University of Montana ScholarWorks at University of Montana Biological Sciences Faculty Publications Biological Sciences 9-1-2009 Desulfovibrio idahonensis sp. nov., sulfatereducing bacteria isolated from a metal(loid)-contaminated freshwater sediment H. Sass Cardiff University S. Ramamoorthy Cardiff University C. Yarwood University of Montana H. Langner University of Montana P. Schumann Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH See next page for additional authors Follow this and additional works at: https://scholarworks.umt.edu/biosci_pubs Part of the Biology Commons Let us know how access to this document benefits ou.y Recommended Citation Sass, H.; Ramamoorthy, S.; Yarwood, C.; Langner, H.; Schumann, P.; Kroppenstedt, R. M.; Spring, S.; and Rosenzweig, R. Frank, "Desulfovibrio idahonensis sp. nov., sulfatereducing bacteria isolated from a metal(loid)-contaminated freshwater sediment" (2009). Biological Sciences Faculty Publications. 439. https://scholarworks.umt.edu/biosci_pubs/439 This Article is brought to you for free and open access by the Biological Sciences at ScholarWorks at University of Montana. It has been accepted for inclusion in Biological Sciences Faculty Publications by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. Authors H. Sass, S. Ramamoorthy, C. Yarwood, H. Langner, P. Schumann, R. M. Kroppenstedt, S. Spring, and R. Frank Rosenzweig This article is available at ScholarWorks at University of Montana: https://scholarworks.umt.edu/biosci_pubs/439 International Journal of Systematic and Evolutionary Microbiology (2009), 59, 2208–2214 DOI 10.1099/ijs.0.016709-0 Desulfovibrio idahonensis sp. nov., sulfate- reducing bacteria isolated from a metal(loid)- contaminated freshwater sediment H. Sass,1 S. Ramamoorthy,2 C. Yarwood,2 H. Langner,3 P. Schumann,4 R. M. Kroppenstedt,4 S. Spring4 and R. F. Rosenzweig2 Correspondence 1School of Earth, Ocean and Planetary Sciences, Cardiff University, Main Building, Park Place, R. F. Rosenzweig Cardiff CF10 3YE, UK [email protected] 2Division of Biological Sciences, University of Montana, Missoula, MT 59812-4824, USA 3Department of Geology, University of Montana, Missoula, MT 59812, USA 4DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7B, D-38124 Braunschweig, Germany Two novel sulfate-reducing bacteria, strains CY1T and CY2, were isolated from heavy-metal- contaminated sediments of Lake Coeur d’Alene, Idaho, USA. Strains CY1T and CY2 were found to contain c-type cytochromes and to reduce sulfate, sulfite, thiosulfate, elemental sulfur, DMSO, anthraquinone disulfonate and fumarate using lactate as an electron donor. In a comparison of 16S rRNA gene sequences, CY1T and CY2 were found to be 100 % identical, but only 97 and 92.4 % similar, respectively, to the type strains of Desulfovibrio mexicanus and Desulfovibrio aminophilus. Unlike these species, however, CY1T was neither able to disproportionate thiosulfate nor able to use yeast extract or amino acids as electron donors. These data, considered in conjunction with differences among strain CY1T and the two related type strains in chemotaxonomy, riboprint patterns, temperature and pH optima, support recognition of a distinct and novel species within the genus Desulfovibrio, Desulfovibrio idahonensis sp. nov., with the type strain CY1T (5DSM 15450T 5JCM 14124T). The genus Desulfovibrio ranks among the most speciose, (Goldstein et al., 2003). Desulfovibrios facilitate metal phenotypically diverse genera in the Proteobacteria. corrosion (Neria-Gonzalez et al., 2006) and have been Diagnostic traits include dissimilatory sulfate reduction, repeatedly isolated from oil and gas production facilities absence of sporulation, presence of polar flagella and the (Miranda-Tello et al., 2003; Magot et al., 2004). presence of desulfoviridin and cytochrome c (Postgate & Desulfovibrio ribosomal gene sequences have been Campbell, 1965). Ribosomal gene sequence analyses retrieved from submerged mine timbers (Labrenz & indicate that the group’s origin lies deep in the Banfield, 2004), gas hydrate mounds (Mills et al., 2003) Deltaproteobacteria (Devereux et al., 1990). To date, names and floating macrophyte rhizospheres (Acha et al., 2005), of some 57 Desulfovibrio species have been validly while desulfovibrio-specific dissimilatory sulfite reductase published (http://www.dsmz.de/microorganisms/bacterial_ genes (dsrAB) have been recovered from temperate sulfide- nomenclature_info.php?genus=Desulfovibrio; four of these rich streams (Elshahed et al., 2003) and frozen Antarctic species have since been reclassified), enriched from such lakes (Karr et al., 2005). diverse environments as sediments (Bale et al., 1997; Sass As a group, desulfovibrios tolerate extreme ranges of et al., 1998), wastewater sludge (Baena et al., 1998; temperature (Bale et al., 1997; Vandieken et al., 2006), pH Hernandez-Eugenio et al., 2000) and animal intestines (Abildgaard et al., 2006; Fro¨hlich et al., 1999) and salinity (Sass & Cypionka, 2004; Ito et al., 2002), and productively Abbreviation: AQDS, anthraquinone disulfonate. use a multitude of electron donors, from aromatic The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene compounds (Reichenbecher & Schink, 1997) and haloge- sequences of strains CY1T and CY2 are AJ582755 and AJ582758, nated hydrocarbons (Sun et al., 2000) to amino acids respectively. (Baena et al., 1998; Hernandez-Eugenio et al., 2000). In An electron micrograph of a cell of strain CY1T and PvuII riboprints of addition to dissimilatory sulfate reduction, certain the novel strains and D. mexicanus DSM 13116T are available as Desulfovibrio species use alternative terminal electron supplementary material with the online version of this paper. acceptors such as U(VI) (Payne et al., 2002), Fe(III) 2208 016709 G 2009 IUMS Printed in Great Britain Desulfovibrio idahonensis sp. nov., from mine-impacted sediment (Vandieken et al., 2006) and Cr(VI) (Humphries & after three successive passages. Negative controls consisted Macaskie, 2005), though these electron acceptors do not of (i) live cells in medium without lactate or sulfate, (ii) generally support growth. It has long been known that autoclaved cells added to complete medium (Madigan et sulfidogenic microbes biomineralize aqueous-phase metals al., 1997) and (iii) live cells filtered through a 0.2 mm sterile and metalloids (Tuttle et al., 1969). Now, this activity is filter, with the filtrate added to complete medium. being harnessed to bioremediate soils and sediments Reduction of sulfate, sulfite, sulfur and thiosulfate to contaminated by mining and radionuclide processing sulfide was tested by adding 0.5 % Fe(NH4)2(SO4)2 to (Lovley, 2001). Because the genomes of several desulfovi- culture tubes. For accurate determination of sulfide (as brios have been fully sequenced (Heidelberg et al., 2004; hydrogen sulfide, H2S) concentrations, the spectrophoto- http://cmr.tigr.org/tigr-scripts/CMR/GenomePage.cgi?org= metric methylene blue assay of Cline (1969) was used. ntds01), autecological studies of Desulfovibrio species are Reduction of anthraquinone disulfonate (AQDS) was now possible using genome-enabled technologies (e.g. Clark measured photometrically at 450 nm, as described by et al., 2006). Lovley et al. (1996). As CY1T and CY2 were isolated from To understand metal(loid) cycling better in mining- metal(loid)-contaminated sediments, we tested for reduc- impacted freshwater sediments, our group has enriched tion of Mn(IV), As(V), Fe(III) and Se(VI) presented for and studied iron-, arsenic- and sulfate-reducing respectively as MnO2,Na2HAsO4 .7H2O, Fe4(P2O4)3 and bacteria, as the characteristic activities of such microbes Na2SeO4. MnO2 reduction was scored by the characteristic are likely to influence contaminant fate and stability colour change from black MnO2 to white MnCO3, while (Cummings et al., 2000; Niggemyer et al., 2001; formation of red elemental selenium precipitate was scored [ Ramamoorthy et al., 2009). Strains CY1T and CY2 were as indicating Se(VI) reduction. Reduced arsenic as ] isolated from the highest positive tubes of an MPN series arsenite, As(III) was quantified photometrically from the inoculated with sediment (pH 6.8–7.2) from Lake Coeur A865 using the procedure originally described by Johnson & d’Alene, Idaho, USA, a freshwater system historically Pilson (1972) and later modified by Niggemyer et al. impacted by lead, zinc and antimony mining (2001). Sulfate, thiosulfate, lactate and acetate concentra- (Ramamoorthy et al., 2009). For enrichment and isolation, tions were determined using ion chromatography we used selective dithionite-reduced medium with sodium (Ramamoorthy et al., 2006). Culture density was estimated photometrically as OD . Fluorimetry after staining with lactate (10 mM) and Na2SO4 (10 mM) (Widdel & Pfennig, 420 1977; Widdel, 1980). Bacteria were isolated by three SybrGreenI (Martens-Habbena & Sass, 2006) or direct cell passages in 1.5 % agar shake tubes. counting was used to confirm growth on selenate, manganese oxide and Fe(III). Cells were stained with Gram staining, oxidase and catalase tests, electron 10 mg49,6-diamidino-2-phenylindole (DAPI; Sigma) ml21, microscopy and assessment of spore formation were and cells were then counted by epifluorescence microscopy performed as described previously (Magee et al., 1975; using a Zeiss Axioskop. Growth yield was defined as Cappuccino & Sherman, 1998; Ramamoorthy