INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1993, p. 135-142 Vol. 43, No. 1 0020-7713/93/010135-08$02.00/0 Copyright 0 1993, International Union of Microbiological Societies Thauera selenatis gen. nov., sp. nov., a Member of the Beta Subclass of Proteobacteria with a Novel Type of Anaerobic Respiration J. M. MACY,’” S. RECH,’? G. AULING,2 M. DORSCH,3 E. STACKEBRANDT,3 AND L. I. SLY3 Department of Animal Science, University of California, Davis, Davis, Cali ornia 95616’; Institut fur Mikrobiologie, Universitat Hannover, 3000 Hannover 1, Germany4 and Centre for Bacterial Diversity and Identification, Department of Microbiology, The University of Queensland, Brisbane, Australia 40723 A recently isolated, selenate-respiring microorganism (strain AXT [T = type strain]) was classified by using a polyphasic approach in which both genotypic and phenotypic characteristics were determined. Strain AXT is a motile, gram-negative, rod-shaped organism with a single polar flagellum. On the basis of phenotypic characteristics, this organism can be classified as a Pseudomonas sp. However, a comparison of the 16s rRNA sequence of strain AXT with the sequences of other organisms indicated that strain AXT is most similar to members of the beta subclass (level of similarity, 86.8%) rather than to members of the gamma subclass (level of similarity, 80.2%) of the Pruteobacteriu. The presence of the specific polyamine 2-hydroxyputrescine and the presence of a ubiquinone with eight isoprenoid units in the side chain (ubiquinone Q-8) excluded strain AXT from the authentic genus Pseudumnas and allowed placement in the beta subclass of the Pruteobacteria. Within the beta subclass, strain AXT is related to ZodobacterjZuvatiZe.The phylogenetic distance (level of similarity, less than 90%), as well as a lack of common phenotypic characteristics between these organisms, prevents classification of strain AXT as a member of the genus Zodobacter. In addition, strain AXT possesses a unique mechanism for anaerobic respiration, which allows it to utilize selenate as an electron acceptor without interference by nitrate. Therefore, we propose that strain AXT should be the first member of a new genus and species, Thauera selenatis. In the past, gram-negative, aerobic, rod-shaped bacteria placed in other genera (e.g., the genera Xanthomonas [35] with polar flagella have frequently been assigned to the and Cornamonas [13]) or elevated to genus rank (e.g., the genus Pseudomonas. The genetic heterogeneity of this genus genera Sphingomonas [44], Hydrogenophaga [36], and Aci- became apparent when levels of rRNA cistron similarity dovorax [38]) by using this approach. were determined, which revealed the presence of several The same polyphasic strategy was used in this study to clusters of related organisms (28). These results were later classify an organism that was tentatively and incorrectly confirmed by the results of the extensive DNA-DNA and classified as a Pseudomonas strain (22). Only one strain of DNA-rRNA hybridization studies of De Vos et al. (11-14) this organism has been isolated, and this strain has the and by 16s rRNA cataloging results (41, 43), which revealed unique characteristic of reducing selenate to selenite by that members of the genus Pseudomonas branch off within anaerobic respiration. the alpha, beta, and gamma subclasses of the Proteobacteria (34). Only the members of the Pseudomonas JEuorescens branch in the gamma subclass represent the authentic MATERIALS AND METHODS pseudomonads. As pointed out by Willems et al. (36), all of Media and growth conditions. The media which were used the other Pseudomonas species are considered to be mis- (defined minimal medium and minimal medium containing classified. Chemotaxonomy provides a rapid alternative ap- 0.4% yeast extract) have been described previously (22). proach for excluding organisms from or including organisms Strain AXT (T = type strain) was grown as described in the authentic pseudomonad taxon on the basis of the previously (22). This organism has been deposited with the results of fatty acid, ubiquinone, and polyamine analyses (1, American Type Culture Collection as strain ATCC 55363T. 2, 8-10, 27). Below, those organisms that have the genus Bacterial strains tested for anaerobic growth with selenate name Pseudomonas, but are not part of the phylogenetic or nitrate. Strains were obtained from the American Type nucleus of the true pseudomonad taxon (see above) are Culture Collection (ATCC) and from the Culture Collection indicated by enclosing the genus name in single quotation of the Department of Microbiology, University of Queen- marks (e.g., ‘Pseudomonas’ cepacia). sland, and were tested for the ability to grow by using In a polyphasic approach to bacterial systematics, geno- selenate or nitrate for respiration in anaerobic ATCC media typic and phenotypic properties are combined to delineate (16) containing 5 or 10 mM selenate or 10 mM nitrate (22). taxa, and it was not until recently that several species of the The following bacterial strains were obtained from the obviously heterogeneous taxon Pseudomonas were either ATCC: Cornamonas terrigena ATCC 8461 (tested in ATCC medium 386 containing glucose [final concentration, 2 dli- ter]), Janthinobacterium lividum ATCC 12473 (tested in * Corresponding author. ATCC medium 3 containing glucose [final concentration, 2 t Present address: Department of Microbiology and Molecular diter]), Hydrogenophaga flava ATCC 33667 (tested in Genetics, University of California, Los Angeles, Los Angeles, CA ATCC medium 1246 containing gluconate [final concentra- 90024-1489. tion, 2 diter]), Chrornobacterium violaceum ATCC 12472 135 136 MACY ET AL. (tested in ATCC medium 3 containing acetate [final concen- of minimal medium broth in closed 20-ml serum bottles; 10 tration, 1.4 &liter]), Iodobacterfluviatile ATCC 33051 (test- ml of H2 and 10 ml of air were added to the bottle after ed in ATCC medium 3 containing acetate [final concentra- inoculation, and CO, was supplied by the HC0,- present in tion, 1.4 &liter]), ‘Pseudomonas’ cepacia ATCC 25416 the medium (22). Growth on methanol, isobutyrate, and (tested in ATCC medium 3 containing acetate [final concen- propionate was tested in 10-ml portions of minimal medium tration, 1.4 &liter]), Alcaligenes faecalis ATCC 8750 (tested broth in closed roll tubes under a 100% N, atmosphere (22). in ATCC medium 3 containing gluconate [final concentra- The following concentrations were used: methanol, 1 and 3 tion, 2 &liter]), and Alcaligenes xylosoxydans subsp. xy- mM; isobutyrate, 5 mM; propionate, 5 mM. Zusoxydans ATCC 27061 (tested in ATCC medium 3 contain- The agar method described above, which was used to test ing gluconate [final concentration, 2 &liter]). The bacterial the range of substrates that could support growth of strain strains obtained from the Culture Collection of the Depart- AXT, was used for the following reasons: (i) with certain ment of Microbiology, University of Queensland, were substrates, cells tended to lyse after growth ceased, and ‘Pseudomonas’ mkta UQM 1762 (= ATCC 49108) (tested in therefore, it was desirable not to do the tests in broth ATCC medium 416 containing sucrose [final concentration, cultures; and (ii) neither the optimum concentrations nor the 20 @liter]), Acidovorux facilis UQM 1918 (= ATCC 11228) inhibitory concentrations of the substrates were known, and (tested in ATCC medium 72 containing glucose [final con- therefore, use of a concentration gradient permitted the centration, 2 &liter]), Variovorax paraduxus UQM 1905 (= organism to grow at the concentrations that best suited it. ATCC 17713) (tested in ATCC medium 3 containing acetate DNA base composition. DNA was extracted and the melt- [final concentration, 1.4 g/liter]), and Delxia gumosa UQM ing temperature was determined by using the method of 2144 (= ATCC 15994) (tested in ATCC medium 165 contain- Nelson et al. (25), with some modifications. Strain AXT was ing glucose [final concentration, 2 gliter]). grown aerobically on a shaker in 100 ml of minimal medium Phenotypic characterization. The presence of oxidase, containing 0.4% yeast extract, 20 mM nitrate, and 20 mM catalase, arginine dihydrolase, and lysine decarboxylase and acetate. After 24 h of incubation at 28”C, 1g (wet weight) of the ability to hydrolyze starch and gelatin were determined cells was collected by centrifugation at 10,000 x g for 15 as described by Smibert and Krieg (31). The presence of a min. DNAs isolated from Escherichia coli and Pseudomonas diffusible, fluorescent pigment was determined by using putida were used as controls; both organisms were grown in King media A and B (28). The denitrification test was carried nutrient broth (Difco) at 37”C, and after 24 h of incubation, 2 out in anaerobic minimal medium containing 0.4% yeast g (wet weight) of cells was harvested by centrifugation at extract, 20 mM nitrate, 20 mM acetate, and a Durham tube; 10,000 x g for 15 min. All of the cells were broken by using growth and gas production were recorded within 48 h after a French press (16,000 lb/in2; Aminco Chance, Silver Spring, inoculation (31). The nature of the gas produced during Md.). DNA from each resulting cell extract was isolated, and denitrification was determined by using the method of Jen- the melting temperature was determined and used to calcu- neman et al. (18). Strain AXT was grown for 48 h in an late the moles percent G+C content as described by Nelson anaerobic chamber (Coy Laboratory Products, Inc., Ann et al. (25). Arbor, Mich.) on minimal medium containing 2% agar, 20 Scanning electron microscopy. Strain AXT was grown at mM acetate, 40 mM nitrate, and 0.0001% resazurin. The 28°C for 6 to 12 h in minimal medium supplemented with 10 production of N,O was detected by the formation of a pink mM acetate and 10 mM nitrate. The cells either were filtered color around the colonies, which was due to oxidation of the onto Nuclepore filters (pore size, 0.4 pm) and fixed in resazurin by N,O; N, production does not cause the oxida- Sorenson buffer (17) containing 2.5% glutaraldehyde or were tion of resazurin.