Abstract
Abbreviations: ASW, artificial seawater; DOM, dissolved organic material; HMM, high molecular mass
Footnotes
†Present address: UMR6197, Laboratoire de Microbiologie des Environnements Extrêmes, IUEM, Technopôle Brest-Iroise, F-29280 Plouzané, France.The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Eudoraea adriatica AS06/20aT is AM745437.
A scanning electron micrograph of cells of strain AS06/20aT and graphs showing the effects of temperature, pH and salinity on the maximum growth rate of the novel isolate are available as supplementary material with the online version of this paper.
Bacteria belonging to the phylum Bacteroidetes are widely distributed in nature and have been found in almost every habitat of the biosphere, the lithosphere and the hydrosphere (Bernardet et al., 2002; Kirchman, 2002). They seem particularly common and abundant in marine surface waters, as indicated by fluorescence in situ hybridization analyses (Glöckner et al., 1999; Cottrell & Kirchman, 2000a; Simon et al., 1999; Alonso & Pernthaler, 2005). Cultured isolates of Bacteroidetes are all heterotrophs and especially efficient in degrading complex biomacromolecules such as protein, chitin, pectin, agar, starch and cellulose.
In marine ecosystems, the heterotrophic prokaryotes are the main consumers of dissolved organic material (DOM) and represent a very dynamic compartment in global biogeochemical cycles. Uptake of DOM is the first step in the microbial loop that ultimately mineralizes over half of the organic matter formed by photosynthetic bacteria and algae (Cole et al., 1988). Although the importance of DOM uptake is well recognized, the relative contributions of the different phylogenetic groups to DOM uptake remain to be elucidated. Because of their abundance and heterotrophic metabolism, Bacteroidetes are thought to occupy a special place in the carbon cycling of oceans. They are obviously involved in the degradation and uptake of DOM and probably contribute to the degradation of the high-molecular-mass (HMM; >1 kDa) fraction of the DOM (Kirchman, 2002). Indeed, the HMM fraction is composed of biopolymers; hydrolysis of these macromolecules requires specific extracellular enzymes, which are produced by numerous known members of the Bacteroidetes. Using a method combining microautoradiography and hybridization of fluorescent rRNA-targeted oligonucleotide probes to whole cells from natural marine assemblages (Micro-FISH), Cottrell & Kirchman (2000b) demonstrated that Bacteroidetes represented, for the considered habitats, a large fraction of the community that was efficient in degrading chitin, N-acetylglucosamine and protein, part of the HMM fraction of the DOM. All these data together suggest strongly that, in marine environments, Bacteroidetes are key players in the degradation of the HMM fraction of the DOM, which has been demonstrated to be more bioreactive and more bioavailable than the low-molecular-mass fraction (Amon & Benner, 1996). As relevant work on cultured representatives from marine origin is lacking, there is great interest in isolating and describing phenotypic and genotypic characteristics of marine Bacteroidetes known only by their 16S rRNA gene sequences. For example, there are notably very few sequence data on enzymes catalysing biopolymer hydrolysis (Kirchman, 2002). The isolation of strains is still the easiest way to access their physiological and genomic capital. This is probably the reason why the number of strains of Bacteroidetes from marine ecosystems described in the last five years has increased exponentially.
In this study, a novel marine bacterium belonging to the phylum Bacteroidetes is described. Based on the results of a polyphasic taxonomic analysis, strain AS06/20aT represents a novel species in a new genus, Eudoraea adriatica gen. nov., sp. nov.
In April 2006, marine surface waters were collected on the coast of the Adriatic Sea, Italy (44.69 ° N 12.52 ° E). One subsample collected at a depth of 10 m was spread on a marine agar 2216 (MA; Difco) plate and then incubated at 25 °C. After 2 weeks, a small non-pigmented colony was picked and purified by repeated streaking on MA plates. It was referenced as strain AS06/20aT. Stock cultures were stored at –80 °C in marine broth 2216 (MB; Difco) supplemented with 5 % (v/v) DMSO or 35 % (v/v) glycerol until characterization.
The almost complete 16S rRNA gene (1393 bp) of the strain was a double-strand sequenced from one single colony, as described elsewhere (Agogué et al., 2005). This sequence was compared to those in available databases by using the program BLAST (Altschul et al., 1990) and then aligned to its nearest neighbours using the program CLUSTAL_X (Thompson et al., 1997). Alignments were refined manually using the program SEAVIEW (Galtier et al., 1996). Phylogenetic trees were constructed using the PHYLIP version 3.63 software () on the basis of evolutionary distance (neighbour-joining method with Jukes and Cantor corrections) (Saitou & Nei, 1987) and maximum-likelihood (Felsenstein, 1981). The robustness of the inferred topologies was assessed by bootstrap analyses based on 1000 bootstrap resamplings for the neighbour-joining and 100 replications for the maximum-likelihood method (Felsenstein, 1985). The 16S rRNA gene-based analysis placed strain AS06/20aT within the phylum Bacteroidetes. The novel isolate was affiliated to the family Flavobacteriaceae, one of the main phyletic lines of the phylum Bacteroidetes (Reichenbach, 1989; Bernardet et al., 1996, 2002) (Fig. 1). The results of different phylogenetic reconstructions performed with different treeing algorithms were in accordance with each other. The novel isolate formed a distinct lineage together with Zeaxanthinibacter enoshimensis TD-ZE3T and Robiginitalea biformata HTCC2501T, but this lineage did not cluster robustly with any of the recognized genera in the family. Within this lineage, the novel isolate showed only a distant relatedness to its nearest phylogenetic neighbours, the most closely related genus being Zeaxanthinibacter (93 % 16S rRNA gene sequence similarity), followed by Robiginitalea (92 %). These low 16S rRNA gene sequence similarities between strain AS06/20aT and the genera described so far suggest that the novel isolate represents a member of a new genus within the family Flavobacteriaceae.
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The DNA G+C content was determined by the Identification Service of the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) by HPLC analysis of deoxyribonucleosides as described by Mesbah et al. (1989). The G+C content of strain AS06/20aT was 38.9 mol%.
Colonies on MA were non-pigmented and cream in colour. They were circular, punctiform, opaque with an entire edge and possessed a smooth surface. After 1 week incubation, colonies were less than 1 mm in diameter. Morphological characteristics of the cells were determined by light microscopy (Olympus AX70) and by transmission electron microscopy (Hitachi H-7500) after negative staining with uranyl acetate (Raguénès et al., 1997). Gram-staining was determined using standard procedures. Gliding motility was determined using the hanging drop method on cells grown at 30 °C in low nutrient medium [0.1 % (w/v) MB solidified with 1 % agar], as described by Bernardet et al. (2002). In summary, cells of strain AS06/20aT were Gram-negative, non-motile, straight rods of 1.48–2.99 µm in length (mean 2.22±0.54 µm; n=8) and 0.6–0.78 µm in width (mean 0.70±0.07 µm; n=8) in the mid-exponential phase of growth (see Supplementary Fig. S1 in IJSEM Online). Gliding motility was not observed under the conditions tested. Cells swelled into balloon-like shapes during the late stationary growth phase.
In order to analyse respiratory quinones and polar lipids, strain AS06/20aT was grown for 5 days on MB medium at 30 °C and checked for purity. Analyses of isoprenoid quinones and polar lipids were carried out by the Identification Service of the DSMZ (B. Tindall, DSMZ Culture Collection). As described for the other members of the family Flavobacteriaceae, the main respiratory quinone was menaquinone-6 (MK-6; 83 %) (Bernardet et al., 2002); MK-7 was also detected (17 %). Phosphatidylethanolamine was the only major phospholipid identified in strain AS06/20aT. Three unidentified polar lipids, two containing amino groups, were also present as major components.
Determination of the whole-cell fatty acid composition was performed on cultures grown at 30 °C for 72 h on MA. This analysis was carried out at the DSMZ according to the standard protocol of the Microbial Identification System (MIDI, 2001). Extracts were analysed using a Hewlett Packard model HP6890A GC equipped with a flame-ionization detector as described by Kämpfer & Kroppenstedt (1996). Results are summarized in Table 1. The dominant fatty acids in strain AS06/20aT were iso-C15 : 1 G, iso-C15 : 0, iso-C15 : 0 3-OH, iso-C17 : 1ω9c, iso-C17 : 0 3-OH and the fatty acids in summed feature 3.
Table 1. Phenotypic and genotypic characteristics of strain AS06/20aT (Eudoraea adriatica gen. nov., sp. nov.)
Unless stated otherwise, physiological characterization was carried out aerobically in MB in triplicate and incubation was done in the dark and under agitation. Growth was routinely monitored by measuring the increase in optical density at 600 nm using a spectrophotometer. Cell numbers were determined by flow cytometry in order to calculate calibration curves [cell number=f(OD600)]. Growth rates were calculated using linear regression analysis of four to nine points along the logarithmic portions of the resulting growth curves. Growth temperatures were tested over the range 9–44 °C (i.e. 9, 15, 20, 25, 30, 33, 37 and 44 °C). The temperature range for growth was 15–33 °C, with optimum growth rate at 30 °C (see Supplementary Fig. S2a in IJSEM Online). No growth was observed at 9 or 37 °C. The pH range for growth was tested at 30 °C in MB medium, buffered and adjusted to the required pH as described elsewhere (Alain et al., 2002). The pH range for growth was rather narrow (see Supplementary Fig. S2b in IJSEM Online); growth was observed at pH 6.5–8.5, the optimum being around pH 7.5–8.0. Salt tolerance was tested at 30 °C in MB medium prepared with various concentrations of NaCl (0.02, 0.5, 1, 2, 3, 4, 5, 6, 7 and 9 %, w/v). The isolate required NaCl for growth (see Supplementary Fig. S2c in IJSEM Online). This is congruent with the fact that this strain was isolated from brackish waters of the North Adriatic Sea. Growth was observed at salt concentrations ranging from 2 to 6 % (w/v) NaCl, the optimum salinity being around 2 %. No growth was observed at 1 or 7 % (w/v) NaCl.
Strain AS06/20aT was aerobic. Conventional phenotypic tests, including those for oxidase, catalase, Tween esterase and nitrate reductase, were performed according to standard methods (Smibert & Krieg, 1994). The results are given in Table 1. Biochemical tests were performed at 30 °C using API ZYM (bioMérieux) and Biolog GN2 microplates (Oxoid). These tests were inoculated with cells grown on MA plates, swabbed from the surface of the agar plates and then suspended in half-strength artificial seawater (ASW) to the density specified by the manufacturer. Supplementary biochemical tests were also performed using API 20NE strips (bioMérieux), following the manufacturer's instructions. The data obtained are given in Table 1. Testing for oxidation of carbon sources with Biolog GN2 plates indicated that the strain was able to oxidize a range of sugars, organic acids and amino acids. To confirm these results and to test for the capability of the strain to catabolize different substrates as sole carbon and energy sources, with oxygen as a terminal electron acceptor, the strain was grown aerobically in the dark on a mineral medium supplemented with one substrate. The defined medium had the following composition (l–1): phosphate buffer, 30 mM; NaCl, 20 g; MgCl2 . 6H2O, 3 g; CaCl2 . 2H2O, 1.0 g; NH4Cl, 0.3 g; KCl, 0.5 g; Na2SO4, 3 g; NaNO3, 1 g; trace element solution, 1 ml; selenite-tungstate solution, 1 ml; vitamin solution, 1 ml. The strain grew chemo-organoheterotrophically on a variety of carbon compounds, including pentoses, hexoses, polyols, organic acids, amino acids and complex organic substrates (Table 1).
Antibiotic sensitivity tests were performed by using susceptibility discs (Bio-Rad) or filter-paper discs impregnated with different antibiotics. Discs were placed on MA plates spread with a culture of the isolate and were then incubated at 30 °C for 1 week. Susceptibility was scored as positive at zone diameters above 10 mm. The results are summarized in Table 1.
The phenotypic and genotypic characteristics of the novel isolate generally met the minimal standards for the family Flavobacteriaceae (Bernardet et al., 2002). Nevertheless, strain AS06/20aT differed from members of other genera of the Flavobacteriaceae with validly published names in a number of phylogenetic, genotypic, chemotaxonomic, morphological and physiological features (Table 2). In brief, in addition to the phylogenetic distance, the novel taxon can be distinguished from its closest relatives by some of its phenotypic features, including its absence of colonial pigmentation, the narrow NaCl and pH ranges for growth, and its restricted degrading capabilities regarding the tested macromolecules. In addition to these phenotypic differences, the novel isolate is unambiguously distinct from its two closest neighbours by its genomic DNA G+C content, which is more than 5 mol% lower than those of Zeaxanthinibacter enoshimensis and Robiginitalea biformata. In conclusion, in view of all the above-mentioned distinctive features, it is proposed that isolate AS06/20aT represents a novel species in a new genus, Eudoraea adriatica gen. nov., sp. nov.
Table 2. Differential characteristics of strain AS06/20aT (Eudoraea adriatica gen. nov., sp. nov.) and members of related genera of the family Flavobacteriaceae Strains: 1, AS06/20aT (data from this study); 2, Zeaxanthinibacter enoshimensis TD-ZE3T(Asker et al., 2007); 3, Robiginitalea biformata HTCC2501T (Cho & Giovannoni, 2004); 4, Pibocella ponti KMM 6031T (Nedashkovskaya et al., 2005); 5, Maribacter sedimenticola KMM 3903T (Nedashkovskaya et al., 2004); 6, Zobellia galactanivorans DsijT (Barbeyron et al., 2001); 7, Arenibacter latericius KMM 426T (Ivanova et al., 2001); 8, Sediminicola luteus CNI-3T (Khan et al., 2006). +, Positive; –, negative; ND, not determined; W, weak.
Description of Eudoraea gen. nov.
Eudoraea (Eu.do.ra'e.a. N.L. fem. n. Eudora a sea goddess in Greek mythology; N.L. fem. n. Eudoraea named after Eudora).
Cells are Gram-negative, non-spore-forming, non-motile, non-gliding rods. Pigments are not produced. Aerobic and chemo-organoheterotrophic. Catalase- and oxidase-positive. Mesophilic. Neutrophilic. Require NaCl for growth. The predominant quinone is MK-6. Polar lipids include phosphatidylethanolamine. Major fatty acids are iso-C15 : 1 G, iso-C15 : 0, iso-C15 : 0 3-OH, iso-C17 : 1ω9c, iso-C17 : 0 3-OH and summed feature 3. The G+C content of the DNA is close to 39 mol%. Phylogenetically, the genus Eudoraea belongs to the family Flavobacteriaceae, phylum Bacteroidetes, showing a distant relatedness to the marine genera Zeaxanthinibacter and Robiginitalea. The type species is Eudoraea adriatica.
Description of Eudoraea adriatica sp. nov.
Eudoraea adriatica (a.dri.a'ti.ca. L. fem. adj. adriatica of the Adriatic Sea, where the type strain was isolated).
In addition to the characters described for the genus, the following properties apply. Colonies on MA medium are punctiform, cream in colour, opaque and smooth. Optimal growth occurs at 30 °C; grows at 15–33 °C. pH and NaCl ranges for growth are 6.5–8.5 (optimum 7.5–8.0) and 2–6 % (w/v) (optimum 2 %, w/v), respectively. Aesculin is hydrolysed, but agar, cellulose, gelatin, starch and urea are not. β-Galactosidase-positive. Glucose is not fermented. Nitrate is not reduced. Various carbon compounds are used as sole carbon sources, including pentoses, hexoses, polyols, organic acids, amino acids and complex substrates (Table 1).
The type strain, AS06/20aT (=DSM 19308T=CIP 109577T=OOB 358T), was isolated from coastal waters of the Adriatic Sea, Italy (44.69 ° N 12.52 ° E). The DNA G+C content of strain AS06/20aT is 38.9 mol%.
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