Reclassification of Marine Agrobacterium Species: Proposals

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 species. Agrobacterium atlanticum IAM 14463T (a superscript Ttype strain), Agrobacterium ferrugineum 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 physiological studies, we propose the transfer of A. atlanticum, A. gelatinovorum, and Roseobacter algicola 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., respectively; 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. stellulatum 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 performed 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 pontiacus (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 to 4.0 mm long. Motile cells were observed in all the strains except A. atlanticum IAM 14463T, A. ferrugineum IAM 12616T, and A. meteori IAM 14464T and had several polar ﬂagella (Fig. 1). A star-shaped aggregate formation was sometimes observed, but not always. The physiological and chemotaxonomic characteristics of the strains are summarized in Table 2. “A. agile” IAM 12615 grew well on a medium without sodium ions, indicating it did not require sodium ions to grow. Because the strain A. ferrugineum IAM 12616T scarcely grew on the BM medium, the requirement of this strain for sodium ions could not be con- ﬁrmed. Other strains required sodium ions for their growth. All strains tested had oxidase and catalase activities.

Chemotaxonomic characteristics The major quinone of “A. agile” IAM 12615 was ubiquinone 9, and that of the other strains was T ubiquinone 10. A. atlanticum IAM 14463 and A. me- Fig. 1. Electron micrographs of whole cells of strain IAM teori IAM 14464T showed a similarity in their cellular 12616T stained with phosphotungstic acid. fatty acid compositions except for the presence of oc- A, Agrobacterium gelatinovorum IAM 12617T; B, “Agrobacterium T tadecanoic acid (18 : 0) in A. meteori IAM 14464T. The kieliense” IAM 12618; C, Agrobacterium stellulatum IAM 12621 . Scale bars0.2 mm. fatty acid compositions of A. stellulatum IAM 12621T and IAM 12614 were the same. In the other strains they were different from one another (Table 2). close to the genus Rhodobacter (Fig. 2), and A. atlanticum IAM 14463T, A. gelatinovorum IAM 12617T, Detection of photosynthetic activities and A. meteori IAM 14464T formed a cluster with the The 16S rDNA sequence analysis suggested that A. aerobic, photosynthetic species of the genus ferrugineum IAM 12616T was phylogenetically very Roseobacter. However, these four species did not 204 UCHINO et al. Vol. 44

Table 2. Biochemical, physiological, and chemotaxonomic characteristics of marine Agrobacterium species.a

Fatty acid (%)b Requirement Species IAM No. Morphology Motility Catalase Oxidase Quinone Non-polar 2-OH 3-OH for Na 16 : 0 16 : 1 18 : 0 18 : 1 16 : 0 10 : 0 12 : 0 14 : 0 14 : 1

Agrobacterium 14463T rods Q-10 80 90 77 19c atlanticum Agrobacterium 12616T rods ND Q-10 76 — 54 38 ferrugineum Agrobacterium 12617T rods Q-10 28 56 — 96 gelatinovolum Agrobacterium 14464T rods Q-10 24 58 96 79 15 meteori Agrobacterium 12614 rods Q-10 23 71 — 94 stellulatum Agrobacterium 12621T rods Q-10 22 72 — 62 stellulatum “Agrobacterium 12615 rods Q-9 18 33 19 — 34 53 agile” “Agrobacterium 12618 rods Q-10 81 — 85 kieliense”

a , positive; , negative; ND, no data. b Only major components were indicated. c 3-OH 14:1 was identiﬁed by gas chromatography-mass spectrometry.

Table 3. Comparison of the genus Ruegeria and Roseobacter with genera belonging to the a-3 subgroup of the Proteobacteria.

Major hydroxy fatty acids Oxidase Catalase Photo- Bacterio- GC content Flagella Quinone activity activity synthesis chlorophyll a (mol%) 2-OH 3-OH

Roseobacter subpolar ()a none 10 : 0, 14 : 1 Q-10 56–60 Ruegeria polar/subpolar/ 16 : 0/none 12 : 0, 10 : 0, Q-10 55–64 nonmotile (14 : 1)b Amaricoccus nonmotile or ND ND ND 51–63 Paracoccus nonmotile/polar + none 10 : 0, (14 : 0) Q-10 64–70 Rhodobacter polar/nonmotile NDc ND none 10 : 0, 14 : 1 Q-10 64–70 Rhodovulum nonmotile/polar ND ND ND ND Q-10 62–69 Sagittula nonmotile none 12 : 1 ND 65 Sulﬁtobacter subpolar ND ND ND ND

a Aerobic phototrophic activity. b The components in parentheses are the minor components. c No data. grow phototrophically and did not contain bacteri- calculation, were generally similar to those in the pre- ochlorophyll a (Tables 3, 4). To investigate the photo- vious study (Fig. 2) (Uchino et al., 1997). Four species synthetic activity of these four species, we tried to de- of the genus Amaricoccus (Maszenan et al., 1997) tect genes for photosynthesis, e.g., pufL and pufM, were grouped in a single cluster whose independence but none could be found. Furthermore, we were un- was supported by a high bootstrap value (100%). Sul- able to detect intracytoplasmic membrane systems in fitobacter pontiacus (Sorokin, 1995) formed a cluster A. ferrugineum IAM 12616T (Fig. 3). These results in- with Roseobacter litoralis and R. denitrificans, and the dicate that A. ferrugineum IAM 12616T is not closely bootstrap value was relatively high (80%). Roseobac- related to Rhodobacter species. ter algicola formed a cluster with A. atlanticum IAM 14463T, A. gelatinovorum IAM 12617T, and A. meteori A. atlanticum, A. ferrugineum, A. gelatinovorum, and IAM 14464T, and the bootstrap value was 52%. Sagit- A. meteori in the Proteobacteria a-3 subgroup tula stellata (Gonzalez et al., 1997) formed a cluster The results of the neighbor-joining (NJ) phyloge- with three species of the genus Roseobacter, three netic analysis of the species within the Proteobacteria species of the genus Agrobacterium, and S. ponti- a-3 subgroup, to which six species were added for the acus. The confidence of unity of these groups was 1998 Reclassification of marine Agrobacterium species 205

Fig. 2. Neighbor-joining tree of species of Proteobacteria a-3 subgroup inferred from 16S rDNA. The percentage of bootstrap confidence level was derived from 1,000 resamplings: bold lines indicate branches having greater than 95% bootstrap confidence. supported by a high bootstrap value (100%). The species could be separated into three groups; first, R. bootstrap value of the cluster of these seven species, litoralis, R. denitrificans, and S. pontiacus; second, A. excluding S. stellata, was 57%. The relationship of atlanticum IAM 14463T, A. gelatinovorum IAM 12617T, these eight species in the neighbor-joining analysis A. meteori IAM 14464T, and R. algicola; and third, S. was identical to parsimony analysis (data not shown). stellata. Two different analyses indicated that these eight The 16S rDNA sequence similarity values were 206 UCHINO et al. Vol. 44

teori IAM 14464T and IFO 15793T (identical with IAM 14464T) was nonmotile and catalase-positive (Table 2). Although the discrepancy between these reports has not been well explained, based on the 16S rDNA similarity and the level of DNA-DNA hybridization, we considered that A. meteori is a synonym of A. atlanticum. Rüger and Höfle (1992) reported that A. ferrugineum IAM 12616T required seawater, but this was not confirmed in our study. This result is consistent with the fact that the natural habitat of the species of Rhodobacter is freshwater (Hiraishi and Ueda, 1994). As for cellular fatty acids, A. ferrugineum IAM 12616T has 3-hydroxy decanoic acids (3-OH 10 : 0) and 3-hy- Fig. 3. Electron micrograph of a thin section of strain IAM droxy tetradecanoic acids (3-OH 14 : 1), which are 12616T stained with uranyl acetate and lead citrate. common in Rhodobacter species. However, it differs Scale bars0.1 mm. from the species of Rhodobacter in the GC content (A. ferrugineum IAM 12616T: 58%; Rhodobacter: about 70%), by involving the insertion of 15 bases be- below 97% among A. ferrugineum IAM 12616T and tween positions 189 and 203 (E. coli numbering sys- the species of the genus Rhodobacter; among A. at- tem) (Brosius et al., 1981) in the 16S rRNA gene, and lanticum IAM 14463T, A. gelatinovorum IAM 12617T, by the absence of photosynthetic abilities, bacteri- A. meteori IAM 14464T, and the species of the genus ochlorophyll a, and intracytoplasmic membrane sys- Roseobacter; and between A. atlanticum IAM 14463T tems, which are characteristics for the definition of the (or A. meteori IAM 14464T) and A. gelatinovorum IAM genus Rhodobacter (Table 4, Fig. 3). A. ferrugineum 12617T. These values are low enough to indicate that IAM 12616T should be excluded from the genus these strains do not hybridize with one another or with Agrobacterium, but further studies are necessary to any bacteria at the species level (Stackebrandt and give adequate taxonomic position for it. Goebel, 1994). The 16S rDNA similarity value be- Based on the results described above, we propose tween A. atlanticum IAM 14463T and A. meteori IAM that A. atlanticum IAM 14463T, A. gelatinovorum 14464T was 100%, and the level of DNA-DNA homol- IAM12617T, and A. meteori IAM 14464T should be ogy between them was 99%, which indicates that transferred to the new genus Ruegeria as species of these two strains belong to the same species. new combination. A. meteori IAM 14464T was as- A. atlanticum IAM 14463T, A. gelatinovorum IAM signed to be a synonym of A. atlanticum. We also pro- 12617T, and A. meteori IAM 14464T formed a cluster pose to transfer Roseobacter algicola to the genus with Roseobacter algicola. The 16S rDNA sequence Ruegeria as Ruegeria algicola comb. nov. A. ferru- similarity value between R. algicola and A. gelatinovo- gineum IAM 12616T was deferred an official nomen- rum IAM 12617T was 96.1%, which is higher than that clatural proposal to other new species until more phe- between R. algicola and R. denitrificans (95.1%), or notypic data are available for differentiation. between R. algicola and R. litoralis (94.9%). The genus Roseobacter was originally defined as the A. stellulatum and “A. kieliense” in the Proteobacteria genus containing aerobic photosynthetic species, in- a-2 subgroup cluding R. denitrificans and R. litoralis (Shiba, 1991), Our phylogenetic analysis revealed that A. stellula- but R. algicola, a species with no bacteriochlorophyll a tum IAM 12621T and IAM 12614 formed a single clus- and no aerobic photosynthesis, has been added to ter located independently from the genera in the Pro- this genus (Lafay et al., 1995). Therefore the definition teobacteria a-2 subgroup, and that “A. kieliense” IAM of the genus Roseobacter is ambiguous at present. 12618 is clearly separated from the other genera in Based on the physiological, chemotaxonomic, and the Proteobacteria a-2 subgroup. The independence 16S rDNA sequence data, we propose that A. at- of these species was assured by several other kinds lanticum IAM 14463T, A. gelatinovorum IAM 12617T, of phenotypic and genotypic evidence (Table 5). The A. meteori IAM 14464T, and R. algicola should be 16S rDNA sequence homology value between A. stel- classified in a new genus Ruegeria (Table 3). lulatum IAM 12621T and the strain IAM 12614 was Rüger and Höfle (1992) reported that A. meteori lower than 97%. This value is low enough to indicate DSM 5824T (identical with IAM 14464T) is motile and that these bacteria do not hybridize with each other at catalase-negative. However, in our experiment, A. me- the species level (Stackebrandt and Goebel, 1994). 1998 Reclassification of marine Agrobacterium species 207

Table 4. Comparison of taxonomic characteristics between A. ferrugineuma and species of the genus Rhodobacter.b,c

A. ferrugineum Characteristic R. veldkampii R. capsulatus R. blasticus R. azotoformans R. sphaeroides IAM 12616T

Morphology ovoid or rod ovoid or rod ovoid or rod ovoid or rod variable rodd Arrangement of ﬂagella polar polar nonmotile polar polar nonmotiled Optimum growth temperature (°C) 30–35 30–35 30–35 30–35 30–34 20–30 Nitrate assimilation ND Utilization of Citrate NDe ND Glycerol ND Mannitol ND NaCl or seawater requirement Aerobic dark growth d Growth occurs photoheterotrophically d under anaerobic condition Production of bacteriochlorophyll a d GC content of DNA (mol%) 64–67 68–69 65 69–70 70–73 58 3-OH fatty acid composition ND 10 : 0, 14 : 1 14 : 1, 10 : 0 ND 10 : 0, 14 : 1 10 : 0, 14 : 1d 16S rRNA signature(s) at position(s)d 66–103 G-C G-C G-C G-C G-C A-T 190–204 — — — — — CACTGTAGTGGTGGG 835–851 G-C G-C G-C G-C G-C A-T 841 C C C C C T 845 G G G G G A 1011–1018 C-G C-G C-G C-G C-G T-A 1012–1017 A-T A-T A-T A-T A-T G-C

a Rüger and Höﬂe (1992). b Imhoff (1984). c Hiraishi et al. (1996). d Determined in this study. e ND, no data.

Table 5. Comparison of the genus Ahrensia and Stappia with genera belonging to the a-2 subgroup of the Proteobacteria.

Major hydroxy fatty acids Oxidase Catalase GC content Flagella Quinone activity activity (mol%) 2-OH 3-OH

Ahrensia peritrichous none 12 : 0 Q-10 48 Stappia peritrichous none 14 : 0 Q-10 59 Devosia polar none 24 : 1, 26 : 1 Q-10 61 Agrobacterium peritrichous or (18 : 1) 14 : 0, 16 : 0 (i15 : 0)c Q-10 55–65 Bradyrhizobium polar NDa ND none 12 : 0, 14 : 0 Q-10 61–65 Brevundimonas polar (12 : 0) 12 : 0 (10 : 0) Q-10 65–68 Methylobacterium lateral/polar or w b none 14 : 0 Q-10 60–70 Mycoplana peritrichous ND none 12 : 0 (14:0) Q-10 63–68 Phyllobacterium lateral/polar ND ND ND ND ND 60–61 Rhizomonas lateral/polar 14 : 0 none Q-10 58–65 Rhizobium peritrichous/polar ND ND (18 : 1) 14 : 0, 16 : 0 (i13 : 0/ai15 : 0) Q-10 59–64 Sphingomonas polar ND 14 : 0 none Q-10 59–68

a ND, no data. b w, weakly positive. c The components in parentheses are the minor components. i, iso-branched chain fatty acid; ai, anteiso-branched chain fatty acid.

A. stellulatum IAM 12614 was formerly labeled as long to different species (Stackebrandt and Goebel, “A. aggregatum,” but it was regarded as a synonym of 1994). Therefore we propose to transfer A. stellulatum A. stellulatum by Rüger and Höfle (1992). However, in IAM 12614 and A. stellulatum IAM 12621T to the new our analysis the 16S rDNA sequence similarity value genus Stappia as Stappia aggregata sp. nov., nom. between A. stellulatum IAM 12614 and IAM 12621T rev. and Stappia stellulata comb. nov., respectively. was 95.1%, which suggests that these two strains be- Based on 16S rDNA sequencing, and morphologi- 208 UCHINO et al. Vol. 44 cal and biochemical characteristics, “A. kieliense” is Description of Ahrensia gen. nov. Ahrensia also relatively isolated in the Proteobacteria a-2 sub- (Ahrens.i.a. M.L. dim. ending -ia; M.L. fem. n. Ahren- group. We therefore propose that this bacterium sia, honoring Ahrens, a German microbiologist, for his should be classified in a new genus, Ahrensia as contribution to the taxonomy of marine species of Ahrensia kieliense sp. nov., nom. rev. Agrobacterium). The cells are gram-negative rods 0.6 to 1.0 mm wide and 2.0 to 4.0 mm long. They do not Description of Stappia gen. nov. Stappia (Stappi. form spores and are motile by means of polar flagella. a. M. L. dim. ending -ia; M. L. fem. n. Stappia, honor- They grow aerobically. Oxidase-positive and catalase- ing Stapp, a Belgian microbiologist, for his contribution positive. The major quinone is ubiquinone 10. The to the taxonomy of marine species of Agrobacterium). major fatty acid is 18 : 1. The 3-hydroxy fatty acid is 3- The cells are gram-negative rods, 0.6 to 1.0 mm wide OH 12 : 0. The 2-hydroxy fatty acids are absent. The and 2.0 to 4.0 mm long. They do not form spores and GC content of the DNA is 48 mol%. Belong to Pro- are motile by means of polar flagella. They grow aero- teobacteria a-2 subgroup. bically; photosynthetic growth does not occur. Oxi- The type species is Ahrensia kieliense. dase-positive and catalase-positive. Isolated from marine sediment and seawater. Require seawater or Na Description of Ahrensia kieliense (ex Ahrens 1968) for growth. The major quinone is ubiquinone 10. The sp. nov., nom. rev. Ahrensia kieliense (kiel.i.ense. major fatty acids are 18 : 1 and 18 : 0. The 3-hydroxy M.L. fem. n. Kiel, pertaining to Kiel, the source of the fatty acid is 3-OH 14 : 0. The 2-hydroxy fatty acids are soil from which the organism was isolated). The fol- absent. The GC content of the DNA is 59 mol%. Be- lowing description is based on our own observations long to Proteobacteria a-2 subgroup. and previous descriptions of the species by Rüger and The type species is Stappia stellulata. Höfle (1992). Nitrate is not reduced to nitrite or gas. Acids are produced from fructose and xylose after 4 to Description of Stappia aggregata (ex Ahrens 1968) 6 weeks of incubation. None of 34 organic substrates sp. nov., nom. rev. Stappia aggregata (ag.gre. is utilized at 20°C. Isolated from seawater of the Baltic ga’ta. L. adj. aggregatus joined together, referring to Sea. The major quinone is ubiquinone 10. The major the frequent formation of rosettes). The following de- fatty acid is 18 : 1. The 3-hydroxy fatty acid is 3-OH scription is based on our own observations and on 12 : 0. The 2-hydroxy fatty acids are absent. The GC previous descriptions of the species by Rüger and content of the DNA is 48 mol%. Höfle (1992). Nitrate is not reduced to nitrite, but it is The type strain is IAM 12618T. reduced to gas. No acid is produced from glucose or xylose; weak or variable acid production from fruc- Description of Ruegeria gen. nov. Ruegeria tose, glycerol, and maltose occurs after 4 to 6 weeks (Rueger.ia. M.L. ending -ia; M.L. fem. n. Ruegeria, of incubation. The major quinone is ubiquinone 10. honoring Rueger, a German microbiologist, for his The major fatty acids are 18 : 1 and 18 : 0. The 3-hy- contribution to the taxonomy of marine species of droxy fatty acid is 3-OH 14 : 0. The 2-hydroxy fatty Agrobacterium). The cells are gram-negative ovoid acids are absent. The GC content of the DNA is shape or rods 0.6 to 1.6 mm wide and 1.0 to 4.0 mm 59 mol%. long, and they do not form spores. They are motile or The type strain is IAM 12614. nonmotile, and the motile strains are motile by means of a polar flagella. They are oxidase- and catalase- Description of Stappia stellulata (Rüger and Höfle positive, and they grow aerobically. Photosynthetic 1992) comb. nov. Stappia stellulata (stel’lu.la.ta. growth does not occur. Bacteriochlorophyll a is ab- M.L. fem. n. stella star, stellulata star-shaped morphol- sent. The major quinone is ubiquinone 10. The GC ogy of cells). The following description is based on our content of the DNA is 55 to 59 mol%. Belongs to Pro- own observations and previous descriptions of the teobacteria a-3 subgroup. species by Rüger and Höfle (1992). Nitrate is not re- The type species is Ruegeria atlantica. duced to nitrite, but it is reduced to gas. No acid is produced from glucose, maltose, or mannitol after 4 to Description of Ruegeria atlantica (Rüger and Höfle 6 weeks of incubation. The major quinone is 1992) comb. nov. Ruegeria atlantica (at.lan’ti.ca ubiquinone 10. The major fatty acids are 18 : 1 and M.L. adj. atlantica, pertaining to the Atlantic Ocean as 18 : 0. The 3-hydroxy fatty acid is 3-OH 14 : 0. The 2- the locality). The following description is based on our hydroxy fatty acids are absent. The GC content of own observations and on previous descriptions of the the DNA is 59 mol%. species by Rüger and Höfle (1992). Nonmotile. Nitrate The type strain is IAM 12621T. is reduced to nitrite. Isolated from marine sediments of the northwestern Atlantic Ocean. Requires seawater 1998 Reclassification of marine Agrobacterium species 209 or Na for growth. The major quinone is ubiquinone bacteria in the marine environment. Doctor Thesis, the Tsukuba 10. The major fatty acid is 18 : 1. The 3-hydroxy fatty University. acids are 3-OH 12 : 0 and 3-OH 14 : 1. The 2-hydroxy Baumann, P., Baumann, L., and Mandel, M. (1971) Taxonomy of marine bacteria: The genus Beneckea. J. Bacteriol., 107, 268– fatty acid is 2-OH 16 : 0. The G+C content of the DNA 294. is 55 to 58 mol%. Brosius, J., Dull, T. J., Sleeter, D. D., and Noller, H. F. (1981) Gene The type strain is IAM 14463T. organization and primary structure of a ribosomal RNA operon from Escherichia coli. Mol. Biol.,148, 107–127. Description of Ruegeria gelatinovora (Rüger and Gonzalez, J. M., Mayer, F., Moran, M. A., Hodson, R. E., and Whit- man, W. B. (1997) Sagittula stellata gen. nov., sp. nov., a lignin- Höfle 1992) comb. nov. Ruegeria gelatinovora transforming bacterium from a coastal environment. Int. J. Syst. (ge.la.ti.no’vor.a. M.L. n. gelatinum gelatin, that which Bacteriol., 47, 773–780. stiffens; L. v. voro to devour; M.L. n. gelatinovora de- Hiraishi, A., Muramatsu, K., and Ueda, Y. (1996) Molecular genetic vours gelatin). The following description is based on analyses of Rhodobacter azotoformans sp. nov. and related our own observations and on previous descriptions of species of phototrophic bacteria. Syst. Appl. Microbiol., 19, 168–177. the species by Rüger and Höfle (1992). Motile by Hiraishi, A. and Ueda, Y. (1994) Intrageneric structure of the genus means of polar flagella. Nitrate is reduced to nitrite, Rhodobacter: Transfer of Rhodobacter sulfidophilus and re- but not to gas. Acids are produced from glycerol after lated marine species to the genus Rhodovulum gen. nov. Int. J. 4 to 6 weeks of incubation, but not from glucose, fruc- Syst. Bacteriol., 44, 15–23. tose, maltose, or xylose. Only 12 of 34 organic sub- Imhoff, J. F. (1984) Genus Rhodobacter Imhoff, Trüper and Pfennig 1984.342VP. In Bergey’s Manual of Systematic Bacteriology, strates are utilized. Isolated from seawater of the Vol. 3, ed. by Krieg, N. R. and Holt, J. G., The Williams & Baltic Sea. Requires seawater or Na . The major Wilkins Co., Baltimore, pp. 1668–1672. quinone is ubiquinone 10. The major fatty acids are Kawasaki, H., Hoshino, Y., Hirata, A., and Yamasato, K. (1993) Is 18 : 1 and 18 : 0. The 3-hydroxy fatty acid is 3-OH intracytoplasmic membrane structure a generic criterion? It 12 : 0. The 2-hydroxy fatty acids are absent. The GC does not coincide with phylogenetic interrelationships among phototrophic purple nonsulfur bacteria. Arch. Microbiol., 160, content of the DNA is 59 mol%. 358–362. T The type strain is IAM 12617 . Kawasaki, H., Hoshino, Y., Kuraishi, H., and Yamasato, K. (1992) Rhodocista centenaria gen. nov., sp. nov., a cyst-forming Description of Ruegeria algicola (Lafay et al. 1995) anoxygenic phototrophic bacterium and its phylogenetic posi- comb. nov. Ruegeria algicola (al.gi’co.la L. n. alga, tion in the Proteobacteria alpha group. J. Gen. Appl. Microbiol., 38, 541–551. L. subst. cola, dweller; M.L. n. algicola, algae dweller). Kersters, K. and De Ley, J. (1984) Genus III Agrobacterium Conn The following description is based on our own obser- 1942, 359. In Bergey’s Manual of Systematic Bacteriology, Vol. vation and previous descriptions of the species by 1, ed. by Krieg, N. R. and Holt, J. G., The Williams & Wilkins Lafay et al. (1995). The cells are ovoid in the logarith- Co., Baltimore, pp. 244–254. mic growth phase. Motile by means of one or two sub- Lafay, B., Ruimy, R., Rausch, C., Breittmayer, V., Gauthier, M. J., and Christen, R. (1995) Roseobacter algicola sp. nov., a new polar flagella. Colonies on salt-containing agar media marine bacterium isolated from the phycosphere of the toxin- are beige when the cultures are young and pinkish producing dinoflagellate Prorocentrum lima. Int. J. Syst. Bacte- beige after 96 h of incubation. Optimum growth tem- riol., 45, 290–296. peratures are from 25 to 30°C. Not able to denitrify. Marmur, J. (1961) A procedure for the isolation of deoxyribonucleic Oxidase-, catalase-, gelatinase-, esculinase-, -galac- acid from microorganisms. J. Mol. Biol., 3, 208–218. b Maszenan, A. M., Seviour, R. J., Patel, B. K. C., Rees, G. N., Mc- tosidase-, and (to a lesser extent) amylase-positive. Dougall, B. M. (1997) Amaricoccus gen. nov., a gram-negative Do not accumulate polyhydroxybutyrate. Isolated from coccus occurring in regular packages or tetrads, isolated from a culture of the toxin-producing dinoflagellate Proro- activated sludge biomass, and descriptions of Amaricoccus centrum lima PLV2, obtained from Vigo, Spain. Re- veronensis sp. nov., Amaricoccus tamworthensis sp. nov., quires Na for growth. Bacteriochlorophyll a is absent. Amaricoccus macauensis sp. nov., and Amaricoccus kaplicensis sp. nov. Int. J. Syst. Bacteriol., 47, 727–734. The major quinone is ubiquinone 10. The major cellu- Nagashima, K. V. P., Hiraishi, A., Shimada, K., and Matsuura, K. lar fatty acid is 18 : 1. The 3-hydroxy fatty acids, 3-OH (1997) Horizontal transfer of genes coding for the photosyn- 12 : 0, 3-OH 10 : 0, and 3-OH 14 : 1, are present, but thetic reaction centers of purple bacteria. J. Mol. Evol., 45, the 2-hydroxy fatty acids are absent. 131–136. The type strain is IAM 14591T. Rüger, H.-J. and Höfle, M. G. (1992) Marine star-shaped-aggregate- forming bacteria: Agrobacterium atlanticum sp. nov.; Agrobac- terium meteori sp. nov.; Agrobacterium ferrugineum sp. nov., We would like to thank Dr. Y. Nakagawa, Institute for Fermenta- nom. rev.; Agrobacterium gelatinovorum sp. nov., nom. rev.; tion, Osaka (Osaka, Japan) for determining the fatty acid methyl and Agrobacterium stellulatum sp. nov., nom. rev. Int. J. Syst. ester composition by gas chromatography-mass spectrometry. Bacteriol., 42, 133–143. Sawada, H., Ieki, H., Oyaizu, H., and Matsumoto, S. (1993) Pro- References posal for rejection of Agrobacterium tumefaciens and revised descriptions for the genus Agrobacterium and for Agrobac- Akagawa, M. (1994) Systematic study on the culturable aerobic terium radiobacter and Agrobacterium rhizogenes. Int. J. Syst. 210 UCHINO et al. Vol. 44

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