INTERNATIONAL JOURNALOF SYSTEMATIC BACTERIOLOGY,OCt. 1997, p. 1249-1252 Vol. 47, No. 4 0020-7713/97/$04.00 +0 Copyright 0 1997, International Union of Microbiological Societies Proposal To Reclassify Zoogloea ramigera IAM 12670 (P. R. Dugan 115) as Duganella zoogloeoides gen. nov., sp. nov. AKIRA HIRAISHI,132*YONG KOOK SHIN,’? AND JUNTA SUGIYAMA’ Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113,’ and Department of Ecological Engneering Toyohashi University of Technology, Tenpaku, Toyohashi 441, Japan The taxonomic position of a misclassified strain, Zoogloea ramigera IAM 12670T (= ATCC 25925T = P. R. Dugan lUT),was reevaluated. A phylogenetic analysis based on 16s ribosomal rDNA sequences revealed that this organism was located in the beta subclass of the class Proteobacteria with members of the genus TeZZuria as its closest relatives. On the basis of phenotypic and phylogenetic information, we propose that this organism should be reclassified in a new taxon with the name Duganella zoogloeoides gen. nov., sp. nov. Zoogloea ramigera, which is at this time the only species of ical groups of colonies appeared and were designated strains the genus Zoogloea Itzigsohn 1868, is an aerobic, chemoor- 12670A and 12670B. The colonies of strain 12670A, which ganotrophic, gram-negative, rod-shaped bacterium that forms accounted for more than 70% of the colonies recovered, were characteristic cell aggregates surrounded by gelatinous matri- glistening, convex with entire margins, viscous, and cream to ces, the so-called zoogloeal matrices. This organism has been straw colored. The viscous appearance of this strain became isolated from wastewater environments, such as activated more pronounced with time of incubation. The colonies of sludge and trickling filters, and it has been suggested that 2. strain 12670B were tough, leathery, dry, wrinkled, and pale ramigera plays an important role in these environments. The yellow. We examined both of the newly isolated clones in this following three strains of 2. ramigera have historically been study. All phenotypic tests, DNA-DNA hybridization assays, well-known through a number of experimental studies: type and 16s rDNA sequencing were performed as previously de- strain ATCC19544 (= N. C. Dondero 106) (22), ATCC 19623 scribed (9-11, 19). A distance matrix tree based on 16s rDNA (= K. T. Crabtree I-16-M) (2), and ATCC 25935T (= P. R. sequences was constructed by the neighbor-joining method Dugan 115T) (4, 6, 12). During studies to taxonomically revise (18) with the CLUSTAL W program (21). Randomly amplified 2. ramigera, however, Unz (22, 23) mentioned that there were polymorphic DNA (RAPD) analysis (24, 27) was performed major phenotypic differences among these three strains of 2. with a Ready-To-Go RAPD analysis kit (Pharmacia, Uppsala, ramigera and that strains ATCC 19623 and ATCC 25935T Sweden) and with crude cell lysates as sources of template should be removed from the genus Zoogloea. Recent research DNA, these crude cell lysates were prepared by a previously on polyamine patterns (8), fatty acid and quinone profiles (9), described protocol (11). The PCR primers used were the fol- and 16s ribosomal DNA (rDNA) sequences of Zoogloea lowing six RAPD primers offered as a set by the manufacturer: strains (17, 19) provided evidence that there has been chemo- primer 1, 5 ‘-GGTGCGGGAA-3’; primer 2, 5’-GTTTCGCT taxonomic and phylogenetic divergence between 2. rarnigera CC-3‘; primer 3, 5‘-GTAGACCCGT-3’; primer 4, 5’-AAGA ATCC 19544T and the other two strains. The chemotaxonomic GCCCGT-3’; primer 5, 5’-AACGCGCAAC-3’; and primer 6, and phylogenetic data, together with the earlier phenotypic 5’-CCCGTCAGCA-3’. The RAPD reaction and detection of information, led to a reevaluation of the taxonomic criteria for RAPD patterns by gel electrophoresis were performed accord- identification of 2. ramigera and emendation of the genus ing to the manufacturer’s instructions. Zoogloea (19). The data require that each of the misnamed 2. The two strains that were repurified differed in floc forma- ramigera strains be reclassified as a member of an appropriate tion in addition to colony appearance. Strain 12670A exhibited known genus or a new genus. Here we report the results of a dispersed growth with no or little formation of visible flocs reexamination of one of the misnamed strains, strain JAM when it was cultured with shaking in complex liquid media 12670T (= ATCC 25935T), and propose that this bacterium containing peptone. This strain formed visible flocs only when should be reclassified as a strain of Duganella zoogloeoides gen. it was grown in chemically defined medium supplemented with nov., sp. nov. organic acids, such as tartrate, as the sole carbon source. The Strain IAM 12670T was obtained from the Culture Collec- flocs formed were amorphous. On the other hand, strain tion Center of the Institute of Applied Microbiology (now 12670B constantly produced cell aggregates both in complex IAM Culture Collection, Institute of Molecular and Cellular media and in chemically defined media. The flocs formed by Biosciences), The University of Tokyo, Tokyo, Japan. After this strain were amorphous but, as is the case in “typical” several transfers of the strain on agar media, two morpholog- Zoogloea strains, were fingerlike occasionally. Strains 12670A and 12670B were indistinguishable from each other in all other characteristics investigated, including cell morphology and * Corresponding author. Mailing address: Department of Ecological physiological, biochemical, and chemotaxonomic characteris- Engineering, Toyohashi University of Technology, Tenpaku, Toyo- tics (for details, see the descriptions of the genus and species hashi 441, Japan. Phone: 81-532-44-6913. Fax: 81-532-44-6929. below). The phenotypic studies indicated that the two strains j- Present address: Korean Collection for Type Cultures, Genetic Resources Center, Korea Research Institute of Bioscience & Biotech- were variants that originated from a single strain. Variations in nology, Korea Institute of Science & Technology, Taejon 305-333, colony appearance of the original strain, 2. rurnigeru P. R. Korea. Dugan 115T, on agar media have been reported previously (4). 1249 1250 NOTES INT.J. SYST.BACTERIOL. 12670T from its recognized phylogenetic neighbors at the ge- neric level. 1 2 3 4 5 6 On the basis of phenotypic, chemotaxonomic, and phyloge- n n n n n n netic evidence noted above and elsewhere (8, 17, 19, 23), we GABABABABABABS propose that 2. rumigeru IAM 12670Tshould be reclassified as a member of a new genus and new species with the name Duganella zoogloeoides. Differential characteristics of D. zoog- bP loeoides and phylogenetically and phenotypically related or- ganisms are summarized in Table 1. Although our proposal allows the existence of only one strain in the new genus at this 1882 -.-- time, this is reasonable considering the necessity for avoiding 1489 - further confusion in Zuogloeu taxonomy and also the impor- tance of the strain in the field of wastewater microbiology and biotechnology (4). A number of strains that show high levels of 16s rDNA sequence similarity to D. zoogloeoides have recently been iso- lated from soil (16). The partial 16s rDNA sequences (ca. 500 bases) of these new strains (DDBJ, EMBL, and GenBank accession no. D84564, D84572, D84574, D84576, and D84577) FIG. 1. RAPD patterns of strains 12670A and 12670B. The patterns obtained have similarity levels of 96.7 to 98.1% with the sequence of with arbitrary PCR primers 1 to 6 (indicated at the top) are shown. Lanes A and D. zoogloeoides IAM 12670T, suggesting that all of these or- B contained strains 12670A and 12670B, respectively. PCR products were sep- ganisms may form a phylogenetically coherent group at the arated by gel electrophoresis (2% agarose) and were detected by ethidium generic level. Further study of the new soil strains noted above bromide straining. Size marker, A-EcoT14 I digest. should provide more criteria to circumscribe the new genus Duganella. Also, the D. zoogloeoides description should be- Also, studies with typical 2. ramigera strains have shown that nonflocculating variants appear spontaneously upon subcul- ture (20). Yariovoraxparadom We confirmed the genetic homogeneity of strains 12670A Comamonas testosteroni and 12670B by DNA-DNA hybridization studies, as they showed 93 to 102% hybridization to each other in two different Burkholderia caryophylli assays. The guanine-plus-cytosine contents of the genomic Burkholderia cepacia DNAs of the two strains varied from 63.4 to 63.8 mol%, but the Ralstonia solanacearum variations appeared to be within the range of analytical error. Ralstonia pickettii We also found that the small-subunit rRNA structures of the Ralstonia eutropha two strains were identical. Moreover, there was no difference II 73 between the two strains in their RAPD patterns with the six arbitrary PCR primers (Fig. l), suggesting that they were de- Zoogloea ramigera LAM 12670 :::! zEg zzt:aes rived from the same strain. 294 Pseudomonas Iemoignei The 16s rDNA sequence analysis of strain IAM 12670T Nitrosolobus multiformis performed in this study revealed that there were some errors in Alcaligenes faecalis 280 the sequence for the strain previously reported by us (DDBJ, Bordetella bronchiseptica EMBL, and GenBank accession no. D14256) (19). Our revised 1- Taylorella equigenitalis Rhodocycluspupureus sequence for strain IAM 12670T differed at only one position 657 Zoogloea ramigera from the sequence of strain ATCC 25935T published by Ros- 0.02 sello-Mora et al. (accession no. X74914) (17). Previous phylo- Spirillum volutans Neisseria gonorrhoeae genetic studies have indicated that strain IAM 12670T 865 (= ATCC 25935T), as well as 2. rumigeru ATCC 19544T, be- Chromobacterium violaceum long to the beta subclass of the class Proteobucteriu, but that Escherichia coli within this subclass, the two strains form different clusters at FIG. 2. Phylogenetic tree derived from an analysis of the 16s rDNA se- the generic level (17,19). We reconstructed a phylogenetic tree quences of strain IAM 12670T and related organisms belonging to the beta subclass of the class Proteobacteria.