Retrieval of the Species Alteromonas Tetraodonis Simidu Et Al. 1990 As Pseudoalteromonas Tetraodonis Comb

International Journal of Systematic and Evolutionary Microbiology (2001), 51, 1071–1078 Printed in Great Britain

Retrieval of the species Alteromonas tetraodonis Simidu et al. 1990 as Pseudoalteromonas tetraodonis comb. nov. and emendation of description

1 Paciﬁc Institute of Bioorganic Elena P. Ivanova,1,2† Ludmila A. Romanenko,1 Maria H. Matte! ,2,3 Chemistry, Far-Eastern 2,3 4 5 6 Branch, Russian Academy of Glavur R. Matte! , Anatolii M. Lysenko, U. Simidu, K. Kita-Tsukamoto, Sciences, 690022 Vladivostok, 7 8 1 pr. 100 let Vladivostok 159, Tomoo Sawabe, Mikhail V. Vysotskii, Galina M. Frolova, Russia Valery Mikhailov,1 Richard Christen9 and Rita R. Colwell2,10 2 Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Columbus Center, Author for correspondence: Elena P. Ivanova. Tel: j61 3 9214 5137. Fax: j61 3 9214 5050. Suite 236, 701 E. Pratt St, e-mail: eivanova!swin.edu.au Baltimore, MD 21202, USA

3 School of Public Health, University of Sao Paulo, Av. A polyphasic taxonomy study was undertaken of three strains of Dr Arnaldo, 715, Sao Paulo 01246-904, Brazil Pseudoalteromonas haloplanktis subsp. tetraodonis (Simidu et al. 1990) Gauthier et al. 1995. DNA was prepared from each of the strains and genomic 4 Institute of Microbiology, T Russian Academy of Sciences, relatedness was measured by DNA–DNA hybridization. Strains KMM 458 and 117811 Moscow, Russia IAM 14160T shared 99% genetic relatedness, but were only 48–49% related to 5 Hikarigaoka 5-2-5-806, the type strain of Pseudoalteromonas haloplanktis subsp. haloplanktis, IAM Nerima-ku, Tokyo 179-0072, T Japan 12915 . The third strain, P. haloplanktis subsp. tetraodonis A-M, showed 83% T 6 Ocean Research Institute, genetic similarity with P. haloplanktis subsp. haloplanktis IAM 12915 and 32% University of Tokyo, with KMM 458T. From these results, it is concluded that strains KMM 458T and 1-15-Minamidai, Nakano-ku, T Tokyo 164, Japan IAM 14160 comprise a separate species, originally described as Alteromonas tetraodonis, whereas strain A-M belongs to the species Pseudoalteromonas 7 Laboratory of Microbiology, Faculty of Fisheries, Hokkaido haloplanktis. Based on phenotypic and chemotaxonomic data, genomic University, 3-1-1 Minato-cho, Hakodate 041-8611, Japan ﬁngerprint patterns, DNA–DNA hybridization data and phylogenetic analysis of 16S rRNA, it is proposed that the species Alteromonas tetraodonis be retrieved 8 Institute of Marine Biology, Far-Eastern Branch, Russian and recognized as Pseudoalteromonas tetraodonis comb. nov. (type strain IAM Academy of Sciences 690041, T T Vladivostok, Russia 14160 l KMM 458 ).

9 Centre National de la Recherche Scientiﬁque et Universite! , Pierre et Marie Curie, Station Zoologique, Keywords: retrieval, Alteromonas tetraodonis, Pseudoalteromonas haloplanktis subsp. Villefranche-sur-Mer 06230, tetraodonis, Pseudoalteromonas tetraodonis France

10 Department of Cell Biology and Molecular Biology, University of Maryland, College Park, MD 20742, USA

INTRODUCTION (Baumann et al., 1984; Gauthier & Breittmayer, 1992). The type species of the genus Pseudoalteromonas is The genus Pseudoalteromonas Gauthier et al. 1995 Pseudoalteromonas haloplanktis, which includes two currently comprises 17 validly described species orig- subspecies, P. haloplanktis subsp. haloplanktis and P. inating from the Alteromonas haloplanktis rRNA haloplanktis subsp. tetraodonis. The latter was de- branch (Van Landschoot & De Ley, 1983) of the scribed as Alteromonas tetraodonis Simidu et al. 1990 former genus Alteromonas Baumann et al. 1972 to accommodate one of four marine bacterial isolates, strain GFCT, that produces tetrodotoxin (Simidu et ...... al., 1990). This bacterium was isolated from the surface † Present address: Swinburne University of Technology, IRIS, 533–545 Burwood Road, Hawthorn, Melbourne, Victoria 3122, Australia. slime of a puﬀer ﬁsh (Fugu poecilonotus) shown to The GenBank accession numbers for the 16S rDNA sequences of Pseudo- produce tetrodotoxin in association with the animals. alteromonas tetraodonis KMM 458T and strain IAM 14160T are AF214729 A few years later, on the basis of DNA–DNA and AF214730, respectively. hybridization data, it was found that A. tetraodonis

01609 # 2001 IUMS 1071 E. P. Ivanova and others

Simidu et al. 1990 should be recognized as a junior Phenotypic analysis. Unless otherwise indicated, the pheno- subjective synonym of A. haloplanktis (ZoBell and typic properties employed to characterize Alteromonas and Upham 1944) Reichelt and Baumann 1973 (Baumann related species were obtained using procedures described by et al., 1984; Akagawa-Matsushita et al., 1993). At Baumann et al. (1972, 1984) and Smibert & Krieg (1994). present, and in accordance with results of phylogenetic Tests for utilization of various organic substrates (Table 1) as sole carbon sources, at concentrations of 0n1% (w\v), and biochemical analyses and the level of genomic were performed as described elsewhere (Ivanova et al., DNA relatedness, this bacterial taxon is recognized as 1996). P. haloplanktis subsp. tetraodonis (Simidu et al. 1990) Gauthier et al. 1995. Sensitivity to antibiotics was tested by the disc-diffusion method using Marine Agar 2216 and discs impregnated with During the last decade, bacterial isolates in the the following antibiotics: kanamycin (30 µg); ampicillin Collection of Marine Micro-organisms, Vladivostok, (10 µg); benzylpenicillin (10 µg); streptomycin (10 µg); Russia, that are related to Alteromonas have been erythromycin (15 µg); gentamicin (10 µg); oxacillin (20 µg); subjected to extensive taxonomic investigation. lincomycin (15 µg); carbenicillin (25 µg); vancomycin Shortly after publication of the article describing the (30 µg); tetracycline (30 µg); oleandomycin (15 µg); and O\129 (150 µg). new species Alteromonas tetraodonis, strain GFCT was subjected to DNA–DNA hybridization experiments to Lipid analysis. Lipids were extracted from wet cells, clarify the taxonomic position of the newly isolated according to the method of Bligh & Dyer (1953). Polar bacteria. In 1991, K. Kita-Tsukamoto, on behalf of lipids were subjected to two-dimensional TLC using 10i U. Simidu, kindly provided strain GFCT, designated 10 cm silica gel plates (KSK). Following development in chloroform\methanol\ammonium hydrate\benzene A. tetraodonis IAM 14160T, and deposited it in our T (65:30:6:10) (first dimension) and chloroform\methanol\ laboratory as strain KMM 458 (KMM; Collection of acetone\acetic acid\benzene\water (70:30:5:4:1:10) (se- Marine Micro-organisms, Pacific Institute of Bio- cond dimension), lipids were detected on chromatograms by organic Chemistry). Results of DNA–DNA hybrid- spraying with both a non-specific reagent (50% sulfuric acid ization studies disagreed with those published by in methanol and heating at 180 mC) and specific reagents Akagawa-Matsushita et al. (1993). After consultation (ninhydrin and Dragendorff’s reagent). Quantification of with M. Akagawa-Matsushita, DNA–DNA hybrid- phospholipids on two-dimensional chromatograms was ization experiments were repeated using strains P. done using the method of Vaskovsky et al. (1975). T haloplanktis subsp. haloplanktis IAM 12915 , P. halo- Fatty acid methyl ester analysis. Analysis of fatty acid methyl planktis subsp. tetraodonis A-M (l KMM 3660), esters was performed by GLC, as described previously by kindly provided by M. Akagawa-Matshushita, P. Svetashev et al. (1995). T haloplanktis subsp. tetraodonis IAM 14160 from the Serology. ELISA was performed, according to the methods Collection of the Institute of Molecular and Cellular of Voller et al. (1979), as described elsewhere (Ivanova et al., Biosciences (formerly Institute of Applied Micro- T 1998). The level of antigen relatedness was estimated, as biology), Japan, and strain KMM 458 (formerly described by Conway de Macario et al. (1982), as the mean A. tetraodonis and provided by U. Simidu). Results value of three independent experiments. T T showed that strains KMM 458 and IAM 14160 are Genetic analysis. DNA was isolated according to the method identical and should be considered as a separate of Marmur (1961). DNA GjC content (mol%) was species, as described originally by Simidu et al. (1990). determined by the thermal denaturation methods of Marmur A polyphasic taxonomy study, including phenotypic, & Doty (1962) and Owen et al. (1969). DNA–DNA biochemical and genomic characteristics, together with hybridization was performed spectrophotometrically and phylogenetic study of the strains, was undertaken. initial renaturation rates were recorded as described by De Results given here have led to the conclusion that the Ley et al. (1970). species initially described as Alteromonas tetraodonis Genomic fingerprints: primers and PCR conditions. PCR (Simidu et al., 1990) is indeed a species separate from genomic fingerprinting was carried out using specific primers P. haloplanktis and should be retrieved as Pseudo- and conditions as follows. REP-directed PCR (Louws et al., alteromonas tetraodonis comb. nov. 1994): REP1R-I, 5h-IIIICGICGICATCIGGC-3h; REP2-I, 5h-ICGICTTATCIGGCCTAC-3h. ERIC-directed PCR (Judd et al., 1993): ERIC 1 (reverse), 5h-ATGTAAGCTC- METHODS CTGGGGATTCAC-3h; ERIC 2 (forward): 5h-AAGTA- AGTGACTGGGGTGAGCG-3h. BOX-directed PCR Bacterial strains. The strains employed in this study are as (Versalovic et al., 1991; Louws et al., 1994): 5 -CTACG- T h follows: P. haloplanktis subsp. haloplanktis IAM 12915 GCAAGGCGACGCTGACG-3h. PCRs were performed (l ATCC 14393T ); P. haloplanktis subsp. tetraodonis IAM using the following programs: one initial cycle at 95 C for T m 14160 ; P. haloplanktis subsp. tetraodonis A-M (l KMM 7 min, 35 cycles of denaturation at 94 mC for 1 min, annealing 3660), obtained from M. Akagawa-Matsushita; strain at 44, 52 or 53 C for 1 min with REP, ERIC or BOX T T m KMM 458 (l IAM 14160 ), originally provided in 1991 by primers, respectively, and extension at 65 mC for 10 min, K. Kita-Tsukamoto, on behalf of U. Simidu, as Alteromonas with a single final extension cycle at 65 mC for 20 min for T tetraodonis IAM 14160 ; Pseudoalteromonas atlantica IAM REP, ERIC and BOX primers, and a final soak at 4 mC. PCR 12376T ; and Pseudoalteromonas carrageenovora IAM products were separated by horizontal electrophoresis on T 12662 . The strains were grown at 25 mC on Marine Agar 2n2% agarose gels, stained with SYBR Green I (FMC 2216 (Difco) and stored frozen at k80 mC in Marine Broth Bioproducts) and visualized by scanning at 500 nm in a (Difco) supplemented with 30% (v\v) glycerol. Fluorimager scanner (Molecular Dynamics). To examine

1072 International Journal of Systematic and Evolutionary Microbiology 51 Retrieval of Alteromonas tetraodonis

Table 1. Phenotypic data for P. haloplanktis subsp. haloplanktis and strains of P. haloplanktis subsp. tetraodonis ...... All strains studied produced gelatinase and lipase, did not produce alginase or agarase, did not grow at 41 mC and utilized succinate and fumarate. j, Positive; k, negative; , no data available; , 11–89% of strains positive; , variable.

Characteristic P. haloplanktis subsp. haloplanktis IAM 12915T P. haloplanktis subsp. tetraodonis IAM 14160T

Baumann Akagawa-Matsushita This study Akagawa-Matsushita Simidu This study et al. (1984) et al. (1993) et al. (1993) et al. (1990)

Production of: Amylase  kkk kk Chitinase k  kk Growth at: 4 mC kj* kj* jj 35 mC  jjj jj Utilization of: -Glucose, pyruvate, j  j  jj ethanol, acetate -Mannose jkjkjk -Fructose  jjk jk Sucrose jkjjjj Trehalose  kkj jk Maltose j  j  jj Cellobiose kjkjjk Lactose, -ribose, -xylose, kkkkjk -arabinose, rhamnose, gluconate, salicin, ketoglutarate, glycerol -Galactose  k  kjj Propionate j  j  j  Butyrate, valerate  j  jjj Caproate kjkjkj -Lactate  kkk jj Citrate jkjkkj Aconitate jkjkjj Caprylate  k  k  k -Malate kkkjkk Mannitol  k  kjk -Sorbitol k  k  jk p-Hydroxybenzoate kkkkj -Alanine k  j  jj -Threonine k  k  jk Aspartate kk Glutamate j  jj -Lysine kkkjjk -Arginine  kkj jk -Ornithine kkkkjk -Histidine j  jk -Proline j  jk -Tyrosine jkjkjj -Phenylalanine kkkkjk

* Growth tested at 5 mC.

the reproducibility of the results, the ERIC and BOX PCRs systems model 373S automated sequencer. Nine sequencing were repeated at least twice. primers were used (Sawabe et al., 1998a). DNA amplification and sequencing. Bacterial DNAs for Phylogenetic analysis. Sequences were aligned and studied PCR were prepared using the Promega Wizard genomic using a set of programs developed by R. Christen. In all DNA extraction kit according to the instruction manual. phylogenetic analyses, the sequences determined in this DNA templates (100 ng) were used in PCR to amplify small- study and small-subunit rDNA sequences obtained from the subunit rRNA genes, as described previously by Sawabe et EMBL database were used. Domains used to construct the al. (1998a, b). PCR conditions were as follows: an initial dendrogram shown in Fig. 2 were regions of the small- denaturation step at 94 mC for 180 s, an annealing step at subunit rDNA sequences available for all sequences and 55 mC for 60 s and an extension step at 72 mC for 90 s. The excluding positions likely to show homoplasy: positions thermal profile then consisted of 30 cycles. The amplification 101–181, 219–837, 850–1133, 1138–1205 (E. coli small- primers used in this study gave a 1n5 kb PCR product and subunit rDNA sequence J01695 numbering). Phylogenetic corresponded to positions 25–1521 of the Escherichia coli analyses were performed using three different methods: sequence. The PCR products were purified using a Promega distances were calculated using Kimura’s two-parameter Wizard PCR preps DNA purification kit and sequenced correction and a modified neighbour-joining method,  directly by using a Taq FS dye terminator sequencing kit (Gascuel, 1997), maximum-likelihood (options QFYG, (ABI) and the protocol recommended by the manufacturer. fastDNAml program; Olsen et al., 1994) and maximum- DNA sequencing was performed with an Applied Bio- parsimony (, Phylogeny Inference Package, version

International Journal of Systematic and Evolutionary Microbiology 51 1073 E. P. Ivanova and others

3.5c; distributed by J. Felsenstein, Department of Genetics, positive. It required Na+ or sea water for growth; good University of Washington, Seattle, WA, USA). The ro- growth was observed in media containing 1–7% NaCl. bustness of each topology was checked using the neighbour- The temperature range for growth was 4–35 mC, with joining method and 100 bootstrap replications. Trees were an optimum between 25 and 30 mC. No growth was drawn using the  program for the Macintosh (M. detected at 40 C. The pH range for growth was Gouy, CNRS URA 243, Universite! Claude Bernard, Lyon, m France). 5n5–9n5, with optimum growth at pH 7n5–8n0. Amylase, agarase, chitinase and alginase were not produced. RESULTS AND DISCUSSION Tween 80, gelatin and DNA were decomposed. Many of these characteristics, and others listed in Table 1, are T T The bacterial strain GFC (l KMM 458 l IAM similar for P. haloplanktis subsp. haloplanktis, P. 14160T), described originally as Alteromonas tetra- haloplanktis subsp. tetraodonis and strains A-M and T T odonis, had all the features characteristic for the genus KMM 458 (l IAM 14160 ). On the other hand, Pseudoalteromonas (formerly an rRNA cluster of diﬀerences in carbohydrate utilization were noted Alteromonas haloplanktis). The strain was motile by when comparing related data published by other means of a single ﬂagellum. It was a chemo-organo- investigators. Strain KMM 458T utilized -lactate, troph, with a respiratory metabolism. It did not form caproate, valerate and butyrate, whereas P. halo- endospores or accumulate polyhydroxybutyrate as an planktis subsp. haloplanktis did not utilize these intracellular reserve product. It did not possess ar- compounds. In contrast, strain KMM 458T did not ginine dihydrolase and was oxidase- and catalase- utilize -mannose, -fructose, mannitol, -histidine or

Table 2. Fatty acid composition of P. haloplanktis and P. tetraodonis ...... Data were from Svetashev et al. (1995) and this study.

Fatty acid P. haloplanktis IAM 12915T P. haloplanktis A-M P. tetraodonis KMM 458T (l IAM 14160T )

11:0 3-OH 0n30n24 0n4 12:0 3-OH 1n91n11 0n9 12:0 2n02n12 1n5 12:1 0n30n54 0n3 i13:0 0n10n00 0n0 13:0 0n20n26 0n5 13:1 0n30n26 0n5 i14:0 0n00n15 0n1 14:0 2n01n62 2n0 14:1(n-7) 0n61n13 1n2 i15:0 0n00n11 0n0 a15:0 0n20n58 0n4 15:0 3n33n47 8n0 15:1(n-8) 2n32n80 6n3 15:1(n-6) 0n20n36 0n6 i16:0 0n22n52 1n4 16:0 30n123n10 18n2 16:1(n-9) 0n00n82 0n0 16:1(n-7) 40n538n37 35n0 16:1(n-5) 0n00n39 0n2 i17:0 0n00n68 0n3 a17:0 0n01n15 0n6 17:0 3n93n76 5n5 17:1(n-8) 6n06n25 10n7 17:1(n-6) 0n20n52 0n8 i18:0 0n00n49 0n2 18:0 1n91n30 0n6 18:1(n-11) 0n10n34 0n4 18:1(n-9) 0n20n11 0n2 18:1(n-7) 2n44n28 2n5 19:1 0n20n28 0n2 Unidentiﬁed fatty acid k 0n56 k Total 99n599n67 99n6

1074 International Journal of Systematic and Evolutionary Microbiology 51 Retrieval of Alteromonas tetraodonis

(a) (b) (c)

...... Fig. 1. Comparative genomic ﬁngerprinting of some Pseudoalteromonas species. REP (a), ERIC (b) and BOX (c) PCR patterns are shown. The ﬁngerprints are as follows: M, 1 kb ladder (Gibco-BRL); 1, P. tetraodonis IAM 14160T ;2,P. haloplanktis IAM 12915T ;3,P. tetraodonis KMM 458T ;4,P. atlantica IAM 12376T ; and 5, P. carrageenovora IAM 12622T.

Table 3. DNA relatedness of P. haloplanktis and P. tetraodonis

Taxon GjC content (mol%) DNA relatedness (%)

P. haloplanktis IAM 12915T P. tetraodonis IAM 14160T

P. haloplanktis IAM 12915T 42n2 100 – P. haloplanktis A-M 41n38332 P. tetraodonis KMM 458T 41n54999 P. tetraodonis IAM 14160T 41n8 48 100

-proline (Table 1). The strain was susceptible and 1n2% and those of lyso-phosphatidyl ethanola- to benzylpenicillin, carbenicillin, vancomycin and mine were 1n9, 0n7 and 0n6%, respectively. Unidentified tetracycline. phospholipids (0n6%) were present in the lipid fractions of P. haloplanktis subsp. tetraodonis IAM Cellular fatty acids of strains A-M and KMM 458T 14160T and KMM 458T. Cardiolipin and glycolipids were characteristic for Pseudoalteromonas species were not detected. (Table 2) and the dominant fatty acids were as follows: 16:1(n-7), 16:0, 17:1(n-8), 17:0 and 18:1(n-8). How- Genomic fingerprinting patterns obtained using ever, the fatty acid profiles of P. haloplanktis subsp. primers corresponding to the REP, ERIC and BOX haloplanktis IAM 12915T and strain A-M were similar elements of P. haloplanktis subsp. tetraodonis IAM and they contrasted with that of strain KMM 458T. 14160T and KMM 458T were identical, whereas those For example, the amounts of 15:0, 15:1(n-8), 17:0 of P. haloplanktis subsp. haloplanktis IAM 12915T and 17:1(n-8) fatty acids for the latter strain were were different. Since a dispersion of repetitive almost twice those in the other two strains. sequences in the bacterial genome reflects a strain- specific characteristic, the identity of the genomic Phospholipids were a significant component of the profiles is interpreted as identity of strains IAM 14160T polar lipids (ca. 80–85%); the phospholipid com- and KMM 458T (Fig. 1). position was generally the same for all strains included in this study. Phosphatidyl ethanolamine and Results of DNA–DNA hybridizations showed that phosphatidyl glycerol were the major components: strains KMM 458T and IAM 14160T are 99% related 73n2 and 23% for P. haloplanktis subsp. haloplanktis to each other, but only 48–49% related to the type T T IAM 12915 ,75n1 and 23n7% for P. haloplanktis strain, P. haloplanktis subsp. haloplanktis IAM 12915 . T subsp. tetraodonis IAM 14160 and 71n7 and 21n7% Strain A-M showed 83% genetic similarity to P. for strain KMM 458T, respectively. The concentrations haloplanktis subsp. haloplanktis IAM 12915T and 32% of the minor phospholipids varied slightly. In the three to KMM 458T (Table 3). Thus, the two strains KMM T T strains above, levels of bis-phosphatidic acid were 1n8, 458 and IAM 14160 represent a distinct species, 2n2 and 1n7%, those of phosphatidic acid were 0n3, 0n7 originally described as Alteromonas tetraodonis by

International Journal of Systematic and Evolutionary Microbiology 51 1075 E. P. Ivanova and others

0·02

* Alteromonas macleodii IAM 12920T / X82145 Colwellia psychrerythraea ATCC 27364T / AF001375 Pseudoalteromonas elyakovii O22 / AF116188 Pseudoalteromonas distincta KMM 638T / AF082564 Pseudoalteromonas elyakovii KMM 162T / AF082562 Pseudoalteromonas antarctica NF3T / X98336 Pseudoalteromonas antarctica N-1 / AF045560 Pseudoalteromonas citrea KMM 216 / AF082563 ‘Pseudoalteromonas gracilis’ B9 / AF038846 Pseudoalteromonas haloplanktis ATCC 14393T / X67024 Pseudoalteromonas nigrifaciens NCIMB 8614T / X82146 Pseudoalteromonas undina NCIMB 2128T / X82140 Pseudoalteromonas tetraodonis IAM 14160T / X82139 * Pseudoalteromonas tetraodonis KMM 458T / AF214729 *100% Pseudoalteromonas atlantica IAM 12927T / X82134 Pseudoalteromonas espejiana NCIMB 2127T / X82143 Pseudoalteromonas carrageenovora ATCC 12662T / X82136 Pseudoalteromonas aurantia ATCC 33046T / X82135 *88% *100% Pseudoalteromonas citrea NCIMB 1889T / X82137 Pseudoalteromonas peptidolytica F12-50-A1T / AF007286 *100% Pseudoalteromonas piscicida C201 CERBOM / X82141 *60% Pseudoalteromonas piscicida Cura-d / AF081498 *90% Pseudoalteromonas piscicida KMM 636 / AF144036 Pseudoalteromonas luteoviolacea NCIMB 1893T / X82144 *98% Pseudoalteromonas rubra ATCC 29570T / X82147 Pseudoalteromonas denitrificans ATCC 43337T / X82138 72% + Pseudoalteromonas tunicata D2T / Z25522 Pseudoalteromonas bacteriolytica IAM 14595T / D89929 Psychromonas antarctica DSM 10704T / Y14697 Moritella marina NCIMB 1144T / X82142 Ferrimonas balearica PATT / X93021 Shewanella hanedai CIP 103207T / X82132 ‘Curacaobacter baltica’ Baltic 166 / AJ002006 Pseudomonas doudoroffi ATCC 27123T / AB019390 Arsenophonus nasoniae SK14T / M90801 * Buchnera aphidicola / M63252 Escherichia coli K-12 / AE000452 Aeromonas hydrophila subsp. hydrophila ATCC 7966T / X60418 Ruminobacter amylophilus ATCC 29744T / Y15992 T *58% *100% Aeromonas jandaei ATCC 49568 / X74678 * Aeromonas media ATCC 33907T / X74679 Tolumonas auensis TA 4T / X92889 Vibrio cholerae CECT 514T / X76337

...... Fig. 2. Unrooted tree obtained using the neighbour-joining algorithm and Kimura’s two-parameter correction for distance calculations. Branches also obtained in the maximum-likelihood analysis are indicated by asterisks (P ! 0n01). j, Branches also found in maximum-likelihood and parsimony analysis; bootstrap percentages retrieved in 500 replicons bootstrap analysis using Kimura’s two-parameter analysis are shown at the nodes. Bar, 0n02 accumulated changes per nucleotide.

Simidu et al. (1990), which is consistent with the and, on the basis of its high DNA–DNA hybridization criteria cited by Wayne et al. (1987). In contrast, it is value with strain IAM 12915T (83%), that it does not concluded that strain A-M belongs to P. haloplanktis merit subspecies status. Results of the DNA–DNA

1076 International Journal of Systematic and Evolutionary Microbiology 51 Retrieval of Alteromonas tetraodonis hybridization experiments were supported by sero- Emended description of Pseudoalteromonas logical data obtained using polyclonal antibodies to haloplanktis cell surface determinants. Antiserum to P. haloplanktis The description of Pseudoalteromonas haloplanktis is subsp. haloplanktis IAM 12915T did not cross-react identical to that of Alteromonas haloplanktis given by with surface antigens of KMM 458T, even though it ZoBell & Upham (1944), with some additional charac- showed 50% surface antigen similarity to strain IAM teristics. It is resistant to benzylpenicillin, vancomycin 14160T. and tetracycline. Cellular fatty acids include 16:1(n-7), In order to clarify the taxonomic affiliation of P. 16:0, 17:1(n-8), 17:0 and 18:1(n-8). The 15:0, 15:1(n- haloplanktis subsp. tetraodonis IAM 14160T and 8), 17:0 and 17:1(n-8) fatty acids are minor com- KMM 458T at the genus level, the 16S rDNA sequences ponents. The major phospholipids, phosphatidyl etha- were aligned by comparison to a database containing nolamine and phosphatidyl glycerol, constitute 73n2 about 10000 already aligned eubacterial small-subunit and 23n0% of the total polar lipids. Cardiolipin and rDNA sequences. The results of broad phylogenetic glycolipids are absent. The type strain is IAM 12915T T analyses showed clearly that the strains studied be- (l ATCC 14393 ). Strain A-M (l KMM 3660), longed to the γ-Proteobacteria of the Bacteria (data assigned previously to P. haloplanktis subsp. tetra- not shown) and, more precisely, to the γ-3 subgroup. odonis, is a strain of P. haloplanktis. More detailed analyses showed that these bacteria were included in the genus Pseudoalteromonas and ACKNOWLEDGEMENTS formed a cluster with P. haloplanktis, P. nigrifaciens, ‘P. gracilis’, P. citrea KMM 216 and P. antarctica. This study was supported by a short-term UNESCO Sequence similarities between P. haloplanktis subsp. Fellowship, by funds from the Russian Fund for Basic haloplanktis ATCC 14393T and both P. haloplanktis Research 99-04-48017 and by a grant of the State Com- subsp. tetraodonis IAM 14610T and KMM 458T were mittee for Science and Technologies of the Russian Fed- 99 9% (Fig. 2). Rather low DNA homology values, eration, 99-03-19. The authors also gratefully acknowledge n the Fundac: a4 o de Amparo a' Pesquisa do Estado de Sa4 o about 50%, against P. haloplanktis subsp. haloplanktis Paulo for postdoctoral fellowship support provided to have enabled recognition of P. haloplanktis subsp. M.H.M. and G.R.M. tetraodonis and P. haloplanktis subsp. haloplanktis as separate species of the genus Pseudoalteromonas. Pseudoalteromonas tetraodonis comb. nov. and an REFERENCES emended species Pseudoalteromonas haloplanktis are Akagawa-Matsushita, M., Koga, Y. & Yamasato, K. (1993). DNA proposed. relatedness among nonpigmented species of Alteromonas and synonymy of Alteromonas haloplanktis (ZoBell and Upham 1944) Reichelt and Baumann 1973 and Alteromonas tetraodonis Description of Pseudoalteromonas tetraodonis comb. Simidu et al. 1990. Int J Syst Bacteriol 43, 500–503. nov. Baumann, L., Baumann, P., Mandel, M. & Allen, R. D. (1972). Basonym Pseudoalteromonas haloplanktis subsp. Taxonomy of aerobic marine eubacteria. J Bacteriol 110, tetraodonis (Simidu et al. 1990) Gauthier et al. 1995. 402–429. Baumann, P., Gauthier, M. J. & Baumann, L. (1984). Genus The description of Pseudoalteromonas tetraodonis Alteromonas Baumann, Baumann, Mandel and Allen 1972, comb. nov. is identical to that given by Simidu et al. 418AL.InBergey’s Manual of Systematic Bacteriology, vol. 1, (1990), with some additional characteristics, including pp. 343–352. Edited by N. R. Krieg & J. G. Holt. Baltimore: growth in media containing 1–10% NaCl, growth at Williams & Wilkins. pH 5n5–9n5, with optimum growth at 7n5–8n0, and Bligh, E. G. & Dyer, W. J. (1953). A rapid method of lipid utilization of -glucose, sucrose, -maltose, -galac- extraction and purification. Can J Biochem Physiol 7, 911–915. tose, -lactate, caproate, valerate, butyrate, citrate, Conway de Macario, E., Macario, A. J. L. & Wolin, M. J. (1982). fumarate, succinate, caprylate, glutamate, acetate, Specific antisera and immunological procedures for charac- pyruvate, ethanol and -tyrosine. It does not utilize - terization of methanogenic bacteria. J Bacteriol 149, 320–328. mannose, -fructose, mannitol, -histidine, -proline De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative or -malate. It is susceptible to benzylpenicillin, measurement of DNA hybridization from renaturation rates. carbenicillin, vancomycin and tetracycline. Cellular Eur J Biochem 12, 133–142. fatty acids include 16:1(n-7), 16:0, 17:1(n-8), 17:0 Gascuel, O. (1997). BIONJ: an improved version of the NJ and 18:1(n-8). The 15:0, 15:1(n-8), 17:0 and 17:1(n- algorithm based on a simple model of sequence data. Mol Biol 8) fatty acids are minor components. The major Evol 14, 685–695. phospholipids, phosphatidyl ethanolamine and Gauthier, M. J. & Breittmayer, V. A. (1992). The genera Altero- phosphatidyl glycerol, constitute 75n1 and 23n7% of monas and Marinomonas.InThe Prokaryotes, pp. 3046–3070. the total polar lipids. Cardiolipin and glycolipids are Edited by A. Balows, H. G. Tru$ per, M. Dworkin, W. Harder & absent. The DNA GjC content of P. tetraodonis is K.-H. Schleifer. New York: Springer. 41n5 mol% (determined by thermal denaturation). The T T T Gauthier, G., Gauthier, M. & Christen, R. (1995). Phylogenetic type strain is GFC (l KMM 458 l IAM 14160 ), analysis of the genera Alteromonas, Shewanella, and Moritella isolated from the surface slime of a puffer fish (Fugu using genes coding for small-subunit rRNA sequences and poecilonotus). division of the genus Alteromonas into two genera, Alteromonas

International Journal of Systematic and Evolutionary Microbiology 51 1077 E. P. Ivanova and others

(emended) and Pseudoalteromonas gen. nov., and proposal of Sawabe, T., Makino, H., Tatsumi, M., Nakano, K., Tajima, K., twelve new species combinations. Int J Syst Bacteriol 45, Iqbal, M. M., Yumoto, I., Ezura, Y. & Christen, R. (1998b). 755–761. Pseudoalteromonas bacteriolytica sp. nov., a marine bacterium Ivanova, E. P., Kiprianova, E. A., Mikhailov, V. V., Levanova, G. F., that is the causative agent of red spot disease of Laminaria Garagulya, A. D., Gorshkova, N. M., Yumoto, N. & Yoshikawa, S. japonica. Int J Syst Bacteriol 48, 769–774. (1996). Characterization and identification of marine Altero- Simidu, U., Kita-Tsukamoto, K., Yasumoto, T. & Yotsu, M. (1990). monas nigrifaciens strains and emendation of the description. Taxonomy of four marine bacterial strains that produce Int J Syst Bacteriol 46, 223–228. tetrodotoxin. Int J Syst Bacteriol 40, 331–336. Ivanova, E. P., Kiprianova, E. A., Mikhailov, V. V. & 8 other Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. authors (1998). Phenotypic diversity of Pseudoalteromonas In Methods for General and Molecular Bacteriology, pp. citrea from different marine habitats and emendation of the 607–654. Edited by F. Gerhardt. Washington, DC: American description. Int J Syst Bacteriol 48, 247–256. Society for Microbiology. Judd, A. K., Schneider, M., Sadowsky, M. J. & de Bruijn, F. J. Svetashev, V. I., Vysotskii, M. V., Ivanova, E. P. & Mikhailov, V. V. (1993). Use of repetitive sequences and the polymerase chain (1995). Cellular fatty acids of Alteromonas species. Syst Appl reaction technique to classify genetically related Bradyrhizobium Microbiol 18, 37–43. japonicum serocluster 123 strains. Appl Environ Microbiol 59, 1702–1708. Van Landschoot, A. & De Ley, J. (1983). Intra- and intergeneric similarities of the rRNA cistrons of Alteromonas, Marinomonas Louws, F. J., Fulbright, D. W., Stephens, C. T. & de Bruijn, F. J. (gen. nov.) and some other Gram-negative bacteria. J Gen (1994). Specific genomic fingerprints of phytopathogenic Microbiol 129, 3057–3074. Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR. Appl Environ Vaskovsky, V. E., Kostetsky, E. Y. & Vasendin, I. M. (1975). A Microbiol 60, 2286–2295. universal reagent for phospholipid analysis. J Chromatogr 114, 129–141. Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218. Versalovic, J., Koeuth, T. & Lupski, J. R. (1991). Distribution of Marmur, J. & Doty, P. (1962). Determination of the base repetitive DNA sequences in eubacteria and application to composition of deoxyribonucleic acid from its thermal fingerprinting of bacterial genomes. Nucleic Acids Res 19, denaturation temperature. J Mol Biol 5, 109–118. 6823–6831. Olsen, G. J., Matsuda, H., Hagstrom, R. & Overbeek, R. (1994). Voller, A., Bidwell, D. E. & Bartlett, A. (1979). Setting up ELISA. fastDNAml: a tool for construction of phylogenetic trees of In The Enzyme-linked Immunosorbent Assay (ELISA). A Guide DNA sequences using maximum likelihood. Comput Appl with Abstracts of Microplate Applications, pp. 38–39. Guernsey: Biosci 10, 41–48. Dynatech Europe. Owen, R. J., Hill, L. R. & Lapage, S. P. (1969). Determination of Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors DNA base compositions from melting profiles in dilute buffers. (1987). Report of the ad hoc committee on reconciliation of Biopolymers 7, 503–516. approaches to bacterial systematics. Int J Syst Bacteriol 37, Sawabe, T., Sugimura, I., Ohtsuka, M., Nakano, K., Tajima, K., 463–464. Ezura, Y. & Christen, R. (1998a). Vibrio halioticoli sp. nov., a non- ZoBell, C. E. & Upham, H. C. (1944). A list of marine bacteria motile alginolytic marine bacterium isolated from the gut of the including description of sixty new species. Bull Scripps Inst abalone Haliotis discus hannai. Int J Syst Bacteriol 48, 573–580. Oceanogr Univ Calif 5, 239–292.

1078 International Journal of Systematic and Evolutionary Microbiology 51