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Department of Microbiology, University of Aarhus, Ny Munkegade Building 1540, DK-8000 Aarhus C, Denmark
Correspondence
Kjeld Ingvorsen
kjeld.ingvorsen{at}biology.au.dk
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ABSTRACT |
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97 %) was shared with the recently described strain ‘Desulfovermiculus halophilus’ VKM B-2364. Strain EtOH3T, however, clearly differed from this strain in both genomic G+C content and in several of its phenotypic properties. On the basis of phenotypic and genotypic characteristics, the novel species Desulfohalobium utahense sp. nov. is proposed, with strain EtOH3T (=VKM B-2384T=DSM 17720T) as the type strain.
The GenBank/EMBL/DDBJ accession number for the dsrAB and the 16S rRNA gene sequences of strain EtOH3T are DQ386236 and DQ067421, respectively.
A transmission electron micrograph of a cell of strain EtOH3T is available as supplementary material in IJSEM Online.
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270 g NaCl l–1. Despite the extreme salinity, the sediment from this part of the lake was recently shown to harbour active populations of sulfate-reducing micro-organisms (Brandt et al., 2001
), although so far the identity of these sulfate reducers has remained unknown. In recent years, growing interest in micro-organisms from hypersaline environments has led to the isolation of a limited number of halophilic sulfate-reducing bacteria (SRB). However, most of these SRB are only weakly halophilic and would hardly be able to proliferate at the elevated salinities in the northern arm of the Great Salt Lake. Thus, members of only five described SRB species are able to grow in vitro at salinities exceeding 200 g NaCl l–1, namely Desulfohalobium retbaense DSM 5692T, which grows at salinities up to 240 g l–1 (Ollivier et al., 1991), ‘Desulfovermiculus halophilus’ VKM B-2364 (Belyakova et al., 2006), growing at salinities up to 230 g l–1, and three members of the genus Desulfovibrio, growing at salinities up to 217–225 g l–1, ‘Desulfovibrio cavernae’ B1T (Sass & Cypionka, 2004), Desulfovibrio indonesiensis strains B1M and H3M (Sass & Cypionka, 2004) and Desulfovibrio oxyclinae DSM 11498T (Krekeler et al., 1997).
In the course of a survey of the diversity of SRB inhabiting the sediment of the northern arm of the Great Salt Lake, several novel sulfate-reducing strains were isolated. One strain, designated EtOH3T, was characterized further and assigned to the deltaproteobacterial genus Desulfohalobium as the type strain of a novel species. Strain EtOH3T and Desulfohalobium retbaense represent the most halophilic SRB described to date and together constitute the only recognized members of the genus Desulfohalobium, which is the type genus of the family Desulfohalobiaceae (Kuever et al., 2005). In addition to these taxa, other species affiliated with this family include Desulfonatronovibrio hydrogenovorans (Zhilina et al., 1997), Desulfothermus naphthae (Kuever et al., 2006), Desulfonauticus submarinus (Audiffrin et al., 2003) and ‘Desulfovermiculus halophilus’ (Belyakova et al., 2006).
Strain EtOH3T was isolated from an anoxic hypersaline (270 g NaCl l–1) sediment sample, obtained from Station 27 (Brandt et al., 2001). Strain EtOH3T was enriched, isolated and routinely cultivated in an anoxic basal medium (BM) consisting of (l–1 Milli-Q water): NaCl, 100 g; MgSO4.7H2O, 10 g; KCl, 6.0 g; CaCl2.2H2O, 0.4 g; NH4Cl, 1.0 g; KH2PO4, 0.1 g; yeast extract, 0.5 g; trace element solution, 1.0 ml (Widdel & Bak, 1992); 20 g l–1 resazurin solution, 50 µl; selenite tungstate solution, 1.0 ml (Widdel & Bak, 1992). The medium was prepared as described by Brandt & Ingvorsen (1997). NaHCO3 (30 mM final concentration) was used as the buffer and the pH was 7.0–7.2. Unless otherwise noted, all incubations were carried out at 30 °C in the dark using a 1 % (v/v) inoculum. Strain EtOH3T was enriched and isolated using ethanol (10 mM) as the electron donor. The isolation was performed by repeated application of the roll-tube technique (Hungate, 1969) using BM solidified by 20 g washed agar l–1. The purity of cultures was checked by phase-contrast microscopy and by performing growth tests in BM containing yeast extract (1 g l–1), glucose (10 mM), fumarate (10 mM), pyruvate (10 mM), lactate (10 mM) and succinate (10 mM) at 10 or 100 g NaCl l–1.
Unless stated otherwise, all growth experiments were performed in triplicate in BM dispensed into 16x125 mm Hungate anaerobic culture tubes (Bellco Glass) with a 3 ml gas phase (90 : 10 N2 : CO2, by vol.) using lactate (10 mM) as the electron donor and sulfate as the electron acceptor (lactate subsequently proved to be a better growth substrate than ethanol). Cell growth was quantified by measuring optical density at 600 nm, by phase-contrast microscopy combined with photometric measurement of sulfide by the method of Cline (1969) or, for pH experiments only, by total counts of SYBR Gold-stained cells as described by Mogensen et al. (2005). The effect of temperature on growth rate was investigated simultaneously at 14 different temperatures between 10 and 45 °C using a temperature gradient block (Elsgaard et al., 1994). The pH range for growth was investigated by titrating a modified BM with sterile, anoxic 1 M HCl or NaOH solution. This medium, in which NaHCO3 was replaced by 10 g MOPS (pKa 7.2) l–1 and 11 g CAPSO (pKa 9.6) l–1, buffered effectively within a pH range of 6.0–10.0 and did not interfere with the ability of strain EtOH3T to grow (results not shown). Growth was tested at 11 different pH values ranging from 6.2 to 9.3; the pH was monitored regularly during these incubations and adjusted if necessary. Because of the development of precipitates in the modified BM at pH values of >7.5, growth was quantified by total counts of cells stained with SYBR Gold (see above). Strain EtOH3T was tested for growth at 15 different concentrations of NaCl (ranging from 0 to 300 g l–1) in BM with and without 0.5 g yeast extract l–1. Furthermore, the effect of Mg2+ on growth was tested at 21 different concentrations in modified BM (100 g NaCl l–1) in which MgSO4.7H2O was replaced by Na2SO4 (5.76 g l–1) and 0–200 g MgCl2.6H2O l–1. Substrate utilization was tested by substituting either lactate or sulfate with potential electron donors or electron acceptors (Table 1). Transmission electron microscopy was performed as described previously (Mogensen et al., 2005). The Gram-staining reaction was determined using standard procedures.
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), represent the most halotolerant SRB described to date. Optimum growth occurred at 80–100 g NaCl l–1 independent of the presence of yeast extract. This NaCl optimum classifies strain EtOH3T as a moderate halophile according to Larsen (1986). The highest MgCl2.6H2O concentration tolerated by strain EtOH3T was 121 g l–1, with an optimum ranging from 2 to 51 g l–1. Despite the high NaCl tolerance of strain EtOH3T compared with most other known SRB, the observed upper NaCl limit for growth for this strain nevertheless indicates that it must be challenged by severe salt stress in its natural habitat, i.e. hypersaline sediment of Great Salt Lake, which contained 270 g NaCl l–1. The moderately halophilic SRB Desulfovibrio halophilus DSM 5663T was shown previously to profit from the uptake of compatible solutes for osmoadaptation (Welsh et al., 1996). The effect of compatible solutes on the growth of strain EtOH3T was tested by adding a mixture consisting of (final concentrations) glycine betaine (5 mM), trehalose (2.6 mM), sucrose (5 mM) and choline (5 mM) to BM containing 100 g NaCl l–1 and lactate as the electron donor. The aforementioned test had shown that strain EtOH3T was unable to utilize any of these compatible solutes as growth substrates (Table 1
). In the presence of yeast extract (0.5 g l–1), the addition of compatible solutes increased the final cell densities and the growth rate of strain EtOH3T by 17 and 13 %, respectively. In the absence of yeast extract, the addition of compatible solutes increased the same two values by 22 and 21 %, respectively. Each of these increments was significant according to a one-way analysis of variance (results not shown). Although the observed effects were minor, the presence of compatible solutes in the surrounding medium thus seems to enhance the growth of strain EtOH3T. In this respect, it is worth mentioning that yeast extract may contain the compatible solute glycine betaine in relatively large amounts (Galinski & Trüper, 1994) in addition to other possible growth-stimulating compounds. The availability of the tested or other more potent osmolytes in the Great Salt Lake sediment may help to explain the above-mentioned discrepancy between the NaCl tolerance of strain EtOH3T in vitro and the salinity of the sediment in situ. The optimum pH for growth of strain EtOH3T was 6.8 and growth was detected between pH 6.5 and 8.3. The strain was mesophilic, growing within a temperature range of 15.0–43.6 °C with an optimum at 37 °C. Unlike other members of the family Desulfohalobiaceae, and other halophilic SRB, strain EtOH3T utilized a broad range of different electron donors (Table 1). For instance, strain EtOH3T grew on propionate, methanol and Casamino acids, which, respectively, are only utilized by the weakly to moderately halophilic SRB Desulfacinum hydrothermale DSM 13146T (Sievert & Kuever, 2000), as well as ‘Desulfovermiculus halophilus’ (Belyakova et al., 2006), ‘Desulfovibrio cavernae’ H1M (Sass & Cypionka, 2004) and ‘Desulfovibrio brasiliensis’ DSM 15816 (Warthmann et al., 2005). In contrast to other members of the family Desulfohalobiaceae, strain EtOH3T was unable to use sulfite as a terminal electron acceptor (Table 1). This finding was unexpected, as the strain harboured genes (dsrAB) encoding the alpha and beta subunits of the dissimilatory (bi)sulfite reductase enzyme (EC number 1.8.99.3) (Fig. 1b). It may be argued that the concentrations of sulfite tested (2.5 and 5.0 mM) were toxic to strain EtOH3T. However, the strain was able to grow when the same concentrations of sulfite were added to BM containing sulfate and, furthermore, sulfite had no effect on the morphology of the cells as evaluated by phase-contrast microscopy (results not shown). These findings indicated that the sulfite concentrations applied were not toxic to strain EtOH3T. It is therefore possible that the strain lacks a transport mechanism for the uptake of sulfite.
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). Phylogenetic 16S rRNA gene sequence-based trees were inferred from neighbour-joining (with Jukes–Cantor distance correction), maximum-parsimony and maximum-likelihood analyses of a sequence dataset consisting of sequences of >1300 nt and including a broad range of proteobacterial taxa. Analyses were performed using the respective algorithms of the ARB program package (Ludwig et al., 2004). Two different filters [the deltaproteobacterial 50 % conservation filter of the ssu_jan04_corr_opt ARB database (available at http://www.arb-home.de) and a custom-made 50 % conservation filter calculated for the Desulfohalobiaceae-affiliated taxa shown in Fig. 1a] were used to select sequence positions for the analyses. Neighbour-joining-based bootstrap analysis was performed in PAUP*, version 4.0b10 (Swofford, 2003). Phylogenetic analyses of DsrAB amino acid sequence (deduced from nucleotide sequences) datasets including 530 unambiguously aligned positions were performed as described previously (Abildgaard et al., 2006). The phylogenetic consensus trees shown in Fig. 1 were constructed as recommended by Ludwig et al. (1998). A range of outgroup taxa were removed from the presented phylogenetic consensus trees; complete trees are available from the corresponding author on request. The genomic G+C content of strain EtOH3T was determined by HPLC analysis at the Identification Service of DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany).
Phylogenetic analyses of the dsrAB and 16S rRNA gene sequences of strain EtOH3T consistently grouped the strain within the deltaproteobacterial family Desulfohalobiaceae (Fig. 1). The phylogenetically closest relative of strain EtOH3T with a validly published name was Desulfohalobium retbaense (Ollivier et al., 1991), sharing 76.0 and 90.5 % dsrAB and 16S rRNA gene sequence similarity, respectively, with strain EtOH3T (Fig. 1). Recently the isolation of a novel moderately halophilic SRB, ‘Desulfovermiculus halophilus’ strain 11, was reported (Belyakova et al., 2006). No dsrAB data are currently available for ‘Desulfovermiculus halophilus’ strain 11, but the 16S rRNA gene sequence of this strain differs by only 3.1 % (1413 nt included) from that of strain EtOH3T (Fig. 1a). The 16S rRNA gene sequence of strain EtOH3T contained a unique 97 nt insert from positions 186 to 191 (Escherichia coli numbering), which is absent in the 16S rRNA gene sequences of all other species affiliated with the family Desulfohalobiaceae. This region was not included in phylogenetic analyses. The DNA of strain EtOH3T differed in G+C content by 5.7 and 3.8 mol% from Desulfohalobium retbaense and ‘Desulfovermiculus halophilus’, respectively (Table 1). This is around the threshold of 5 mol% difference in genomic G+C content empirically observed generally to constitute the common range within a species (Rosselló-Mora & Amann, 2001). In contrast to the comparison of the available genotypic data, strain EtOH3T differed significantly from ‘Desulfovermiculus halophilus’ when comparing their phenotypic characteristics (Table 1). Most notably, ‘Desulfovermiculus halophilus’ differed from strain EtOH3T in its capacity for autotrophic growth, its low Mg2+ tolerance and its limited ability to grow using alcohols (Table 1). Similarly, several phenotypic characteristics differentiated strain EtOH3T from Desulfohalobium retbaense as well as the other species affiliated with the family Desulfohalobiaceae (Table 1). The cellular fatty acid profile of strain EtOH3T was determined at the Identification Service of the DSMZ. Cells used for the analysis were cultivated in BM containing 100 g NaCl l–1 and 10 mM lactate, harvested by centrifugation (16 000 g), washed with 20 g NaCl l–1 solution and freeze-dried. The major fatty acids of strain EtOH3T were (calculation method TSBA50): iso-C15 : 0 (68.47 %), iso-C17 : 0 (11.61 %), iso-C17 : 1
9c (5.92 %), iso-C15 : 0 3-OH (3.19 %), C16 : 0 (2.15 %), C18 : 0 (2.98 %), 10-methyl C18 : 0 (tuberculostearic acid) (1.15 %), iso-C17 : 0 3-OH (0.89 %) and iso-C16 : 0 (0.79 %). No substantial similarities in the fatty acid profiles of strain EtOH3T and Desulfohalobium retbaense (Ollivier et al., 1991) were evident. Unfortunately, fatty acid profiles have not been published for ‘Desulfovermiculus halophilus’.
Several physiological characteristics of Desulfohalobium retbaense, ‘Desulfovermiculus halophilus’ and strain EtOH3T, such as the NaCl, temperature and pH ranges for growth, were very similar (Table 1). This is in agreement with their strong phylogenetic coherence demonstrated by the dsrAB and 16S rRNA gene sequence analyses (Fig. 1). Based on the phenotypic and genotypic data discussed above, it is proposed that strain EtOH3T represents a novel species within the genus Desulfohalobium, Desulfohalobium utahense sp. nov. As the name ‘Desulfovermiculus halophilus’ has not yet been validly published, strain EtOH3T cannot be assigned to its genus, although this might seem justifiable from the available phylogenetic data avoiding making the genus Desulfohalobium paraphyletic (Fig. 1a). In fact, according to its genotypic and phenotypic characteristics (Fig. 1a; Table 1), it may even be warranted to reclassify ‘Desulfovermiculus halophilus’ as a novel member of the genus Desulfohalobium. Future research resulting in the description of additional members of the family Desulfohalobiaceae and in particular the genus Desulfohalobium will allow this issue to be addressed in further detail.
Description of Desulfohalobium utahense sp. nov.
Desulfohalobium utahense (u'tah.en'se. N.L. neut. adj. utahense pertaining to the state of Utah, USA, where the type strain was first isolated).
Cells are non-motile, oval to rod-shaped (1.0–1.2x2.5–3.0 µm) and occur singly or in pairs. Under suboptimal conditions, cell elongation (up to 15 µm) and/or filamentous growth may occur. Cells stain Gram-negative and do not contain endospores. Anaerobic and chemo-organotrophic. Utilizes (with sulfate as the electron acceptor): formate, propionate, lactate, pyruvate, butyrate, succinate, malate, fumarate, methanol, ethanol, 1-propanol, 1-butanol, 1-pentanol, yeast extract, Casamino acids, H2/acetate and H2/yeast extract. Acetate is not oxidized during long-term incubation. Sulfate and thiosulfate are used as electron acceptors with lactate as the electron donor. Sulfur and sulfite are not reduced. Yeast extract improves, but is not required for, growth. Temperature range is 15.0–43.6 °C with an optimum of 37 °C. Sodium and magnesium ions are required for growth, the NaCl and MgCl2.6H2O growth ranges being 20–240 and >0–121 g l–1, respectively. The optimum NaCl and MgCl2.6H2O concentrations are 80–100 and 2–51 g l–1, respectively. The optimum pH is 6.8–7.5. Growth occurs between pH 6.5 and 8.3. The G+C content of the DNA is 51.4 mol%.
The type strain, EtOH3T (=VKM B-2384T=DSM 17720T), was isolated from the hypersaline northern arm of Great Salt Lake, Utah, USA.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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