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Department of Microbiology, University of Massachusetts, Amherst, MA 01003, USA
Correspondence
Derek R. Lovley
dlovley{at}microbio.umass.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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-subclass of the Proteobacteria that is composed of three distinct phylogenetic clusters: Geobacter, Desulfuromonas and Desulfuromusa. The sequence data provided here will make it possible to discriminate better between physiologically distinct members of the Geobacteraceae, such as Pelobacter propionicus and Geobacter species, in geobacteraceae-dominated microbial communities and greatly expands the potential to identify geobacteraceae sequences in libraries of environmental genomic DNA.
. Genetic and synonymous distances for the genes analysed within the genera Desulfuromonas, Desulfuromusa, Geobacter and Pelobacter are available as supplementary material in IJSEM Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Holmes et al., 2002; Roling et al., 2001; Snoeyenbos-West et al., 2000; Stein et al., 2001). Although a wide diversity of bacteria and archaea are capable of dissimilatory Fe(III) reduction (Lovley, 2000a, b), most of these organisms do not appear to be important in Fe(III) reduction in typical subsurface environments, which are at circumneutral pH, freshwater or marine salinities and temperatures of about 10–20 °C. This finding, coupled with the fact that it is possible to recover geobacteraceae in culture, provides the rare potential opportunity to apply knowledge gained from pure culture physiology studies to the subsurface. Furthermore, the finding that geobacteraceae can account for about 40–90 % of the total microbial community under Fe(III)-reducing conditions in some subsurface environments (Anderson et al., 2003; Holmes et al., 2002) suggests that subsurface Fe(III)-reducing communities may provide the type of low-diversity community that is most amenable to environmental genomics studies. This is also true for the surface of electrodes harvesting energy from aquatic sediments, in which geobacteraceae typically comprise about half of the microbial community (Bond et al., 2002; Holmes et al., 2004; Tender et al., 2002).
However, more information than is available from 16S rRNA gene sequence analyses is required to characterize these geobacteraceae-dominated communities fully. Although 16S rRNA gene phylogeny correlates with the physiology of most species of the Geobacteraceae (Lonergan et al., 1996), several organisms in this family have metabolisms that are notably different. Most geobacteraceae have the ability to oxidize acetate and other organic compounds to carbon dioxide with Fe(III) serving as the electron acceptor (Lovley, 2000a). However, Pelobacter species, which are phylogenetically intertwined among these acetate-oxidizing geobacteraceae, are primarily hydrogen-oxidizing Fe(III) reducers and are not able to oxidize organic acids completely (Lonergan et al., 1996; Lovley, 2000a). Other unique physiological traits displayed by Pelobacter species include the ability to grow via the fermentation of unusual substrates, such as polyethylene glycol (Schink & Stieb, 1983) and acetylene (Schink, 1985), and syntrophic growth with methanogens (Schink, 1984; Schink & Pfennig, 1982; Schink & Stieb, 1983), a property not observed in non-Pelobacter species of the Geobacteraceae.
In order to define better the phylogeny of the Geobacteraceae, an intensive phylogenetic analysis of 30 strains of the Geobacteraceae was conducted. The nucleotide and amino acid sequences of the following genes were compared: rpoB, which encodes the 
subunit of RNA polymerase; recA, which encodes the DNA repair protein RecA; gyrB, the structural gene for the DNA gyrase subunit; fusA, which encodes the protein synthesis elongation factor-G (EF-G); nifD, which encodes the 
subunit of the dinitrogenase protein; and the 16S rRNA gene. The results demonstrate that the Geobacteraceae is a phylogenetically and physiologically distinct family within the -subclass of Proteobacteria. This information significantly expands the number of genes that can be used for analysing the distribution of geobacteraceae in sedimentary environments and for identifying environmental genomic DNA that can be assigned to the Geobacteraceae.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Geobacter hydrogenophilus DSM 13691T and Geobacter chapellei DSM 13688T were obtained from the laboratory of John Coates. ‘Desulfuromonas michiganensis’ BB1 was obtained from the laboratory of Frank Loffler (Sung et al., 2003). Desulfuromonas acetexigens ATCC 51529T, Desulfuromonas chloroethenica ATCC 700295T and Pelobacter acidigallici ATCC 49970T were obtained from the ATCC. Desulfuromonas thiophila DSM 8987T, Desulfuromusa bakii DSM 7345T, Desulfuromusa succinoxidans DSM 8270T, Geobacter bremensis DSM 12179T, Geobacter pelophilus DSM 12255T, Pelobacter acetylenicus DSM 3246T, Pelobacter massiliensis DSM 6233T and Pelobacter venetianus DSM 2394T were obtained from the DSMZ. Trichlorobacter thiogenes K1T was obtained from the laboratory of James Tiedje (De Wever et al., 2000).
Standard anaerobic techniques were used throughout (Balch et al., 1979; Nottingham & Hungate, 1969). All media was sterilized by autoclaving and all incubations were at 30 °C, unless otherwise noted. ‘Geobacter bemidjiensis’ BEM, ‘Geobacter humireducens’ JW-3, Geobacter hydrogenophilus, Geobacter metallireducens and Geobacter sulfurreducens were grown in freshwater medium (Lovley & Phillips, 1988) with 50 mM Fe(III) citrate as electron acceptor and 20 mM acetate as electron donor, as were Geobacter bremensis and Geobacter pelophilus with 1 mM ascorbic acid as reductant. Geobacter grbiciae was grown in freshwater medium (Lovley & Phillips, 1988) with either 5 mM anthraquinone 2,6-disulfonate (AQDS) or 100 mM Fe(III) oxide as electron acceptor and 10 mM acetate as electron donor. Geobacter chapellei was grown in freshwater medium (Lovley & Phillips, 1988) with 5 mM Fe(III) nitrilotriacetic acid as electron acceptor and 10 mM acetate as electron donor at 25 °C. Desulfuromonas chloroethenica was grown in ATCC medium 2063 with 40 mM fumarate as electron acceptor and 10 mM acetate as electron donor. Desulfuromonas palmitatis was grown in APW medium (Coates et al., 1995) with 50 mM Fe(III) citrate as electron acceptor and 20 mM acetate as electron donor and 40 % of the recommended amount of MgCl2.6H2O and CaCl2.2H2O. Desulfuromonas thiophila was grown in DSM medium 647 with 20 g elemental sulfur l–1 as electron acceptor and 5 mM pyruvate as electron donor. Medium 647 was steam sterilized at 100 °C for 1 h on three consecutive days. ‘Desulfuromonas michiganensis’ BB1 was grown in either DSM medium 298 or other anaerobic medium (Loffler et al., 2000) with 40 mM fumarate as electron acceptor and 10 mM acetate as electron donor and 0·36 g NaS.9H2O l–1 as reductant at 25 °C. Desulfuromonas acetoxidans, Desulfuromonas acetexigens, Desulfuromusa bakii, Desulfuromusa kysingii and Desulfuromusa succinoxidans were grown in APW medium (Coates et al., 1995) with 40 mM fumarate as electron acceptor, 20 mM acetate as electron donor and 40 % of the recommended amount of MgCl2.6H2O and CaCl2.2H2O and 0·36 g NaS.9H2O l–1 as reductant. ‘Geothermobacter ehrlichii’ SS015 was grown in marine medium (Grayson et al., 1999) with 10 mM nitrate as electron acceptor and 10 mM malate as electron donor at 55 °C. Desulfuromusa sp. strain S1, ‘Geopsychrobacter electrodophilus’ A2 and ‘Geopsychrobacter multivorans’ A1 were grown at 20 °C in APW medium (Coates et al., 1995) with Fe(III) oxide (60 mM) or colloidal S0 (10 mM) as electron acceptor and acetate (5 mM) as electron donor (Holmes et al., 2004). Malonomonas rubra was grown in APW medium (Coates et al., 1995) with 40 mM fumarate, 40 % of the recommended amount of MgCl2.6H2O and CaCl2.2H2O and 0·36 g NaS.9H2O l–1 as reductant. Pelobacter acetylenicus and Pelobacter propionicus were grown in DSM medium 298 with 1 g acetoin l–1 and 0·9 g 2,3-butanediol l–1, respectively. Pelobacter acidigallici, Pelobacter carbinolicus, Pelobacter venetianus and Pelobacter massiliensis were grown in DSM medium 293 with 0·68 g gallic acid l–1, 0·9 g 2,3-butanediol l–1, 1 g polyethylene glycol l–1 and 0·5 g pyrogallol l–1, respectively. Trichlorobacter thiogenes was grown in basal salts medium (De Wever et al., 2000) with 10 mM fumarate as electron acceptor and 5 mM acetate as electron donor and 0·36 g NaS.9H2O l–1 as reductant at 25 °C for DNA sequence studies.
DNA extraction.
Cells were collected by centrifugation and genomic DNA was extracted using the BIO 101 FastDNA Spin kit following the manufacturer's instructions. Cultures grown with poorly crystalline Fe(III) oxide as the electron acceptor were first treated with an equal volume of filter-sterilized oxalate solution (Phillips & Lovley, 1987) to remove Fe(III), which inhibits Taq polymerase.
Determination of nifD.
In order to determine whether or not the various species of the Geobacteraceae carried the nifD gene, a genomic dot blot was carried out on DNA extracted from the 30 different strains. Genomic DNA was denatured at 90 °C for 10 min and 1–10 µg denatured DNA was hybridized to a Zeta-Probe GT charged membrane (Bio-Rad) according to the manufacturer's instructions. A digoxigenin-labelled probe was constructed using primers targeting the nifD gene of Geobacter sulfurreducens with the PCR DIG Probe Synthesis kit (Roche Diagnostics). The probe was denatured and allowed to hybridize to single-stranded genomic DNA immobilized on the membrane. Addition of NBT/BCIP alkaline phosphatase solution (Sigma) to the membrane allowed visualization of bound probe.
Amplification of targeted genes.
The total volume of each PCR mixture was 100 µl. The amount of DNA template varied for each reaction, ranging from 
6 ng for more specific primers to 25 ng for more degenerate primers. Every PCR contained 10 µl Qiagen 10x buffer (15 mM MgCl2), 8 µl of a 0·25 µM dNTP solution (Sigma), 60 pmol forward and reverse primers, 160 µg BSA ml–1 (Sigma) and 3 U Taq polymerase (Qiagen). If Buffer Q (Qiagen) was added to the PCR mixture, the amounts ranged from 5 to 20 µl for each 100 µl reaction; additional MgCl2 was also included at concentrations ranging from 1 to 4 mM (final reaction concentration). The following components (all from Sigma) were sometimes added to improve the PCR: 5 µl DMSO per 100 µl, 1·25–10 % (w/v) formamide, 5 µl Ipegal with 5 % (w/v) acetamide, 15–30 mM (NH4)SO4 (final reaction concentration) or 15–20 % (w/v) glycerol. To ensure sterility, the PCR mixtures were exposed to UV radiation for 8 min prior to the addition of template and Taq polymerase. All PCR amplifications were performed in a DNA Engine thermal cycler (MJ Research, Inc.). PCR products were agarose gel-purified with the QIAquick gel extraction kit (Qiagen), ligated into the TOPO TA cloning vector and transformed into Escherichia coli cells with the TOPO TA cloning kit, version K2 (Invitrogen), according to the manufacturer's instructions. Plasmid inserts were then amplified with M13 forward and reverse primers (Invitrogen) and PCR products were prepared for sequencing using a QIAquick PCR purification kit (Qiagen).
Primers and PCR conditions used to amplify the 16S rRNA gene, gyrB, fusA, nifD, recA and rpoB.
16S rRNA gene sequences for ‘Geopsychrobacter multivorans’ A1, ‘Geopsychrobacter electrodophilus’ A2, ‘Geothermobacter erhlichii’ SS015 and Pelobacter massiliensis were amplified with primers 8 forward (Eden et al., 1991) and 1492 reverse (Amann et al., 1990; Achenbach & Woese, 1995) using the following PCR conditions: an initial denaturation step at 94 °C for 5 min, followed by 35 cycles of 94 °C (45 s), 50 °C (1 min) and 72 °C (1 min), with a final extension at 72 °C for 10 min.
The initial degenerate primers targeting the five protein-coding genes gyrB, fusA, nifD, recA and rpoB were designed using protein sequences from various bacterial and archaeal species available in the GenBank database. An alignment of amino acid sequences for each protein was done in CLUSTAL X (Thompson et al., 1997) and the Genetic Computer Group (GCG) sequence editor (Wisconsin Package, version 10). This alignment was then examined and conserved regions of the proteins were targeted for primer design. More specific primer sets were designed based on alignments constructed from sequences from members of the Geobacteraceae obtained further along in the study.
Gene fragments were amplified by either the primer pairs and PCR conditions outlined in Table 1 or the following primers and conditions: fusA, FUSAF/FUSAR (Berchet et al., 2000); gyrB, UP1E/APRU (Yamamoto et al., 2000); nifD, NIFD883F/NIFD1337R (Ueda et al., 1995); recA, FGPR-476/FGPR-477 (Maréchal et al., 2000); rpoB, BF/BR (Lee et al., 2000).
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). Nucleotide and amino acid sequences for each gene were initially aligned in CLUSTAL X (Thompson et al., 1997) and imported into the GCG sequence editor, where alignments were checked and hypervariable regions were masked.
Similarity matrices for each gene were generated using CLUSTAL W and CLUSTAL X (Thompson et al., 1994, 1997). Translation products were compared using the PAM120 amino acid substitution matrix (Dayhoff, 1978) and the PAM 1 matrix was used to determine similarity values for nucleotide sequences. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 2.1 (Kumar et al., 2001). Genetic distances were calculated by the Kimura two-parameter (Kimura, 1980) and Tamura–Nei models (Tamura & Nei, 1993). The method of Nei & Gojobori (1986) was applied to the various sequences to obtain synonymous distances (multiple substitutions adjusted by the Jukes–Cantor formula).
All distance matrices constructed with 16S rRNA gene sequences considered 1323 nucleotides. The gyrB matrices used 883 nucleotides and 265 amino acids; 590 nucleotides and 182 amino acids were used in the fusA matrices; 412 nucleotides and 170 amino acids were used in the recA matrices; 440 nucleotides and 150 amino acids were used in the nifD matrices; and 540 nucleotides and 176 amino acid sequences were considered in the rpoB matrices.
Construction of phylogenetic trees.
Aligned sequences were imported into PAUP 4.0b 4a (Swofford, 1998), where phylogenetic trees were inferred. Distances and branching order were determined and compared using character-based (maximum-parsimony and maximum-likelihood) and distance-based algorithms [HKY85 (Hasegawa et al., 1985) and Jukes–Cantor (Jukes & Cantor, 1969)]. Chlorobium tepidum TLST and Nostoc sp. PCC7120 were the outgroups used for phylogenetic comparisons of 16S rRNA, gyrB, fusA, nifD, recA and rpoB gene fragments, and bootstrap analyses were performed using ‘Fast’ stepwise-addition, with 1000 replicates. Nucleotide-based phylogenetic trees were generated by a heuristic search with maximum-likelihood settings, while amino acid-based trees were generated with maximum-parsimony settings. Phylogenetic trees constructed with amino acid and nucleotide sequences yielded similar results.
Preliminary sequence data from Geobacter sulfurreducens, Geobacter metallireducens, Desulfuromonas acetoxidans and Pelobacter carbinolicus were obtained from The Institute for Genomic Research (TIGR) website (http://www.tigr.org) and the DOE Joint Genome Institute (JGI) website (http://www.jgi.doe.gov).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Phylogenies based on nifD suggested that the Desulfuromonas and Desulfuromusa species were phylogenetically intertwined, while Geobacter species formed a distinct cluster (Fig. 1d).
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Phylogenetic structure of fusA, gyrB, nifD, recA and rpoB genes within the Geobacteraceae
The genetic distances between the fusA, gyrB, nifD, recA, rpoB and 16S rRNA gene sequences within the same genus were within 0·56 of the Kimura two-parameter (Kimura, 1980) distances and within 0·96 of the synonymous distances (Nei & Gojobori, 1986). According to both phylogenetic models, fusA and rpoB sequences from species within the genus Geobacter showed the greatest variation, whereas nifD, recA and gyrB gene sequences within the genus Pelobacter were most divergent. Overall, sequences from Desulfuromusa varied the least, and the nifD gene seemed to have the fewest nucleotide substitutions across all four genera. A table showing the genetic and synonymous distances for the 16S rRNA, fusA, gyrB, nifD, recA and rpoB genes within the genera Desulfuromonas, Desulfuromusa, Geobacter and Pelobacter is available as supplementary material in IJSEM Online.
In addition, there was a significant correlation between the genetic distances calculated by the model of Tamura & Nei (1993) for gyrB, rpoB and fusA gene fragments and the corresponding 16S rRNA genes within the Geobacteraceae; r values were 0·81, 0·74 and 0·75 (P<0·005) for the gyrB, rpoB and fusA genes. The evolutionary distances calculated for the nifD and recA gene fragments did not correspond as closely with 16S rRNA gene distances; r values were 0·62 and 0·64 (P<0·005) for the nifD and recA gene fragments. However, the distances from concatemers constructed with all five protein-coding genes were correlated with 16S rRNA gene distances (r=0·79; P<0·005) (Fig. 2).
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Further intragenomic comparisons of multiple copy genes within these genomes from geobacteraceae indicated that the fusA gene is relatively divergent within the Geobacter sulfurreducens and Geobacter metallireducens genomes. In the Geobacter sulfurreducens genome, the genetic distances between the three fusA genes were within 0·63 to 0·76 of the Kimura two-parameter distances, within 0·59 to 0·81 of the synonymous distances and the p-distance values (based on amino acid sequences) ranged from 0·52 to 0·64. The Kimura two-parameter distance for the two fusA genes in Geobacter metallireducens was 0·63, while the synonymous and p-distance values were 0·80 and 0·54, respectively.
While these protein-coding genes were relatively divergent within the Geobacteraceae, the 16S rRNA genes within the Geobacter sulfurreducens genome were highly conserved, and very few nucleotide differences between intraspecies 16S rRNA genes were detected; the Kimura two-parameter distance values were 0·00.
The intragenomic heterogeneity observed between multiple copies of the fusA gene in the Geobacter sulfurreducens and Geobacter metallireducens genomes is likely to influence the phylogenies generated with this gene. Therefore, fusA should not be considered definitive in the taxonomic classification of the Geobacteraceae. Although the 16S rRNA gene is also present in multiple copies, this gene appears to have a low nucleotide substitution rate within the genome of geobacteraceae. Therefore, intragenomic heterogeneity should not have a significant impact on the phylogeny predicted by the 16S rRNA gene.
Taxonomic classification of the Geobacteraceae
The Geobacteraceae form a phylogenetic clade within the -subclass of the Proteobacteria that can be divided into three distinct subclades: Desulfuromonas, Desulfuromusa and Geobacter. For the most part, conserved genes within the Desulfuromusa subclade have the fewest nucleotide substitutions. A possible explanation for the low phylogenetic diversity within this genus is that Desulfuromusa species evolved most recently within the family Geobacteraceae. Evolutionary distances within the Geobacter and Desulfuromonas clusters, on the other hand, are relatively large. It is likely that these organisms evolved relatively early and share a common ancestor within the -subclass of the Proteobacteria.
Six genera officially comprise these three taxonomic subclades: Geobacter, Desulfuromonas, Desulfuromusa, Pelobacter, Malonomonas and Trichlorobacter. Analysis of all six genes and concatenated alignments indicates that Pelobacter species other than Pelobacter propionicus are not phylogenetically coherent and are interspersed among Desulfuromonas and Desulfuromusa species. However, unique physiological characteristics such as their ability to ferment a number of unusual substrates (Schink, 1984, 1985, 1992; Schink & Pfennig, 1982; Schink & Stieb, 1983) and to grow syntrophically with methanogens (Schink, 1984; Schink & Pfennig, 1982; Schink & Stieb, 1983) suggest that these Pelobacter species are distinct from Desulfuromonas and Desulfuromusa species.
As previously noted (Lonergan et al., 1996), phylogenetic analysis suggests that Pelobacter propionicus should also be placed in the genus Geobacter. However, Pelobacter propionicus is unique in that, unlike previously described Geobacter species (Lovley, 2000a) and Trichlorobacter thiogenes (Nevin et al., 2003), Pelobacter propionicus cannot utilize acetate as an electron donor (Schink, 1992) or completely oxidize organic acids. Furthermore, although Pelobacter propionicus and other Pelobacter species can reduce Fe(III) (Lonergan et al., 1996; Lovley et al., 1995), unlike Geobacter species (Lovley, 2000a) and Trichlorobacter thiogenes (Nevin et al., 2003), Pelobacter propionicus and other Pelobacter species do not contain the c-type cytochromes that are involved in electron transfer in Geobacter species (Leang et al., 2003; Lloyd et al., 2003). Thus, due to its distinct physiological differences from Geobacter species, a separate genus designation for Pelobacter propionicus may be warranted.
The phylogenies generated from all six genes evaluated, as well as comparisons of concatenated alignments, demonstrate clearly that Trichlorobacter thiogenes falls within the phylogenetically coherent Geobacter cluster of the Geobacteraceae.
Malonomonas rubra, which is the only current representative of this genus, has recently been found to use Fe(III) as a terminal electron acceptor (Nevin et al., 2003), contains c-type cytochromes (Dehning & Schink, 1989) and, like all of the described Desulfuromusa species (Liesack & Finster, 1994), is able to ferment both fumarate and malate (Dehning & Schink, 1989). These similarities with closely related Desulfuromonas and Desulfuromusa species suggests that the taxonomic classification of Malonomonas rubra may need to be re-evaluated.
Application to community analysis and environmental genomics
The data presented here will permit more refined analysis of the composition of subsurface microbial communities in which geobacteraceae predominate. This may aid in a better understanding of the physiological characteristics of the geobacteraceae community. For example, Pelobacter propionicus falls within the Geobacter clade; however, this organism has a significantly different physiology from the Geobacter species. Analysing the distribution of the genes whose phylogenies are described here, in addition to the 16S rRNA gene, should make it easier to use molecular analysis to differentiate between Pelobacter and Geobacter species.
Previous studies have suggested that microbial community analysis with a low-copy-number gene may be more accurate than analysis with high-copy-number genes, such as the 16S rRNA gene (Dahllof et al., 2000; Peixoto et al., 2002). Preliminary analysis of the Geobacter sulfurreducens and Geobacter metallireducens genomes indicated that several of the conserved genes used in this study occur as single-copy or low-copy-number genes in the Geobacteraceae. For example, there is only one copy of the nifD, recA and gyrB genes in both of the genomes analysed from the Geobacteraceae.
Significant information about the physiology of uncultivated geobacteraceae in subsurface environments might also be derived from sequencing genomic DNA extracted from environments of interest (Lovley, 2002). Analysis of the genes reported here will make it possible to design specific PCR primers or probes that can be used to screen for fragments of geobacteraceae genomic DNA that contain these genes. This will significantly expand the amount of geobacteraceae genomic DNA that can be identified in genomic DNA libraries beyond what would be possible with screening only for 16S rRNA genes.
Significance of nifD
Previous physiological studies have demonstrated that Geobacter metallireducens and Geobacter sulfurreducens are capable of nitrogen fixation (Bazylinski et al., 2000; Coppi et al., 2001). The finding that nifD was present in all 30 species of the Geobacteraceae examined suggests that nitrogen fixation is a highly conserved physiological trait within this family. The ability to fix nitrogen might help geobacteraceae to compete successfully in subsurface environments, particularly those undergoing bioremediation of organic compounds or metals. For example, geobacteraceae were the predominant Fe(III)-reducing micro-organisms in subsurface environments contaminated with petroleum (Rooney-Varga et al., 1999) and landfill leachate (Roling et al., 2001). It is likely that the influx of organic carbon into these otherwise nutrient-poor subsurface environments resulted in a limitation of fixed nitrogen relative to the availability of carbon substrates. In a similar manner, geobacteraceae became the predominant dissimilatory metal-reducing micro-organisms when acetate, but no fixed nitrogen, was added to uranium-contaminated subsurface sediments to promote the reductive precipitation of uranium (Anderson et al., 2003; Holmes et al., 2002). The results suggest that the ability to adapt to such nitrogen-limited environments is typically found in geobacteraceae. In contrast, the genomes of other well-studied metal-reducing micro-organisms, such as Shewanella oneidensis and Desulfovibrio vulgaris, do not appear to contain nifD, suggesting that they do not have the adaptive feature of nitrogen fixation.
In summary, these results suggest that, in addition to the 16S rRNA gene, a number of other gene sequences may be useful for analysing the distribution of sequences from geobacteraceae in subsurface environments. Such studies may further aid in understanding why micro-organisms in this family are such successful competitors over other Fe(III)-reducing micro-organisms in many subsurface environments.
Description of Geobacteraceae fam. nov.
Geobacteraceae (Ge.o.bac'te.ra.ce.ae. N.L. masc. n. Geobacter the type genus of the family; -aceae ending to denote a family; N.L. neut. pl. n. Geobacteraceae the Geobacter family).
The family Geobacteraceae is located within the domain Bacteria and the -subclass of the Proteobacteria. This family branches phylogenetically between the orders ‘Desulfovibrionales’ and ‘Desulfobacterales’. The family Geobacteraceae comprises five genera: Geobacter, Desulfuromonas, Desulfuromusa, Pelobacter and Malonomonas. The type genus is Geobacter.
Micro-organisms within this family have mostly been isolated from anoxic sedimentary environments. All species of the Geobacteraceae are able to reduce Fe(III) and some are able to utilize S0 and/or the humics analogue AQDS as terminal electron acceptors. With the exception of Pelobacter and Malonomonas, all genera of the Geobacteraceae contain species that can oxidize acetate and other multi-carbon organic substrates to carbon dioxide. This family includes species with rod-like morphology, and members of all of the genera within this family, except Pelobacter, contain abundant c-type cytochromes. In addition, cells of members of the Geobacteraceae are non-motile or are motile by means of flagella.
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ACKNOWLEDGEMENTS |
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