International Journal of Systematic and Evolutionary Microbiology (2010), 60, 1060–1065 DOI 10.1099/ijs.0.014746-0 Desulfosalsimonas propionicica gen. nov., sp. nov., a halophilic, sulfate-reducing member of the family Desulfobacteraceae isolated from a salt-lake sediment Kasper Urup Kjeldsen,13 Trine Fredlund Jakobsen,1 Jens Glastrup2 and Kjeld Ingvorsen1 Correspondence Kjeld Ingvorsen kjeld.ingvorsen@biology.au.dk 1 Department of Biological Sciences, Microbiology, University of Aarhus, Ny Munkegade 114, Building 1540, DK-8000 Aarhus C, Denmark 2 Department of Conservation, The National Museum of Denmark, Brede 260, DK-2800 Lyngby, Denmark A novel halophilic Gram-negative sulfate-reducing bacterium affiliated with the deltaproteobacterial family Desulfobacteraceae, strain PropAT, was isolated from the extreme hypersaline sediment of the northern arm of Great Salt Lake, Utah, USA. Comparative 16S rRNA gene sequence analysis showed that strain PropAT is the first cultured representative of a clade of phylotypes that have been retrieved from a range of geographically and ecologically distinct hypersaline environments. Strain PropAT shared ¡90 % 16S rRNA gene sequence identity with cultured strains within the family Desulfobacteraceae. Cells of strain PropAT were rod-shaped and sometimes motile. The strain required NaCl for growth and grew at salinities up to 200 g NaCl l”1 (optimum 60 g l”1). Growth was observed at 15–40 6C, optimum growth occurred at about 40 6C, while growth was absent at 10 and 45 6C. The pH range for growth was pH 6.0–8.3. Yeast extract (0.1 g l”1) was required for growth. C2–4 alcohols, C3–4 carboxylic acids, yeast extract and H2/acetate supported growth with sulfate as electron acceptor. Sulfate, thiosulfate and sulfite served as electron acceptors, but not elemental sulfur, nitrate or fumarate. The DNA G+C content of strain PropAT was 54.1 mol%. Based on the genotypic and physiological properties, we propose that strain PropAT represents a novel species within a novel genus, Desulfosalsimonas propionicica gen. nov., sp. nov. The type strain of Desulfosalsimonas propionicica is PropAT (5DSM 17721T 5VKM B-2385T). Aquatic systems such as solar salterns, inland lakes and deepsea brines are characterized by salinities far exceeding 100 g NaCl l21 (Oren, 2002). Sulfate reduction is often an important anoxic mineralization process in these hypersaline environments (Brandt et al., 2001; Daffonchio et al., 2006; Foti et al., 2007), yet relatively few sulfate-reducing bacteria 3Present address: Center for Geomicrobiology, Department of Biological Sciences, University of Aarhus, Ny Munkegade 114, Building 1540, DK-8000 Aarhus C, Denmark. Abbreviations: SPME-GC-MS, solid-phase micro-extraction gas chromatography mass spectrometry; SRB, sulfate-reducing bacteria. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and dsrAB sequences of strain PropAT are DQ067422 and DQ386237, respectively. Transmission electron micrographs of cells of strain PropAT and details of sequences shown as genus groups and sequences constituting outgroups in Fig. 2 are available as supplementary material with the online version of this paper. 1060 (SRB) that are able to grow at such extreme salinities are known. Halophilic SRB primarily comprise members of the families Desulfohalobiaceae (Kuever et al., 2005a) and Desulfonatronaceae (Kuever et al., 2005b) along with certain members of the genus Desulfovibrio of the family Desulfovibrionaceae (e.g., Sass & Cypionka, 2004; Krekeler et al., 1997). Members of the family Desulfohalobiaceae exhibit the highest known in vitro halotolerance and are able to grow in the presence of up to 240 g NaCl l21 (Beliakova et al., 2006; Jakobsen et al., 2006; Ollivier et al., 1991). Here, we describe a novel halophilic SRB, designated strain PropAT, which is affiliated with the deltaproteobacterial family Desulfobacteraceae (Kuever et al., 2005c). Only three recognized members of the family Desulfobacteraceae are capable of growing at NaCl concentrations exceeding 100 g l21, namely Desulfocella halophila (Brandt et al., 1999), Desulfobacter halotolerans (Brandt & Ingvorsen, 1997) and Desulfotignum balticum (Drzyzga et al., 1993; Kuever et al., 2001) (Table 1). The first two species, like strain 014746 G 2010 IUMS Printed in Great Britain Desulfosalsimonas propionicica gen. nov., sp. nov. Table 1. Differential phenotypic characteristics between strain PropAT and the three other members of the family Desulfobacteraceae that grow at salinities ¢100 g NaCl l”1 Strains: 1, Desulfosalsimonas propionicica gen. nov., sp. nov. PropAT; 2, Desulfobacter halotolerans DSM 11383T; 3, Desulfocella halophila DSM 11763T; 4, Desulfotignum balticum DSM 7044T. Data for reference strains were taken from Brandt & Ingvorsen (1997), Brandt et al. (1999), Drzyzga et al. (1993) and Kuever et al. (2001). +, Positive; 2, negative; ND, no data available. Characteristic NaCl concentration for growth (g l ) Range Optimum Temperature for growth (uC) Range Optimum pH for growth Range Optimum Oxidation of: Acetate Propionate Lactate Butyrate Isobutyrate Formate Fumarate Succinate Malate Ethanol Propanol Butanol Benzoate Glucose H2/CO2 H2/acetate Yeast extract Fermentation Reduction of: Sulfite Thiosulfate Elemental sulfur Autotrophic growth Complete oxidation of carbon compounds DNA G+C content (mol%) 1 2 3 4 10–200 60 5–130 10–20 20–200 40–50 10–110 20 15–40 ~40 7–38 33 14–37 34 10–42 28–32 6.0–8.3 ~7.0 6.2–8.1 6.2–7.4 5.8–7.6 6.5–7.3 6.5–8.2 7.3 2 + + + + 2 + 2 2 + + + 2 2 2 + + 2 + 2 2 2 2 2 2 2 2 + 2 2 2 2 2 2 ND + 2 + + 2 + + + + 2 2 2 + 2 + + ND + 2 + (pyruvate) + + 2 2 +/2 + + 2 2 + 2 2 2 2 2 ND 54 49 35 53 21 ND 2 2 + 2 ND 2 ND 2 + 2 PropAT, were isolated from the hypersaline Great Salt Lake (Utah, USA), which is characterized by salinities of 120 and 270 g NaCl l21 in its southern and northern parts, respectively (Brandt et al., 2001; Oren, 2002). In contrast, the aromatic compound-degrading species Desulfotignum balticum, which exhibits the lowest halotolerance of the three above-mentioned species, was isolated from a marine coastal sediment. Being able to grow at salinities up to 200 g NaCl l21, strain PropAT is, together with Desulfocella halophila, the most halotolerant member of the family Desulfobacteraceae described so far. Notably, the 16S rRNA gene sequence of strain PropAT clusters within a clade of uncultured phylotypes derived from various hypersaline http://ijs.sgmjournals.org ND 2 2 2 2 ND + + + + environments. Thus, strain PropAT provides the first insights into the physiology of this distinct group of unknown members of the Desulfobacteraceae. Strain PropAT was isolated from hypersaline (270 g NaCl l21) sediment of the northern arm of Great Salt Lake as part of an investigation of the diversity of SRB in this extreme habitat (Kjeldsen et al., 2007). Strain PropAT was enriched, isolated and routinely cultivated in anoxic basal medium containing 100 g NaCl l21, prepared as described previously (Jakobsen et al., 2006). Propionate served as the electron donor for enrichment and isolation. All incubations were carried out in the dark at 30 uC, 1061 K. U. Kjeldsen and others unless otherwise noted. Growth experiments and transmission electron microscopy were performed essentially as described previously (Jakobsen et al., 2006). Lactate served as the electron donor for NaCl, pH and temperature experiments, whereas propionate was used as the electron donor for electron acceptor experiments. Cells of strain PropAT were rod shaped, and swimming motility was sometimes observed. Cells were 0.8–1.0 mm wide and 2–4 mm long and sometimes formed short filaments under routine growth conditions (Supplementary Fig. S1, available in IJSEM Online). The cells stained Gramnegative and did not form endospores. Growth was tested at 13 different salinities, ranging from 0 to 250 g NaCl l21, and was found to occur at 10–200 g NaCl l21, with an optimum growth rate at 60 g NaCl l21 (Fig. 1). Thus, according to the definitions of Larsen (1986), strain PropAT is a moderate halophile. The observed upper in vitro salinity limit for growth indicates that strain PropAT may be severely salt stressed in the sediment of the northern part of Great Salt Lake (270 g NaCl l21) from where it was isolated. Perhaps the strain is more active in the southern part of the lake, in which the salinity is only 120 g NaCl l21, or perhaps the sediment in the northern part contains niches of lower salinity, possibly arising from freshwater seeps, as previously speculated (Kjeldsen et al., 2007). Discrepancies between the in vitro halotolerance of halophilic SRB and the in situ salinity of their habitat are, however, not unprecedented (Jakobsen et al., 2006; Ollivier et al., 1991; Sass & Cypionka, 2004). The pH dependence of growth was determined at eight different values ranging from pH 5.0 to 8.5. Growth was observed between pH 6.0 and 8.3, with an optimum at about pH 7.0. The temperature dependence of growth was tested at 10, 15, 20, 30, 40, 45, 50 and 60 uC. Growth was observed at 15–40 uC, with the fastest growth occurring at 40 uC. No growth was observed at 45 uC. H2/acetate and yeast extract (2 g l21) as electron donors. The strain required yeast extract (0.1 g l21) for growth irrespective of the type of electron donor provided, including H2/acetate. This requirement was evident despite the routine amendment of the growth medium with the following defined trace element and vitamin solutions (Widdel & Bak, 1992): non-chelated trace element mixture, selenite–tungstate solution, thiamine solution, vitamin mixture (modified by including 30 mg folic acid l21) and vitamin B12 solution. The following substrates did not support growth: D-glucose, D-galactose, trehalose, D-xylose, D-arabinose, L-rhamnose, sucrose, D-fructose, maltose, D-mannose, methanol, 1-pentanol, acetone, formate, acetate, citrate, succinate, DL-malate, glycine, glycerol, choline, benzoate, caprylate and Casamino acids. Besides sulfate, strain PropAT grew with sulfite and thiosulfate as electron acceptors, but not nitrate, elemental sulfur or fumarate. Growth was absent in the presence of oxygen. Fermentative growth was absent on pyruvate, fumarate and lactate. By comparing the substrate spectrum of strain PropAT with those of Desulfobacter halotolerans and Desulfocella halophila, the two other halophilic members of the family Desulfobacteraceae previously isolated from Great Salt Lake, it appears that strain PropAT is metabolically the most versatile of the three (Table 1). This versatility might explain how several strains closely related to strain PropAT seemingly coexist in the Great Salt Lake sediment (Kjeldsen et al., 2007). Fig. 1. Effect of NaCl on the specific growth rate of strain PropAT, when grown at 30 6C at pH 7.0 on lactate and sulfate. The capacity of strain PropAT to oxidize lactate, propionate, ethanol and propanol completely to CO2 was tested in triplicate growth experiments in which the concentration of acetate was measured at the beginning and end of the experiments along with the concentration of sulfate. Sulfate concentrations were determined using a BioLC ion chromatograph (Dionex) equipped with a 46250 mm IonPac AS18 column (Dionex) at an eluent (23 mM KOH) flow rate of 1 ml h–1. Acetate was measured by automated solid-phase micro-extraction gas chromatography mass spectrometry (SPME-GC-MS) directly from the growth medium (Glastrup et al., 2006). In contrast to ion chromatography-based measurements of acetate, SPMEGC-MS measurements are not affected by the high concentration of chloride in the growth medium. Acetate did not accumulate (detection limit 5 mM) with growth on lactate or propionate, despite the consumption of 4–8 mM sulfate, suggesting that these carboxylic acids were oxidized completely. In contrast, the alcohols ethanol and propanol were oxidized incompletely, resulting in the accumulation of approximately stoichiometric amounts of acetate and propionate, respectively (quantified by SPME-GC-MS). Even though propionate and lactate were oxidized completely by strain PropAT, acetate did not support growth, possibly due to the low energy yield gained from the oxidation of this substrate and the high energetic cost of maintaining the osmotic balance in a saline growth medium (Oren, 1999). Similar observations have been made, for example, for the completely oxidizing SRB 1062 International Journal of Systematic and Evolutionary Microbiology 60 Strain PropAT utilized short-chain carboxylic acids (propionate, pyruvate, lactate, butyrate, isobutyrate and fumarate) and alcohols (ethanol, 1-propanol, 1-butanol), Desulfosalsimonas propionicica gen. nov., sp. nov. Desulfatibacillum alkenivorans (Cravo-Laureau et al., 2004), Desulfobacula toluolica (Rabus et al., 1993) and Desulfospira joergensenii (Finster et al., 1997), which also belong to the family Desulfobacteraceae. The sequences for dsrAB, which encodes the two major subunits of dissimilatory (bi)sulfite reductase (Wagner et al., 2005), and the 16S rRNA gene (1946 and 1469 nt, respectively) of strain PropAT were retrieved as described previously (Kjeldsen et al., 2007). The 16S rRNA gene sequence was added to the alignment of the Silva SSURef version 95 ARB database (Pruesse et al., 2007) containing .4500 high-quality deltaproteobacterial 16S rRNA gene sequences. A few additional short or recently published sequences that were found to be related to the 16S rRNA gene sequence of strain PropAT by BLAST searches of the GenBank database were added manually to the ARB database. From this database, 51 sequences affiliated to the Desulfobacteraceae were subsequently selected for phylogenetic analyses. Furthermore, 14 sequences representing other major deltaproteobacterial families were included as an outgroup. The phylogenetic position of strain PropAT among the selected taxa was inferred from Bayesian analysis using MrBayes version 3.1.2 (Ronquist & Huelsenbeck, 2003) with sampling of 1395 unambigously aligned sequence positions. Tree searches were performed with the general time reversible evolutionary model with gamma-distributed rate variation across sites, with a proportion of the sites being kept invariable. Program default priors on model parameters as well as default settings for the Metropolis-coupled Markov chain Monte Carlo runs were applied. Searches included 1 million generations with a sampling of trees for every 100. Likelihood values converged after approximately 100 000 generations. A consensus tree was constructed after removing the first 2500 trees as ‘burn-in’. Distance-matrix-based bootstrap values were calculated in PAUP* version 4.0b10 (Swofford, 2003) using Jukes–Cantor distance correction and 1000 resamplings. Phylogenetic analysis of a dataset consisting of 50 DsrAB amino acid sequences (deduced from nucleotide sequences) with 563 unambiguously aligned sequence positions were performed using the TreePuzzle (quartet puzzling using a JTT amino acid substitution model) algorithm as implemented in the ARB program package (Ludwig et al., 2004). Some taxa were grouped or removed from the trees presented in Fig. 2 in order to enhance clarity (details of grouped and removed sequences are given in Supplementary Table S1). The 16S rRNA gene sequence of strain PropAT shared ¡90 % similarity with sequences originating from named isolates. Its closest cultured relatives (89–90 % identity) were various members of the deltaproteobacterial family Desulfobacteraceae, particularly members of the genera Desulfatibacillum, Desulfobacterium, Desulfococcus, Desulfofaba, Desulfonema and Desulfosarcina, which are mostly associated with marine habitats (Cravo-Laureau et al., 2004; Kuever et al., 2005c). The 16S rRNA gene and DsrAB amino acid sequence-based phylogenetic analyses clearly placed the strain within the family Desulfobacteraceae http://ijs.sgmjournals.org (Fig. 2). This family mainly includes SRB that, like strain PropAT, possess the capacity to oxidize carbon compounds completely (Kuever et al., 2005c). As seen from Fig. 2, neither the 16S rRNA gene sequence nor the DsrAB amino acid sequence of strain PropAT clustered with any recognized member of the family Desulfobacteraceae. Rather, the 16S rRNA gene sequence of strain PropAT formed a clade with a range of environmentally derived sequences that, interestingly, all originate from hypersaline environments, representing different habitat types such as sediments, microbial mats and anoxic basins (Fig. 2a). Thus, strain PropAT is the first cultured representative of what seems to be a geographically and ecologically widely distributed lineage of putative halophilic SRB. The DNA G+C content of strain PropAT was 54.1 mol%, as determined by HPLC analysis by the Identification Service of the Deutsche Sammlung von Mikroorganismen und Zellculturen (Braunschweig, Germany). The DNA G+C content varies considerably, from 35 to 62 mol%, among members of the family Desulfobacteraceae (Kuever et al., 2005c). The mean±SD DNA G+C content for recognized members of the family is 49.6±6.3 mol% (41 species representing 16 genera; results not shown) and, consequently, the value of 54.1 mol% determined for strain PropAT is in good agreement with G+C contents reported for the family Desulfobacteraceae. In conclusion, we propose that strain PropAT should be classified in a novel genus and species as Desulfosalsimonas propionicica gen. nov., sp. nov. within the family Desulfobacteraceae because of its distant phylogenetic relatedness to recognized members of this family and its unique halotolerance. Description of Desulfosalsimonas gen. nov. Desulfosalsimonas [De.sul9fo.sal.si.mo9nas. L. pref. de from; L. n. sulfur sulfur; L. adj. salsus salty, saline; L. fem. n. monas unit, monad; N.L. fem. n. Desulfosalsimonas a sulfatereducing monad that thrives in (hyper)saline environments]. Obligately anaerobic, sulfate-reducing chemoheterotrophs. Cells are rod-shaped, stain Gram-negative and do not produce endospores. Short-chain carboxylic acids, primary C2–4 alcohols and H2 serve as electron donors. Optimal growth occurs in the presence of elevated concentrations of NaCl at neutral pH and at mesophilic temperatures. Phylogenetically, the genus belongs to the deltaproteobacterial family Desulfobacteraceae, as can be recognized by both dsrAB and 16S rRNA gene sequence analyses. The type species is Desulfosalsimonas propionicica. Description of Desulfosalsimonas propionicica sp. nov. Desulfosalsimonas propionicica (pro.pi.o.ni.ci9ca. N.L. n. acidum propionicum propionic acid; L. fem. suff. -ica suffix used with the sense of pertaining to; N.L. fem. adj. propionicica belonging to propionic acid). 1063 K. U. Kjeldsen and others Fig. 2. Trees showing the inferred phylogenetic position of strain PropAT among members of the family Desulfobacteraceae based on comparative analyses of 16S rRNA gene sequences (a) and DsrAB amino acid sequences (b). For the 16S RNA gene sequence tree, bootstrap percentage values and Bayesian posterior probability percentages (.50 %) are shown above and below branch nodes, respectively. For the DsrAB amino acid sequence tree, percentages at nodes are levels of bootstrap support. Numbers in square brackets indicate the number of taxa constituting the group. #, Short sequences subsequently added to the tree without changing the overall tree topology using parsimony criteria. Bars, 10 % sequence divergence. Details of sequences represented as genus groups and outgroups are available in Supplementary Table S1. The species has the following characteristics in addition to those listed for the genus. Cells are 0.8–1.0 mm wide and 2–4 mm long, sometimes forming filaments. Utilizes (with sulfate as electron acceptor) propionate, pyruvate, lactate, butyrate, isobutyrate, fumarate, ethanol, 1-propanol, 1-butanol, yeast extract and H2/acetate. Oxidizes lactate and propionate completely and ethanol and propanol incompletely. Acetate does not support growth. Yeast extract is required for growth. In addition to sulfate, thiosulfate and sulfite serve as electron acceptors. NaCl is required for growth and up to 200 g NaCl l21 is tolerated; the optimum concentration for growth is 60 g l21. Growth occurs within the range pH 6.0–8.3. Growth occurs at 15– 40 uC (optimum about 40 uC); growth is absent at 10 and 45 uC. The DNA G+C content of the type strain is 54.1 mol%. 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