Two Species of Culturable Bacteria Associated With ... - Springer Link

Microb Ecol (2002) 43:242±249 DOI: 10.1007/s00248-001-1011-y Ó 2002 Springer-Verlag New York Inc.

Two Species of Culturable Bacteria Associated With Degradation of Brown Algae Fucus Evanescens E.P. Ivanova,1,2 I.Yu. Bakunina,1 T. Sawabe,3 K. Hayashi,3 Y.V. Alexeeva,4 N.V. Zhukova,5 D.V. Nicolau,2 T.N. Zvaygintseva,1 V.V. Mikhailov1 1 Paci®c Institute of Bioorganic Chemistry of the Far-Eastern Branch of the Russian Academy of Sciences, 690022 Vladivostok, Pr. 100 Let Vladivostoku 159, Russia 2 Industrial Research Institute Swinburne, Swinburne University of Technology, P.O. Box 218, Hawthorn, Vic 3122, Australia 3 Laboratory of Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato-cho, Hakodate 041-8611, Japan 4 Far-Eastern State University, 690000 Vladivostok, Russia 5 Institute of Marine Biology of the Far-Eastern Branch of the Russian Academy of Sciences, 690041 Vladivostok, Russia

Received: 27 December 2000; Accepted: 10 October 2001; Online publication: 23 January 2002

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B S T R A C T

The heterotrophic microbial enrichment community established during degradation of brown algae Fucus evanescens was characterized. A two-species bacterial community of marine culturable gamma-proteobacteria consisted of Pseudoalteromonas and Halomonas. The ®rst member of the community, Pseudoalteromonas sp., was highly metabolically active, had bacteriolytic and hemolytic activities, produced proteinases (gelatinase and caseinase), lipases, DNases, and fucoidanhydrolases, laminaranases, alginases, pustulanases, b-glucosidases, b-galactosidases, b-N-acetylglucosaminidases, and b-xylosidases. The second member of the community, Halomonas marina, produced only caseinase and DNase, and it did not hydrolyze algal polysaccharides. Both members of the studied bacterial community utilized a range of easily assimilable monosaccharides and other low molecular weight organic substances. The results provide an evidence of the complex metabolic interrelations between two members of this culturable community. One of them Pseudoalteromonas sp., most likely plays the major role in the initial stages of algal degradation; the other one, H. marina, resistant to the bacteriolytic activity of the former, is able to utilize the products of degradation of polysaccharides.

Correspondence to: Elena P. Ivanova; Fax: +61 3 9214 5050; E-mail: [email protected]

Degradation of Brown Alga by Pseudoalteromonas sp. and Halomonas marina

Introduction Diverse microbial communities including those residing on the surfaces of macroalgae constitute an important part in the microbial food web and participate in the cycling of organic matter in the marine environment [2, 5, 7, 9]. Microorganisms are capable of degrading a variety of complex polysaccharides that are major constituents of algal cell walls (up to 50±80% of defatted algal mass). The main polysaccharides of brown algae comprise alginic acids, cellulose, laminarans, and fucoidans [34]. The diversity of these polysaccharides is due to the wide variety of monosaccharides and the different glycosidic bonds that linked polysaccharide's monomers [6, 8]. Laminarans (1,3; 1,6-b-D-glucans) and their analogs have been found in fungal cell walls, plants, and lichens, whereas alginic acids and fucoidans were never detected in terrestrial sources. Therefore, in order to hydrolyze the algal polysaccharides, the marine microorganisms produce a multiplicity of speci®c enzymes [34]. These polysaccharides are of signi®cant interest because of their speci®c biological activities [36]. Recently it was shown that the enzymatic transformation of both laminarans and fucoidans resulted in biological products with medical applications [6, 14, 37]. The rates and speci®ty of enzyme activity in marine microorganisms may be limited by the intrinsic properties of both the algal surface environments and the structure of the epiphytic microbial community. Little is known about the enzyme activities of the microbial associations degrading the algal thallus. Although some of the bacterial±algal interactions have been discussed, the ecological signi®cance of most naturally occurring bacterial communities is unclear and in most cases the bacterial species involved have not been identi®ed [1, 15]. The roles of the physiological adaptation and bacterial species selection with respect to algal degradation are not easily investigated in natural systems, but may be investigated in a laboratory-based model system. It is obvious that more complex interactions exist in natural microbial communities, while laboratory model communities are likely to be compositionally simpler [16]. This study aimed to investigate how the substrate availability might be re¯ected in the structure of microbial enrichment community during degradation of the brown alga Fucus evanescens, to characterize culturable, numerically dominant members of the community, and to study metabolic interrelations between species of this community.

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Materials and Methods Isolation Procedure Brown algae (Fucus evanescens) were collected by SCUBA divers in mid-summer (July 1999) at the Kraternaya Bay, Kuril Islands, the Sea of Okhotsk, during the 23rd scienti®c expedition of the R/V Akademician Oparin. The algae were transferred into 10% HClwashed and seawater-prerinsed 5-liter polyethylene containers, transported to the laboratory, and further processed for approximately 3 h. A piece (5 g) of algal thallus was prewashed in sterilized-by-®ltration seawater and placed in a ¯ask with 200 mL of sterilized natural seawater. After 2-months of incubation at room temperature (ca 23°C), 100 ll samples (in duplicate) were taken at 2-week (6 times) intervals and plated on agar plates of marine agar 2216 (Difco) and on plates with medium B, containing 0.2% (w/v) Bacto Peptone (Difco, USA), 0.2% (w/v) casein hydrolysate (Merck, USA), 0.2% (w/v) Bacto Yeast Extract (Difco, USA) 0.1% (w/v) glucose, 0.002% (w/v) KH2PO4, 0.005% (w/v) MgSO4 á 7H2O and 1.5% (w/v) Bacto Agar (Difco, USA), 50% (v/v) of natural seawater and 50% (v/v) distilled water at pH 7.5±7.8, as described elsewhere [19]. All glassware used in the enrichment experiments was combusted or treated with 10% NaCl to avoid contamination. In total, 19 colonies were selected randomly from agar plates and subcultured at least three times under the same conditions to control the purity. Pure cultures were maintained on the same semisolid B medium in tubes under mineral oil, at 4°C, and stored at )80°C in marine broth 2216 (Difco, USA) supplemented with 20% (v/v) of glycerol.

Phenotypic Analysis The phenotypic properties used for characterization of the isolates previously described [19, 20] followed standard procedures [3, 31]. Cell morphology and gram stain were determined after 24 h incubation on medium B. The motility was determined by examining 18-h cultures in Marine Broth. The physiological and biochemical properties examined were oxidation/fermentation of glucose; reduction of nitrate and nitrite, oxidase and catalase activity, gelatin liquefaction, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, acetoin production (Vogues±Proskauer test), accumulation of poly-(b-hydroxybutyrate [27]; sodium requirement (0, 3, 6, 8, 10, 12, 15, 20 (w/v) NaCl; indole and H2S production; and the ability to hydrolyze starch, Tween-80, casein, and DNA. The growth at dierent temperatures (4, 10, 18, 37, 42°C) was determined in liquid medium B. The hemolytic activity of the strains studied was detected on trypticase soy agar with 50 mL of sheep blood [13]. The bacteriolytic activity was performed according to the method of Yumoto et al. [35]. The tests for the utilization of various organic substrates (listed in Table 1) as sole carbon sources at a concentration of 0.1% (wt/vol) were performed in 10 ml per tube of liquid BM medium [3]. The liquid cultures of bacteria were grown by shaking (160 rpm) for 72 h at 26 to 28°C. The analysis of fatty acid methyl esters (FAME) was performed by gas liquid chromatography as described previously by

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Table 1. Comparative phenotypic features of two bacterial populations that degrade brown alga Fucus evanescens Characteristic GC content (mol%) Pigmentation Oxidase Catalase Na required for growth Requirement for organic growth factors Arginine dihydrolase Lysine, ornithine decarboxylases Indole, H2S, V-P Denitri®cation Hemolysis Bacteriolytic activity Production of: Amylase Gelatinase Caseinase DNAase Lipase Chitinase Alginase Growth in NaCl: 1±15% Growth at: 4±37°C 42°C Utilization of: D-Glucose Maltose Lactose D-Arabinose D-Galactose Sucrose D-Rhamnose D-Ribose D-Xylose Glycerol Mannitol Cellobiose Tagatose L-Fucose D-Fructose D-Mannose Trehalose Citrate Susceptibility to: Tetracyclin (15lg) Neomycin (150lg) Kanamycin (10lg) Ampicillin (10lg) Benzyl-penicillin (10lg) Streptomycin (15lg) Neomycin (15lg) Gentamicin (10lg) Carbenicillin (20lg) Polymyxin (50lg) Lincomycin (l0lg) a

Pseudoalteromonas sp. (n = 11)

Halomonas marina (n = 8)

42±43 0 100a 100 100 0 0 0 0 0 100 100

63±665 0 0 100 0 0 0 0 0 100 0 0

0 100 100 100 100 80 100

0 0 100 88 0 0 0

100

100

100 0

100 37

100 100 100 100 100 100 80 0 0 0 0 0 0 0 0 0 0 0

100 100 100 100 100 100 100 100 100 100 100 100 100 0 0 0 0 0

0 0 20 20 0 60 20 100 20 20 0

0 0 60 0 0 30 0 100 0 0 0

Values are the percentage of positive strains.

Svetashev et al. [32]. The strains were grown on B medium at 28°C for 24 h and at 12°C for 4 days.

The DNA was extracted from cells grown over night on B medium following the method of Marmur [24]. The G+C content

Degradation of Brown Alga by Pseudoalteromonas sp. and Halomonas marina of the DNA was determined by the thermal denaturation method of Marmur and Doty [25].

DNA Ampli®cation and Sequencing Bacterial DNAs for PCR were extracted using the Promega Wizard genomic DNA kit (Promega, USA) according to the instruction manual. One hundred ng of DNA templates was used in a PCR to amplify the small-subunit rRNA genes as previously described by Sawabe et al. [28, 29]. PCR conditions were as initial denaturation, 94°C for 60 s, annealing, 55°C for 60 s, and extension at 72°C for 90 s. The thermal pro®le consisted of 30 cycles. The ampli®cation primers yielded a 1.5 kb long product and corresponded to positions 25 to 1521 of E. coli sequence. The

PCR products were puri®ed using the Promega Wizard PCR preps DNA puri®cation kit and directly sequenced using a Taq FS Dye terminator sequencing kit (ABI, USA) following the protocol supplied by manufacturer. The DNA sequencing was performed with an Applied Biosystems model 310 genetic analyzer. Nine sequencing primers were used for sequencing [28]. Phylogenetic Analysis The sequences were aligned and the phylogenetic tree was constructed using a Clustal X program which was a updated version of Clustal W [33]. In all phylogenetic analyses, we used the sequences determined in this study and the small-subunit rDNA sequences obtained from the EMBL/Genebank databank. The nucleotide sequence of Pseudoalteromonas sp. KMM 3549, KMM 3558 and Halomonas sp. KMM 3550 was deposited to GenBank/ DDBJ/EMBL DNA databases with the accession numbers AF 316144, AF 316142, and AF 316143, respectively.

Crude Enzyme Extraction After cultivation in 500 ml of liquid medium B with 1 g L)1 of fucoidan during 24 h at 25°C, cells (1 g of wet weight) were collected by centrifugation for 30 min at 5,000 rpm at 4°C, and disrupted by ultrasonication 20 kHz 20 sec, ´3 in 10 mL of 50 mM phosphate buer, pH 7.2. The suspension was centrifuged at 12,000 ´ g for 20 min to remove cell debris. The supernatant was used as the crude enzyme solution.

Enzyme Assay Fucoidan of a brown alga F. evanescens [37], laminaran from a brown alga Laminaria cichorioides [36], pustulan of the lichen Umbellicaria russica [36], alginate, agar, and cellulose (Serva, USA) were used as substrates for determining the activities of fucoidanhydrolase, laminarinase, alginase, agarase, cellulose, and pustulanase (1,6-b-D-glucanase). The enzyme activities were measured by colorimetric analysis of the reducing sugars re-

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leased [26] and expressed as the amount of enzyme that liberated 1 lmol of correspondent substrate per 1 mg of protein. The amount of protein was measured by the method of Lowry et al. [23] with bovine serum albumin as the standard. One unit of glycanase activity was de®ned as the amount of enzyme that liberated 1 lmol of corresponding monosaccharides in 1 h. Glycosidase activities were tested with the p-nitrophenyl derivatives of relevant monosaccharides (Sigma, USA): p-nitrophenyl-b-D-glucopyranoside, p-nitrophenyl-b-D-galactropyranoside, p-nitrophenyl-b-N-acetyl- D-glucosaminide, p-nitrophenyla-D-galactropyranoside, p-nitrophenyl-a-D-mannopyranoside, pnitrophenyl-a-L-fucoside, p-nitrophenyl-a-N-acetyl-D-galactosaminide, and umbellipheryl-b-D-xylopyranoside. Glycosidase activities of bacterial (cell free) extracts were measured in 0.1 M phosphate buer, pH 7.0 at 20°C. The reaction mixture consisted of 0.05 mL of the bacterial extract and 0.05 mL of corresponding p-nitrophenyl glycoside (1 mg mL)1 in 50 mM phosphate buer, pH 7.0). The reaction was stopped by adding 0.1 mL of 1 M Na2CO3. One unit of glycosidase activity was de®ned as the amount of enzyme that liberated 1 lmol of p-nitrophenol in 1 h.

Results Generic Identi®cation At the end of the 6-month enrichment period, up to 3.7 ´ 107 bacterial cells (CFU) mL)1 were detected. Enrichment cultures were obtained from seawater with added algal thallus to favor growth of hydrolytic enzyme-producing microbes. Only two bacterial phenotypes were clearly observed on the plates during the monitoring of thallus degradation. A total of 19 randomly selected nonpigmented strains of the algal-degrading enrichment community were subsequently isolated and studied in detail. The bacteria of one of the phenotypic group (11 strains) were gram-negative, aerobic, rod-shaped organisms with a single polar ¯agellum. On medium B or Marine agar incubated at 22°C, colonies were circular, with a diameter of 3±5 mm, translucent, smooth and convex with irregular edges. None of the bacteria accumulated poly-b-hydroxybutyrate as an intracellular reserve product and none had arginine dihydrolase, lysine, and ornithine decarboxylases. All were positive for oxidase, catalase, proteinase (gelatinase and caseinase), lipase, alginase, chitinase, DNase, b-hemolysis, and bacteriolytic activity and all required Na ion (1±15% NaCl) or seawater for growth. Other phenotypic features are shown in Table 1. The fatty acid composition of the bacteria was characteristic for the genus Pseudoalteromonas [19]. The major FAs were in the range 38±46% for 16:l(n-7), 12± 16% for 16:0, 9±14% for 17:l(n-8), and 4±8% for 8:l(n-7). The G+C contents of the DNA were 39.2±40.2 mol%.

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The bacteria of the second phenotypic group (8 strains) were gram-negative, strictly aerobic, rod-shaped ¯agellated organisms. On medium B or Marine agar incubated at 22°C, colonies were circular, with a diameter of 2±3 mm, opaque, smooth and convex with regular edges. None of the bacteria accumulated poly-b-hydroxybutyrate as an intracellular reserve product and had an arginine dihydrolase and lysine and ornithine decarboxylases. All were positive for catalase and proteinase (caseinase), and most of the strains studied (88%) produced DNases. The bacteria did not require Na ion or seawater for growth, but were tolerant up to 15% NaCl in culture medium (Table 1). The members of this group had the simplest FA composition with three major components 16:0, 16:1(n-7), and 18:1(n-7), which generally accounted for greater than 90% of the total fatty acids. Branched-chain fatty acids were not detected. Speci®cally, the amount of 16:0, 16:1(n7), and 18:1(n-7) FA reached up to 11±13%, 51±52%, and 26±30%, respectively. In addition, 19:0 cyclo FA was present in minor amounts 0.4±0.6%. The G+C contents of the DNA were 63.1±65.2 mol%. The bacteria of both phenotypes were halophilic and grew at NaCl concentrations up to 15%, at 4 to 37°C (37% of the strains of the second group were able to grow at 42°C) with optimum growth at 22±25°C, and at pH 6±10 (optimum 7±8). According to the phenotypic characteristics the bacteria of the ®rst group were tentatively identi®ed as Pseudoalteromonas sp., and bacteria of the second group as Halomonas sp. Phylogenetic Analysis 16S rDNA gene sequence analysis of two representatives of the ®rst group, Pseudoalteromonas sp. KMM 3558 and KMM 3549, and one strain Halomonas sp. KMM 3550 of the second group revealed that the organisms are members of gamma subclass of the class Proteobacteria (Fig. 1). These data clearly indicate that the strains KMM 3558 and KMM 3549 represent a lineage closely related to other species of Pseudoalteromonas, which is consistent with the phenotypic characteristics. The 16S rRNA sequence of KMM 3550 was compared with the 16S RNA sequences of type strains of the genus Halomonas and exhibited high level of homology (99.6%) with the sequences of Halomonas marina. This phylogenetic relationship is consistent with the results of the phenotypic and chemotaxonomic evidence and allowed us to conclude that the second taxon of algal thallus degradation community belonged to H. marina.

E.P. Ivanova et al.

Activity of glycoside-hydrolases All strains of Pseudoalteromonas sp. produced an array of enzymes that catalized the hydrolysis of the complex polysaccharides found in brown alga thallus. Some of the corresponding glycanases detected in cell extracts of Pseudoalteromonas sp. are shown in Table 2, namely: highly active laminaranases (b-1,3-glucanase), alginases, fucoidan-hydrolases, pustulanases, agarases, and cellulases were not active. In addition, glycosidases (b-D-galactosidases, bD-xylosidases, b-D-glucosidases, and b-N-acetylglucoaminidases), which hydrolized mainly b-O-glycosidic bonds, were found in cell extracts of Pseudoalteromonas sp. Other glycosidases (a-galactosidases, a-N-Ac-galactosidases, a-mannosidases, a-L-fucosidases) which hydrolyzed a-Oglycosidic bonds were not detected. In contrast to Pseudoalteromonas sp., which had complex enzyme systems for the hydrolysis of algal polysaccharides, Halomonas marina did not produce any of those enzymes (Table 2).

Discussion The monitoring of algae degradation resulted in the recovery of two morphologically dierent bacterial groups directly from the samples examined using a culture-mediated approach. It is likely that enrichment conditions have led to signi®cant change in community structure, i.e., from a mixed bacterial assemblage residing on the surface of algal thallus to a community dominated by two c-proteobacterial lineages. Our study extends previous ®ndings that readily culturable marine c-proteobacteria can be rapidly enriched in substrate-amended seawater or media with a relatively low carbon component [11, 12]. Our result also agrees with the hypothesis of Cottrel and Kirchman that only small, less diverse groups of bacteria from natural assemblages are capable of hydrolyzing biopolymers due to the presence of corresponding extracellular enzymes [8]. In this study one group of metabolically highly active c-proteobacteria was found to belong to the genus Pseudoalteromonas. Numerous bacteria of this genus (more than 20 species) are frequently isolated from marine waters, are found in association with marine invertebrates, plants, and algae, and have been intensively studied during past decades [3, 4, 18, 30]. Pseudoalteromonas are readily culturable bacteria highly capable of surviving in nutrient-poor marine environments by adjustment of their biochemical pathways. This capacity allows them to obtain nutrients and eliminate their


247

Fig. 1. Phylogenetic position of isolates Pseudoalteromonas sp. KMM 3549, KMM 3558 and Halomonas marina KMM 3550 within the gamma subclass of Proteobacteria. The tree was constructed using the neighbor-joining analysis based on 1,387 unambiguously aligned 16S rRNA positions. The scale bar represents 0.1 nucleotide substitution per position.

competitors. In current experiments Pseudoalteromonas sp. was considered as a saprophytic organism, a degrader of the algal thallus. However, its ability to produce hemolytic substances and bacteriolytic enzymes may also allow this bacterium to live as a pathogen such as P. bacteriolytica, which proved to be the causative agent of red spot disease of brown alga Laminaria japonica [29]. Bacteria of the second taxonomic group, Halomonas marina, are moderately halophilic halomonads that are widely distributed in dierent natural habitats, namely; seawater, estuarine water, hypersaline soils, Antarctic lakes, and the Dead Sea [10]. Bacteria of this genus also notable by the lack of ability to hydrolyze complex polysaccharides. To our knowledge, this is the ®rst report describing the c-proteobacterial microbial community found to degrade the thallus of the brown alga Fucus evanescens. Later, we consider the possible process of alga degradation.

Typically, the Pseudoalteromonas sp. cells have single ¯agellum and can move according to nutrients' signals. In the initial phase of the thallus degradation, the bacteria migrate toward the detected nutrients' source (i.e., thallus) and attach on the surface of the algae. At this stage the production of bacteriolytic enzymes and hemolysins may be responsible for the elimination of the other bacteria [17]. Speci®c glycoside-hydrolases, i.e., b-N-acetylglucosaminidases, produced by Pseudoalteromonas sp., for example, also play an important role in decomposition of bacteria [16, 21, 34]. A later phase of degradation of the thallus by Pseudoalteromonas sp. results in the breakdown of the algal cell walls and all membrane systems by producing proteinases and lipases. The wide range of glycanases (fucoidan-hydrolase, laminarinase, alginase, pustulanase) and glycosidases (b-D-glucosidase, b-D-galactosidase, b-D-xylosidase) produced by Pseudoalteromonas sp. facilitated the

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Table 2. Enzyme activities of bacterial association that degrades brown alga Fucus evanescens Substratea

Pseudoalteromonas sp. (n = 11)

Fucoidan Laminaran Alginic acid Pustulan Np-b-D-Glucopyranoside Np-b-D-Galactopyranoside Np-b-N-Ac-Glucoaminopyranoside Np-b-D-Xylanoside Np-a-D-Galactopyranoside Np-a-N-Ac-Galactopyranoside Np-a-Manppyranoside Np-a-L-Fucopyranoside

0.03±0.4 1.77±4.9 0.25±0.37 0.06±0.16 1.2±2.4 1.2±2.4 0.6±0.4 5.4 0 0 0 0

Halomonas marina (n = 18) 0 0 0 0 0 0 0 0 0 0 0 0

a

One unit of glycanase activity is the amount of enzyme that liberated 1 lmol of corresponding monosaccharides in 1 h; one unit of glycosidase activity is the amount of enzyme that liberated 1 lmol of p-nitrophenol-in 1 h.

access to and degradation of the polysaccharides that constitute the matrix of the algal thallus and provided an important carbon source for both groups of the association. During the ®nal phase of thallus degradation the damage caused by Pseudoalteromonas sp. is extended into thallus to a signi®cant depth because of utilization by the H. marina and Pseudoalteromonas sp. of mono- and disaccharides released after degradation of complex polysaccharides. In conclusion, our results suggest that the metabolic interactions for nutrition between Pseudoalteromonas sp. and H. marina has led to an enhanced degradation of brown algal thallus. Although it is dicult to ascertain the degree to which such a culture-mediated approach represents the actual community structure, the replication of recovered bacterial community in this study suggests that the observed community structure is real. The interactions between two aerobic heteroorganothrophic species are rather complex and particularly interesting. While one of them, Pseudoalteromonas sp., most likely plays the major role in the initial stages of algal degradation, the other one, H. marina, resistant to bacteriolytic activity of the former, is able to utilize the products of polysaccharide degradation. Our study thus enhances our understanding of the complex structure±function interactions in marine bacterial communities during algal degradation when substratum availability controls the taxonomic structure of culturable heterotrophic bacteria.

Acknowledgments This study was supported by funds from the Russian Foundation for Basic Research #99-04-48017, and by a grant of the State Committee for Science and Technologies

of the Russian Federation #00-03-19 and in part by Award #REC-003 of US CRDF.

References 1.

Ashen JB, Goff LJ (1996) Molecular identi®cation of a bacterium associated with gall formation in the marine red algae Priontis lanceolata. J Phycol 32:286±297 2. Azam F, Cho BC (1987) Bacterial utilization of organic matter in the sea. In: Ecology of Microbial Communities. Cambridge University Press, Cambridge, UK pp 261±281 3. Baumann L, Baumann P, Mandel M, Allen RD (1972) Taxonomy of aerobic marine eubacteria. J Bacteriol 110:402±429 4. Baumann P, Baumann L (1981) The marine Gram-negative eubacteria; genera Photobacterium, Beneckea, Alteromonas, Pseudomonas, and Alcaligenes. In: MP Starr, H Stolp, HG TruÈper, A Balows, HG Schlegel (eds) The prokaryotes. A handbook on Habitats, Isolation, and Identi®cation of Bacteria, vol 2. Springer-Verlag AG, Berlin, pp 1302±1330 5. Boetius A, Lochte K (1994) Regulation of microbial enzymatic degradation of organic matter in deep-sea sediments. Mar Ecol Prog Ser 104:299±307 6. Chizhov AO, Dell A, Morris HR (1999) A study of fucoidan from the brown seaweed Chorda ®lum. Carbohydr Res 320:108±119 7. Cho BC, Azam F (1988) Major role of bacteria in biogeochemical ¯uxes in the ocean's interior. Nature 332:441±443 8. Cottrell MT, Kirchman DL (2000) Natural assemblages of marine proteobacteria and members of the Cytophaga± Flavobacter cluster consuming low- and high-molecularweight dissolved organic matter. Appl Environ Microbiol 66:1692±1697 9. Daniel R, Berteau O, Jozefonvicz J, Goasdoue N (1999) Degradation of algal (Ascophyllum nodosum) fucoidan by an enzymatic activity contained in digestive glands of the marine mollusc Pecten maximus. Carbohydr Res 322:291±297 10. Dobson SJ, Franzmann PD (1996) Uni®cation of the genera Deleya (Baumann et al., 1983), Halomonas (Vreeland et al.,


11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

1080), and Halovibrio (Fendrich 1988) and the species Paracoccus halodenitri®cans (Robinson and Gibbons 1952) into a single genus, Halomonas, and placement of the genus Zymobacter in the family Halobacteriaceae. Int J Syst Bacteriol 46:550±558 Eilers H, Pernthaler J, Glokner FO, Amann R (2000) Culturability and in situ abundance of pelagic bacteria from the North Sea. Appl Environ Microbiol 66:3044±3051 Eilers H, Pernthaler J, Amann R (2000) Succession of pelagic marine bacteria during enrichment: a close look at cultivation-induced shifts. Appl Environ Microbiol 66:4634±4640 Fanner III JJ, Arduino MJ, Hickman-Brenner FW (1992) The genera Aeromonas and Plesiomonas In: A Balows, HG Truper, M Dworkin, W Harder and K-H Schleifer (eds) The Prokaryotes. Springer-Verlag, New York, pp 3012±3045 Furukawa S, Fujikawa T, Koga D, Ide A (1992) Puri®cation and some properties of exo-type fucoidanases from Vibrio sp. N-5. Biosci Biotech Biochem 56:1829±1834 Fisher MM, Wilcox LW, Graham LE (1998) Molecular characterization of epiphytic bacterial communities on Cherophycean green algae. Appl Environ Microbiol 64:4384±4389 Gonzalez JM, Whitman WB, Hodson RE, Moran MA (1996) Identifying numerically abundant culturable bacteria from complex communities: an example from a lignin enrichment culture. Appl Environ Microbiol 62:4433±4440 Grant WD, Rhodes LL, Prosser BA, Asher RA (1986) Production of bacteriolytic enzymes and degradation of bacteria by ®lamentous fungi. J Gen Microbiol 132:2353±2358 Holmstrom C, Kjelleberg S (1999) Marine Pseudoalteromonas species are associated with higher organisms and produce biologically active extracellular agents. FEMS Microbiol Ecol 30:285±293 Ivanova EP, Kiprianova EA, Mikhailov VV, Levanova GF, Garagulya AG, Gorshkova NM, Yumoto N, Yoshikawa S (1996) Characterization and identi®cation of marine Alteromonas nigrifaciens strains and emendation of the description. Int J Syst Bacteriol 46:223±228 Ivanova EP, Kiprianova EA, Mikhailov VV, Levanova GF, Garagulya AD, Gorshkova NM, Vysotskii MV, Nicolau DV, Yumoto Y, Yoshikawa S (1998) Phenotypic diversity of Pseudoalteromonas citrea from different marine habitats and emendation of the description. Int J Syst Bacteriol 48:247±256 Ivanova EP, Zhukova NV, Svetashev VI, Gorshkova NM, Kurilenko VV, Frolova GM, Mikhailov VV (2000) Evaluation of phospholipid and fatty acid compositions as chemotaxonomic markers of Alteromonas-like proteobacteria. Curr Microbiol 41:341±345. Jaffray AE, Anderson RJ, Coyne VE (1997) Investigation of bacterial epiphytes of the agar-producing ref seaweed Gracilaria gracilis (Stackhouse) Steentoft, Irvine et Farnham from Saldanha Bay, South Africa and LuÈderitz, Namibia. Botanica Marina 40:569±576 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265±275

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24. Marmur J (1961) A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3:208± 218 25. Marmur J, Doty P (1962) Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5:109±118 26. Nelson TE (1944) A photometric adaptation of the Somogyi method for the determination of glucose. J Biol Chem 153:375±381 27. Ostle A, Holt JG (1975) Nile blue as a ¯uorescent stain for poly-b-hydroxybutyrate. Appl Environ Microbiol 44:238± 240 28. Sawabe T, Sugimura I, Ohtsuka M, Nakano K, Tajima K, Ezura Y, Christen R (1998a) Vibrio halioticoli sp. nov., a non-motile alginolytic marine bacterium isolated from the gut of the abalone Haliotis discus hannai. Int J Syst Bacteriol 48:573±580 29. Sawabe T, Makino H, Tatsumi M, Nakano K, Tajima K, Iqbal MM, Yumoto I, Ezura Y, Christen R (1998b) Pseudoalteromonas bacteriolytica sp. nov., a marine bacterium that is causative agent or red spot disease of Laminaria japonica. Int J Syst Bacteriol 48:769±774 30. Sawabe T, Tanaka R, Iqbal MM, Tajima K, Ezura Y, Ivanova EP, Christen R (2000) Assignment of Alteromonas elyakovii KMM 162T and ®ve strains isolated from spot-wounded frond of Laminaria japonica to Pseudoalteromonas elyakovii comb. nov. and the extended description of the species. Int J Evol Syst Microbiol 50:267±271 31. Smibert RM, Krieg NR (1994) Phenotypic characterization In: F Gerhardt (ed) Methods for General and Molecular Bacteriology. American Society for Microbiology, Washington DC, pp 607±654 32. Svetashev VI, Vysotskii MV, Ivanova EP, Mikhailov VV (1995). Cellular fatty acid of Alteromonas species. System Appl Microbiol 18:37±43 33. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-speci®c gap penalties and weight matrix choice. Nucleic Acids Res 22:4673±4680 34. Warren J (1996) Microbial hydrolysis of polysaccharides. Annu Rev Microbiol 50:83±212 35. Yumoto I, Yamaguchi K, Yamada K, Ezura Y, Kimura T (1989) Relationship between bacterial ¯ora and occurrence of the Alteromonas sp., a nonobligate bacterial predator, and bacterial prey in arti®cial sea water. Bull Jpn Soc Microb Ecol 3:101±108 36. Zvyagintseva TN, Shirokova NM, Elyakova LA (1994) The structure of laminarans from brown seaweeds. Bioorg Khim 20:349±1358 37. Zvyagintseva TN, Shevchenko NM, Popivnich IB, Isakov W, Scobun AS, Sundukova EV, Elyakova LA (1999) A new procedure for the separation of the water-soluble polysaccharides from brown seaweeds Carbohydrate Res 322: 32±39