International Journal of Systematic and Evolutionary Microbiology (2002), 52, 2211–2217 DOI: 10.1099/ijs.0.02255-0 Shewanella denitrificans sp. nov., a vigorously denitrifying bacterium isolated from the oxic–anoxic interface of the Gotland Deep in the central Baltic Sea 1 GBF–German Research Ingrid Brettar,1 Richard Christen2 and Manfred G. Ho$ fle1 Centre for Biotechnology, Department of Environmental Author for correspondence: Microbiology, Ingrid Brettar. Tel: j49 531 6181 440. Fax: j49 531 6181 411. Mascheroder Weg 1, e-mail: inb!gbf.de D-38124 Braunschweig, Germany Three strains of denitrifying estuarine bacteria, OS217T, OS220 and OS226, were 2 UMR 6078 CNRS and characterized for their physiological and biochemical features, fatty acid Universite! Nice Sophia Antipolis, Batiment Jean profiles and their phylogenetic position based on 16S rDNA sequences. The Maetz, F-06230 strains were isolated from the oxic–anoxic interface of an anoxic basin of the Villefranche sur Mer, central Baltic Sea. Phylogenetic analyses of the 16S rDNA sequences revealed a France clear affiliation with members of the genus Shewanella of the γ- Proteobacteria. The closest sequence similarity was seen with Shewanella baltica, Shewanella putrefaciens and Shewanella frigidimarina (95–96%). The dominant fatty acids were 16:1ω7c, 15:0 iso, 16:0 and 13:0 iso. The GMC content of the DNA ranged from 468to481 mol%. The strains were unpigmented, polarly flagellated, mesophilic, facultatively anaerobic and able to use nitrate, nitrite and sulphite as electron acceptors. Growth was observed at salinities from 0 to 6%, with an optimum between 1 and 3%. According to their morphology, physiology, fatty acid composition and 16S rRNA sequences, the described bacteria fitted well into the genus Shewanella, but could be easily distinguished from the Shewanella species described to date. Because of their capacity for vigorous denitrification, the name Shewanella denitrificans sp. nov. is suggested for the Baltic isolates, for which the type strain is OS217T (l DSM 15013T l LMG 21692T). Keywords: marine, estuarine bacteria, Baltic Sea, denitrifier, Shewanella INTRODUCTION survival and competition in aquatic ecosystems with oxic–anoxic interfaces (Brettar & Ho$ fle, 1993). The genus Shewanella (MacDonell & Colwell, 1985) comprises 13 species of facultatively anaerobic γ- In this study, we describe three denitrifying strains of Proteobacteria at the time of writing, with a major the genus Shewanella that were isolated from the fraction isolated from aquatic habitats, including the oxic–anoxic interface of the Gotland Deep, a basin most recently described species, Shewanella japonica with anoxic deep water in the central Baltic. The (Ivanova et al., 2001). A comprehensive study on the strains were recognized by their unique low-molecular- phylogenetic relationships and the taxonomy of the weight (LMW) RNA fingerprints (Ho$ fle & Brettar, genus Shewanella was provided by Venkateswaran et 1996). Investigations on in situ denitrification in the al. (1999). A notable feature of members of the genus water column of the Gotland Deep provided strong Shewanella is their ability to use a variety of different evidence that the oxic–anoxic interface was the layer in electron acceptors that can be of special relevance for which most of the denitrification occurred in anoxic basins of the central Baltic (Brettar & Rheinheimer, ................................................................................................................................................. 1991, 1992). The three novel strains are vigorous Detailed fatty acid composition data for the novel isolates are available as supplementary material in IJSEM Online (http://ijs.sgmjournals.org/). denitrifiers present in the denitrifying layer of the The GenBank/EMBL/DDBJ accession numbers for the 16S rDNA sequences interface, and thus could have contributed to nitrogen of S. denitrificans sp. nov. strains OS217T, OS220 and OS226 are respectively elimination by denitrification in the central Baltic AJ311964, AJ457093 and AJ457092. (Brettar & Ho$ fle, 1993; Brettar et al., 2001). In addition 02255 # 2002 IUMS Printed in Great Britain 2211 I. Brettar, R. Christen and M. G. Ho$ fle to the three novel strains, a larger set of Shewanella cation was studied in nutrient broth (Difco) (plus nitrate at −" strains were isolated from the same samples of the 20 mmol l and sea salt at 8=,w\v) as described in more interface and were described earlier as another novel detail by Brettar & Ho$ fle (1993). As a positive control, S. Shewanella species, Shewanella baltica (Ziemke et al., baltica OS155, a strain isolated concomitantly with the novel 1998). Phylogenetic analyses of the 16S rDNA se- strains from the central Baltic (Ziemke et al., 1998), was used for a comparison. S. baltica OS155 was able to use the quences showed that the three novel strains were electron acceptors provided. included in the robust cluster formed by the genus Shewanella and suggest, concomitantly with the phy- Phylogenetic analysis based on 16S rRNA gene sequence siological features, fatty acid composition and LMW comparison. Genomic DNAs were prepared from individual RNA fingerprints, that the strains represent a novel colonies as described by Moore et al. (1996). 16S rRNA genes were amplified by PCR (Mullis & Faloona, 1987) and species of the genus Shewanella. the PCR products were sequenced directly as described previously (Moore et al., 1999). METHODS The 16S rDNA sequences of strains OS217T, OS220 and Bacterial strains, isolation and growth conditions. During a OS226 were aligned automatically and then manually by cruise aboard the RV Poseidon in August 1986, three strains, reference to a database of 37000 already-aligned bacterial OS217T, OS220 and OS226, were isolated from the water 16S rDNA sequences. The same sequences were then column of a basin with anoxic, sulphide-containing deep submitted to a search against the current content of water in the central Baltic Sea (Gotland Deep: BY 15, the EMBL database (Bacteria division) to check for the 57n1920m N, 20n0302m E). The strains were isolated from the presence of newly submitted, related sequences. Phylogen- oxic–anoxic interface (120–130 m depth) of the Gotland etic trees were constructed using three different methods Deep. The medium for isolation was nutrient broth (Difco) (bioNJ, maximum-likelihood and maximum-parsimony). −" plus nitrate (2 g KNO$ l ). The strains were isolated from For the neighbour-joining (NJ) analysis, a matrix distance liquid cultures inoculated with 1 and 10 ml sea water, i.e. the was calculated according to Kimura’s two-parameter cor- abundance of the novel strains according to the viable rection. Bootstraps were done using 500 replications, bioNJ counts was estimated to be as low as one cell per ml (in and Kimura’s two-parameter correction; bioNJ was used comparison, S. baltica showed viable counts, from the same according to Gascuel (1997) and the maximum-likelihood samples, that were three orders of magnitude higher; Brettar and maximum-parsimony programs were from et al., 2001). All details of the environmental conditions, version 3.573c (distributed by J. Felsenstein, Department of sampling and isolation procedures have been given elsewhere Genetics, University of Washington, Seattle). The phylo- (Brettar & Rheinheimer, 1992; Brettar & Ho$ fle, 1993; Ho$ fle genetic trees were drawn using (Perrie' re & Gouy, & Brettar, 1995, 1996). Strains grew well on ZoBell agar 1996) and software for Apple Macintosh. The (Oppenheimer & ZoBell, 1952) and half-strength as well as domains used to construct phylogenetic trees were regions of full-strength marine broth or agar (Difco). the small-subunit rDNA sequences available for all se- quences and excluding positions likely to show homoplasy. Physiological and biochemical tests and morphology. The isolates were tested for a number of key characteristics by For the tree shown in Fig. 2, the retention of only sequences using standard procedures (Gerhardt et al., 1994), such as of related genera, mostly from type strains (26 sequences), the Gram reaction (Gram staining, Gram string test), cell allowed the inclusion in the analysis of almost the entire 16S size and morphology (phase-contrast microscopy and elec- rDNA sequences, corresponding to positions 1–1194 of the T tron microscopy after negative staining with 1% uranyl OS217 sequence. The topology shown is that of the acetate) and tests for cytochrome oxidase and catalase (3% bootstrap analysis, as it has been demonstrated that this H#O#). Furthermore, production of hydrogen sulphide (Dye, topology is often better than that of a simple NJ analysis 1968), haemolysis (bovine-blood agar), acid production (Berry & Gascuel, 1996). As a result, there is no distance bar from glucose, ribose and arabinose and hydrolysis of starch, in this tree; note, also, that one should consider the distance gelatin, Tween 80 and lecithin were tested. Strains were bar with caution in a simple tree, as the distance bar repre- additionally characterized by the whole test spectrum of the sents the distances calculated after corrections (Kimura’s identification systems API 50CH, API 20NE and API ZYM two-parameter; Jukes & Cantor, 1969), and that the (bioMe! rieux) at 20 mC. Growth at different temperatures lengths of the branches do not simply represent the real was tested at 4, 10, 20, 25, 30 and 37 mC. Growth at different numbers of differences between the sequences themselves. salinities was tested at 0, 1, 3, 6 and