Integrated multi-omics analyses reveal the biochemical mechanisms and phylogenetic relevance of anaerobic androgen biodegradation in the environment

Abstract

Steroid hormones, such as androgens, are common surface-water contaminants. However, literature on the ecophysiological relevance of steroid-degrading organisms in the environment, particularly in anoxic ecosystems, is extremely limited. We previously reported that Steroidobacter denitrificans anaerobically degrades androgens through the 2,3-seco pathway. In this study, the genome of Sdo. denitrificans was completely sequenced. Transcriptomic data revealed gene clusters that were distinctly expressed during anaerobic growth on testosterone. We isolated and characterized the bifunctional 1-testosterone hydratase/dehydrogenase, which is essential for anaerobic degradation of steroid A-ring. Because of apparent substrate preference of this molybdoenzyme, corresponding genes, along with the signature metabolites of the 2,3-seco pathway, were used as biomarkers to investigate androgen biodegradation in the largest sewage treatment plant in Taipei, Taiwan. Androgen metabolite analysis indicated that denitrifying bacteria in anoxic sewage use the 2,3-seco pathway to degrade androgens. Metagenomic analysis and PCR-based functional assays showed androgen degradation in anoxic sewage by Thauera spp. through the action of 1-testosterone hydratase/dehydrogenase. Our integrative 'omics' approach can be used for culture-independent investigations of the microbial degradation of structurally complex compounds where isotope-labeled substrates are not easily available.

Figure 1

Proposed catabolic pathways of testosterone. (a) Aerobic 9,10-seco pathway established in Comamonas testosteroni TA441. (b) Anaerobic 2,3-seco pathway established in Steroidobacter denitrificans DSMZ18526. The compound in bracket is presumed. Suggested signature metabolites are enclosed in boxes.

Figure 2

(a) Structure and distribution of the putative catabolic genes for androgen degradation on the chromosome of Sdo. denitrificans. The genes involved in molybdopterin biosynthesis and molybdate transport are also shown. Proposed gene products are enclosed in gray boxes. (b) Genes coding for a steroid C25 dehydrogenase-like enzyme are found in the genomes of Sdo. denitrificans, Stl. denitrificans and Thauera terpenica. The latter was identified as an anaerobic androgen-degrader in this study. Homologous open reading frames (colored arrows) between different bacterial strains are connected with dotted lines. Numbers (%) indicate the identity of the deduced amino acid sequences with those of the genes of Sdo. denitrificans. (c) Genes encoding β-oxidation enzymes are conserved in androgen-degrading anaerobes and aerobes (e.g., C. testosteroni). The numbers (%) indicate the identity of the amino acid sequences with those of the functionally confirmed genes of C. testosteroni TA441. For the detailed catabolic functions of individual genes of the strain TA441, refer Horinouchi et al. (2012). (d) Genes coding for the molybdoproteins of the xanthine oxidase family. The numbers (%) indicate the identity of deduced amino acid sequences with those of the corresponding genes of Sdo. denitrificans, whose product was characterized as 1-testosterone hydratase/dehydrogenase in this study.

Figure 3

Global gene expression profiles (RNA-Seq) of Sdo. denitrificans grown under different conditions. (a) The COG classification of the upregulated (greater than twofold) genes during anaerobic growth on testosterone, compared with anaerobic growth on heptanoic acid and aerobic growth on testosterone. Note that some genes are assigned into multiple COG groups. (b) Aerobic versus anaerobic growth on testosterone. (c) Anaerobic growth on testosterone and heptanoic acid. Each spot represents a gene. Diagonal lines indicate the identical thresholds. The house keeping genes include glucose-6-phosphate 1-dehydrogenase (ACG33_00890), chaperone htpG (ACG33_01365), isocitrate dehydrogenase (ACG33_02930), enolase (ACG33_09515), pyruvate dehydrogenase (ACG33_11195), malate dehydrogenase (ACG33_14070) and type II citrate synthase (ACG33_14495).

Figure 4

Purification and characterization of 1-testosterone hydratase/dehydrogenase (AtcABC) from Sdo. denitrificans grown anaerobically on testosterone. (a) SDS–polyacrylamide gel electrophoresis (4% to 12%) of active pools during the fast protein liquid chromatography purification of AtcABC. (b) APCI-MS spectrum of the reaction product 17-hydroxy-androstan-1,3-dione. (c) Substrate preference of AtcABC determined using a DCPIP-based enzyme assay. The assay mixtures (0.9 ml) contained 20 mm Tris-HCl (pH 7.5), 5 μg purified enzyme, 50 μm DCPIP, 125 μm of individual substrates and 1.25% 2-propanol. 2-Propanol was used as the solvent. Abbreviations: DEAE, DEAE sepharose; Butyl, butyl sepharose; Sephacryl, Sephacryl S-300.

Figure 5

Phylogenetic tree of the members of the xanthine oxidase family based on the amino acid sequences of molybdopterin-containing subunits. Supplementary Table S3 provides detailed information on these molybdoenzymes. The phylogenetic trees were constructed using the neighbor-joining method with Jukes–Cantor parameter and a bootstrap value of 1000. Abbreviations: CHO, aldehyde; CO, carbon monoxide; DH, dehydrogenase; 1-t, 1-testosterone.

Figure 6

UPLC-APCI-MS/MS analysis of the ethyl acetate extracts of the anoxic DHSTP sewage treatments. (a) Total ion chromatograms of anoxic sewage incubated with testosterone but without nitrate. (b) Total ion chromatograms of anoxic sewage incubated with testosterone and nitrate. (c) Extracted ion chromatograms (m/z=305.21 for 2,3-SAOA) of anoxic sewage incubated with testosterone and nitrate. (d) MS/MS spectra of the authentic standard (top) and 2,3-SAOA extracted from anoxic sewage incubated with testosterone and nitrate (bottom). See Figure 1 for the abbreviations of androgens.

Figure 7

(a) Genus-level phylogenetic analysis (Illumina MiSeq) revealed the enrichment of Thauera spp. in anoxic sewage incubated with testosterone and nitrate. See Supplementary Figure S6 for detailed information. (b) PCR-based functional assay using degenerate primers (see Supplementary Figure S1 for sequences) derived from the atcA genes. (bI) Agarose gel electrophoresis showed that the atcA genes are harbored only by androgen-degrading anaerobes. (bII) Temporal increase in atcA-specific PCR products was observed only in anoxic DHSTP sewage incubated with testosterone and nitrate. (bIII) Neighbor-joining tree of atcA gene fragments obtained from anoxic DHSTP sewage incubated with testosterone and nitrate for 120 h. atcA-like sequences are shown in Supplementary Figure S8. The gene encoding the large subunit (MhyADHL) of 3-hydroxycyclohexanone dehydrogenase from Ali. denitrificans served as an outgroup sequence.