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Rutgers University, Department of Biochemistry and Microbiology, School of Environmental and Biological Sciences, 76 Lipman Drive, New Brunswick, NJ 08901, USA
Correspondence
Max M. Häggblom
haggblom{at}aesop.rutgers.edu
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Pelobacter seleniigenes sp. nov. KMT is DQ991964.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Five novel micro-organisms, which included two species of the class Gammaproteobacteria and a member of each of the classes Deferribacteres, Chrysiogenetes and Deltaproteobacteria, capable of dissimilatory selenate reduction were isolated from different sediments. Here, we provide a detailed description of strain KMT which was isolated for its selenate-respiring capability from wetland sediment, Kearny Marsh, NJ, USA.
Strain KMT groups within the family Geobacteraceae in the class Deltaproteobacteria. The class Deltaproteobacteria houses a number of genera which play a highly significant role in the environment, such as the Fe(III)-respiring members of the genus Geobacter and mainly fermentative members of the genus Pelobacter. The Geobacteraceae family has recently been grouped into three phylogenetic clusters, Desulfuromusa, Desulfuromonas and Geobacter (Holmes et al., 2004), based on a number of conserved genes, including recA, gyrB, rpoB, nifA and fusA. Within this family, the genus Pelobacter comprises a group of organisms fermenting unusual substrates (Schink, 2005, 2006). Species of the genus Pelobacter are highly diverse and are scattered in the three phylogenetic clusters. Since strain KMT shares similar characteristics with Malonomonas rubra Gra Mal 1T and only two of the Pelobacter species, we compared the physiological properties of strain KMT with closely related organisms and investigated the taxonomic position of strain KMT and related species.
Strain KMT is a dissimilatory selenate-respiring bacterium isolated from an enrichment culture of sediment from Kearny Marsh, a wetland system in NJ, USA (Narasingarao & Häggblom, 2007). After sequential transfers into fresh media, strain KMT was isolated using strict anaerobic techniques in soft agar (0.4 % Noble agar; Difco) shake tubes with selenate (10 mM) as the electron acceptor and pyruvate (20 mM) as the electron donor. Strain KMT was cultivated and maintained in a minimal salts medium (Fennell et al., 2004) with pyruvate as the carbon source and selenate as the electron acceptor under a head space of N2/CO2 (30 % : 70 %, v/v).
Strain KMT was grown with selenate (10 mM) and pyruvate or fermentatively with only pyruvate (10 mM) to examine cell morphology. Transmission electron microscopy and scanning electron microscopy were performed as described previously (Narasingarao & Häggblom, 2006). Strain KMT is a rod-shaped bacterium, approximately 2x0.5 µm (Fig. 1a). The cells formed large aggregates when grown in the presence of selenate and formed clumps along with precipitated elemental selenium (Fig. 1b) that settled at the bottom of the flask as a slimy mat. Strain KMT is a Gram-negative, motile bacterium that formed bright red colonies in soft agar shake tubes when grown on selenate (10 mM) due to the precipitation of red elemental selenium. Cells of selenate-respiring cultures produce abundant elemental selenium granules which are closely associated with the cells. Strain KMT has unique physiological and taxonomic characteristics that distinguish it from closely related genera and thus warrants classification as a novel species.
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) was used to test the metabolic capabilities of strain KMT. Strain KMT respires selenate stoichiometrically to elemental selenium with acetate as the electron donor and the carbon source. The utilization of 5.4 mM acetate was accompanied by reduction of 7.3 mM selenate to 4.6 mM selenite and 2.7 mM elemental selenium (estimated from the difference in total and soluble selenium measured). Selenate reduction was followed by a transient accumulation of selenite and this was finally reduced to elemental selenium, as evidenced by a bright red precipitate and further confirmed using X-ray absorption near edge structure analysis (XANES; Narasingarao & Häggblom, 2007). Strain KMT can also grow fermentatively on short-chain organic acids, such as pyruvate, citrate and lactate. Most dissimilatory selenate-reducing bacteria known so far are facultative or strictly anaerobic respiratory micro-organisms, unknown for their fermentative capability (Stolz & Oremland, 1999).
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The fatty acid content of strain KMT was determined after growth under fermentative conditions with pyruvate (10 mM) as the sole carbon and energy source at 28 °C using the method described by Narasingarao & Häggblom (2006). The bulk of the fatty acids (65 %) consisted of straight-chain fatty acids, such as C15, C16 and C17 (Table 2). Strain KMT, being Gram-negative, possessed, as expected, some 3-hydroxy fatty acids from the lipopolysaccharide of the outer membrane. Unsaturated fatty acids, predominantly C16 : 1
7c and C17 : 18c, were found in small amounts. A fairly high amount (9.5 %) of C17 : 0 cyclo was also detected. The fatty acid content of strain KMT is substantially different from that of Desulfuromusa ferrireducens (Vandieken et al., 2006), the only closely related species for which this information is available at the time of writing.
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with the following modifications. A Synergi 4U Fusion-RP 80A C18 reverse-phase column (Phenomenex) was used in a HPLC (1100; Agilent) system. The solvent system consisted of eluent A, 20 mM ammonium acetate (pH 4.5) and eluent B, acetonitrile. With a 1 ml min–1 flow rate, a gradient was established starting with 95 % eluent A, gradually decreasing to 60 % over 10 min. The nucleosides were detected at a wavelength of 260 nm. Salmon sperm DNA was used for calibration and Sedimenticola selenatireducens AK4OH1T was used as a control for verification. The G+C content of strain KMT was found to be 54.1±1.1 mol%.
The 16S rRNA gene sequence analysis was performed using primers described in Narasingarao & Häggblom (2006). The sequence data were complied in Contig Express (Vector NTI Suite; Informax). The 16S rRNA gene sequences of related micro-organisms were identified using a BLAST search (Altschul et al., 1997) and downloaded from GenBank. The sequences were aligned using CLUSTAL_X (Thompson et al., 1997). A similarity matrix was constructed using the Ribosomal Database Project version 8.1 small-subunit similarity matrix calculator (http://rdp8.cme.msu.edu/html/analyses.html). Phylogenetic trees were constructed using the neighbour-joining, maximum-parsimony and maximum-likelihood methods with PHYLIP (Felsenstein, 1989) and PHYML (Guindon et al., 2005). Bootstrap analysis was performed for all completed trees.
Strain KMT falls into the family Geobacteraceae within the class Deltaproteobacteria, based on the nearly complete 16S rRNA gene sequence. The 16S rRNA gene of strain KMT was 97.3 % similar to that of Malonomonas rubra Gra Mal 1T, 96.1 % to Pelobacter acidigallici Ma Gal 2T, 96.2 % to Pelobacter massiliensis and 96.4–96.6 % similar to that of species of the genus Desulfuromusa, but highly dissimilar to other species of the genus Pelobacter (<90 % in some cases), reflecting their complex taxonomy. The family Geobacteraceae has recently been grouped into three clusters, Desulfuromusa, Desulfuromonas and Geobacter (Holmes et al., 2004), based on sequence analysis of a number of genes such as nifD, recA, gyrB, rpoB and fusA, along with the 16S rRNA gene. A phylogenetic tree (Fig. 2) of the family Geobacteraceae constructed incorporating the 16S rRNA gene sequence of strain KMT showed the same topology as that described by Holmes et al. (2004), with strain KMT grouping within the Desulfuromusa cluster. Strain KMT is most closely related to M. rubra Gra Mal 1T, along with two of the Pelobacter species, P. acidigallici Ma Gal 2T and P. massiliensis, and a group of species of the genus Desulfuromusa. This distinct grouping is also supported by neighbour-joining and maximum-parsimony methods (not shown).
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shows a comparison of the metabolic characteristics of strain KMT with closely related genera. The ability of strain KMT to ferment short-chain fatty acids such as pyruvate, citrate and lactate is not shared by any of the closely related genera, although members of the genera Malonomonas and Pelobacter are strict fermenters. This also contrasts with species of the genus Desulfuromusa that are primarily respiratory and ferment only fumarate and malate (Liesack & Finster, 1994). Furthermore, the fermentative capability of members of the genera Malonomonas and Pelobacter is restricted to only a very narrow range of compounds. P. acidigallici Ma Gal 2T can ferment only gallic acid, 2,4,6-trihydroxybenzoic acid and phloroglucinol (Schink & Pfennig, 1982). P. massiliensis can ferment only hydroquinones (Schnell et al., 1991) and M. rubra Gra Mal 1T can ferment only malonate, fumarate and malate (Dehning & Schink, 1989).
The 16S rRNA gene sequence of M. rubra Gra Mal 1T is about 96 % similar to those of the two species of the genus Pelobacter and about 95 % to the species of the genus Desulfuromusa. Thus, reclassifying M. rubra Gra Mal 1T as a species of genus Pelobacter would result in better grouping as opposed to grouping with species of the genus Desulfuromusa, as suggested by Vandieken et al. (2006). This is further supported by 16S rRNA phylogeny (Fig. 2) in which, M. rubra Gra Mal 1T groups within this cluster consistently with strain KMT. This is affiliation is further supported by phylogenetic analysis of other conserved genes (Holmes et al., 2004).
In terms of its electron acceptor utilization, strain KMT is very versatile in its ability to reduce nitrate, fumarate, selenate, anthraquinone disulfonate (AQDS), Fe(III) and elemental sulfur, along with its fermentative capability, which is in contrast to species of the genera Malonomonas and Pelobacter. Furthermore, P. acidigallici Ma Gal 2T and P. massiliensis do not possess any cytochromes (Schnell et al., 1991). Species of the genus Malonomonas can however use Fe(III), as recently shown by Nevin et al. (2003), and also possess high amounts of periplasmic cytochromes (Kolb et al., 1998). The enzymes of the citric acid cycle were detected in M. rubra Gra Mal 1T with a possible explanation that it could be involved in assimilatory metabolism. Unfortunately none of these strains have been tested for growth on selenate as electron acceptor, which could be one of the distinguishing characters. Strain KMT can grow at a wide range of NaCl concentrations (0.5–3.5 %, w/v) whereas all other related strains have a strict requirement for NaCl, primarily due to their marine origin.
Prior to the 16S rRNA gene sequencing era, species of the genus Pelobacter were grouped in the same genus because of their unique ability to ferment a narrow range of compounds. However, based on 16S rRNA gene phylogeny (Fig. 2), it is clear that species of the genus Pelobacter are scattered throughout the phylogenetic tree of the family Geobacteraceae. This heterogeneity is further supported by other conserved genes (Holmes et al., 2004) and evidence arising from DNA–DNA hybridization [J. P. Touzel & B. Schink, unpublished data as cited in Schink (2005)]. Thus, P. acidigallici Ma Gal 2T and P. massiliensis, which group in the Desulfuromusa cluster, should be clearly distinguished from the other species of the genus Pelobacter.
Based on the priority of publication, P. acidigallici Ma Gal 2T, the type species of the genus (Schink & Pfennig, 1982), should retain the original genus name and the other species of the genus Pelobacter not belonging to the Desulfuromusa cluster may need to be reclassified. Since M. rubra Gra Mal 1T and strain KMT group with the genus Pelobacter sensu strictu (P. acidigallici Ma Gal 2T and P. massiliensis), it is suggested that the genus Malonomonas and its only recognized species, Malonomonas rubra, could be included in the genus Pelobacter. As the type strain of Malonomonas rubra, Gra Mal 1T, is currently only available in one culture collection (as DSM 5091T), it is not possible to make a formal proposal for this reclassification at this time. Strain KMT is classified as representing a novel species, for which the name Pelobacter seleniigenes sp. nov. is proposed.
Description of Pelobacter seleniigenes sp. nov.
Pelobacter seleniigenes (sel.en'ii.gen.es. N.L. n. selenium selenium; Gr. v. gennao produce; N.L. part. adj. seleniigenes selenium-producing).
Gram-negative, motile rod-shaped bacterium approximately 0.5x2 µm. Strictly anaerobic. Ferments pyruvate, citrate and lactate and can respire selenate to elemental selenium coupled to acetate utilization. Is able to respire other inorganic electron acceptors such as Fe(III), nitrate, AQDS and elemental sulfur. In agar shake tubes, colonies are round, about 5 mm in diameter and bright red coloured due to the elemental selenium formed during selenate reduction. Tolerates a wide range of NaCl concentrations (0.5–3.5 %, w/v) and grows robustly. The predominant cellular fatty acids are penta, hexa- and heptadecanoic acid and their corresponding monounsaturated fatty acids, C17 : 0 cyclo fatty acid and 3-hydroxyl fatty acids. Groups within the family Geobacteraceae of the class Deltaproteobacteria. The genomic DNA G+C content of the type strain is 54.1±1.1 mol%.
The type strain, strain KMT (=DSM 18267T=ATCC BAA-1388T) was isolated for its selenate-respiring capability from a freshwater wetland system in New Jersey, USA.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Dehning, I. & Schink, B. (1989). Malonomonas rubra gen. nov., sp. nov., a microaerotolerant anaerobic bacterium growing by decarboxylation of malonate. Arch Microbiol 151, 427–433.[CrossRef]
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Holmes, D. E., Nevin, K. P. & Lovley, D. R. (2004). Comparison of 16S rRNA, nifD, recA, gyrB, rpoB and fusA genes within the family Geobacteraceae fam. nov. Int J Syst Evol Microbiol 54, 1591–1599.
Kolb, S., Seeliger, S., Springer, N., Ludwig, W. & Schink, B. (1998). The fermenting bacterium Malonomonas rubra is phylogenetically related to sulfur-reducing bacteria and contains a c-type cytochrome similar to those of sulfur and sulfate reducers. Syst Appl Microbiol 21, 340–345.[Medline]
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Vandieken, V., Mußmann, M., Niemann, H. & Jørgensen, B. B. (2006). Desulfuromonas svalbardensis sp. nov. and Desulfuromusa ferrireducens sp. nov., psychrophilic, Fe (III)-reducing bacteria isolated from Arctic sediments, Svalbard. Int J Syst Evol Microbiol 56, 1133–1139.
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