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Alignment of the amino acid sequences of the putative ACAD gene in Pseudomonas sp. strain Chol1 (Acad_Chol1) and its homologous ACAD gene in P. haloplanktis TAC 125 (PSHAa0888) performed with the software DNASTAR. Conserved residues are in boldface.
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Bile acids are surface-active steroid compounds with toxic effects for bacteria. Recently, the isolation and characterization of a bacterium, Pseudomonas sp. strain Chol1, growing with bile acids as the carbon and energy source was reported. In this study, initial reactions of the aerobic degradation pathway for the bile acid cholate were investiga...
Citations
... We also tested whether Nov2c221 and Nov2c222 might be able to take over the function of the second ACAD pair in P. stutzeri Chol1 by heterologous expression in P. stutzeri Chol1 R1, which is a transposon mutant defective in Scd2AB (16). However, expression of nov2c221 and nov2c222 in P. stutzeri Chol1 R1 did not restore growth and degradation of cholate (data not shown). ...
... The mass of 1,136.5 Da indicates that this compound is the CoA-ester of P4 (387 Da of P4 1 767.5 Da of CoA -18 Da H 2 O, which is removed for thioester formation) (Fig. 5B), and the mass of 1,134.5 Da accordingly indicates that (Fig. 5C). The UV-absorption spectra of these compounds are also indicative of CoAesters (16). Both CoA-esters were not formed when the cell extract was omitted and when cell extracts of E. coli empty vector controls were used. ...
... In cell suspensions supplied with androsta-1,4-diene-3,17-dione (ADD; XIX in Fig. S9), E. coli MG1655 pBBR1MCS-5::kshA Chol1 -kshB Chol1 cleaved the substrate and formed the 9,10-seco-steroid 3-hydroxy-9,10-secoandrosta-1,3,5-triene-9,17-dione (HSATD; XX in Fig. 8) (Fig. 8A). These reactions were not observed in suspensions of the vector control (Fig. 8B), indicating that this is a functional platform for observing the activities of KshAB complexes (16,17,22). To further explore the substrate spectrum of KshAB Chol1 , a variety of metabolites with the D 1,4 -3-keto structure, which had either no or different side chains, were tested in this platform. ...
The reaction sequence for aerobic degradation of bile salts by environmental bacteria resembles degradation of other steroid compounds. Recent findings show that bacteria belonging to the Sphingomonadaceae use a pathway variant for bile-salt degradation. This study addresses this so-called Δ 4,6 -variant by comparative analysis of unknown degradation steps in Sphingobium sp. strain Chol11 with known reactions found in Pseudomonas stutzeri Chol1. Investigations with strain Chol11 revealed an essential function of the acyl-CoA dehydrogenase Scd4AB for growth with bile salts. Growth of the scd4AB deletion mutant was restored with a metabolite containing a double bond within the side chain which was produced by the Δ ²² -acyl-CoA dehydrogenase Scd1AB from P. stutzeri Chol1. Expression of scd1AB in the scd4AB deletion mutant fully restored growth with bile salts while expression of scd4AB only enabled constricted growth in P. stutzeri Chol1 scd1A or scd1B deletion mutants. Strain Chol11 Δ scd4A accumulated hydroxylated steroid metabolites which were degraded and activated with coenzyme A by the wild type. Activities of five Rieske type monooxygenases of strain Chol11 were screened by heterologous expression and compared to the B-ring cleaving KshAB Chol1 from P. stutzeri Chol1. Three of the Chol11 enzymes catalyzed B-ring cleavage of only Δ 4,6 -steroids while KshAB Chol1 was more versatile. Expression of a fourth KshA homolog, Nov2c228 led to production of metabolites with hydroxylations at an unknown position. These results indicate functional diversity of β-proteobacterial enzymes for bile-salt degradation and suggest a novel side-chain degradation pathway involving an essential ACAD reaction and a steroid hydroxylation step.
Importance
This study highlights the biochemical diversity of bacterial degradation of steroid compounds in different aspects. First, it further elucidates an unexplored variant in the degradation of bile-salt side chains by Sphingomonads, a group of environmental bacteria that is well-known for their broad metabolic capabilities. Moreover, it adds a so-far unknown hydroxylation of steroids to the reactions Rieske monooxygenases can catalyze with steroids. Additionally, it analyzes a proteobacterial ketosteroid-9α-hydroxylase and shows that this enzyme is able to catalyze side reactions with non-native substrates.
... Aerobic bile salt degradation proceeds similarly to the degradation of other steroids such as cholesterol and can be divided into different phases ( Fig. 1) (7,8,15): (1) oxidation of the A-ring, (2) side chain degradation, (3) oxygenolytic cleavage of ring B, and (4) oxygenolytic and hydrolytic degradation of the remaining seco-steroid. The first three steps may occur simultaneously (16)(17)(18). ...
... In phase 1, oxidative reactions at the A-ring generate intermediates with a D 1,4 -3-keto structure of the steroid skeleton (7,13,14). During degradation of the trihydroxy bile salt model-substrate cholate (I in Fig. 1), this leads to formation of D 1,4 -3-ketocholate (III in Fig. 1) (16). In phase 2, the bile salt side chain is degraded by the successive release of acetyl coenzyme A (acetyl-CoA) and propionyl-CoA (16,19,20). ...
... During degradation of the trihydroxy bile salt model-substrate cholate (I in Fig. 1), this leads to formation of D 1,4 -3-ketocholate (III in Fig. 1) (16). In phase 2, the bile salt side chain is degraded by the successive release of acetyl coenzyme A (acetyl-CoA) and propionyl-CoA (16,19,20). In actinobacteria such as R. jostii RHA1, acetyl-CoA is predicted to be released by b-oxidation (13). ...
Bile salts are amphiphilic steroids chain with digestive functions in vertebrates. Upon excretion, bile salts are degraded by environmental bacteria. Degradation of the bile-salt steroid skeleton resembles the well-studied pathway for other steroids like testosterone, while specific differences occur during side-chain degradation and the initiating transformations of the steroid skeleton. Of the latter, two variants via either Δ 1,4 - or Δ 4,6 -3-ketostructures of the steroid skeleton exist for 7-hydroxy bile salts. While the Δ 1,4 - variant is well-known from many model organisms, the Δ 4,6 -variant involving a 7-hydroxysteroid dehydratase as key enzyme has not been systematically studied. Here, combined proteomic, bioinformatic and functional analyses of the Δ 4,6 -variant in Sphingobium sp. strain Chol11 were performed. They revealed a degradation of the steroid rings similar to the Δ 1,4 -variant except for the elimination of the 7-OH as a key difference. In contrast, differential production of the respective proteins revealed a putative gene cluster degradation of the C 5 carboxylic side chain encoding a CoA-ligase, an acyl-CoA dehydrogenase, a Rieske monooxygenase, and an amidase, but lacking most canonical genes known from other steroid-degrading bacteria. Bioinformatic analyses predicted the Δ 4,6 -variant to be widespread among the Sphingomonadaceae , which was verified for three type strains which also have the predicted side-chain degradation cluster. A second amidase in the side-chain degradation gene cluster of strain Chol11 was shown to cleave conjugated bile salts while having low similarity to known bile-salt hydrolases. This study signifies members of the Sphingomonadaceae remarkably well-adapted to the utilization of bile salts via a partially distinct metabolic pathway.
Importance
This study highlights the biochemical diversity of bacterial degradation of steroid compounds, in particular bile salts. Furthermore, it substantiates and advances knowledge of a variant pathway for degradation of steroids by sphingomonads, a group of environmental bacteria that are well-known for their broad metabolic capabilities. Biodegradation of bile salts is a critical process due to the high input of these compounds from manure into agricultural soils and wastewater treatment plants. In addition, these results may also be relevant for the biotechnological production of bile salts or other steroid compounds with pharmaceutical functions.
... All these strains belong to the Proteobacteria or Actinobacteria. Among the Proteobacteria, individual strains of the genera Sphingomonas, Sphingobium, Novosphingobium (α-Proteobacteria; ( [18,23])), Comamonas, Azoarcus, Zoogloea (β-Proteobacteria; [18,24,25]), Pseudomonas, Pseudoalteromonas and Shewanella (γ-Proteobacteria; [17,19,26,27]) have been identified to be able to degrade bile acids. Among the Actinobacteria, several members of the genus Rhodococcus [28,29], as well as individual strains from the genera Thermomonospora, Amycolatopsis [27], Dietzia [18], Gordonia [17], and Nocardia [30] are able to grow with and metabolize bile acids. ...
... For P. stutzeri Chol1 and Sphingobium sp. strain Chol11, NAD + dependent 3α-HSD activity has been detected in cell extracts with cholic acid [26,40]. The formation of 3-keto intermediates in other bile acid-degrading bacteria suggests that this step is conserved in aerobic and anaerobic bile acids degradation [25,62]. ...
... In analogy to the β-oxidation of fatty acids, coenzyme A (CoA) activation of the carboxylic group of free bile acids is the initial side-chain degradation step [26,35,41]. StdA1, a CoA-ligase encoded in the bile acid degradation gene cluster of P. putida DOC21 converted cholic acid and the degradation intermediates 3-ketocholic acid (IX), ∆ 1/4 -3-ketocholic acid (e.g., X) and ∆ 1,4 -3-ketocholic acid (VI) into the corresponding CoA-thioesters, while it did not activate bile acid derivatives with a C 3 side chain [35]. ...
Bile acids are surface-active steroid compounds with a C5 carboxylic side chain at the steroid nucleus. They are produced by vertebrates, mainly functioning as emulsifiers for lipophilic nutrients, as signaling compounds, and as an antimicrobial barrier in the duodenum. Upon excretion into soil and water, bile acids serve as carbon- and energy-rich growth substrates for diverse heterotrophic bacteria. Metabolic pathways for the degradation of bile acids are predominantly studied in individual strains of the genera Pseudomonas, Comamonas, Sphingobium, Azoarcus, and Rhodococcus. Bile acid degradation is initiated by oxidative reactions of the steroid skeleton at ring A and degradation of the carboxylic side chain before the steroid nucleus is broken down into central metabolic intermediates for biomass and energy production. This review summarizes the current biochemical and genetic knowledge on aerobic and anaerobic degradation of bile acids by soil and water bacteria. In addition, ecological and applied aspects are addressed, including resistance mechanisms against the toxic effects of bile acids.
... BaiCD and BaiH from C. scindens have been described to catalyze this reaction with specificity for 7α-hydroxy bile salts such as cholate or chenodeoxycholate and 7β-hydroxy bile salts such as ursodeoxycholate, respectively (Kang et al., 2008). Although 5β-Δ 4 -KSTD activity can easily be measured in cell extracts of bile-salt degrading bacteria P. stutzeri Chol1 (Birkenmaier et al., 2007) and Sphingobium sp. strain Chol11 with artificial electron acceptors, no 5β-Δ 4 -KSTDs for bile salts that are involved in the first steps of steroid degradation and, thus, bile-salt detoxification, have been found and characterized so far. ...
... Enzyme activity was measured at 436 nm in a spectrophotometer (UV-2600, Shimadzu, Kyoto, Japan) at 30°C for cuvettes and at 450 nm in a heatable microplate reader (EL808, Biotek, Winooski, VT, United States) at 30°C for microplates. Extinction coefficients for K 3 Fe(CN) 6 were 0.7 cm −1 mM −1 at 436 nm and 0.262 cm −1 mM −1 at 450 nm according to literature and own measurements (Singer, 1974;Birkenmaier et al., 2007). For determination of optimal pH, assays were performed with 50 mM Tris buffer adjusted to various pH values with NaOH or HCl or McIlvaine buffer (McIlvaine, 1921) 5α-3-Ketolithocholate, 5β-3-ketolithocholate, 5α-androstan-3,17-dione, and 5β-androstan-3,17-dione solutions were prepared in DMSO with a concentration of about 25 mM. ...
... This conclusion is in agreement with earlier enzymatic studies with cell extracts of Sphingobium sp. strain Chol11 and of P. stutzeri Chol1 (Birkenmaier et al., 2007) in which likewise no oxidation of 3-ketocholate was observed with NAD + but rather the requirement of K 3 Fe(CN) 6 or PMS was reported. In contrast, BaiCD from C. scindens uses NAD + as electron acceptor. ...
In contrast to many steroid hormones and cholesterol, mammalian bile salts are 5β-steroids, which leads to a bent structure of the steroid core. Bile salts are surface-active steroids excreted into the environment in large amounts, where they are subject to bacterial degradation. Bacterial steroid degradation is initiated by the oxidation of the A-ring leading to canonical Δ ⁴ -3-keto steroids with a double bond in the A-ring. For 5β-bile salts, this Δ ⁴ -double bond is introduced into 3-keto-bile salts by a 5β-Δ ⁴ -ketosteroid dehydrogenase (5β-Δ ⁴ -KSTD). With the Nov2c019 protein from bile-salt degrading Sphingobium sp. strain Chol11, a novel 5β-Δ ⁴ -KSTD for bile-salt degradation belonging to the Old Yellow Enzyme family was identified and named 5β-Δ ⁴ -KSTD1. By heterologous production in Escherichia coli , 5β-Δ ⁴ -KSTD function could be shown for 5β-Δ ⁴ -KSTD1 as well as the homolog CasH from bile-salt degrading Rhodococcus jostii RHA1. The deletion mutant of 5β-Δ ⁴ -kstd1 had a prolonged lag-phase with cholate as sole carbon source and, in accordance with the function of 5β-Δ ⁴ -KSTD1, showed delayed 3-ketocholate transformation. Purified 5β-Δ ⁴ -KSTD1 was specific for 5β-steroids in contrast to 5α-steroids and converted steroids with a variety of hydroxy groups regardless of the presence of a side chain. 5β-Δ ⁴ -KSTD1 showed a relatively low K m for 3-ketocholate, a very high specific activity and pronounced substrate inhibition. With respect to the toxicity of bile salts, these kinetic properties indicate that 5β-Δ ⁴ -KSTD1 can achieve fast detoxification of the detergent character as well as prevention of an overflow of the catabolic pathway in presence of increased bile-salt concentrations.
... acids is the so-called 9,10-seco-pathway of steroid degradation via Δ 1,4 -3-keto-intermediates 14-17 . Degradation is initiated by oxidation of the A-ring and splitting off the C 5 -carboxylic side chain yielding C 19 steroids with Δ 1,4 -3,17-diketo structure 14,15,18,19 , which are called androstadienediones (ADDs). ADDs are also central intermediates in the degradation of other steroids such as cholesterol and testosterone 17,20 (Supplemental Fig. S1).The further degradation of ADDs proceeds by oxygenation of C9, which results in cleavage of the B-ring 21,22 . ...
Bile acids are steroid compounds from the digestive tracts of vertebrates that enter agricultural environments in unusual high amounts with manure. Bacteria degrading bile acids can readily be isolated from soils and waters including agricultural areas. Under laboratory conditions, these bacteria transiently release steroid compounds as degradation intermediates into the environment. These compounds include androstadienediones (ADDs), which are C19-steroids with potential hormonal effects. Experiments with Caenorhabditis elegans showed that ADDs derived from bacterial bile acid degradation had effects on its tactile response, reproduction rate, and developmental speed. Additional experiments with a deletion mutant as well as transcriptomic analyses indicated that these effects might be conveyed by the putative testosterone receptor NHR-69. Soil microcosms showed that the natural microflora of agricultural soil is readily induced for bile acid degradation accompanied by the transient release of steroid intermediates. Establishment of a model system with a Pseudomonas strain and C. elegans in sand microcosms indicated transient release of ADDs during the course of bile acid degradation and negative effects on the reproduction rate of the nematode. This proof-of-principle study points at bacterial degradation of manure-derived bile acids as a potential and so-far overlooked risk for invertebrates in agricultural soils.
... However, a parallel study demonstrated that both systems act in the reassimilation of transiently accumulated cholic acid intermediates, previously secreted to the culture broth by bacteria during bile acid catabolism [84]. The accumulation of metabolic intermediates in culture broths by steroid-degrading bacteria has been documented [85]; Pseudomonas stutzeri strain Chol1 metabolising bile acids extracellularly accumulates intermediates, probably to regulate their intracellular levels or due to overflowing of the downstream metabolic pathway [85]. Later, during growth, these metabolites disappear from the culture broth, which implies: (i) the existence in these bacteria of an exclusion system, and (ii) a catabolic route involved in the reassimilation of these compounds [85]. ...
... However, a parallel study demonstrated that both systems act in the reassimilation of transiently accumulated cholic acid intermediates, previously secreted to the culture broth by bacteria during bile acid catabolism [84]. The accumulation of metabolic intermediates in culture broths by steroid-degrading bacteria has been documented [85]; Pseudomonas stutzeri strain Chol1 metabolising bile acids extracellularly accumulates intermediates, probably to regulate their intracellular levels or due to overflowing of the downstream metabolic pathway [85]. Later, during growth, these metabolites disappear from the culture broth, which implies: (i) the existence in these bacteria of an exclusion system, and (ii) a catabolic route involved in the reassimilation of these compounds [85]. ...
... The accumulation of metabolic intermediates in culture broths by steroid-degrading bacteria has been documented [85]; Pseudomonas stutzeri strain Chol1 metabolising bile acids extracellularly accumulates intermediates, probably to regulate their intracellular levels or due to overflowing of the downstream metabolic pathway [85]. Later, during growth, these metabolites disappear from the culture broth, which implies: (i) the existence in these bacteria of an exclusion system, and (ii) a catabolic route involved in the reassimilation of these compounds [85]. ...
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.
... Related compounds were detected in an aerobic degradation study of bile acid cholate by a Pseudomonas sp. [52]. ...
... These mainly include taxa not previously known to contain steroid degraders, including Rhizobiales and Hyphomonadaceae, Rhodobacteraceae, Halieaceae, Spongiibacteraceae, and Alteromonadales. However, these also include the genera Sphingomonas, Novosphingobium, and Pseudoalteromonas previously shown to degrade steroids (18,52). Two MAGs from oxygen minimum zones were classified only to the phylum level Proteo-bacterium, indicating that the respective organisms belong to novel taxonomic lineages. ...
Steroids are abundant growth substrates for bacteria in natural, engineered, and host-associated environments. This study analyzed the distribution of the aerobic 9,10-seco steroid degradation pathway in 346 publically available metagenomes from diverse environments. Our results show that steroid-degrading bacteria are globally distributed and prevalent in particular environments, such as wastewater treatment plants, soil, plant rhizospheres, and the marine environment, including marine sponges. Genomic signature-based sequence binning recovered 45 metagenome-assembled genomes containing a majority of 9,10-seco pathway genes. Only Actinobacteria and Proteobacteria were identified as steroid degraders, but we identified several alpha- and gammaproteobacterial lineages not previously known to degrade steroids. Actino- and proteobacterial steroid degraders coexisted in wastewater, while soil and rhizosphere samples contained mostly actinobacterial ones. Actinobacterial steroid degraders were found in deep ocean samples, while mostly alpha- and gammaproteobacterial ones were found in other marine samples, including sponges. Isolation of steroid-degrading bacteria from sponges confirmed their presence. Phylogenetic analysis of key steroid degradation proteins suggested their biochemical novelty in genomes from sponges and other environments. This study shows that the ecological significance as well as taxonomic and biochemical diversity of bacterial steroid degradation has so far been largely underestimated, especially in the marine environment.
IMPORTANCE
Microbial steroid degradation is a critical process for biomass decomposition in natural environments, for removal of important pollutants during wastewater treatment, and for pathogenesis of bacteria associated with tuberculosis and other bacteria. To date, microbial steroid degradation was mainly studied in a few model organisms, while the ecological significance of steroid degradation remained largely unexplored. This study provides the first analysis of aerobic steroid degradation in diverse natural, engineered, and host-associated environments via bioinformatic analysis of an extensive metagenome data set. We found that steroid-degrading bacteria are globally distributed and prevalent in wastewater treatment plants, soil, plant rhizospheres, and the marine environment, especially in marine sponges. We show that the ecological significance as well as the taxonomic and biochemical diversity of bacterial steroid degradation has been largely underestimated. This study greatly expands our ecological and evolutionary understanding of microbial steroid degradation.
... StdA1 DOC21 is known to be able to activate both cholate and 3-ketocholate (II) with CoA (15). In cell extracts of P. stutzeri Chol1, Δ 1/4 -3-ketocholate and Δ 1,4 -3-ketocholate are thioesterified with CoA, probably by StdA1 Chol1 , although the in vitro oxidation of cholate to Δ 1,4 -3-ketocholate does not require a thioesterification step (22). Our data strongly suggest that a 3-keto-7-deoxy-Δ 4,6 steroid skeleton is the preferred substructure for SclA, indicating that DOCDA (XII) is the physiological substrate of this enzyme in strain Chol11. ...
... However, similar to the deletion of hsh2 (20), the deletion of sclA strongly impaired but did not abolish the growth of strain Chol11 with cholate. This phenotype is in contrast to that of P. stutzeri Chol1, in which the deletion of genes involved in initial reactions of bile salt degradation results in a complete lack of growth (9,21,22), and supports the notion that strain Chol11 has a broad metabolic repertoire. Our bioinformatics analysis revealed that the genome of strain Chol11 encodes several potential isoenzymes for some steroid degradation reactions. ...
Bile salts such as cholate are steroid compounds with a C5 carboxylic side chain and occur ubiquitously in vertebrates. Upon their excretion into soils and waters, bile salts can serve as growth substrates for diverse bacteria. Novosphingobium sp. strain Chol11 degrades 7-hydroxy bile salts via 3-keto-7-deoxy-Δ4,6 metabolites by the dehydration of the 7-hydroxyl group catalyzed by the 7α-hydroxysteroid dehydratase Hsh2. This reaction has not been observed in the well-studied 9-10-seco degradation pathway used by other steroid-degrading bacteria indicating that strain Chol11 uses an alternative pathway. A reciprocal BLASTp analysis showed that known side chain degradation genes from other cholate-degrading bacteria (Pseudomonas stutzeri Chol1, Comamonas testosteroni CNB-2, and Rhodococcus jostii RHA1) were not found in the genome of strain Chol11. The characterization of a transposon mutant of strain Chol11 showing altered growth with cholate identified a novel steroid-24-oyl–coenzyme A ligase named SclA. The unmarked deletion of sclA resulted in a strong growth rate decrease with cholate, while growth with steroids with C3 side chains or without side chains was not affected. Intermediates with a 7-deoxy-3-keto-Δ4,6 structure, such as 3,12-dioxo-4,6-choldienoic acid (DOCDA), were shown to be likely physiological substrates of SclA. Furthermore, a novel coenzyme A (CoA)-dependent DOCDA degradation metabolite with an additional double bond in the side chain was identified. These results support the hypothesis that Novosphingobium sp. strain Chol11 harbors an alternative pathway for cholate degradation, in which side chain degradation is initiated by the CoA ligase SclA and proceeds via reaction steps catalyzed by so-far-unknown enzymes different from those of other steroid-degrading bacteria.
IMPORTANCE
This study provides further evidence of the diversity of metabolic pathways for the degradation of steroid compounds in environmental bacteria. The knowledge about these pathways contributes to the understanding of the CO2-releasing part of the global C cycle. Furthermore, it is useful for investigating the fate of pharmaceutical steroids in the environment, some of which may act as endocrine disruptors.
... In addition, during oxic degradation of bile acids by the Gram-negative Pseudomonas sp. strain Chol1, catabolic intermediates are exported to the media (4,51). Similar extracellular accumulation of metabolites has been observed in aerobically cholate-grown Rhodococcus jostii RHA1 (52). ...
Cholesterol catabolism by actinobacteria has been extensively studied. In contrast, the uptake and catabolism of cholesterol by Gram-negative species is poorly understood. Here we investigated microbial cholesterol catabolism at the subcellular level. (13)C-metabolomic analysis revealed that anaerobically grown Sterolibacterium denitrificans, a betaproteobacterium, adopts an oxygenase-independent pathway to degrade cholesterol. S. denitrificans cells did not produce biosurfactants upon growth on cholesterol and exhibited high cell surface hydrophobicity. Moreover, S. denitrificans did not produce extracellular catabolic enzymes to transform cholesterol. Accordingly, S. denitrificans accessed cholesterol through direction adhesion. Cholesterol is imported through the outer membrane via a putative FadL-like transport system, which is induced by neutral sterols. The OM steroid transporter is able to selectively import various C27 sterols into the periplasm. S. denitrificans spheroplasts exhibited a significantly higher efficiency in cholest-4-en-3-one-26-oic acid uptake than in cholesterol uptake. We separated S. denitrificans proteins into four fractions, namely the outer membrane, periplasm, inner membrane, and cytoplasm, and observed the individual catabolic reactions within them. Our data indicated that, in the periplasm, various periplasmic and peripheral membrane enzymes transform cholesterol into cholest-4-en-3-one-26-oic acid. The C27 acidic steroid is then transported into cytoplasm, in which side-chain degradation and the subsequent sterane cleavage occur. This study sheds light into microbial cholesterol metabolism under anoxic conditions.
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