**1. Introduction**

Endocrine disruptors (EDCs) are chemicals that interfere with the endocrine system and produce adverse effects in both humans and wildlife. Numerous studies have reported the feminization and/or masculinization of freshwater wildlife exposed to estrogens and/or androgens in polluted rivers [1]. Natural estrogens and androgens enter the environment through the excretions of humans, domestic or farm animals, and wildlife. 17β-estradiol (E2) (estrogen) and testosterone (TES) (androgen), are the most ubiquitously sexual hormones found as pollutants in soil and water systems [2–5]. These compounds contaminate the waste water treatment plant effluents and occur at low concentration (ng/L to μg/L) [5,6]. Microbial degradation is a crucial mechanism to eliminate steroid hormones from contaminated systems and the persistence and fate of TES and E2 have been studied previously [3,5] showing that only 6% of E2 and 63% of TES could be mineralized in native soils under aerobic conditions. Similar results were obtained in water treatment plants [4]. This indicates that either a limited number of organisms can mineralize E2 [7–14] (Table S1) or that this compound is mineralized at lower rates compared to TES [15].

Although some bacteria are able to partially degrade/transform E2, only few of them, isolated mainly during the last 20 years, have been described to be able to completely metabolize E2 and use it as a sole carbon and energy source, either aerobically or anaerobically [15] (Table S1). In some cases, these bacteria need to grow in a rich medium to metabolize E2 (e.g., *Vibrio* sp. strain H5 [16] and

*Buttiauxella* sp. strain S19-1 [17]). In other cases, a defined mixed culture of co-degraders are required to eliminate this compound (e.g., *Achromobacter xylosoxidans* and *Ralstonia* sp.) [8]. Nevertheless, there are only few bacteria described so far that can catabolize both E2 and TES [16–23] (Table S1).

*N. tardaugens* NBRC 16725 (strain ARI-1) is a Gram-negative, aerobic, rod-shaped and non-motile α-Proteobacterium isolated from a sewage treatment plant in Tokyo [7,24]. It was isolated due to its capacity to mineralize E2 and it has been used when immobilized in alginate to remove estrogens from sewage and cow dung [25]. Nevertheless, nobody has described so far its capacity to degrade TES or other androgens.

The complete oxic mineralization of TES has been studied in detail in *Comamonas testosteroni* [26,27] (Figure 1). The aerobic catabolism of TES is initiated by dehydrogenation of the 17β-hydroxyl group to produce androst-4-en-3,17-dione (AD), which undergoes a further dehydrogenation to form androsta-1,4-diene-3,17-dione (ADD). The subsequent cleavage of the core ring system is catalysed by several oxygenases that utilize oxygen as co-substrate [26,28] (Figure 1).

**Figure 1.** Proposed TES degradation pathway in *N. tardaugens*. Compound names are shown or indicated with an abbreviation: (I) androst-5-ene-3,17-dione; (3-HSA) 3-hydroxy-9,10-secoandrosta-1,3,5(10)- triene-9,17-dione; (3,4-DHSA) 3,4-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione; (4,9-DHSA) 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-diene-4-oic acid; (II) 2-hydroxyhexa-2,4 -dienoate; (III) 4-hydroxy-2-oxohexanoate; (HIP) 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid CoA ester; (HIPE-CoA) 9-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid; (9OH-HIC-CoA) 9α-hydroxy-17-oxo-1,2,3,4,5,6,10,19-octanorandrostan-7-oic acid, (HIEC-CoA) 9,17-dioxo-1,2,3,4,5,6,10, 19-octanorandrost-8(14)-en-7-oic acid; (COCHEA-CoA) 9-oxo-1,2,3,4,5,6,10,19-octanor-13,17-secoandrost -8(14)-ene-7,17-dioic acid; (IV) 4-methyl-5-oxo-octane-1,8-dioic acid; and (MOODA-CoA) 4-methyl-5- oxo-oct-2-ene-1,8-dioic acid. Enzyme names are: (3,17β-*hsd*) 3,17β-hydroxysteroid dehydrogenase; (Δ5,3-*ksi*) 3-ketosteroid Δ4(5)-isomerase; (*kstD*) 3-ketosteroid-delta1-dehydrogenase; (*kshAB*) 3-ketosteroid 9alpha-hydroxylase; (*tesA1A2*) 3-HSA hydroxylase; (*tesB*) 3,4-DHSA 4,5-dioxygenase; (*tesE*) 2-hydroxyhexa-2,4-dienoate hydratase; (*tesG*) 4-hydroxy-2-oxovalerate aldolase; (*tesF*) propionaldehyde

dehydrogenase; (*tesD*) 4,9-DHSA hydrolase; (*ORF18 scdA*) HIP-CoA ligase; [*IpdF*] 5-oxo HIC-CoA oxidase; (*ORF28 ORF30 scdC1C2*) acyl-CoA dehydrogenase; (*ORF32 scdD*) enoyl-CoA hydratase; (*ORF27 scdE*) dehydrogenase; (*ORF23 scdF*) CoA acetyl transferase; (*ORF31 scdG*) hydroxylacyl dehydrogenase; (*ORF4 scdK*) acyl-CoA dehydrogenase; (*ORF5 scdY*) enoyl-CoA hydratase; (*ORF1,2 scdL1L2*) β -ketoacyl-CoA-transferase; and (*ORF3 scdN*) CoA-hydratase. The proposed catabolic genes from *N. tardaugens* are indicated in italics by their locus code (*EGO55\_XXXX*), in green other possible candidates are considered. The homologous genes from model bacteria *C. testosteroni* TA441 and *M. tuberculosis* H37Rv are shown in brackets and square brackets, respectively.

In this work, we have analysed the growth of *N. tardaugens* NBRC 16725 (strain ARI-1) with TES and other C-19 steroids as the sole carbon and energy sources. The complete genome sequence of this bacterium has been recently reported [29] allowing us to predict a SD gene cluster presumably involved in the catabolism of TES and other C-19 steroids. We have determined the expression of the catabolic genes by using a transcriptomic approach. The enzyme 17β-hydroxysteroid dehydrogenase, encoded outside of the predicted SD cluster, involved in the first step of TES catabolism in *N. tardaugens* ARI-1, has been identified by biochemical analyses and a metabolic pathway for the degradation of TES in this strain has been proposed.

#### **2. Materials and Methods**
