Showing papers by "Junta Sugiyama published in 1997"

Polyphyletic origins of species of the anamorphic genus Geosmithia and the relationships of the cleistothecial genera: Evidence from 18S, 5S and 28S rDNA sequence analyses

Abstract: The anamorphic genus Geosmithia, with the type species G. lavendula, includes species strictly lacking a teleomorph as well as species associated with the teleomorphs Talaromyces and Chromocleista. Our 18S rDNA sequence-based tree, inferred from 1586 alignable sites from 57 selected taxa within the Ascomycota and using two basidiomycetes as out- groups, clearly demonstrates that Geosmithia is a poly- phyletic taxon with evolutionary affinities to at least three groups of the euascomycete lineage within the

Proposal to reclassify Zoogloea ramigera IAM 12670 (P. R. Dugan 115) as Duganella zoogloeoides gen. nov., sp. nov.

Abstract: The taxonomic position of a misclassified strain, Zoogloea ramigera IAM 12670T (= ATCC 25925T = P. R. Dugan 115T), 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 Telluria 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.

Phylogenetic relationships of filamentous cyanobacterial taxa inferred from 16S rRNA sequence divergence.

Abstract: Cyanobacteria are autotrophic bacteria which perform oxygenic photosynthesis. Since cyanobacteria have been traditionally treated as a cluster of the plant kingdom in the past, they have been classified mainly based on cell morphology. Woese (1987) analyzed the phylogeny of the organisms by means of small subunit ribosomal RNA gene sequence divergence, and placed cyanobacteria within the eubacteria of prokaryotes. The taxonomy of cyanobacteria has been based only on their morphology up to now, and cyanobacteria are divided into five orders at present (Castenholz and Waterbury, 1989). These are Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales, and Stigonematales. Cyanobacteria which are filamentous and do not form heterocyst are accommodated in the order Oscillatoriales (Castenholz, 1989a). One of the differential characteristics to classify the genera of Oscillatoriales is whether or not a sheath is present, but this characteristic is known to change due to culturing conditions (Pearson and Kingsbury, 1966). Consequently, the definition of the genera belonging to Oscillatoriales is obscure. In this study, to grasp a general image of Oscillatoriales, the phylogenetic position of the eight genera of Oscillatoriales was investigated based on 165 rRNA gene sequences which have proven to be very useful for the study of bacterial phylogeny (e.g., Giovannoi et al., 1988; Stackebrandt,1992; Woese,1987). The axenic cyanobacterial strains used in this study are Oscillatoria neglects M-82, Oscillatoria rosea M220, Phormidium ambiguum M-71, Phormidium sp. M99, Phormidium sp. M-117, Phormidium mucicola M221, and Spirulina subsalsa M-223; these were obtained from the IAM culture collection. Oscillatoria neglecta M-82, Ph. ambiguum M-71, Phormidium sp. M99, and Phormidium sp. M-117 were grown in medium A-1 (Yamasato et al., 1993), 0. roses M-220 in medium f/2 (Watanabe and Nozaki, 1994), Ph. mucicola M-221 in medium Csi (Watanabe and Nozaki, 1994), and Sp. subsalsa M-223 in medium MA (Watanabe and Nozaki, 1994). These seven strains were cultured by incubation in test tubes without aeration at 20°C with fluorescent lamp illumination of 500 lux. Cells from 1.5 ml culture broth were harvested by centrifugation, washed with 1 ml of deionized water, and suspended in 100 μl of 20 mM Tris (pH 8.0) containing 0.1 mM EDTA, 0.5% Tween 20, and 0.1% Nonidet P-40 (Boehringer Mannheim, Germany). Final lysis was achieved by adding 10μg of Proteinase K and incubating for 20 min at 60°C. The almost complete 165 rDNA from the genomic DNA of the respective strains was amplified by PCR using oligonucleotide primers by the combination of 1 and 11, 8, and 16 as described by Wilmotte et al. (1993). PCR was performed for 5 min at 94°C at first and consisted of 33 cycles with the following features: 1 min at 94°C, 1 min at 52°C, and 2 min at 72°C followed by a final elongation step for 10 min at 72°C. The PCR product generated with Takara EX Taq polymerase (Takara Shuzo Co., Ltd., Japan) was cloned using an Original TA Cloning Kit® (Invitrogen Corporation, CA, U.S.A.). The sequencing reaction was performed with a SequiThermTM Long-ReadTM Cycle Sequencing Kit-LC (Ar Brown Co., Ltd., Tokyo, Japan) using IRD-41 labeled primers, M13 forward primer and M13 reverse primer. Sequencing was carried out on a LI-COR * Address reprint requests to: Dr. Akira Yokota, ular and cellular Biosciences, The University Yayoi, Bunkyo-ku, Tokyo 113, Japan. Institute of Molec-

Phylogenetic position of the marine subdivision of Agrobacterium species based on 16S rRNA sequence analysis.

Abstract: In the genus Agrobacterium, terrestrial and plant pathogenic species and marine star-shaped-aggregate-forming species were reported (Conn, 1942; Jordan, 1984; Ophel and Kerr, 1990; Ruger and Hofle, 1992; Starr and Weiss, 1943). The phylogenetic positions of the terrestrial and plant pathogenic species have already been made clear, but those of the marine species have not been published yet. Since Stapp and Knosel (1954) grouped a marine species as Agrobacterium stellulatum based on their formation of star-shaped aggregation, many marine isolates resemble to A, stellulatum as a species of the genus Agrobacterium (Ahrens, 1968; Ahrens and Rheinheimer, 1967). However, intrageneric relationships between marine and terrestrial Agrobacterium species remain uncertain. Ruger and Hofle (1992) performed a phenotypic analysis on these Agrobacterium species in addition to their marine isolates, and they insisted that among these marine strains, five species could be recognized as a distinct taxon. They believe these five species form a group independently from the terrestrial Agrobacterium species, but there were no distinctive phenotypic characteristics to separate the marine species from the genus Agrobacterium. Therefore, they concluded that the genus Agrobacterium must be divided into two subdivisions, accommodating the terrestrial and plant pathogenic species in subdivision 1 and the marine star-shaped-aggregate-forming five species Agrobacterium atlanticum, Agrobacterium ferrugineum, Agrobacterium gelatinovorum, Agrobacterium meteori and A. stellulatum in subdivision 2, until further taxonomic data are available (Ruger and Hofle,1992). In this communication, we determined 165 ribosomal RNA gene sequences of these marine subdivision species, including the non-validated species "Agrobacterium agile" and "Agrobacterium kieliense ," and investigated their phylogenetic position to clarify the classification of marine Agrobacterium species. The bacterial strains used in this study were A. atlanticum IAM 14463T, A, ferrugineum IAM 12616T, A. gelatinovorum IAM 12617T, A. meteori IAM 14464T, A. stellulatum IAM 12614, A. stellulatum IAM 12621T , "A. agile" IAM 12615 and "A. kieliense" IAM 12618. All the strains were grown aerobically on the plate of Difco marine agar 2216 for 2 days at 25°C. Cells grown in the late exponential phase were harvested, washed with sterile water and stored at -20°C until use. The 165 rRNA gene was amplified by PCR using Takara 7 aq (Takara Shuzo, Kyoto, Japan), the three forward primers 8F (5'-AGAGTTTGATCCTGGCTCAG-3'), 520F (5'-CAGCAGCCGCGGTAATAC-3') and 926F (5'-AAACTCAAAGGAATTGACGG-3'), and three reverse primers 704R (5'-TCTACGCATTTCACC-3'), 1110R (5'GGGTTGCGCTCGTTG-3') and 1510R (5'-GGCTACCTTGTTACGA-3') [Escherichia coli numbering system], by the combination of 8F-704R, 520F1110R and 926F-1510R. Amplified fragments were cloned using an Invitrogen TA CloningTM Kit (Invitrogen, CA, U.S.A.). Extraction and purification of the plasmids were carried out using a QlAprep Spin Plasmid Kit (Qiagen, Hilden, Germany). The cloned plasmids were used for sequencing. Sequencing was carried out using an ABI PRISMTM Dye Primer Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Co., CA, U.S.A.) and a model ABI 3735 automatic DNA sequencer (Perkin-Elmer Co.). Previously published 165 rRNA gene sequences, which were already aligned, * Address reprint requests to: Dr . Akira Yokota, ular and cellular Biosciences, The University Yayoi; Bunkyo-ku, Tokyo 113, Japan. Institute of Molec-

Phylogenetic relationships of the helical-shaped bacteria in the alpha Proteobacteria inferred from 16S rDNA sequences.

Abstract: 16S ribosomal RNA gene sequences from seven strains of Aquaspirillum peregrinum, Aqu. itersonii, Aqu. polymorphum, and Oceanospirillum pusillum were compared with homologous sequences from other members of helical-shaped bacteria. The bootstrapped neighbor-joining tree, inferred from 887 aligned sites, placed the spirillum taxa assigned to Aquaspirillum, Oceanospirillum, Azospirillum, Magnetospirillum, Rhodospirillum, and Rhodocista of the Proteobacteria in seven clusters of alpha Proteobacteria separately from other shapes of bacteria. Aqu. peregrinum and Aqu. itersonii grouped together in 88% bootstrap support. They were more related to Rhodospirillum rubrum and Rsp. photometricum than Aqu. polymorphum. Aqu. polymorphum was close to Magnetospirillum gryphiswaldense, Mag. magnetotacticum, Rsp. fulvum, and Rsp. molischianum, and more close to Mag. gryphiswaldense. Oce. pusillum was not related to other spirillum taxa and was placed in a separate branch. Rhodocista was very closely related to Azospirillum. Photosynthesis and magnetotaxis, as phenotypic characters, were not important in the classification of helical bacteria.