J. Gen. Appl. Microbiol., 44, 201–210 (1998) Reclassification of marine Agrobacterium species: Proposals of Stappia stellulata gen. nov., comb. nov., Stappia aggregata sp. nov., nom. rev., Ruegeria atlantica gen. nov., comb. nov., Ruegeria gelatinovora comb. nov., Ruegeria algicola comb. nov., and Ahrensia kieliense gen. nov., sp. nov., nom. rev. Yoshihito Uchino, Aiko Hirata, Akira Yokota,* and Junta Sugiyama Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113–0032, Japan (Received April 17, 1998; Accepted June 15, 1998 ) The bootstrapped 16S rDNA sequence-based neighbor-joining phylogeny has suggested that the marine species of the genus Agrobacterium have no relation to the terrestrial Agrobacterium -type strain), Agrobacterium ferru؍species. Agrobacterium atlanticum IAM 14463T (a superscript T gineum IAM 12616T, Agrobacterium gelatinovorum IAM 12617T, Agrobacterium meteori IAM 14464T, Agrobacterium stellulatum IAM 12621T and IAM 12614, and the invalidly published marine species “Agrobacterium kieliense” IAM 12618 occupy an independent position in the a-subclass of the Pro- teobacteria. Based on 16S rDNA sequencing and on chemotaxonomic, morphological, and physio- logical studies, we propose the transfer of A. atlanticum, A. gelatinovorum, and Roseobacter algi- cola to the genus Ruegeria gen. nov. as Ruegeria atlantica comb. nov., Ruegeria gelatinovora comb. nov., and Ruegeria algicola comb. nov., respectively; of strains of A. stellulatum to the genus Stap- pia gen. nov. as Stappia stellulata comb. nov. and Stappia aggregata sp. nov., nom. rev., respec- tively; and of “A. kieliense” to the genus Ahrensia gen. nov. as Ahrensia kieliense sp. nov., nom. rev. Agrobacterium meteori is assigned to be a synonym of A. atlanticum. Key Words——Ahrensia kieliense gen. nov., sp. nov., nom. rev.; bacterial systematics; marine Agrobac- terium spp.; Ruegeria algicola comb. nov.; Ruegeria atlantica gen. nov., comb. nov.; Ruegeria gelatinovora comb. nov.; Stappia aggregata sp. nov., nom. rev.; Stappia stellulata gen. nov., comb. nov. The genus Agrobacterium has been reported to in- able, as suggested by Rüger and Höfle (1992), that clude terrestrial and plant-pathogenic species and ma- they were classified only tentatively until further taxo- rine species that form star-shaped aggregates (Ker- nomic data were available. sters and De Ley, 1984; Rüger and Höfle, 1992; Our phylogenetic studies based on 16S rDNA se- Sawada et al., 1993; Stapp and Knösel, 1954). Rüger quences revealed the heterogeneity of the marine and Höfle proposed that the genus Agrobacterium subdivision species of the genus Agrobacterium must be divided into two subdivisions: i.e., subdivision (Uchino et al., 1997). These species have no relation 1 accommodates the terrestrial plant pathogens A. rhi- to the terrestrial Agrobacterium species, and the taxo- zogenes, A. rubi, A. tumefaciens, and A. vitis, nomic position of the marine subdivision of Agrobac- whereas subdivision 2 accommodates the marine terium should therefore be reassessed. We showed star-shaped aggregate-forming species A. atlanticum, that A. ferrugineum IAM 12616T formed a cluster with A. ferrugineum, A. gelatinovorum, A. meteori, and A. the photosynthetic bacteria of the genus Rhodobacter; stellulatum. Although these five marine species were A. atlanticum IAM 14463T, A. meteori IAM 14464T, listed in the Validation List of Bacterial Names, the and A. gelatinovorum IAM 12617T formed a cluster taxonomic positions of the species are so question- with the species of the genus Roseobacter; A. stellu- latum IAM 12621T and IAM 12614 formed a single cluster located independently from other genera in the * Address reprint requests to: Dr. Akira Yokota, Institute of Molec- Proteobacteria a-2 subgroup and “A. kieliense” IAM ular and Cellular Biosciences, The University of Tokyo, 1–1–1 12618 was clearly separated from the other genera in Yayoi, Bunkyo-ku, Tokyo 113–0032, Japan. 202 UCHINO et al. Vol. 44 the Proteobacteria a-2 subgroup. paring growth on a BM medium (Akagawa, 1994; Bau- In this report, we propose new taxonomic treat- mann et al., 1971) with the potassium ions replaced ments for the marine Agrobacterium species on the by sodium ions. Oxidase activity was determined by basis of the 16S rDNA sequence analysis and of oxidation of 1% tetramethyl-p-phenylenediamine on chemotaxonomic, morphological, and physiological filter paper, and catalase activity was determined by data. bubble production in a 3% hydrogen peroxide solu- tion. The detection of bacteriochlorophyll a was per- Materials and Methods formed both in vivo and in vitro. In vivo spectra were Bacterial strains. The bacterial strains used in this determined on cell suspension in a 60% sucrose solu- study were A. atlanticum IAM 14463T, A. ferrugineum tion from 3-day cultures grown aerobically on marine IAM 12616T, A. gelatinovorum IAM 12617T, A. meteori agar. In vitro spectra were determined by using IAM 14464T, A. stellulatum IAM 12614, A. stellulatum methanol extracts of 3-day cultures grown aerobically IAM 12621T, “A. agile” IAM 12615, and “A. kieliense” on marine broth. The absorbance of the cell suspen- IAM 12618 (Table 1). Furthermore, Rhodobacter cap- sions and methanol extracts was examined by using a sulatus IAM 14232T, Rhodobacter sphaeroides IAM Shimadzu spectrophotometer UV-3000 (Shimadzu, 14237T, Roseobacter algicola IAM 14591T, Roseobac- Kyoto, Japan). The photosynthetic activity was deter- ter denitrificans IAM 14592T, and Roseobacter litoralis mined by observing the growth under anaerobic con- IAM 14593T were used as reference strains. All the ditions with light. marine strains were grown aerobically on plates of Analyses of isoprenoid quinones and fatty acids. Difco marine agar 2216 (Difco, Detroit, MI, USA) for 2 Isoprenoid quinones were extracted from 100 mg of days at 25°C or in Difco marine broth 2216 for 2 days freeze-dried cells with chloroform/methanol (2 : 1, v/v) at 25°C. The phototrophic bacteria were also grown and purified by thin-layer chromatography (TLC) by phototrophically at 30°C in an SA medium (Kawasaki using n-hexane-diethyl ether (85 : 15, v/v). The et al., 1992). ubiquinone fraction was analyzed by high-perfor- Phenotypic characteristics. Cells in the early ex- mance liquid chromatography (HPLC). ponential growth phase grown on solid media were Fatty acids were extracted from 50 mg of freeze- used for morphological and physiological tests. Motil- dried cells after methylating with 10% hydrogen chlo- ity was observed under a light microscope by using ride-methanol. Isolation of the hydroxy and nonhy- cells in the logarithmic growth phase grown on Difco droxy fatty acid methyl esters was performed by TLC marine agar 2216. Flagellation was observed with a using n-hexane-diethyl ether (1 : 1, v/v). The fatty acid model JEOL 1210 transmission electron microscope methyl ester composition was determined by gas after negative-staining with phosphotungstic acid. Ob- chromatography. servations of the cells by transmission electron mi- DNA-DNA hybridization. Chromosomal DNA was croscopy were performed as follows: The cells were extracted and purified by the method of Marmur fixed in 3% glutaraldehyde and 4% osmium tetroxide (1961). DNA-DNA homology assays were performed (OsO4), dehydrated through a graded ethanol series, by microplate hybridization with photobiotin labeling then embedded in Epon 812. Thin sections were and fluorometric detection (Shatha et al., 1993). The stained with uranyl acetate and lead citrate and exam- level of hybridization was determined by measuring ined under an electron microscope, model JEOL 1210 the fluorescence intensity with a Shimadzu model CS- (Kawasaki, 1993). 9300PC. Sodium ion requirements were determined by com- Detection of genes related to photosynthesis. Table 1. Bacterial strains studied. Species IAM No.a Other designation Reclassified as Agrobacterium atlanticum IAM 14463T DSM 5823T Ruegeria atlantica Agrobacterium ferrugineum IAM 12616T ATCC 25652T Agrobacterium gelatinovorum IAM 12617T ATCC 25655T Ruegeria gelatinovora Agrobacterium meteori IAM 14464T DSM 5824T Ruegeria atlantica Agrobacterium stellulatum IAM 12614 ATCC 25650 Stappia aggregata Agrobacterium stellulatum IAM 12621T ATCC 15215T Stappia stellulata “Agrobacterium agile” IAM 12615 ATCC 25651T “Agrobacterium kieliense” IAM 12618T ATCC 25656T Ahrensia kieliense a A superscript “T” indicates the type strain. 1998 Reclassification of marine Agrobacterium species 203 Chromosomal DNA was extracted and purified by the method of Marmur (1961). The pufL and pufM genes were detected by amplifying their parcel DNA frag- ments by a polymerase chain reaction (PCR) using chromosomal DNA, Taq polymerase (Takara Shuzo, Kyoto, Japan), and the primer sets described by Nagashima et al. (1997). Phylogenetic analysis. The phylogenetic analysis based on the 16S rDNA sequence of the species within the Proteobacteria a-3 subgroup was per- formed as described in the previous report (Uchino et al., 1997). The 16S rDNA sequences of the following six species were newly obtained from the DNA data bank. These were: Amaricoccus kaplicensis (acces- sion number: U88041), Amaricoccus macauensis (U88042), Amaricoccus veronensis (U88043), Amari- coccus tamworthensis (U88044), Sulfitobacter pontia- cus (Y13155), and Sagittula stellata (U58356). Results and Discussion Morphological and physiological characteristics All eight marine strains were gram-negative, rod- shaped organisms whose cells were 0.6 to 1.0 mm wide and 2.0