INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1997, p. 566-568 Vol. 47, No. 2 0020-7713/97/$04.00 + 0 Copyright 0 1997, International Union of Microbiological Societies

Transfer of Blastobacter nataton’us (Sly 1985) to the Genus Blastomonas gen. nov. as Blastomonas natatoria comb. nov.

L. I. SLY* AND MARIAN M. CAHILL Centre for Bacterial Diversity and Identification, Department of Microbiology, University of Queensland, Brisbane, Australia

The budding bacterium Blastobacter natatorius belongs to the alpha-4 group of the and clusters phylogenetically on a deep branch with Sphingomonas capsulata, with which it shares 93.9% 16s rRNA sequence similarity. On phylogenetic, phenotypic, and chemotaxonomic grounds a proposal is made to transfer B. natatorius to the genus Blastomonas gen. nov. as Blastomonas natatoria comb. nov. __ The of the genus Blastobacter Zavarzin 1961 is in branch distinct from Blastobacter aggregatus and Blastobacter a state of confusion. This confusion has arisen due to the lack capsulatus. The relationship of Blastobacter denitrificans to Bra- of a type strain for the type , Blastobacter henricii (14, dyrhizobium japonicum confirmed previous observations of 25), and to the fact that morphological characteristics, such as Willems and Collins (23) based on 16s rRNA sequence simi- budding cell division, which define the genus may not be phy- larities and of Green and Gillis (3) based on rRNA cistron logenetically useful features at the genus level (5) and may similarities. Blastobacter natatorius, on the other hand, was group distantly related species in the genus as currently de- shown by Hugenholtz et al. (5) to be a member of the alpha-4 fined (15, 21, 25). Cell division by non-prosthecate budding is group. confined to the alpha subclass of the Proteobacteria but is Hugenholtz et al. (5)recommended that a new type species widely distributed in genera within the subclass. for the genus Blastobacter be selected and described because of The genus Blastobacter was proposed by Zavarzin (25) to the lack of a type strain of the type species. However, given the include rosette-forming, budding, rod-shaped or wedge-shaped absence of physiological and phylogenetic information about which were observed in a filter paper enrichment of the type species, Blastobacter henn’cii, it would be difficult to reduced iron-containing water from a northern Russian forest designate one of the other species as the type species of the brook. Zavarzin was unable to isolate the cells in pure culture, genus Blastobacter with confidence. There is no way of knowing and the description of Blastobacter henricii is based on draw- which phylogenetic line the true blastobacters as described by ings and observations. Zavarzin (25) belong to, and such action might cause further Several additional Blastobacter species have been validly de- taxonomic confusion in the future. Consequently, the genus scribed since 1961 (8). These include Blastobacter aggregatus, Blastobacter should be reserved at this time for Blastobacter Blastobacter capsulatus, Blastobacter denitrificans (4, 6), and henricii in case a culture matching the description can be iso- Blastobacter natatorius (15, 17). Other taxonomically unvali- lated from the same habitat in the future. New genera need to dated species which have been reported on include “Blasto- be described to include the remaining validated species. bacter V~SCOSUS’~(7), “Blastobacter aminooxidans” (l),and In this paper we propose that Blastobacter natatorius be “Blastobacternovus” (10). Several authors have demonstrated that there is a high de- transferred to a new genus as it is phylogenetically (5), pheno- gree of heterogeneity in the genus Blastobacter with respect to typically (15, 21), and chemotaxonomically (13) distinct from phenotype (21), cellular fatty acids and phospholipids (13), and the other species of the genus Blastobacter as currently defined molecular phylogeny (3, 5, 11, 23). (15, 21, 25). Hugenholtz et al. (5) found that Blastobacter There is a need to revise the taxonomy of the genus Blasto- natatorius was most closely related, as determined by 16s bacter, and the phylogenetic evidence of Hugenholtz et al. (5) rRNA sequence similarity, to Caulobacter subvibrioides, Por- provides a framework with which to commence the taxonomic phyrobacter neustonensis (2), and Eiythrobacter longus (12) in revision. An analysis of the 16s rRNA sequences of the vali- the alpha-4 group of the Proteobacteria. We have recently re- dated species Blastobacter aggregatus, Blastobacter capsulatus, ported (16) that the sequence (accession number M83797) (18) Blastobacter denitr@cans, and Blastobacter natatorius by Hu- for C. subvibn’oides used by Hugenholtz et al. (5) and recently genholtz et al. (5) showed that the species clustered in three by Nohynek et al. (9) in their analyses appears to be the separate groups within the alpha subclass of the Proteobacteria. sequence of an organism closely related to Sphingomonas Blastobacter aggregatus and Blastobacter capsulatus were closely adhaesiva and not the sequence of C. subvibrioides. Resequenc- related to each other (96.6% sequence similarity) and to ing of the 16s ribosomal DNA of the type strain of C. subvib- Agrobacterium tumefaciens (96.0 to 96.9% sequence similarity) rioides showed that this species clusters with the Caulobacter in the alpha-2a group. This confirmed the previous finding of group outside the alpha-4 group and lacks 2-hydroxymyristic Rothe et al. (11) concerning the close relationship between acid, which is characteristic of the genus Sphingomonas (16). C. Blastobacter aggregatus and A. tumefaciens determined by subvibrioides should no longer be considered a member of the rRNA oligonucleotide catalog analysis. Blastobacter denitrifi- Sphingomonas group. Because of this change and the increas- cans grouped with Rhodopseudomonas palustris and Bradyrhi- ing number of genera and species belonging to the alpha-4 zobium japonicum in the alpha-2b group, a phylogenetic group, we undertook a phylogenetic analysis in which we used previously described methods (16); these methods included analyzing the 16s rRNA sequences of a more complete set of * Corresponding author. Phone: 61 7 3365 2396. Fax: 61 7 3365 1566. species which have become available since the analysis done by E-mail: [email protected]. Hugenholtz et al. (5) but do not include C. subvibrioides to

566 VOL.47, 1997 NOTES 567

Rhodopseudomonas palustris of 16s rRNA cistron and sequence similarity values (22). It was

Rhadobacter capsulatus also suggested by van Bruggen et al. (22) that Sphingomonas yanoikuyae may be a separate species of the genus Rhizornonas, Rhodobacter sphaeroides but the deep branch point indicates that this species may be the Brevundimonns diminuta first member of another new genus. Sphingomonas terrae and 11 Sphingomonas macrogoltabidus,with a level of sequence simi- Caulobacter bacteroides larity of 96.7%, cluster together and are most likely separate 7Zymomonas mobilis subsp. mobilis species in another new genus. Phenotypic characteristics to 477 describe and differentiatethese new genera need to be deter- mined before the new taxa can be proposed formally. S. capsulata is considered to be representative of a separate

Sphingomonas terrae genus on the basis of rRNA cistron similarity values and partial sequence similarities (22). Given the low level of 16s rRNA Sphingomonas rnacrogoltabidus similarity between Blastobacter natatorius and S. capsulata and

1- I I41 1- Sphingomonas capsulata the distinctive morphological features, Blastobacter natatorius L must also be considered a member of a separate genus. 48 Bhtomoncrs natatoria Members of the major Sphingomonas group, including spe- Sphingomonas adhaesiva cies of the genera Sphingomonas and Rhizomonas, are charac- - 100 terized by the presence of 2-hydroxymyristicacid (2-OHn14:O) 24 -Sphingomonas paucimobilis -99 in their cellular fatty acids (20, 24). Blastobacter natatorius has Sphingomonas sanguis been shown by Sittig and Hirsch (13) to contain more than

21 Sphingomonas parapaucimobilis 50% 2-hydroxymyristic acid but no 3-hydroxy fatty acids, which supports its close affinity with the alpha-4 group. These fea- Rhizomonas suberifaciens tures differentiate Blastobacter natatorius from Blastobacter ag- gregatus and Blastobacter capsulatus, which do not contain 2-hy- droxymyristic acid or other 2-hydroxy fatty acids, but contain 3-hydroxy fatty acids, including high levels of 3-OHn14:O (>81%) (13). Sittig and Hirsch (13) in a chemotaxonomic study of the budding and/or hyphal bacteria determined that the major cellular fatty acids of Blastobacter natatorius were n16:ld9 (13.3%), n16:O (17.2%), and n18:ldll (61.5%). The major hydroxy fatty acids were 2-OHn14:O (52.2%), 2-OHn16:ldll (16.2%), and 2-OHn16:O (25.7%). It was also shown that the major phospholipids were phosphatidylglycerol, phosphatidyl- ethanolamine, phosphatidyldimethylethanolamine, and phos- phatidylcholine and that Blastobacter natatorius contained ubiquinone QlO. On the basis of its phenotypic and chemotaxonomic features and its phylogenetic position, we propose that Blastobacter natatorius be transferred to the new genus Blastomonas as Blastomonas natatoria comb. nov. Description of Bhstomonas gen. nov. Blastomonas (Blas.to. mo'nas. Gr. n. blastos, bud shoot; Gr. n. monas, a unit, monad; M.L. fem. n. Blastomonas, a budding monad). Cells are gram negative and rod shaped or wedge shaped with a straight or slightly curved axis. Cells are usually 0.5 to 0.8 by 1 to 3 pm. Older cells may become elongated and reach lengths of 10 pm more accurately determine the position of Blastobacter natato- or more. Some cells may have a swollen or bloated appearance. rius in the alpha-4 group. Cells occur singly or in pairs and may form rosettes. Each In the phylogenetic tree (Fig. 1) Blastobacter natatorius clus- rosette-forming cell has a simple mucilaginous holdfast at its ters in a major group containing species of the genus Sphin- nonreproductive pole by which it attaches to other cells or to gomonas and Rhizomonas and joins deeply with Sphingomonas solid surfaces. No stalks, prosthecae, or other holdfast struc- capsulata at a level of sequence similarity of 93.9%. Nohynek tures are present. Reproduction occurs by asymmetric division et al. (9) recently showed that the S. capsulata branch also (budding) of the mother cell to produce a shorter daughter contains the new species Sphingomonas rosa and Sphingomo- cell. Buds may be spherical or ovoid and are usually 0.5 to 0.8 nas subarctica, which exhibit only 91.9 to 92.4% sequence sim- by 0.5 to 1 pm. The free bud cells are motile with a single polar ilarity with Blastobacter natatorius, which continues to be the flagellum. only representative of a deep branch. The Sphingomonas group Chemoorganotrophic. Good growth occurs on peptone- is characterized by a number of deeply branching phylogenetic yeast extract agar or peptone-yeast extract-glucose agar. lines which may represent separate genera. The branch which Growth occurs on glucose-ammonium sulfate agar. No vitamin contains Sphingomonaspaucimobilis, Sphingomonasparapauci- requirement. Colony pigmentation is yellow. mobilis, Sphingomonas sanguis, and S. adhaesiva most likely Aerobic. Catalase and oxidase positive. Acid but no gas is represents the true species of the genus Sphingomonas. Rhi- produced from glucose. Nitrate is not reduced. zomonas suberifaciens, on a separate deep branch, was pro- Contains ubiquinone QlO. Contains 2-hydroxymyristic acid posed as a member of the new genus Rhizomonas on the basis as a major component of the cellular fatty acids. 568 NOTES INT. J. SYST. BACTERIOL.

The guanine-plus-cytosine content of genomic DNA is 65 capsulatus sp. nov., and Blastobacter denitri5cans sp. nov., new budding bac- mol% (as determined by the thermal denaturation method). teria from freshwater habitats. Syst. Appl. Microbiol. 6281-286. 5. Hugenholtz, P., E. Stackebrandt, and J. A. Fuerst. 1994. A phylogenetic Member of the alpha-4 group of the alpha subclass of the analysis of the genus Blastobacter with a view to its future reclassification. Proteobacteria. Syst. Appl. Microbiol. 1751-57. Found in freshwater. The type species is Blastomonas nata- 6. International Journal of Systematic Bacteriology. 1986. Validation of the toria. publication of new names and new combinations previously effectively pub- lished outside the IJSB. List no. 20. Int. J. Syst. Bacteriol. 36354-356. Description of Blastomonas natatoria (Sly 1985) comb. nov. 7. Loginova, N. V., and Y. A. Trotsenko. 1979. Blastobacter viscosus-a new The description of Blastomonas natatoria (na.ta.to'ri.a. M.L. species of autotrophic bacteria utilizing methanol. Mikrobiologiya 48:644- fem. adj. nataton'a, of a swimming place [pool]) is the same as 651. (Translated from Russian.) the genus description with the following additional character- 8. Moore, W. E. C., and L. V. H. Moore. 1992. Index of the bacterial and yeast istics described in the effective publication of Blastobacter na- nomenclatural changes. American Society for Microbiology. Washington, D.C. tatorius (15) and chemotaxonomic data from the study of Sittig 9. Nohynek, L. J., E.-L. Nurmiaho-Lassila, E. L. Suhonen, H.-J. Busse, M. and Hirsch (13). Mohammadi, J. Hantula, F. Rainey, and M. S. Salkinoja-Salonen. 1996. Colonies grown on peptone-yeast extract agar for 3 days at Description of chlorophenol-degrading Pseudomonas sp. strains KFl T, KF3, 28°C are yellow and round and have entire edges, diameters of and NKFl as a new species of the genus Sphingomonas, Sphingomonas subarctica sp. nov. Int. J. Syst. Bacteriol. 46:1042-1055. 0.5 to 1.0 mm, and high convex elevations. The colony surface 10. Rezanka, T., 1. V. Zlatkin, I. Viden, 0. I. Slabova, and D. I. Nikitin. 1991. is shiny, and the growth is easily emulsified. After further Capillary gas chromatography-mass spectrometry of unusual and very long- incubation the colonies have a rubbery consistency and may be chain fatty acids from soil oligotrophic bacteria. J. Chromatogr. 558215-221. removed intact from the agar surface by touching with a wire 11. Rothe, B., A. Fischer, P. Hirsch, M. Sittig, and E. Stackebrandt. 1987. The phylogenetic position of the budding bacteria Blastobacter aggregafus and loop. Colonies on Staley PYG medium (19) after 4 days at Gemmobacter aquatilis gen. nov., sp. nov. Arch. Microbiol. 147:92-99. 28°C are pale pink and 0.5 mm in diameter, 12. Shiba, T., and U. Simidu. 1982. Erythrobacter longus gen. nov., sp. nov., an Catalase, oxidase, phosphatase, and DNase are produced. aerobic bacterium which contains bacteriochlorophyll a. Int. J. Syst. Bacte- Gelatin and Tween 80 are hydrolyzed, but cellulose, chitin, rial. 32:211-217. 13. Sittig, M., and P. Hirsch. 1992. Chemotaxonomic investigation of budding alginate, starch, tributyrin, casein, and dextran are not hydro- and/or hyphal bacteria. Syst. Appl. Microbiol. 15209-222. lyzed. There is no hemolytic activity. Urease, phenylalanine 14. Skerman, V. B. D., V. McGowan, and P. H. A. Sneath. 1989. Approved lists deaminase, arginine dihydrolase, indole, and H,S are not pro- of bacterial names, amended edition. American Society for Microbiology, duced. Nitrate is not reduced. An alkaline reaction occurs in Washington, D.C. Hugh-Leifson medium. Acid but no gas is produced from fruc- 15. Sly, L. I. 1985. Emendation of the genus Blasfobacfer Zavarzin 1961 and description of Blastobacternatatonus sp. nov. Int. J. Syst. Bacteriol. 354045. tose, glucose, glycerol, maltose, mannose, melezitose, and su- 16. Sly, L. I., M. Cahill, K. Majeed, and G. Jones. 1997. Reassessment of the crose in media containing Andrades indicator. phylogenetic position of Caulobacter subvibiioides. Int. J. Syst. Bacteriol. The following chemotaxonomic information was determined 47211-213. by Sittig and Hirsch (13) for the type strain. Contains ubiqui- 17. Sly, L. I., and M. H. Hargreaves. 1984. Two unusual budding bacteria isolated from a swimming pool. J. Appl. Bacteriol. 56479486. none QlO. The major cellular fatty acids are n16:ld9, n16:0, 18. Stahl, D. A., R. Key, B. Flesher, and J. Smit. 1992. The phylogeny of marine and n18:ldll. The major hydroxy fatty acids are 2-OHn14:07 and freshwater caulobacters reflects their habitat. J. Bacteriol. 1742193- 2-OHn16:ldl17and 2-OHn16:O. The major phospholipids are 2198. phosphatidylglycerol, phosphatidylethanolamine, phosphatidyl- 19. Staley, J. T. 1981. The genus Pasteun'a, p. 490-492. In M. P. Starr, H. Stolp, H. G. Triiper, A. Balows, and H. G. Schlegel (ed.), The prokaryotes, vol. 1. dimethylethanolamine, and phosphatidylcholine. Springer-Verlag, Berlin, Germany. The guanine-plus-cytosine content of the DNA of the type 20. Takeuchi, M., H. Sawada, H. Oyaizu, and A. Yokota. 1994. Phylogenetic strain is 65 mol% (as determined by the thermal denaturation evidence for Sphingomonas and Rhizomonas as nonphotosynthetic members method). of the alpha-4 subclass of the Proteobacteria. Int. J. Syst. Bacteriol. 44:308- The type strain is strain ACM 2507 (= ATCC 35951 = DSM 314. 21. Trotsenko, Y. A., N. V. Doronina, and P. Hirsch. 1989. Genus Blastobrrcter, 3183 = NCIMB 12085), which was isolated from a freshwater p. 1963-1968. In J. T. Staley, M. P. Bryant, N. Pfennig, and J. G. Holt (ed.), swimming pool. Bergey's manual of systematic bacteriology, vol. 3. Williams & Wilkins, Baltimore, Md. 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