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

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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: j81 138 40 5569. Fax: j81 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 , which had never been observed among 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 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 l 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, GjC 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 2n5 ml melted agar (0n8% 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 GjC content and DNA–DNA hybridization. METHODS DNA from bacterial strains was prepared by the procedures of Marmur (12), with minor modification. GjC 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 n T soytone 1n0 g, Difco yeast extract 1n0 g, ferric ammonium rubra ATCC 29570 were labelled with photobiotin (Vector citrate 0n1 g, and 1000 ml natural seawater, pH 7n5 (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 (6i10 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 "! " n 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 GjC content Reassociation (%) with biotinylated (mol%) DNA from:

P. bacteriolytica P. rubra ATCC IAM 14595T 29570T

P. bacteriolytica IAM 14595T 46 100n03n4 P.bacteriolytica No. 8R 44 92n4  P. bacteriolytica E-1 45 99n3  P. bacteriolytica E-2 44 98n3  P. bacteriolytica A-3 46 93n5  P. rubra ATCC 29570T 46 5n6 100n0 A. macleodii IAM 12920T 46n2* 2n63n9 P.espejiana IAM 12640T 41n4* 3n34n1 P.atlantica NCIMB 301T 41n2* 4n53n2 P.carrageenovora NCIMB 302T 39n5* 3n92n2 P.marinovulgaris ATCC 14394  3n13n4 P.nigrifaciens IAM 13010T 40n6* 4n14n5 P.haloplanktis IAM 12915T 41n6* 3n75n8 P.haloplanktis ATCC 19648 40n5* 4n16n0 P.undina IAM 12922T 40n1* 3n94n3 P.piscicida NCIMB 645T 43–46* 3n77n5 M.communis IAM 12914T 47n0* 3n32n2 M.vaga IAM 12923T 48n4* 3n12n8 , 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 / X82140 T Pseudoalteromonas nigrifaciens / X82146 marinus’ ATCC 15831 , X74709; Vibrio sp., U145842; Pseudoalteromonas haloplanktis / X67024 Moritella marinas NCIMB 1144T, X82142; Ferrimonas / X82137 balearica, X93021; hanedai CIP 103207T, 36 %/ *+ Pseudoalteromonas aurantia / X82135 X82132; Shewanella algae, X81621; Salinivibrio costicola Pseudoalteromonas rubra / X82147 T T 81 %/ *+ Pseudoalteromonas luteoviolaceae / X82144 NCIMB 701 , X95527; Vibrio campbellii ATCC 25920 , Pseudoalteromonas denitrificans / X82138 X74692; symbiont Anomolops katoptron, Z19081; Vibrio 94 %/ *+ T 97 %/ *+ Pseudoalteromonas piscicida / X82141 hollisae ATCC 33564 , X74707; Photobacterium leiognathi Gamma proteobacterium / Z25522 ATCC 25521T, X74686; and Photobacterium damsela subsp. Pseudoalteromonas bacteriolytica Vibrio sp. / U14582 piscicida NCIMB 2058, X78105. Moritella marinus / X82142 Domains used to construct the dendrogram shown in Fig. 1 Ferrimonas balearica / X93021 Shewanella hanedai / X82132 were regions of the small-subunit rDNA sequences available Shewanella algae / X81621 for all sequences and excluding positions likely to show Salinivibrio costicola / X95527 homoplasy: positions 251–434, 493–590, 653–835, 855–997, Alteromonas macleodii subsp. macleodii / X82145 1045–1114, 1156–1354 (E. coli small-subunit rDNA se- Vibrio campbellii / X74692 quence J01695 numbering). Phylogenetic analyses were Symbiont (Anomalops katoptron) / Z19081 Vibrio hollisae / X74707 performed by using three different methods, neighbour- Photobacterium leiognathi / X74686 joining (16), maximum-likelihood (options QFYG, fast- Photobacterium damsela subsp. piscicida / X78105 DNAml program of G. J. Olsen, University of Illinois, Urbana, USA) and maximum-parsimony [PAUP 3.0s for ...... the Macintosh, heuristic search (18)]. The robustness of each Fig. 1. Unrooted phylogenetic tree showing phylogenetic topology was checked by using the neighbour-joining relationships for a selection of bacteria belonging to the method and 100 bootstrap replications. Trees were drawn by gamma 3 subclass, Pseudoalteromonas and Alteromonas. The figure combines the results of three analyses, neighbour- using the njplot program for the Macintosh (M. Gouy, joining, maximum-parsimony and maximum-likelihood. The CNRS URA 243, Universite! Claude Bernard, Lyon, topology shown was obtained by neighbour-joining, and the France). percentages are the result of a bootstrap analysis using 100 replications. Branches also obtained in the maximum-likelihood RESULTS AND DISCUSSION analysis are indicated by * (P ! 0n01). Monophyletic units also obtained in the most parsimonious tree are indicated by j. The five strains isolated from red-spotted culture beds Scale bar, 0 0071 accumulated changes per nucleotide. n appeared as polarly flagellated, Gram-negative, non- fermentative rods (Table 2). The bacterium required salt for its growth, did not accumulate poly-β-hydroxy- used in a PCR to amplify the small-subunit rRNA genes as butyrate and did not reduce nitrate (Table 2). No previously described by Sawabe et al. (17). PCR conditions peritrichous flagella were observed when the bacterium were as follows; the initial denaturation step at 94 m C for was cultivated on solid media. The GjC contents of 180 s, an annealing step at 55 mC for 60 s and an extension the strains were 44–46 mol% (Table 1), suggesting step at 72 mC for 90 s. The thermal profile then consisted of that this bacterium should be assigned either to the 30 cycles. The amplification primers used in this study gave genus Alteromonas or to the genus Pseudoalteromonas a1n5 kbp PCR product and corresponded to positions (2, 10). 25–1521 in the Escherichia coli sequence. The PCR products Specific biochemical and physiological features of were purified by PEG 6000 and directly sequenced by using T a Taq FS dye terminator sequencing kit (ABI) and the strain IAM 14595 , No. 8R, E-1, E-2 and A-3 are protocol recommended by the manufacturer. DNA se- shown in Table 2. Morphological, biochemical and quencing was performed with an Applied Biosystems model physiological characteristics of these strains including 373A automated sequencer (17). Nine sequencing primers IAM 14595T were most closely related to that of were used for sequencing (17). Pseudoalteromonas rubra ATCC 29570T among Al- Phylogenetic analysis. The sequences were aligned and teromonas, Pseudoalteromonas, Marinomonas and re- studied using a set of programs developed by R. Christen. In lated species. Phenotypic characteristics of Pseudo- all phylogenetic analyses, we used the sequences determined alteromonas haloplanktis subsp. haloplanktis IAM in this study and small-subunit rDNA sequences obtained 12915T, type species of Pseudoalteromonas (8), or from the EMBL database. For Fig. 1, the following Alteromonas macleodii IAM 12920T, type species of sequences were used: Pseudoalteromonas denitirificans T Alteromonas (8), are also quite different from our ATCC 43337 , X82138; Pseudoalteromonas citrea NCIMB strains (Table 2). Ten traits were different between 1889T, X82137; Pseudoalteromonas aurantia ATCC 33046T, X82135; Pseudoalteromonas atlantica IAM 12927T, X82134; these species: bacteriolytic activity, growth at 37 mC, Pseudoalteromonas espejiana NCIMB 2127T, X82143; requirement for organic growth factors, utilization of Pseudoalteromonas carrageenovora ATCC 12662T, X82136; sucrose, N-acetylglucosamine, fumarate, succinate, - Pseudoalteromonas undina NCIMB 2128T, X82140; Pseudo- galactose, -proline and acetate (Table 2). Pseudo- alteromonas haloplanktis ATCC 14393, X67024; Pseudo- alteromonas denitrificans also produced prodigiosin- alteromonas nigrifaciens NCIMB 8614T, X82146; Pseudo- like pigment (3), but a significant number of bio-

International Journal of Systematic Bacteriology 48 771 T. Sawabe and others

Table 2. Phenotypic characteristics for distinguishing Pseudoalteromonas bacteriolytica from P. rubra, P. haloplanktis subsp. haloplanktis and Alteromonas macleodii ...... O, Oxidative; pol, polar flagellum; , weakly positive; d, different reactions; ( ), result of P. bacteriolytica IAM 14595T.

Characteristic P. bacteriolytica P. rubra ATCC P. haloplanktis A. macleodii IAM 14595T, No. 29570T subsp. IAM 12920T 8R, E-1, E-2 and haloplanktis IAM A-3 12915T

Pigmentation jj kk Water-insoluble Red Red Salt requirement jj jj OF test O O O O Flagellar arrangement pol pol pol pol Growth at: 4 mC kk jk 37 mC kj kj 40 mC kk kj Oxidase jj jj Catalase  jjj Production of: Amylase jj kj Alginase kk kj Agarase kk kk Lipase jj jj Chitinase kk kk Gelatinase jj jj Caseinase jj jj κ-Carrageenase kk kk Bacteriolytic activity jk kk − − NO$ reduced to NO$ kk kk Requirement for organic growth factors jk kk Utilization of: -Mannose jj kk -Fructose jj jj Sucrose jk kj Maltose d (k) jjj -Gluconate kk jj -Glucosamine jj kk N-Acetylglucosamine kj kj -Mannitol d (k) kjj Fumarate jk jk Succinate jk jj -Sorbitol\citrate kk kk meso-Erythritol\glycerol\ kk kk m-hydroxybenzoate\-malate\ α-ketoglutarate -Galactose jk kj Cellobiose d (k) kjj Melibiose kk kj Lactose kk kj Xylose kk kj Trehalose d (j) jjk γ-Aminobutyrate kk kj -Proline kj jj Acetate jk jj Pyruvate d (j) jjj

772 International Journal of Systematic Bacteriology 48 Pseudoalteromonas bacteriolytica sp. nov.

Table 2 (cont.)

Characteristic P. bacteriolytica P. rubra ATCC P. haloplanktis A. macleodii IAM 14595T, No. 29570T subsp. IAM 12920T 8R, E-1, E-2 and haloplanktis IAM A-3 12915T

-Tyrosine d (k) kkj Aconitate kk kk -Glutamate d (j) kjj -Glucose\propinonate jj jj -Glucronate\putrescine\δ-aminovarate kk kk Growth on KCN kk kk PHB accumulation kk kk chemical characteristics including GjC content of the Description of Pseudoalteromonas bacteriolytica sp. species were different from our strains. nov. DNA–DNA hybridization results (Table 1) showed Pseudoalteromonas bacteriolytica (bac.te.rio.lyhti.ca. that the four other isolates were more than 90% T Gr. n. baktron rod or staff; Gr. adj. lytica dissolving; similar to IAM 14595 . DNA–DNA homology values M.L. fem. adj. bacteriolytica bacteria-dissolving). between IAM 14595T and representatives of the genera Pseudoalteromonas, Marinomonas and Alteromonas, Gram-negative, strictly aerobic, polarly flagellated used in this study were distinctively low (Table 1). bacterium isolated from red-spotted culture beds of There was no significant homology when Pseudo- Laminaria japonica. Cells are rod-shaped, with alteromonas rubra ATCC 29570T was photobiotinyl- rounded ends, and are 0n6–0n9 µm in diameter and ated and hybridization was performed between 1n9–2n5 µm long when the organism is grown on CSY- Pseudoalteromonas rubra ATCC 29570T and IAM 3 agar medium; the cells occur singly or in pairs. No 14595T (Table 1). endospores or capsules are formed. Peritrichous flagel- T lation is not observed when the organism is cultivated The 16S rDNA sequence of strain IAM 14595 was on solid media. Colonies on CSY-3 agar medium are aligned by comparison to a database containing about red (although non-pigmented colonies frequently oc- 5000 already aligned eubacterial small-subunit rDNA curred), circular, and smooth and convex with entire sequences. The results of broad phylogenetic analyses edges. Mesophilic and neutrophilic chemo-organo- clearly showed that the sequence which we studied troph which grows at temperatures ranging from 15 to belonged to the gamma subclass of the 35 mC. No growth occurs at 37 mC. Positive for acid (data not shown), and more precisely, to the gamma 3 production from glucose, hydrolysis of starch, Tween subclass. More detailed analyses showed that it could 80 and casein; weakly positive for catalase; positive be included in the Pseudoalteromonas genus, with for oxidase; assimilates -mannose, -fructose, su- which it formed a robust clade (and in agreement with crose, -glucosamine, fumarate, succinate, -glucose, phenotypic data) although it was the deepest branch of -galactose, acetate and propionate. Negative for the genus Pseudoalteromonas (Fig. 1). Homology levels luminescence, production of fluorescein and pyo- for nucleotides of 16S rDNAs from Pseudoalteromonas cyanin; hydrolysis of agar, chitin and κ-carrageenan; bacteriolytica and closely related species were very low, nitrate reduction; growth on KCN; accumulation of ranging from 87n7% (against gamma proteobacterium poly-β-hydroxybutyrate; assimilation of -gluconate, Z25522) to 90n3% (against Pseudoalteromonas carra- N-acetylglucosamine, -sorbitol, citrate, meso-eryth- geenovora and Pseudoalteromonas espejiana). This ritol, glycerol, -malate, α-ketoglutarate, m-hydroxy- finding was also revealed by the DNA–DNA hom- T benzoate, melibiose, lactose, -glucronate, xylose, ology results, in which IAM 14595 showed a very low putrescine, δ-aminovarate, γ-aminobutyrate, -pro- level of relatedness with the other species of Pseudo- line, aconitate (Table 2). The GjC content of the alteromonas (Table 1). It was also separated from DNA is 44–46 mol%. The type strain is IAM 14595T Alteromonas macleodii subsp. macleodii (Fig. 1). From (Table 1). the results of the DNA–DNA hybridization (Table 1) and 16S rRNA (Fig. 1) experiments, it was revealed that the strain IAM 14595T and the other four strains, REFERENCES which are the causative agents of red spot disease of 1. Baumann, L., Baumann, P., Mandel, M. & Allen, R. D. (1972). L. japonica, should be recognized as a new species. of aerobic marine eubacteria. J Bacteriol 110, The name proposed for this bacterium with a unique 402–429. bacteriolytic activity and that induces damages in 2. Baumann, P., Gauthier, M. J. & Baumann, L. (1984). Genus Laminaria seed supply is Pseudoalteromonas bacteri- Alteromonas Baumann, Baumann, Mandel and Allen 1972, olytica. 418.AL In Bergey’s Manual of Systematic Bacteriology, vol.

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