lnternational Journal of Systematic Bacteriology (1 999), 49, 1177-1 180 Printed in Great Britain ~~~ ~~ ~. Sulfurospirillum barnesii sp. nov. and Sulfurospirillum arsenophilum sp. nov., new members of the Sulfurospirillum clade of the iz Proteobacteria Department of Biological Sciences, Duquesne University, Pittsburgh, Department of Microbiology, University of Massachusetts, Amherst, M A 01003, USA Water Resources Division, US Geological Survey, 345 Middlefield Road, Menlo Park, CA 94025, USA Nicholas School of the Environment, Duke University, Durham, NC 27708, USA PA 15202-1 521, USA John F. Stolz,’ Debra J. Ellis,* Jodi Switzer B l ~ m , ~ Dianne Ahmann,4 Derek R. Lovley2 and Ronald S. Oremland3 Author for correspondence: John F. Stolz. Tel: + 1 412 396 6333. Fax: + 1 412 396 5907. e-mail : stolz@,duq3.cc.duq.edu Two strains of dissimilatory arsenate-reducing vibrio-shaped bacteria are assigned to the genus Sulfurospirillum. These two new species, Sulfurospirillum barnesii strain SES-3T and Sulfurospirillum arsenophilum strain MIT-13T, in addition to Sulfurospirillum sp. SM-5, two strains of Sulfurospirillum deleyianum, and Sulfurospirillum arcachonense, form a distinct clade within the E subclass of the Proteobacteria based on 165 rRNA analysis. Keywords: arsenate reduction, selenate reduction, E Proteobacteria, Sulfurospirillum barnesi, Sulfurospirillum arsenophilum Two new species of bacteria have recently been identified by their ability to grow as a result of the dissimilatory reduction of arsenate to arsenite. The first, strain SES-3T, was isolated from a selenate- contaminated freshwater marsh in western Nevada (Oremland et al., 1989, 1994). The second, strain MIT- 1 3T, was isolated from arsenic-contaminated water- shed sediments in eastern Massachusetts (Ahmann et al., 1994). As these organisms were among the first to be described with this unique physiology, the genus name Geospirillum was proposed and several of the initial papers describing their physiology and bio- chemistry were published using that genus name (Lonergan et al., 1996; Lovley et al., 1997; Stolz et al., 1997). It was later discovered that a bacterium with very similar morphology and several shared physio- logical characteristics had already been used to de- scribe a new genus Sulfurospirillum (Schumacher et al., 1992). This organism, Sulfurospirillurn deleyianum, is the type species for the genus (Schumacher et al., 1992). Recently, Finster and colleagues, in describing another new member of the genus, Sulfurospirillum arcachonense, suggested that SES-3T should be in- cluded in the genus (Finster et al., 1997). Here we have The GenBank accession number for the 165 rDNA sequence of strain SES-3T is AF038843. used 16s rRNA and DNA-DNA hybridization analy- ses as well as comparison of other characteristics to clarify the taxonomic status of the arsenate-reducing strains SES-3T and MIT-13*. This is the first formal description of these bacteria. The 16s rRNA gene from SES-3Twas amplified using PCR with the EubA and EubB primers (Giovannoni, 1991), cloned using the TA cloning system (Invitro- gen), and sequenced using the methods of Sanger et al. (1977) with forward and reverse sequencing primers (Giovannoni, 1991 ; Lane, 1991). The 16s rRNA gene sequences from MIT-13T and SM-5 were obtained as previously described (Lonergan et al., 1996). Ad- ditional sequences were obtained from GenBank and the Ribosomal Database Projects (Maidak et al., 1996; Van der Peer et al., 1997). Alignment was done using CLUSTAL w (Higgins & Sharp, 1989). Maximum- parsimony and neighbour-joining trees were generated and sequence similarities were determined using a version of PHYLIP (Felsenstein, 1988).The ability of the type strain of S. deleyianum (ATCC 5 I 1 33T)to respire arsenate and selenate was tested using the medium described in Schumacher et al. (1992) with no cysteine and less potassium phosphate (140 mg 1-l). Formate, fumarate or hydrogen were used as electron donors with acetate as the carbon source. Stock cultures of S. deleyianum were maintained on medium containing 00788 0 1999 IUMS 1177