Microarray and functional gene analyses of sulfate-reducing prokaryotes in low-sulfate, acidic fens reveal cooccurrence of recognized genera and novel lineages

Abstract

Low-sulfate, acidic (approximately pH 4) fens in the Lehstenbach catchment in the Fichtelgebirge mountains in Germany are unusual habitats for sulfate-reducing prokaryotes (SRPs) that have been postulated to facilitate the retention of sulfur and protons in these ecosystems. Despite the low in situ availability of sulfate (concentration in the soil solution, 20 to 200 microM) and the acidic conditions (soil and soil solution pHs, approximately 4 and 5, respectively), the upper peat layers of the soils from two fens (Schlöppnerbrunnen I and II) of this catchment displayed significant sulfate-reducing capacities. 16S rRNA gene-based oligonucleotide microarray analyses revealed stable diversity patterns for recognized SRPs in the upper 30 cm of both fens. Members of the family "Syntrophobacteraceae" were detected in both fens, while signals specific for the genus Desulfomonile were observed only in soils from Schlöppnerbrunnen I. These results were confirmed and extended by comparative analyses of environmentally retrieved 16S rRNA and dissimilatory (bi)sulfite reductase (dsrAB) gene sequences; dsrAB sequences from Desulfobacca-like SRPs, which were not identified by microarray analysis, were obtained from both fens. Hypotheses concerning the ecophysiological role of these three SRP groups in the fens were formulated based on the known physiological properties of their cultured relatives. In addition to these recognized SRP lineages, six novel dsrAB types that were phylogenetically unrelated to all known SRPs were detected in the fens. These dsrAB sequences had no features indicative of pseudogenes and likely represent novel, deeply branching, sulfate- or sulfite-reducing prokaryotes that are specialized colonists of low-sulfate habitats.

FIG. 1.

Consumption of supplemental sulfate (500 μM) in anoxic microcosms of soil obtained from Schlöppnerbrunnen I (A) and II (B). The values are averages ± standard deviations for triplicate determinations.

FIG. 2.

Effect of the consumption of supplemental sulfate (500 μM) on the production of methane in anoxic microcosms of soil obtained from Schlöppnerbrunnen I. The values are averages ± standard deviations for triplicate determinations. Open symbols, methane production with supplemental sulfate; solid symbols, controls (no sulfate added); circles, 0 to 10 cm, triangles, 10 to 20 cm; squares, 20 to 30 cm.

FIG. 3.

(A) Use of SRP-PhyloChip for surveys of SRP diversity at four different depths at Schlöppnerbrunnen I. Each probe was spotted in duplicate. The specificity and microarray position of each probe have been described previously (44). Probe spots having a signal-to-noise ratios equal to or greater than 2.0 are indicated by boldface boxes and were considered to be positive. The dotted boldface boxes indicate that only one of the duplicate spots had a signal-to-noise ratio equal to or greater than 2.0. (B) Flow chart illustrating the presence of distinct SRP groups in Schlöppnerbrunnen I as inferred from positive signals for sets of probes with nested and/or parallel specificity. For each probe the position on the microarray is indicated by a superscript.

FIG. 4.

16S rRNA gene phylogenetic consensus tree based on neighbor-joining analysis performed with a 50% conservation filter for the Deltaproteobacteria. The tree shows the affiliations of clone sequences from Schlöppnerbrunnen I and II soils (indicated by boldface type). Bar = 10% estimated sequence divergence. Polytomic nodes connect branches for which a relative order could not be determined unambiguously by applying distance matrix, maximum-parsimony, and maximum-likelihood treeing methods. Parsimony bootstrap values for branches are indicated by solid circles (>90%) and open circles (75 to 90%). Branches without circles had bootstrap values of less than 75%. Brackets indicate the perfect-match target organisms of the probes. The microarray position is indicated by a superscript after each probe designation. Cadagno Lake clones were not sequenced at the target site for probe DSMON1421. TCB, trichlorobenzene.

FIG. 5.

(A) Use of SRP-PhyloChip for surveys of SRP diversity at four different depths at Schlöppnerbrunnen II. See the legend to Fig. 3 for additional details. (B) Flow chart illustrating the presence of distinct SRP groups in Schlöppnerbrunnen II soil as inferred from positive signals for sets of probes with nested specificity. For each probe the position on the microarray is indicated by a superscript. The asterisk indicates that the mean signal-to-noise ratios of the duplicate SYBAC986 spots for 7.5 to 15, 15 to 22.5, and 22.5 to 30 cm were just below the threshold value of 2.0 (1.88, 1.95, and 1.70, respectively).

FIG. 6.

Phylogenetic consensus tree (based on FITCH analysis) for DsrAB amino acid sequences deduced from dsrAB sequences longer than 1,750 bases, showing the affiliation of OTUs from Schlöppnerbrunnen fen soils (indicated by boldface type). DsrAB sequences deduced from dsrAB sequences shorter than 1,750 bases (indicated by dashed branches) were individually added to the distance matrix tree without changing the overall tree topology by using the ARB treeing tool PARSIMONY_INTERAKTIV. Bar = 10% estimated sequence divergence. See the legend to Fig. 4 for additional details.