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1 National Research Institute of Fisheries Science, Fisheries Research Agency, Yokohama, 236-8648, Japan
2 California Institute of Technology, Jet Propulsion Laboratory, Biotechnology and Planetary Protection Group, 89-2, Oak Grove Dr., Pasadena, CA 91109, USA
3 Danish Institute for Fisheries Research, Department of Seafood Research, Søltofts Plads, DTU bldg 221, DK-2800 Kgs. Lyngby, Denmark
Correspondence
Kasthuri Venkateswaran
kjvenkat{at}jpl.nasa.gov
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ABSTRACT |
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98 %, gyrB gene sequence similarity of 89 % and DNA–DNA reassociation values of 20–34 %. Based on the evidence presented, two novel species, Shewanella hafniensis sp. nov. (type strain P010T=ATCC BAA-1207T=NBRC 100975T) and Shewanella morhuae sp. nov. (type strain U1417T=ATCC BAA-1205T=NBRC 100978T), are described.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and gyrB gene sequences for Shewanella hafniensis P010T are AB205566 and AB208056 and for Shewanella morhuae U1417T are AB205576 and AB208062, respectively; accession numbers for other new isolates are indicated in Figs 1 and 2
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). We have, in particular, focused on bacteria that reduce trimethylamine and produce hydrogen sulphide (H2S) as these are the main spoilage agents of chilled, stored fresh fish. These bacteria were non-fermentative, motile rods and initial screening tentatively identified them as members of the genus Shewanella, species of which are common in aquatic habitats. The phenotypic boundaries of and within this group are currently being redefined and the need for a more diagnostically informative pattern of phenotypic characteristics is pressing. Combining genetic and phenotypic analyses in a polyphasic taxonomic approach, 30 Shewanella species have been recognized at the time of writing (http://www.bacterio.cict.fr/s/shewanella.html).
The gene sequence of the 16S rRNA molecule has been used extensively to define phylogenetic relationships between organisms (Woese, 1987), but this molecule, at times, lacks the specificity required for the differentiation of close relatives (Fox et al., 1992; Venkateswaran et al., 1998). To circumvent this limitation, the more rapidly evolving gyrB gene has been employed as a high-resolution molecular identification marker for distinguishing several species (Satomi et al., 2003; Venkateswaran et al., 1998; Yamamoto & Harayama, 1995, 1998; Yamamoto et al., 1999).
In a previous report we found that the majority of H2S-producing strains isolated from Baltic fish were identified as Shewanella baltica (Fonnesbech Vogel et al., 2005). However, a number of strains differed from the Shewanella species included in that report (S. baltica, Shewanella algae, Shewanella putrefaciens, Shewanella oneidensis, Shewanella colwelliana and Shewanella affinis) based on phenotypic testing and 16S rRNA gene sequence analysis. A detailed taxonomic characterization of these H2S-producing organisms is warranted with regard to our understanding of potential human pathogens (related to S. algae) and of organisms important in fish spoilage (S. baltica). In the present study we characterize two of the non-identifiable groups from the previous report (Fonnesbech Vogel et al., 2005) using phenotypic characterization, phylogenetic analysis of 16S rRNA and gyrB gene sequences and DNA–DNA hybridization, and two novel species are described.
A total of 47 strains of novel H2S-producing bacteria were isolated from cod, plaice or flounder caught between August 1995 and September 2001 from the Baltic Sea off Denmark. Bacterial isolation was carried out as reported elsewhere (Fonnesbech Vogel et al., 2005). Briefly, samples were taken from the belly flap area and pour-plated in iron agar (Oxoid CM964) from which H2S-producing bacteria were isolated. Of the 47 strains tested for phenotypic analysis, representative strains were selected for further molecular characterization in order to determine their phylogenetic affiliation (Table 1). In addition to these newly described strains, the type strains of closely related species were purchased from several established culture collections and were used as reference strains. All isolates were maintained in stabs on trypticase soy agar (TSA) at room temperature for short-term analysis and in a medium containing skimmed milk powder and glycerol at –80 °C for long-term storage. Liquid cultures were grown in trypticase soy broth (TSB; BD Biosciences) and were incubated at 25 °C for 2–7 days. Representative strains have been deposited in two public culture collections, namely the American Type Culture Collection (ATCC) and the National Institute of Technology and Evaluation, Biological Resource Center (NBRC), Japan.
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), motility and cell shape [phase-contrast microscopy after growth in veal infusion broth (Difco; 0344-17-6) for 24 h], cytochrome oxidase (BBL DrySlide oxidase, 231746; BD Biosciences), catalase-reaction (3 % H2O2), reduction of trimethylamine oxide (TMAO) in TMAO medium (Gram et al., 1987) and production of H2S from thiosulphate (Gram et al., 1987). Fermentation of glucose was tested in O-F medium (Merck) at 25 °C. Growth at various temperatures (4, 37 and 42 °C) and in 6 % NaCl (Fonnesbech Vogel et al., 1997) and assimilation of several carbon and energy sources (citrate, gluconate, glucose, lactate and sucrose; Ziemke et al., 1998) were used to differentiate the Shewanella isolates further. Type strains of S. putrefaciens, S. baltica and S. algae were included in each trial and served as controls. The genomic G+C content was determined by HPLC (Fonnesbech Vogel et al., 1997). The ability to degrade gelatin (Frazier, 1926), DNA (Difco DNase test agar with methyl green; 0220-17-5) and ornithine (Difco decarboxylase base Moeller; 289020) was also tested.
All of the new isolates were non-fermentative, Gram-negative, motile rods, with positive oxidase and catalase reactions and the ability to reduce TMAO and produce H2S. Under optimum growth conditions, cells were 0·5–0·7 µm in diameter and 1·0–1·2 µm in length. Colonies were round, undulate, beige coloured, non-luminescent and had irregular margins when grown on TSA plates incubated at 25 °C for 1 day. All strains were able to grow between 4 and 25 °C. Based on these traits the strains were classified as belonging to the genus Shewanella, but these characteristics do not allow for species differentiation, for instance between S. putrefaciens and S. algae (Fonnesbech Vogel et al., 1997), or for differentiation between some of the psychrophilic shewanellae (Ziemke et al., 1997).
The first group (group 1; group A3 of Fonnesbech Vogel et al., 2005) of 14 strains included the proposed type strain P010T and were phenotypically similar to the psychrotolerant S. baltica NCTC 10735T. However, unlike the type strain of S. baltica, group 1 strains were unable to utilize sucrose as a sole carbon source (Table 2). Strains of this group degraded gelatin, DNA and ornithine. The DNA G+C content of these strains was 47 mol%. In combination, phenotypic results for these strains were similar but distinguishable from those of S. baltica. A second group (group 2; groups C3 and C4 of Fonnesbech Vogel et al., 2005) encompassing 33 strains and represented by the proposed type strain U1417T grew well at 4 °C, but only assimilated three of the carbohydrates tested (Table 2). This group was phenotypically different from the type strains of S. putrefaciens (ATCC 8071T), S. oneidensis (ATCC 700550T) Shewanella frigidimarina (ACAM 591T; Bowman et al., 1997), Shewanella denitrificans (DSM 15013T; Brettar et al., 2002), Shewanella livingstonensis (LMG 19866T; Bozal et al., 2002) and S. colwelliana (ATCC 39565T). Strains of group 2 did not grow in 6 % NaCl but degraded gelatin, DNA and ornithine. DNA G+C content for these strains was 44 mol%, indicating that they were different from S. frigidimarina (40–43 mol%), S. denitrificans (47 mol%) and S. colwelliana (47 mol%). Although the phenotypic traits of group 2 strains were somewhat similar to those of S. putrefaciens, they differed in several characteristics (Table 2): they liquefied gelatin, assimilated gluconate, did not utilize lactate or sucrose as sole carbon source and did not grow at 37 °C.
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and Yamamoto & Harayama (1995) and subsequently sequenced. To extract DNA, cells were cultured in TSB and collected by centrifugation. Cell pellets were suspended in TE buffer (pH 8·0) and treated with SDS (final concentration, 10 mg ml–1) for lysis. Procedures for extracting chromosomal DNA and subsequent purification steps were carried out according to standard methods (Johnson, 1981; Sambrook et al., 1989). The identity of a given PCR product was verified by bidirectional sequencing analysis. The phylogenetic relationships of organisms covered in this study were determined by comparison of individual 16S rRNA or gyrB gene sequences with other existing sequences in the public databases using the BLAST algorithm (Altschul et al., 1990). Multiple alignment calculation of nucleotide substitution rates (Knuc values) described by Kimura (1980) and construction of phylogenetic trees by the neighbour-joining method (Saitou & Nei, 1987) were performed using the CLUSTAL W program (Thompson et al., 1994). Alignment gaps, primer regions for PCR amplification and unidentified base positions were not taken into consideration for the calculations. Topological robustness of the phylogenetic trees was evaluated by a bootstrap analysis of 1000 replications. GenBank nucleotide accession numbers for the 16S rRNA and gyrB gene sequences are shown in Figs 1 and 2, respectively.
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) indicated that the new isolates clustered with members of the genus Shewanella. Based on 16S rRNA gene sequences, the nearest neighbours of the group 1 strains (represented by P010T) were S. putrefaciens ATCC 8071T and S. baltica NCTC 10735T with sequence similarities of 99 and 98·3 %, respectively. By contrast, the sequences of the group 2 strains (represented by U1417T) shared less than 97 % similarity with recognized Shewanella species, the closest neighbour being S. frigidimarina ACAM 591T (96·6 %). These values suggested that the phylogenetic distances between the group 2 isolates and recognized Shewanella species was enough to clarify them as representing a distinct species (Stackebrandt & Goebel, 1994), but it was unclear whether the group 1 strains were distinguishable from S. putrefaciens as a different species. However, the topology based on the gyrB gene showed these latter strains to cluster monophyletically, distinct from S. baltica, S. oneidensis or S. putrefaciens, and clearly delineating them as representing a distinct species (Fig. 2). Sequence similarity values used to separate species based on the gyrB gene vary depending on the genus (Satomi et al., 2002, 2003, 2004; Venkateswaran et al., 1999) and, therefore, DNA–DNA hybridization experiments were carried out to confirm the novelty of these isolates.
DNA–DNA hybridization was studied by microplate hybridization methods (Ezaki et al., 1989) with photobiotin labelling and colorimetric detection, using 1,2-phenylenediamine (Sigma) as the substrate and streptavidin–peroxidase conjugate (Boehringer Mannheim) as the colorimetric substrate (Satomi et al., 1997). Table 3 shows the results of DNA–DNA hybridization between the putative novel species and type strains of closely related Shewanella species. Strain P010T showed a DNA–DNA hybridization value of 38 % with S. putrefaciens ATCC 8071T and 43 % with S. baltica NCTC 10735T. Similarly, the group 2 strains exhibited levels of DNA–DNA relatedness of 9–34 % with several S. putrefaciens ATCC 8071T, S. frigidimarina ACAM 591T and S. baltica NCTC 10735T. Furthermore, strains of groups 1 and 2 shared only 20–34 % DNA–DNA relatedness. Within each of the groups themselves, DNA–DNA relatedness values were significantly higher, with groups 1 and 2 exhibiting relatedness values of 80–92 and 78–83 %, respectively. This strongly supports the suggestion that the isolates of groups 1 and 2 (respectively represented by strains P010T and U1417T) represent novel species within the genus Shewanella (Wayne, 1988).
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Description of Shewanella hafniensis sp. nov.
Shewanella hafniensis [haf.ni.en'sis. M.L. fem. adj. hafniensis pertaining to Hafnia, the medieval name of København (Copenhagen), the capital of Denmark].
Mesophilic, aerobic, chemoheterotrophic Gram-negative rods that are motile by means of polar flagella. Cells are 0·5–0·7 µm in diameter and 1·0–1·2 µm in length. Growth occurs at 0–6 % (w/v) NaCl and the temperature range for growth is 4–25 °C (25 °C being optimal). Growth does not occur at temperatures >37 °C. Colonies are round, undulate, white-dull, non-luminescent and have irregular margins on TSA plates incubated at 25 °C for 24 h. Cells are positive for oxidase and catalase reactions. They are unable to ferment glucose but reduce TMAO and produce H2S. Cells reduce nitrate, hydrolyse gelatin and are positive for the production of DNase and ornithine decarboxylase. D-Glucose, gluconate, lactate, maltose, N-acetylglucosamine, malate and citrate are readily utilized as energy sources. Sucrose is not utilized as an energy source. Cells do not grow on minimal media, indicative of a required growth factor. The DNA G+C content is 47 mol%.
The type strain, P010T (=ATCC BAA-1207T=NBRC 100975T), was isolated from cod from the Baltic Sea off Denmark. Strains P14 (=NBRC 100976) and R1418 (=ATCC BAA-1208=NBRC 100977) are reference strains.
Description of Shewanella morhuae sp. nov.
Shewanella morhuae (mo.rhu'ae. N.L. gen. n. morhuae of morhua, the specific epithet of Gadus morhua, the Atlantic cod).
Mesophilic, aerobic, chemoheterotrophic Gram-negative rods that are motile by means of polar flagella. Cells are 0·5–0·7 µm in diameter and 1·0–1·2 µm in length. None of the strains is able to grow at 6 % (w/v) NaCl. The temperature range for growth is 4–25 °C (25 °C being optimal). Growth does not occur at temperatures >37 °C. Colonies are round, undulate, white-dull, non-luminescent and have irregular margins on TSA plates incubated at 25 °C for 24 h. Cells are positive for oxidase and catalase reactions. Unable to ferment glucose but reduce TMAO and produce H2S. Cells reduce nitrate, hydrolyse gelatin and are positive for the production of DNase and ornithine decarboxylase. Cells are unable to use most of the carbon substrates tested, although gluconate, N-acetylglucosamine and malate are readily utilized as energy sources. D-Glucose, citrate and sucrose are not utilized as sole carbon sources, but more than half of the strains tested assimilate arabinose. Cells do not grow on minimal media, indicative of a required growth factor. The DNA G+C content is 44 mol%.
The type strain, U1417T (=ATCC BAA-1205T=NBRC 100978T), was isolated from cod from the Baltic Sea off Denmark. Strains T214 (=NBRC 100979), U212 (=NBRC 100980) and U1414 (=ATCC BAA-1206=NBRC 100981) are reference strains.
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ACKNOWLEDGEMENTS |
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-D-galactopyranoside) and do not assimilate phenylacetic acid, mannose, mannitol or adipic acid. +, Positive; –, negative; numbers refer to percentages of strains that are positive for the particular characteristic.
