Molecular analysis of the diversity of sulfate-reducing and sulfur-oxidizing prokaryotes in the environment, using aprA as functional marker gene

Abstract

The dissimilatory adenosine-5'-phosphosulfate reductase is a key enzyme of the microbial sulfate reduction and sulfur oxidation processes. Because the alpha- and beta-subunit-encoding genes, aprBA, are highly conserved among sulfate-reducing and sulfur-oxidizing prokaryotes, they are most suitable for molecular profiling of the microbial community structure of the sulfur cycle in environment. In this study, a new aprA gene-targeting assay using a combination of PCR and denaturing gradient gel electrophoresis is presented. The screening of sulfate-reducing and sulfur-oxidizing reference strains as well as the analyses of environmental DNA from diverse habitats (e.g., microbial mats, invertebrate tissue, marine and estuarine sediments, and filtered hydrothermal water) by the new primer pair revealed an improved microbial diversity coverage and less-pronounced template-to-PCR product bias in direct comparison to those of the previously published primer set (B. Deplancke, K. R. Hristova, H. A. Oakley, V. J. McCracken, R. Aminov, R. I. Mackie, and H. R. Gaskins, Appl. Environ. Microbiol. 66:2166-2174, 2000). The concomitant molecular detection of sulfate-reducing and sulfur-oxidizing prokaryotes was confirmed. The new assay was applied in comparison with the 16S rRNA gene-based analysis to investigate the microbial diversity of the sulfur cycle in sediment, seawater, and manganese crust samples from four study sites in the area of the Lesser Antilles volcanic arc, Caribbean Sea (Caribflux project). The aprA gene-based approach revealed putative sulfur-oxidizing Alphaproteobacteria of chemolithoheterotrophic lifestyle to have been abundant in the nonhydrothermal sediment and water column. In contrast, the sulfur-based microbial community that inhabited the surface of the volcanic manganese crust was more complex, consisting predominantly of putative chemolithoautotrophic sulfur oxidizers of the Betaproteobacteria and Gammaproteobacteria.

FIG. 1.

Preferential amplification of aprA gene fragments from defined genomic DNA ratio mixtures of the four SRP species Desulfosarcina variabilis (DSM 2060), Desulfobacter sp. (DSM 2035), Desulfovibrio profundus (DSM 11384), and Desulfotomaculum thermobenzoicum (6193) by the primer sets used for DGGE analysis. (A) Primer set AprA-1-FW/AprA-5-RV (GC clamp). (B) Primer set AprA-3-FW/APS-RV (GC clamp). In the PCR assays, the DNA concentration of each SRB species was varied to mixing ratios of 1:1, 1:0.1, or 1:0.01.

FIG. 2.

Phylogenetic tree based on 411 SRP/SOB reference strain and environmentally derived partial AprA sequences. The tree was inferred using the maximum-likelihood method. Sequences of the SOB Apr lineage I and Pyrobaculum aerophilum were used as outgroup references. The 16S rRNA gene-based taxonomic classification of SOB and SRP reference strains is indicated. The environmental AprA sequences are highlighted in boldface type and colored according to the habitat they were retrieved from (Spiekeroog Island, orange [Spkg]; Toyoha Mine, red [MatA/B/D]; Hydrate Ridge, green [HR28Begg]; Echinodermata cordatum, blue [Ec2, -4, -9]; Mono Lake, yellow [HS2, -3, -7, -8]; Vilm Island, brown; Vail, violet). The environmental AprA sequences received with primer set AprA-3-FW/APS-RV are termed with a “D” while those obtained with the new primer set AprA-1-FW/AprA-5-RV are termed with a “N” in the sequence name. For comparison of the microbial diversity coverage of both aprA gene-targeting primer pairs, identical phylotypes detected with both primer pairs are marked with open boxes, whereas those phylotypes that were obtained with only one primer pair are highlighted by dark gray (AprA-3-FW/APS-RV) and light gray (AprA-1-FW/AprA-5-RV) shaded boxes. The scale bar corresponds to 10% estimated sequence divergence.

FIG. 2.

Phylogenetic tree based on 411 SRP/SOB reference strain and environmentally derived partial AprA sequences. The tree was inferred using the maximum-likelihood method. Sequences of the SOB Apr lineage I and Pyrobaculum aerophilum were used as outgroup references. The 16S rRNA gene-based taxonomic classification of SOB and SRP reference strains is indicated. The environmental AprA sequences are highlighted in boldface type and colored according to the habitat they were retrieved from (Spiekeroog Island, orange [Spkg]; Toyoha Mine, red [MatA/B/D]; Hydrate Ridge, green [HR28Begg]; Echinodermata cordatum, blue [Ec2, -4, -9]; Mono Lake, yellow [HS2, -3, -7, -8]; Vilm Island, brown; Vail, violet). The environmental AprA sequences received with primer set AprA-3-FW/APS-RV are termed with a “D” while those obtained with the new primer set AprA-1-FW/AprA-5-RV are termed with a “N” in the sequence name. For comparison of the microbial diversity coverage of both aprA gene-targeting primer pairs, identical phylotypes detected with both primer pairs are marked with open boxes, whereas those phylotypes that were obtained with only one primer pair are highlighted by dark gray (AprA-3-FW/APS-RV) and light gray (AprA-1-FW/AprA-5-RV) shaded boxes. The scale bar corresponds to 10% estimated sequence divergence.

FIG. 3.

DGGE analysis of aprA gene fragments (amplified with primer pair AprA-1-FW/AprA-5-RV) using DNA samples from sediment (sed.), manganese crust, and seawater of the Caribbean Sea as templates. Lanes 1 to 8, DNA samples from Kahouanne Basin sediment (horizon 0 to 2 cm, lanes 1 to 4; horizon 4 to 6 cm, lanes 5 to 8); lanes 9 to 12, DNA samples from Montserrat Ridge sediment (horizon 0 to 2 cm); lanes 13 to 16, DNA samples from Montserrat Ridge manganese crust; lanes 17 to 19, DNA samples from aerobic sulfur oxidizer enrichment (enrichm.) cultures inoculated with manganese crust; lanes 20 and 21, DNA samples from St. Lucia Bay filtered (filt.) seawater. Bands indicated with numbers were excised from the gels and sequences.

FIG. 4.

Phylogenetic tree based on 411 SRP/SOB reference strain and environmentally derived partial AprA sequences. The tree was inferred using the maximum-likelihood method. Sequences of the Apr lineage I and Pyrobaculum aerophilum were used as outgroup references. The environmental AprA sequences of sediment, seawater, and manganese crust samples of the Caribbean Sea are highlighted in boldface type (for abbreviations used for study sites and enrichment cultures, see Table 1 and Table 3). The 16S rRNA gene-based taxonomic classification of SOB and SRP reference strains is indicated. The scale bar corresponds to 10% estimated sequence divergence.