Genetic Mechanisms of Bacterial Biodegradation Pathways
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G C A T T A C G G C A T genes Review Steroids as Environmental Compounds Recalcitrant to Degradation: Genetic Mechanisms of Bacterial Biodegradation Pathways Elías R. Olivera * and José M. Luengo Departamento Biología Molecular (Área Bioquímica y Biología Molecular), Universidad de León, 24007 León, Spain * Correspondence: [email protected]; Tel.: +34-987-29-1229 Received: 2 June 2019; Accepted: 3 July 2019; Published: 6 July 2019 Abstract: Steroids are perhydro-1,2-cyclopentanophenanthrene derivatives that are almost exclusively synthesised by eukaryotic organisms. Since the start of the Anthropocene, the presence of these molecules, as well as related synthetic compounds (ethinylestradiol, dexamethasone, and others), has increased in different habitats due to farm and municipal effluents and discharge from the pharmaceutical industry. In addition, the highly hydrophobic nature of these molecules, as well as the absence of functional groups, makes them highly resistant to biodegradation. However, some environmental bacteria are able to modify or mineralise these compounds. Although steroid-metabolising bacteria have been isolated since the beginning of the 20th century, the genetics and catabolic pathways used have only been characterised in model organisms in the last few decades. Here, the metabolic alternatives used by different bacteria to metabolise steroids (e.g., cholesterol, bile acids, testosterone, and other steroid hormones), as well as the organisation and conservation of the genes involved, are reviewed. Keywords: sterols; bile acids; steroid hormones; biodegradation; 9,10-seco pathway; 4,5-seco pathway; 2,3-seco pathway 1. Introduction Steroids are tetracyclic triterpenoid lipids containing a perhydro-1,2-cyclopentanophenanthrene structure, and include sterols, bile acids, steroid hormones, cardenolides, sapogenins, saponins, and vitamin D derivatives. Classically, they have been studied based on their physiological role. In mammals, the steroid hormones, bile acids, and other essential steroids are produced from cholesterol (Figure1), a molecule that is also an important component of membranes, maintaining their fluidity, and participating in cell differentiation and proliferation. The major constituents of plant sterols are sitosterol, stigmasterol, campesterol, and brassicasterol, while in yeast and filamentous fungi, ergosterol is an important component of cell walls [1–3]. It is generally accepted that the biosynthesis of steroids is one of the hallmarks of the evolutionary progression of eukaryotes [4]. However, reports on the biosynthesis of sterols and modified sterols in methanotrophic bacteria (Methylococcus capsulatus, Methylosphaera hansonii, and Methylobacterium organophilum), Gemmata obscuriglobus, Eudoraea adriatica, and in a variety of myxobacteria have been published, though there is limited information about the metabolic machinery involved [5–15]. Genes 2019, 10, 512; doi:10.3390/genes10070512 www.mdpi.com/journal/genes Genes 2019, 10, 512 2 of 34 Genes 2019, 10, x 2 of 35 FigureFigure 1. Chemical 1. Chemical structure structure of of cholesterol cholesterol andand some of of its its mammalian mammalian derivatives, derivatives, and andselected selected syntheticsynthetic steroids. steroids. FromFrom an ecological an ecological point point of view,of view, the the release release into into the the environment environment of of vertebrate vertebrate steroids, steroids, such such as androgens,as androgens, estrogens, estrogens, progestogens, progestogens, cholesterol, cholesterol, and and bile bile acids, acids, during during excreta or or decomposition decomposition of biomassof biomass is constantly is constantly occurring occurring [16 –[1622–].22]. In In addition, addition, notnot only natural natural steroids steroids are are released. released. Along Along withwith antibiotics, antibiotics, synthetic synthetic steroids steroids represent represent aa significantsignificant sector sector of of the the glob globalal pharmaceutical pharmaceutical market, market, and theyand they have have found found their their way way into into environmental environmental niches, niches, as as in in the the case case of ofsynthetic synthetic hormones hormones (ethynylestradiol and anti-inflammatory drugs like dexamethasone) (Figure 1) [19,20,23–27]. With (ethynylestradiol and anti-inflammatory drugs like dexamethasone) (Figure1)[ 19,20,23–27]. With their widespread presence, they can affect endocrine activity, even at low concentrations, with their widespread presence, they can affect endocrine activity, even at low concentrations, with potential potential adverse effects for both the environment and human health. As such, there is an increasing adverseneed e ffforects new for approaches both the environmentin the bioremedia andtion human of steroids health. from As the such, environment. there is an increasing need for new approachesAlthough in steroid the bioremediation synthesis is mostly of steroids restricted from to eukaryotic the environment. organisms [4], the biodegradation Althoughof these compounds steroid synthesis seems to isbe mostly carried restricted out exclus toively eukaryotic by bacteria. organisms Even so, [steroids4], the biodegradation are highly of theserecalcitrant compounds to microbial seems degradation to be carried because out of exclusively the low number by bacteria. of functional Even groups so, steroidspresent in are their highly recalcitrantstructure to and microbial their extremely degradation low solubility because in of wate ther. low Nevertheless, number of the functional persistence groups of these present molecules in their structurein the and environment their extremely has triggered low solubility a response in water.by some Nevertheless, bacteria, either the efficiently persistence detoxifying of these moleculesthese in thehydrophobic environment molecules has triggered or metabolising a response their carbon by some and bacteria, energy-rich either scaffold. efficiently Thus, the detoxifying isolation of these microorganisms able to degrade or modify steroids has been reported from soil [28–30], and from hydrophobic molecules or metabolising their carbon and energy-rich scaffold. Thus, the isolation of freshwater [31] and marine [32–34] environments. This indicates the importance of bacteria in microorganisms able to degrade or modify steroids has been reported from soil [28–30], and from reducing the adverse impact of environmentally released steroids, as well as emphasising their freshwaterimportance [31]and in introducing marine [32 –these34] environments. molecules into Thisthe carbon indicates cycle. the Moreover, importance for of some bacteria pathogenic in reducing the adversebacteria, impact such as ofMycobacterium environmentally tuberculosis released and Rhodococcus steroids, as equii well, catabolism as emphasising of cholesterol their importance has been in introducingidentified these as a trait molecules involved into in their the carbonpathogenicity cycle. and Moreover, persistence for in some the host pathogenic [35,36]. bacteria, such as MycobacteriumIn 1913, tuberculosis Söhngen reportedand Rhodococcus that a Mycobacterium equii, catabolism strain ofcould cholesterol use phytosterols has been as identified a sole carbon as a trait involvedand energy in their source pathogenicity [37]. Thereafter, and persistence many bacterial in thestrains host have [35 ,been36]. isolated that are able to degrade Insteroids, 1913, Söhngenincluding reported species thatinitially a Mycobacterium classified as strainbelonging could to usegenera phytosterols Mycobacterium, as a sole Gordonia, carbon and energyTsukamurella, source [37]. Rhodococcus, Thereafter, Azotobacter, many bacterial Nocardia, strains Flavobacterium, have been isolated Arthrobacter, that areBacillus, able toBrevibacterium, degrade steroids, including species initially classified as belonging to genera Mycobacterium, Gordonia, Tsukamurella, Rhodococcus, Azotobacter, Nocardia, Flavobacterium, Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Streptomyces, Microbacterium, Serratia, Achromobacter, Protaminobacter, and Pseudomonas [29,38–54]. Genes 2019, 10, 512 3 of 34 However, it has not been until the last two decades of the 20th century that studies have been initiated to elucidate the physiological and metabolical determinants, and the mechanisms involved in the aerobic catabolism of different steroid molecules. These studies have included sterols, chiefly cholesterol, testosterone and 17β-estradiol, and bile acids, mainly cholic acid, using selected model organisms from Actinobacteria, comprising different strains of Mycobacterium, Rhodococcus, and Gordonia [36,55–59] (Table1), and some selected Gram negative strains, the α-proteobacteria Comamonas testosteroni and a few Pseudomonas strains [28,29,60–62]. In addition, the catabolism of steroids under anoxic conditions has been studied in Sterolibacterium (β-proteobacteria) and in Steroidobacter (U-proteobacteria) [63–65] (Table1). Table 1. Model strains widely used in the studies that have allowed the characterization of the metabolic mechanisms used for steroid degradation. Steroids used in these studies are also indicated. Strain Steroids Used Reference Genome tuberculosis H37Rv Cholesterol NC_000962 Mycobacterium smegmatis mc2 155 Cholesterol NC_008596 Cholesterol β-sitosterol neoaurum ATCC 25795 NZ_JMDW00000000 Stigmasterol Campesterol equi 103S Cholesterol NC_014659 Rhodococcus Cholesterol rhodochrous DSM43269 β-sitosterol unpublished Campesterol