Identity of major sulfur-cycle prokaryotes in freshwater lake ecosystems revealed by a comprehensive phylogenetic study of the dissimilatory adenylylsulfate reductase

Abstract

Adenylylsulfate reductase is a heterodimeric complex of two subunits, AprB and AprA, and is a key enzyme in dissimilatory sulfate reduction and sulfur oxidation. Common use of aprA as a functional marker gene has revealed the diversity of sulfur-cycle prokaryotes in diverse environments. In this study, we established a comprehensive sequence set of apr genes and employed it to reanalyze apr phylogeny, evaluate the coverage of a widely used primer set (AprA-1-FW/AprA-5-RV), and categorize environmental aprA sequences. Phylogenetic tree construction revealed new members of Apr lineage II and several previously unrecognized lateral gene transfer events. Using the established phylogenetic tree, we classified all previously reported aprA sequences amplified from freshwater lakes with the primer pair AprA-1-FW/AprA-5-RV in addition to the aprA sequences newly retrieved from freshwater lakes; the obtained results were complemented by 16S rRNA clone library analysis. Apr-based classifications of some of operational taxonomic units were supported by 16S rRNA-based analysis. This study updates our knowledge on the phylogeny of aprBA and shows the identities of several sulfur-cycle bacteria, which could not be classified to a known taxa until now. The established apr sequence set is publicly available and can be applied to assign environmental sequences to known lineages.

Figure 1. Consensus phylogenetic tree of AprBA sequences.

The displayed tree is a maximum likelihood tree constructed based on 320 AprBA sequences of the core database. Remaining 154 sequences of sorter AprA or AprBA and 225 AprA sequences of OTUs obtained in this study were added to the consensus tree by the parsimony interactive tool in ARB, and were included in the clusters. Bootstrap values ≥50% for ML (first) and MP (second) based on 100 resamplings are indicated near the branches. Uncultured marine group II/III euryarchaeote KM3_67_G08 was obtained from a metagenomic library of deep-Mediterranean water.

Figure 2. Distribution of clone number per each phylogenetic group defined according to the AprBA consensus tree.

Backgrounds of clone numbers are colored according to relative abundances of sulfate reducer-related clones (blue) and sulfur oxidizer-related clones (red) per respective total clone numbers in each library. Group numbers are corresponding to dashed-line boxes in Figure S3. The groups Ox3 and Ox6 contain the clones named as “uncultured Thiobacillus spp.” (according to the GenBank record), but evidence to assign these clones to Thiobacillus have not been reported. Therefore, Ox3 and Ox6 are most likely to be Sulfuriferula and Sulfuritalea, respectively. Miscellaneous: 1, partial AprA of Desulfofaba gelida DSM 12344, Desulfonema ishimotonii DSM 9680 and symbiont of Olavius algarvensis; 2, full-length AprBA of Desulfococcus oleovorans Hxd3 and partial AprA of Desulfofaba fastidiosa; 3, full-length AprBA of Desulfotalea psychrophila LSv54 and partial AprBA of Desulfobulbus sp. LB2 and Desulforhopalus vacuolatus DSM 9700; 4, full-length AprBA (lineage II) of Thiobacillus thioparus DSM 505, T. denitrificans DSM 12475, T. denitrificans DSM 739 and partial AprA (lineage II) of uncultured Thiobacillus sp. clone and endosymbionts of Oligobrachia mashikoi.