Abstract
Abbreviations: RDX, hexahydro-1,3,5-trinitro-1,3,5-triazine; TMAO, trimethylamine N-oxide
Published online ahead of print on 23 September 2005 as DOI 10.1099/ijs.0.63829-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and gyrB gene sequences of strain HAW-EB4T are AY579751 and AY842131.
The genus Shewanella was first described by MacDonell & Colwell (1985) to accommodate Gram-negative, oxidase-positive, rod-shaped, aquatic and marine bacteria with genomic DNA G+C contents of 3953 mol%. Twenty-nine species of Shewanella were recognized at the time of writing (Bozal et al., 2002; Brettar & Hofle, 2002; Ivanova et al., 2001, 2003a, b, 2004a, b, c; Leonardo et al., 1999; Skerratt et al., 2002; Satomi et al., 2003; Toffin et al., 2004; Venkateswaran et al., 1999; Xu et al., 2005; Yoon et al., 2004a, b). Deep-sea Shewanella species such as Shewanella violacea, S. benthica, S. woodyi, S. hanedai and S. marinintestina require Na+ and low temperature for growth and produce polyunsaturated fatty acids (Bowman et al., 1997; Deming et al., 1984; Jensen et al., 1980; Kato et al., 1998; Kato & Nogi, 2001; MacDonell & Colwell, 1985; Makemson et al., 1997; Nogi et al., 1998; Russell & Nichols, 1999; Satomi et al., 2003). Mesophilic and non-Na+-requiring Shewanella species such as Shewanella algae, S. amazonensis, S. decolorationis, S. japonica, S. oneidensis and S. putrefaciens are usually found in coastal regions, estuaries and/or non-marine environments (Ivanova et al., 2001; Long & Hammer, 1941; Nozue et al., 1992; Simidu et al., 1990; Venkateswaran et al., 1998b, 1999; Xu et al., 2005; Zhao et al., 2005). Some species of Shewanella are known for their potential to degrade organic pollutants (Petrovskis et al., 1994; Semple & Westlake, 1987; Xu et al., 2005; Zhao et al., 2004b). The strain described herein, designated HAW-EB4T, was initially isolated as an effective RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)-mineralizing bacterium from marine sediment obtained from a munitions-dumping area located in Emerald Basin, Atlantic Ocean, offshore Halifax Harbour, Novo Scotia, Canada (Zhao et al., 2004b). In the present study, we found that this bacterium represented a novel obligately respiratory and denitrifying species of Shewanella.
As we reported previously (Zhao et al., 2004b), strain HAW-EB4T was a psychrophilic bacterium (optimum growth at 10 °C) and grew well aerobically on Brewer anaerobic agar (Becton Dickinson) supplemented with sea salts (4 %; Sigma), producing a slightly dark orange or pinkish pigmentation similar to other species of Shewanella (Venkateswaran et al., 1999). Cells harvested after 3 days aerobic incubation in marine broth 2216 (Difco) at 10 °C (spun at 180 r.p.m.) were used for transmission (TEM; Hitachi H7500) or scanning (SEM; Hitachi S3000N) electron microscopic image analyses. For TEM, cells were negatively stained with phosphotungstic acid (0·5 %, w/v; pH 6·0) as described by Bozal et al. (2002) and Beveridge et al. (1994). For SEM, cells were collected on poly-L-lysine-coated coverslips, treated with glutaraldehyde/formaldehyde, tannic acid and osmium tetroxide as described by Prevost et al. (1992), dried using hexamethyldisilazane and sputter-coated with gold/palladium. Like those of most Shewanella species, cells of strain HAW-EB4T were Gram-negative, straight or slightly curved rods. Electron microscopic image analyses showed that cells were 1·53 µm long with a diameter ranging from 0·55 to 0·65 µm (Fig. 1a) and had a single polar flagellum (Fig. 1b).
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To determine the phylogenetic affiliation of strain HAW-EB4T, we purified its genomic DNA (15 mg) using the Marmur method (Johnson, 1994), and amplified its 16S rRNA and gyrB (coding for the β-unit of DNA topoisomerase II) genes using universal primers (Yamamoto & Harayama, 1995; Venkateswaran et al., 1998a) and standard molecular protocols (Sambrook & Russell, 2001). The partial 16S rRNA (1293 bases) and gyrB (938 bases) genes were subsequently sequenced and compared with published sequences using BLAST (NCBI GenBank; ). The gene sequences of strain HAW-EB4T and those of closely related species were aligned using CLUSTAL_X (version 1.81). The neighbour-joining method included in the MEGA2 package (Kumar et al., 2001), based on pairwise nucleotide distances with the Kimura two-parameter correction, was used to construct the phylogenetic tree. The number of bootstrap repetitions was 4000.
The genomic DNA G+C content of strain HAW-EB4T, determined using UV absorbance (A269/A280) at pH 3·0 and its thermal melting profile (Tm) (Johnson, 1985a), was found to be 45 mol%, consistent with reported values for other species of Shewanella (3953 mol%). Phylogenetic analyses of the 16S rRNA gene sequence clearly showed that strain HAW-EB4T belonged to the genus Shewanella (9399 % similarity to recognized Shewanella species); it was related most closely to Shewanella pealeana (99 %), found in Atlantic squid accessory nidamental gland (Leonardo et al., 1999), S. marinintestina, Shewanella schlegeliana and Shewanella sairae, which are marine intestinal species (98·699 %) (Satomi et al., 2003), and Shewanella gelidimarina, found in Antarctic congelation ice (97·9 %) (Bowman et al., 1997) (Fig. 2). Among the above related species, S. pealeana was found to be phylogenetically most closely related to HAW-EB4T (99 % similarity, bootstrap value of 99 %) (Fig. 2). In comparing the gyrB gene sequences, strain HAW-EB4T showed 67·388·4 % similarities to species of Shewanella, and was most similar to S. pealeana (Fig. 3). However, gyrB gene sequence similarity between HAW-EB4T and S. pealeana was 88·4 %, lower than the 90 % species cut-off value recommended for Shewanella species (Venkateswaran et al., 1999), suggesting that the two bacteria belong to different species.
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Strain HAW-EB4T was also found to be phylogenetically distant from another Halifax sediment Shewanella isolate, Shewanella sediminis HAW-EB3T (Zhao et al., 2005), with low 16S rRNA (96·4 %; Fig. 2) and gyrB (83·3 %; Fig. 3) gene sequence similarities.
We conducted genomic DNADNA hybridization between strain HAW-EB4T and S. pealeana using the spectrophotometric method as described by Johnson (1985b) and Bowman et al. (1998). Strain HAW-EB4T and S. pealeana had a level of genomic DNA relatedness of 17·9 %, which was much lower than the 70 % species cut-off value accepted for bacteria (Wayne et al., 1987; Stackebrandt & Goebel, 1994; Gillis et al., 2001), demonstrating that strain HAW-EB4T represents a novel species of Shewanella.
To compare the chemotaxonomic properties of strain HAW-EB4T with those of recognized species of Shewanella, we characterized the respiratory quinone and membrane fatty acid composition of strain HAW-EB4T (grown aerobically in marine broth 2216 at 10 °C) using previously described chromatographic methods (Collins, 1985, 1994; Nishijima et al., 1997; Akagawa-Matsushita et al., 1992; Bowman, 2001; Fay & Richli, 1991; Zhao et al., 2005). Briefly, strain HAW-EB4T produced mainly straight-chain fatty acids such as palmitic (C16 : 0, 20 %), myristic (C14 : 0, 6 %) and palmitoleic (C16 : 1ω7, 37 %) acid, branched fatty acids (iso-C13 : 0, 3 %; iso-C15 : 0, 12 %) and polyunsaturated eicosapentaenoic acid [C20 : 5ω3 (EPA), 7 %], similar to related species of Shewanella (Table 1). The major respiratory quinones of strain HAW-EB4T were ubiquinones (Q7, 28·1 %) and menaquinones (MK-7, 60·9 %), consistent with Shewanella species (Akagawa-Matsushita et al., 1992; Venkateswaran et al., 1999; Bozal et al., 2002; Zhao et al., 2005).
Table 1. Fatty acid composition (percentages of total) of strain HAW-EB4T and related Shewanella species Taxa: 1, HAW-EB4T; 2, S. pealeana; 3, S. gelidimarina; 4, S. schlegeliana; 5, S. marinintestina; 6, S. sairae. , Not detected; ND, no data. Data for strain HAW-EB4T from this study; data for reference species taken from Bowman et al. (1997), Leonardo et al. (1999) and Satomi et al. (2003).
NaCl tolerance was tested on Brewer anaerobic agar that contained 0·5, 1·5, 2·0, 2·5, 3·0, 4·0, 6·0 or 8·0 % NaCl (aerobic growth conditions) using a previously described protocol (Zhao et al., 2005). Growth in the absence of Na+ was tested in a Na+-free agar that contained 0·3 % Bacto beef extract (Difco) and 0·5 % Bacto peptone (Difco). Strain HAW-EB4T required Na+ for growth and was slightly halophilic, with growth at 0·54 % NaCl. However, strain HAW-EB4T appeared to have a lower tolerance to NaCl (optimum growth at 2 % NaCl) than S. pealeana (3 %).
We also identified the electron acceptors of strain HAW-EB4T and compared them with those of recognized Shewanella species. Reduction of the following potential electron acceptors by strain HAW-EB4T was tested in anaerobic jars on Brewer anaerobic agar supplemented with 2 % NaCl and one of the following substrates: MnO2 (40 mM), ferric citrate (40 mM), amorphous iron oxide (FeOOH, 40 mM) or elemental sulfur (40 mM). Anaerobic respiration on trimethylamine N-oxide (TMAO) (10 mM), nitrate (20 mM) or nitrite (5 mM) as an electron acceptor (0·1 % Casamino acids as carbon and energy sources) was tested in sea salts mineral medium (5 ml in 20 ml sealed serum bottles, initial biomass of 0·07 OD600) under anaerobic conditions (the medium was made anoxic by degassing under vacuum and charging the headspace with argon, and with no reducing agents added). Clear zones around colonies were used to indicate iron(III), manganese(IV) and sulfur reduction (Myers & Nealson, 1988). Enhanced bacterial growth in the presence of electron acceptors (compared with that in their absence) was used as an indicator for dissimilatory reduction of nitrate, nitrite and TMAO. Bacterial growth on agar was estimated by multiplying the mean area of colonies and total number of colony-forming units (Zhao et al., 2005). Similar to S. pealeana ATCC 700345T (Leonardo et al., 1999), strain HAW-EB4T was a respiratory bacterium that did not grow by fermentation but grew (0·1 % Casamino acids, 616 days, 10 or 21 °C) by respiring on oxygen, nitrate, nitrite or TMAO, with a final biomass increase of 0·120·6 OD600. Strain HAW-EB4T reduced MnO2 as was the case with S. pealeana, but did not reduce two other electron acceptors of S. pealeana, iron(III) and elemental sulfur (Table 2). Strain HAW-EB4T reduced nitrate and nitrite to nitrous oxide (Casamino acids as carbon source), confirming its identity as a denitrifying bacterium probably involved in denitrification in the Halifax harbour sediment.
Table 2. Phenotypic properties of strain HAW-EB4T and related species of Shewanella Taxa: 1, HAW-EB4T; 2, S. pealeana; 3, S. gelidimarina; 4, S. schlegeliana; 5, S. marinintestina; 6, S. sairae. All data for strain HAW-EB4T and growth substrate data for S. pealeana were from the present study. Other data were from Bowman et al. (1997), Satomi et al. (2003), Leonardo et al. (1999) and Kato & Nogi (2001). All listed species grew on N-acetyl-D-glucosamine, ribose and valerate as sole carbon sources. +, Positive; W+, weakly positive; , negative; ND, no data. Values in parentheses for HAW-EB4T and S. pealeana indicate the increase in biomass (OD600; as a percentage relative to initial value of 0·07) after 616 days incubation in basic sea-salts medium at 10 or 21 °C.
Using previously described protocols (Bowman, 2001; Zhao et al., 2005) and marine broth 2216 as a basal medium, we characterized strain HAW-EB4T for spore formation, acid production from sugars (in Leifson modified O-F medium; Smibert & Krieg, 1994), H2S formation from thiosulfate (1 %) and hydrolysis of DNA (in BBL DNA agar plus 4 % sea salts), casein (skimmed milk, 50 %), gelatin (1 %), Tweens 20 (1 %), 40 (1 %) and 80 (1 %), olive oil (1 %), lecithin (5 % egg yolk), pure chitin (0·3 %), alginate (1 %) and starch (1 %). Additional enzyme activities and substrate metabolisms were tested by using API Rapid 20E and ID32A (bioMérieux) test kits (10 and 37 °C) and GN2 microplates (Biolog; cell biomass of 0·9 OD600 suspended in sea-salts medium for 7 days at 10 °C). Utilization of carbon substrates (0·1 %) by strain HAW-EB4T and S. pealeana ATCC 700345T (purchased from the American Type Culture Collection) was tested aerobically at both 10 °C (optimum growth of HAW-EB4T) and 21 °C (optimum growth of S. pealeana) in basic marine salts medium (pH 7·0, 0·1 % NH4Cl) in the presence or absence of 0·02 % yeast extract or 0·001 % choline chloride. Bacterial growth (OD600) was monitored every 36 days over 1 month (initial biomass of 0·07 OD600). All phenotypic tests were run in triplicate.
Cluster analyses of phenotypic properties (enzyme activities, growth substrates, salt and temperature tolerance) of strain HAW-EB4T and recognized species of Shewanella using the method of Zhao et al. (2005) also indicated that strain HAW-EB4T was similar to deep-sea species of Shewanella. Like S. pealeana (Leonardo et al., 1999), strain HAW-EB4T grew better in the presence of choline chloride (peptone, Casamino acids or yeast extract as carbon source; 10001300 % increase in OD600) than in its absence (600900 % increase in OD600) (10 °C, 16 days). However, strain HAW-EB4T differed from S. pealeana (Table 2) and three other known marine fish or scallop intestinal Shewanella species, S. marinintestina, S. schlegeliana and S. sairae, on activities of some enzymes and utilization of several carbon sources (Satomi et al., 2003). For example, strain HAW-EB4T produced much less biomass (15150 % increase in OD600) than S. pealeana (150700 % increase in OD600) at both 10 and 21 °C when growing on single compounds as sole carbon sources (Table 2). Yeast extract appeared to improve utilization of several carbon sources by strain HAW-EB4T (Table 2).
Finally, strain HAW-EB4T was able to utilize the biodegradation product of RDX, formate, as sole carbon and energy source for growth (at 21 °C) (Table 2), suggesting that RDX can be utilized by strain HAW-EB4T during potential occurrence of in-situ natural attenuation of RDX at the contaminated site (Zhao et al., 2004a, b).
The phenotypic, chemotaxonomic and genetic data presented here demonstrate that strain HAW-EB4T is a distinct marine bacterium, representing a novel species of Shewanella, for which the name Shewanella halifaxensis sp. nov. is proposed.
Description of Shewanella halifaxensis sp. nov.
Shewanella halifaxensis (ha.li.fax.en'sis. N.L. fem. adj. halifaxensis from Halifax, Nova Scotia, a harbour city near the sediment sampling site, Emerald Basin, where the type strain was isolated).
Cells are Gram-negative, non-spore-forming, straight or slightly curved rods generally 23 µm long and 0·550·65 µm in diameter. Cells 6 µm long also occur. Motile by a single polar flagellum. Biomass is slightly dark yellow and non-bioluminescent. Psychrophilic growth at temperatures of 425 °C (optimum at 10 °C). No growth at 30 °C. Na+-requiring and slightly halophilic (0·54 % NaCl; optimum at 2 %). Growth at 4 % but not at 6 % NaCl. MnO2, nitrate, nitrite, TMAO, thiosulfate and RDX are reduced. No reduction of Fe3+ (iron oxide or ferric citrate) or elemental sulfur. Strong production of H2S from thiosulfate. Anaerobic growth by reducing nitrate (to N2O), nitrite or TMAO, but not by fermentation. Positive for catalase, oxidase, nitroreductase, chitinase, DNase, gelatinase, urease, arginine dihydrolase, ornithine decarboxylase, lysine decarboxylase, glutamic acid decarboxylase, N-acetyl-β-D-glucosaminidase, β-galactosidase, β-galactosidase-6-phosphate, alkaline phosphatase, leucine arylamidase and alanine arylamidase; weakly positive for hydrolysis of Tweens 20, 40 and 80, proline arylamidase and arginine arylamidase; negative for caseinase, alginase, amylase, α-galactosidase, α-glucosidase, β-glucosidase, α-arabinosidase, β-glucuronidase, histidine arylamidase, serine arylamidase, phenylalanine arylamidase, pyroglutamic acid arylamidase, tyrosine arylamidase, glycylarylamidase, leucine glycine arylamidase, α-fucosidase and glutamyl glutamic acid arylamidase. On GN2 microplate (10 °C, 7 days), positive for metabolism of N-acetyl-D-glucosamine, α-D-glucose, methylpyruvate, DL-lactic acid, L-alanine, L-alanyl glycine, glycyl L-aspartic acid, glycyl L-glutamic acid, L-serine, inosine, uridine and thymidine; weakly positive for Tweens 40 and 80, acetic acid, β-hydroxybutyric acid, α-ketobutyric acid, L-leucine and L-threonine; negative for α-cyclodextrin, dextrin, glycogen, N-acetyl-D-galactosamine, adonitol, L-arabinose, D-arabitol, D-cellobiose, i-erythritol, D-fructose, D-fucose, D-galactose, gentiobiose, myo-inositol, α-D-lactose, lactulose, maltose, D-mannitol, D-mannose, D-melibiose, methyl β-D-glucoside, D-psicose, D-raffinose, L-rhamnose, D-sorbitol, sucrose, D-trehalose, turanose, xylitol, succinic acid monomethyl ester, cis-aconitic acid, citric acid, formic acid, D-galactonic acid lactone, D-galacturonic acid, D-gluconic acid, D-glucosaminic acid, D-glucuronic acid, α- or γ-hydroxybutyric acid, p-hydroxyphenylacetic acid, itaconic acid, α-ketoglutaric acid, α-ketovaleric acid, malonic acid, propionic acid, quinic acid, D-saccharic acid, sebacic acid, succinic acid, bromosuccinic acid, succinamic acid, glucuronamide, L-alaninamide, D-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-histidine, hydroxy-L-proline, L-ornithine, L-phenylalanine, L-proline, L-pyroglutamic acid, D-serine, DL-carnitine, γ-aminobutyric acid, urocanic acid, phenylethylamine, 2-aminoethanol, 2,3-butanediol, glycerol, DL-α-glycerol phosphate, α-D-glucose 1-phosphate and D-glucose 6-phosphate. Weak aerobic acid formation from N-acetyl-D-glucosamine and glucose. No anaerobic acid formation from N-acetyl-D-glucosamine, fructose, sucrose, galactose, lactose, mannose or glucose. Peptone, yeast extract, Casamino acid, Tweens (20, 40 and 80), propionate, pyruvate, serine and proline are sole carbon and energy sources for growth. N-Acetyl-D-glucosamine, ribose, lactate, acetate, valerate, glutamate, formate and glycine are weak growth substrates. Alanine, threonine, leucine, butyrate and glucose are poorly utilized. Succinate, citrate, malate and fumarate are not utilized. Choline and yeast extract are not required but improve growth on certain substrates. Fatty acids iso-C13 : 0 (3 %), C14 : 0 (6 %), C14 : 1 (1 %), anteiso-C15 : 0 (1 %), C15 : 0 (1 %), iso-C15 : 0 (12 %), C16 : 0 (20 %), C16 : 1ω7 (37 %), C16 : 1ω9 (1 %), C18 : 0 (2 %), C18 : 1ω7 (7 %), C20 : 5ω3 (7 %) and C20 : 4ω3 (1 %) are produced. Quinone composition is Q7 (28·1 %), Q8 (4·0 %), MK-7 (60·9 %) and MMK-7 (6·9 %). The DNA G+C content is 45 mol%.
The type strain is HAW-EB4T (=NCIMB 14093T=DSM 17350T), isolated from marine sediment.
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