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Pseudoalteromonas Bacteriolytica Sp. Nov., a Marine Bacterium That Is the Causative Agent of Red Spot Disease of Laminaria Japonica

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Pseudoalteromonas Bacteriolytica Sp. Nov., a Marine Bacterium That Is the Causative Agent of Red Spot Disease of Laminaria Japonica Pseudoalteromonas bacteriolytica sp. nov., a marine bacterium that is the causative agent of red spot disease of Title Laminaria japonica Author(s) Sawabe, T.; Makino, H.; Tatsumi, M.; Nakano, K.; Tajima, K.; Iqbal, MM; Yumoto, I.; Ezura, Y.; Christen, R. Citation International Journal of Systematic Bacteriology, 48, 769-774 Issue Date 1998-11 Doc URL http://hdl.handle.net/2115/5798 Type article File Information IJSB48.pdf Instructions for use Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP International Journal of Systematic Bacteriology (1998), 48, 769–774 Printed in Great Britain Pseudoalteromonas bacteriolytica sp. nov., a marine bacterium that is the causative agent of red spot disease of Laminaria japonica Tomoo Sawabe,1 Hideyuki Makino,1 Masahiro Tatsumi,1 Kazuaki Nakano,1 Kenichi Tajima,1 Mohammed Mahbub Iqbal,1 Isao Yumoto,2 Yoshio Ezura1 and Richard Christen3 Author for correspondence: Tomoo Sawabe. Tel: ­81 138 40 5569. Fax: ­81 138 40 5569. e-mail: sawabe!pop.fish.hokudai.ac.jp 1 Laboratory of An aerobic, polarly flagellated marine bacterium that produces a prodigiosin- Microbiology, Faculty of like pigment was isolated from the red-spotted culture beds of Laminaria Fisheries, Hokkaido University, 3-1-1 Minato- japonica. Five isolates had unique bacteriolytic activity for both Gram-positive cho, Hakodate 041, Japan and -negative bacteria, which had never been observed among Alteromonas or 2 Bioscience and Chemistry related species. The isolates were identified as the causative agent of red spot Division, Hokkaido disease of L. japonica seeds. The phenotypic features of the isolates were National Industrial similar to these of Pseudoalteromonas rubra ATCC 29570T, but they could be Research Institute, 2-17-2-1 Tsukisamu-Higashi, differentiated using 10 traits (growth at 37 mC, requirement for organic growth Toyohira-ku, Sapporo 062, factors, bacteriolytic activity, utilization of sucrose, N-acetylglucosamine, Japan fumarate, succinate, D-galactose, L-proline and acetate). The GMC content of 3 Centre National de la DNAs from the isolates was 44–46 mol%. The isolates constitute a new species, Recherche Scientifique et distinct from the other Alteromonas and Pseudoalteromonas species, as shown Universite! , Pierre et Marie Curie, Station Zoologique, by DNA–DNA hybridization experiments and phylogenetic clustering of 16S Villefranche-sur-Mer rRNA gene sequences, for which the name Pseudoalteromonas bacteriolytica 06230, France sp. nov. (type strain ¯ IAM 14595T) is proposed. A set of phenotypic features which differentiate this new species from closely related Pseudoalteromonas and Alteromonas species is provided. Keywords: Pseudoalteromonas bacteriolytica sp. nov., bacteriolytic activity, red spot disease, Laminaria INTRODUCTION Originally consisting of four species, the genus Altero- monas included Gram-negative, aerobic, non-pig- In 1984, a bacterium producing a prodigiosin-like red mented, polarly flagellated species of marine bacteria, pigment was isolated from red-spotted culture beds of which differed from the genus Pseudomonas by a lower Laminaria japonica (7). The bacterium was an aerobic, G­C content (1, 2). Recently, following phylogenetic polarly flagellated marine bacterium, and it was analyses of 16S rDNA sequences, the genus was suggested that the bacteria could be assigned to the divided into two new genera, the emended genus genus Alteromonas (7). In addition, the bacterium Alteromonas and the new genus Pseudoalteromonas. showed a broad spectrum of bacteriolytic activity. Pre- Now, at least 14 species, most of which were previously sently, at least six bacteriolytic substances, including Alteromonas species, are included in the genus Pseudo- bacteriolytic enzymes, have been detected in culture alteromonas, and the emended genus Alteromonas is supernatants and cell-bound fractions (19, 20). An restricted to a single species, Alteromonas macleodii, ecological function of this bacteriolytic activity has with two subspecies (8, 15). Bacteriolytic Pseudo- been proposed, which would allow maintenance of the alteromonas or Alteromonas strains have never been bacterial population in oligotrophic aquatic environ- described previously. The precise taxonomic position ments (21). of the aforementioned bacteriolytic bacterium there- fore remains uncertain. In this study, DNA–DNA ................................................................................................................................................. hybridizations, phenotypic characterization and phy- The DDBJ/GenBank/EMBL accession number for the sequence of Pseudo- logenetic analyses were performed to clarify the alteromonas bacteriolytica (IAM 14595T) is D89929. taxonomic assignment of the causative agent of red 00619 # 1998 IUMS 769 T. Sawabe and others spot disease of L. japonica seeds. All of the data natural seawater) was added to 2±5 ml melted agar (0±8% in suggest that this bacterium is a new species of natural seawater) at 45 mC. It was then poured onto a base of Pseudoalteromonas, for which we propose the name CSY-3 agar medium. After 5 d incubation, the formation of Pseudoalteromonas bacteriolytica. a clear zone around the spotted culture was determined. Determination of G­C content and DNA–DNA hybridization. METHODS DNA from bacterial strains was prepared by the procedures of Marmur (12), with minor modification. G­C contents of Bacterial strains. Strains used in this study are listed in Table DNA were determined according to the melting temperature 1. Five strains (No. 8R, E-1, E-2 and A3), including the type (Tm) of the DNA. DNA–DNA hybridization experiments strain IAM 14595T had been isolated from red-spotted were performed in microdilution wells using a fluorometric culture beds of L. japonica (7). These isolates of Pseudo- direct binding method (5) under conditions previously de- alteromonas bacteriolytica were maintained on CSY-3 agar scribed by Sawabe et al. (17). DNAs from Pseudoaltero- T medium containing casitone (Difco) 1 0 g, Difco bacto- monas bacteriolytica IAM 14595 and Pseudoalteromonas ± T soytone 1±0 g, Difco yeast extract 1±0 g, ferric ammonium rubra ATCC 29570 were labelled with photobiotin (Vector citrate 0±1 g, and 1000 ml natural seawater, pH 7±5 (19). The Laboratories). The hybridization of the biotinylated DNA stock cultures were maintained in CSY-3 broth containing to immobilized DNAs was performed under optimal con- 20% glycerol (v}v). All reference strains were maintained on ditions following pre-hybridization, and then biotinylated ZoBell 2216E agar medium (13). DNA that hybridized to immobilized DNA was detected by a fluorimetric method after binding streptavidin-β-galacto- Morphological, biochemical and physiological charac- sidase to labelled DNA. 4-Methylumbelliferyl-β--galacto- terization. Conventional phenotypic characteristics of P. −% pyranoside (6¬10 M; Wako) was added to each well as bacteriolytica, P. rubra, P. haloplanktis subsp. haloplanktis fluorogenic substrate for β-galactosidase prior to incubation and A. macleodii were determined by the methods described at 30 mC. Fluorescence intensity of the well was then by Baumann et al. (2), Hidaka & Sakai (9), Holt et al. (10), measured using the MicroFluoro reader (MTP-22; Corona Leifson (11), Oppenheimer & ZoBell (13), Ostle & Holt (14) Electric) at wavelengths of 360 nm for excitation and 450 nm and West et al. (22). Bacteriolytic activity was determined by for emission. DNA–DNA homology was calculated ac- the formation of a clear zone on the plate using the CSY-3 cording to the method of Ezaki et al. (6). agar medium including freeze-dried cells of Micrococcus luteus (Seikagaku Kogyo). This medium was prepared DNA amplification and sequencing. Bacterial DNAs for according to the methods of Yumoto et al. (20). Briefly, 0 5 PCR were prepared according to the methods of Enright et "! " ± ml viable cell suspension of M. luteus (approx. 10 ml− in al. (4). One hundred nanograms of DNA templates were Table 1. DNA relatedness among Pseudoalteromonas, Alteromonas and Marinomonas strains Strain G­C content Reassociation (%) with biotinylated (mol%) DNA from: P. bacteriolytica P. rubra ATCC IAM 14595T 29570T P. bacteriolytica IAM 14595T 46 100±03±4 P.bacteriolytica No. 8R 44 92±4 P. bacteriolytica E-1 45 99±3 P. bacteriolytica E-2 44 98±3 P. bacteriolytica A-3 46 93±5 P. rubra ATCC 29570T 46 5±6 100±0 A. macleodii IAM 12920T 46±2* 2±63±9 P.espejiana IAM 12640T 41±4* 3±34±1 P.atlantica NCIMB 301T 41±2* 4±53±2 P.carrageenovora NCIMB 302T 39±5* 3±92±2 P.marinovulgaris ATCC 14394 3±13±4 P.nigrifaciens IAM 13010T 40±6* 4±14±5 P.haloplanktis IAM 12915T 41±6* 3±75±8 P.haloplanktis ATCC 19648 40±5* 4±16±0 P.undina IAM 12922T 40±1* 3±94±3 P.piscicida NCIMB 645T 43–46* 3±77±5 M.communis IAM 12914T 47±0* 3±32±2 M.vaga IAM 12923T 48±4* 3±12±8 , Not tested. * Data from reference 2. 770 International Journal of Systematic Bacteriology 48 Pseudoalteromonas bacteriolytica sp. nov. T 0·0071 ‘Vibrio marinus’ / X74709 alteromonas tetraodonis IAM 14160 , X82139; Pseudo- Pseudoalteromonas tetraodonis / X82139 alteromonas piscicida C201 CERBOM, X82141; Pseudo- Pseudoalteromonas espejiana / X82143 alteromonas rubra ATCC 29570T, X82147; Pseudoaltero- Pseudoalteromonas atlantica / X82134 T Pseudoalteromonas carrageenovora / X82136 monas luteoviolacea NCIMB 1893 , X82144; Alteromonas 97 %/ *+ macleodii subsp. macleodii IAM 12920T, X82145; ‘Vibrio Pseudoalteromonas undina / X82140 T Pseudoalteromonas nigrifaciens / X82146 marinus’ ATCC 15831 , X74709; Vibrio
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