dooren de Jong 1926) Tamaoka et al. 1987. Cells are straight characterization, allow identification of these three groups. to slightly curved rods that are 0.4 to 0.8 by 1.1 to 4.0 pm The strains assigned to each of the three groups are shown in (occasionally up to 7 pm). The previous descriptions (28,38) Table 1. The strains of C. terrigena group 1have Tm(e)values are emended with the phenotypic characteristics shown in of at least 78.4"C versus rRNA from C. terrigena NCIB Table 7. The data in Table 7 corroborate the data reported by 8193T; the G+C contents of their DNAs range from 64.0 to Palleroni (28) except for the following characteristics, which 66.6 mol%. Type strain NCIB 8193 belongs to this group. C. were negative according to Palleroni (28): in our hands, terrigena group 2 comprises strains with Tm(e)values of at growth occurred on L-valine (9 of the 21 strains tested), L- least 80.0"C versus rRNA from C. terrigena ATCC 11330 threonine (20 of the 21 strains tested), heptanoate (18 of the (the former [Aquaspirillum aquaticum] type strain); the 21 strains tested), and benzoate (3 of the 21 strains tested). In G+C contents of their DNAs range from 62.8 to 66.7 mol%. addition, growth on malonate, which occurred in all strains C.terrigena group 3 comprises strains having T,(e) values of according to Palleroni (28), was detected by us in only 9 of 77.4 to 773°C versus rRNA from C. terrigena ATCC 11330 the 21 strains tested. and 75.1 to 763°C versus rRNAs from C. acidovorans Additional description of Comamonus testosteroni (Marcus Stanier 14T, C. testosteroni NCTC 10698T, and C.terrigena and Talalay 1956) Tamaoka et al. 1987. Cells are straight to NCIB 8193T; the G+C contents of their DNAs range from slightly curved rods that are 0.5 to 0.7 by 1.1 to 2.4 pm 59.7 to 63.3 mol%. (occasionally up to 5 pm). The previous descriptions (28, 38) VOL. 41, 1991 COMAMONAS TAXONOMY 441

TABLE 7. Phenotypic characteristics of the three Cornamonas species C. acidovorans C. testosteroni C. terrigena No. of strains Result for No. of strains Result for No. of strains Result for Characteristic" positivelno. type strain positiveho. type strain positivelno. type strain of strains of strains NCTC of strains NCIB Stanier 14 tested tested 10698 tested 8193 Growth at 37°C 18/21 + 11/11 42/42 Growth at 42°C 212 1 - 011 1 26/42 Growth on Drigalski-Conradi agar 21/21 + 11/11 41/42 Growth with: 3% NaCl 16/20 + 519 30137 4.5% NaCl 412 1 - 011 1 814 1 Susceptibility to penicillin (10 pg) 0121 - 0111 6/42 Growth on cetrimide 412 1 - 211 1 1/42 Alpha-hemol y sis 612 1 + 0111 0142 Hydrolysis of Starch 012 1 - 011 1 2/42 Tween 80 12/21 + 9/11 0142 Acetamide 19/21 + 011 1 0142 Lysine and ornithine decarboxylase 1/21" - 1/11' 2/42' Christensen urease 212 1 - 211 1 2/42 Nitrate reduction 21/21 + 711 1 40142 Nitrite reduction 0121 - 011 1 3/42 Growth on: Glycerol 20121 + 8/11 4/42 rn-Erythritol 3/21 - 0111 0142 D-Fructose 21/21 + 011 1 0142 L-Fucose 5/21 - 0111 0142 D-Tagat o se 15/21 + 011 1 0142 m-Inositol 10121 - 011 1 0142 D-Mannitol 21/21 + 011 1 0142 Gluconate 21/21 + 11111 18/42 2-Ketogluconate 012 1 - 011 1 4/42 5-Ketogluconate 0121 - 1/11 0142 Am ygdalin 1/21 - 011 1 0142 Gly cine 5/21 + 2/11 2/42 D-a- Alanine 15/21 + 9/11 8/42 L- Alanine 16/21 + 611 1 5/42 P- Alanine 15/21 + 011 1 3/42 DL-2-Aminobutyrate 2012 1 + 011 1 0142 L-Valine 912 1' + 911 1 7/42 DL-Norvaline 21/21 + 3/11 13/42 L-Leucine 2112 1 + 11111 38/42 L-Isoleucine 2012 1 + 11/11 29/42 L-Norleucine 2012 1 + 10111 17/42 DL-Norleucine 19/21 + 911 1 11/42 L-Serine 012 1 - 1/11 0142 L-Threonine 2012 1 + 611 1 3/42 L-Methionine 19/21 + 1/11 0142 L-Phen ylalanine 2012 1 + 11/11 20142 L-T yrosine 2 112 1 + 11/11 14/42 L-Histidine 21/21 + 11/11 4/42 D-Tryptophan 17/21 + 011 1 0142 L-Tryptophan 17/21 + 711 1 0142 L- Aspart ate 21/21 + 11111 39/42 L- Arginine 012 1 - 011 1 2/42 DL-K ynurenine 13/21 + 211 1 0142 DL-3-Aminobutyrate 2012 1 + 011 1 0142 ~~-4-Aminobut yrate 15/21 + 011 1 0142 DL-5-Aminovalerate 11/21 + 011 1 0142 2-Aminobenzoate 212 1 - lot11 0142 4-Aminobenzoate, sarcosine 012 1 - 011 1 1/42 Ace tamide 13/21 + 011 1 0142 Acetate 21/21 + 11/11 40142 Propionate, n-valerate, levulinate 21/21 + 11111 41/42 Isobutyrate, isovalerate 21/21 + 11/11 39/42 n-Caproate 20121 + lot11 40142 Heptanoate 1812 1 + 911 1 28/42 Caprylate 4/21 - 411 1 10142 Pelargonate 10121 - 611 1 4/42 Continued on following page 442 WILLEMS ET AL. INT. J. SYST.BACTERIOL.

TABLE 7-Continued C. acidovorans C. testosteroni C. terrigena No. of strains No. of strains Result for No. of strains Result for Characteristic" positive/no. for positiveho. type strain positive/no. type strain of type strain of strains NCTC of strains NCIB Stanier 14 tested tested 10698 tested 8193 Caprate 16/21 - 411 1 7/42 Maleate 14/21 + 0111 0142 Malonate 912 1 + 011 1 0142 Glycolate 21/21 + 11/11 3/42 DL-Glycerate 21/21 + 10111 16/42 D-Malate 20121 + 11/11 38/42 D-Tartrate 012 1 - 1/11 0142 L-Tartrate 14/21 + 211 1 0142 m-Tartrate 20121 + 511 1 1/42 Pyruvate 17/21 + 911 1 37/42 2-Ketoglutarate 21/21" + 11/11" 18/42 Citraconate 21/21 + 11/11 32/42 Itaconate 2 112 1 + lot11 30142 Mesaconate 2 112 1 + 611 1 26/42 Aconitate 21/21 + 5/11 14/42 Citrate 13/21 + 11/11 2/42 Phenylacetate 2012 1 + 011 1 0142 Benzoate 3/21 + 10111 0142 m-H ydroxybenzoate 21/21 + loll0 6/40 p-H y droxybenzoate 21/21 + 10111 28/42 L-Mandelate 12/21" + 5/11' 0142 Phthalate 612 1 - 511 1 3/42 Isophthalate 0121 - 411 1 2/42 Terephthalate 0121 5/11 4/42 Hydrolysis of: 2-Naphthylphosphate (pH 8.5) 16/21 1/11 4/42 2-Naphthylphosphate (pH 5.4) 10121 7/11 7/42 2-Naph thy1bu tyrate 19/21 11/11 40142 2-Naphth ylcaprylate 21/21 11/11 37/42 ~-Leucyl-2-naphthylamide 20121 11111 41/42 ~-Cystyl-2-naphthylamide 2/21 1/11 014 2 Naphthol-AS-BI-phosphodiamided 0121 811 1 40142 The following characteristics are present in all Comamonas strains: growth at 30"C, growth in the presence of 0.5 or 1.5% NaC1, catalase activity, and growth on L-proline, L-glutamate, butyrate, succinate, fumarate, glutarate, adipate, pimelate, suberate, azelate, sebacate, DL-lactate, ~~-3-hydroxybutyrate,and L- malate. The following characteristics are absent in all Cornamonas strains: growth in the presence of 6.5% NaCI, 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, hydrolysis of esculin, gelatin, and DNA, arginine dihydrolase activity, indole production, p-galactosidase activity, growth on D-arabinose, L-arabinose, D-lyXOSe, D-ribose, D-xylose, L-xylose, adonitol, D-arabitol, L-arabitol, rn-xylitol, D-fucose, D-galactose, D-glucose, D-mannose, L-rhamnose, L-sorbose, dulcitol, sorbitol, D-cellobiose, p-gentiobiose, lactose, maltose, D-melibiose, sucrose, trehalose, D-turanose, D-melezitose, D-raffinose, methyl-p-D-xyloside, methyl-a- D-glucoside, methyl-a-~-mannoside, N-acetylglucosamine, arbutin, salicin, esculin, glycogen, inulin, starch, L-cysteine, trigonelline, L-ornithine, L-lysine, L-citrulline, 3-aminobenzoate, ethanolamine, ethylamine, butylamine, amylamine, benzylamine, diaminobutane, urea, betaine, creatine, spermine, histamine, tryptamine, glucosamine, oxalate, o-hydroxybenzoate, and D-mandelate, and hydrolysis of 2-naphthylmyristate, ~-valyl-2-naphthylamide, N-benzoyl-DL- arginine-2-naphthylamide,N-glutaryl-phenylalanine-2-naphthylamide, 6-bromo-2-naphthyl-a-~-galactopyranoside, 2-naphthyl-p-~-galactopyranoside, naphthol- AS-BI-P-D-glucuronate (= 6-bromo-2-hydroxy-3-naphthoic acid-2-methoxyanilide-p-~-glucuronate),2-naphthyl-a-~-glucopyranoside, 6-bromo-2-naphthyl-p- D-glucopyranoside, 1-naphthyl-N-acetyl-p-D-glucosaminide,6-bromo-2-naphthyI-a-~-mannopyranoside, and 2-naphthyl-a-~-fucopyranoside. NC, no comparison. ' Late reaction (positive after 3 to 5 days). Naphthol-AS-BI-phosphodiamideis 6-bromo-2-phosphodiamide-3-naphthoicacid-2-methoxyanilide.

are emended with the phenotypic characteristics shown in ACKNOWLEDGMENTS Table 7. The data in Table 7 corroborate the data reported by Palleroni (28) except for the following characteristics, which J.D.L., K.K., and M.G. are indebted to the Fund for Medical were negative according to Palleroni (28): in our hands, Scientific Research, Belgium, for research and personnel grants. A.W. and P.V. are indebted to the National Fund for Scientific (8 L- growth occurred on glycerol of the 11 strains tested), Research, Belgium, for positions as research assistants. B.P. is threonine and pelargonate (6 of the 11 strains tested), L-tryp- indebted to the Instituut tot Aanmoediging van het Wetenschappel- tophan (7 of the 11 strains tested), heptanoate (9 of the 11 ijk Onderzoek in Nijverheid en Landbouw, Belgium, for a scholar- strains tested), and caprylate and caprate (4 of the 11 strains ship. Part of this research was carried out within the framework of tested). In addition, according to Palleroni (28) pyruvate and contract BAP-0138-B with the Biotechnology Action Program of the aconitate were used by all strains, but in our hands these Commission of the European Communities. substrates were used by 9 of 11 and 5 of 11 strains, E.F. is grateful to Lars Nehls, Ann Borjesson, and Marie respectively. Blomqvist for excellent technical assistance. We thank API System VOL.41, 1991 COMAMONAS TAXONOMY 443

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