Isolation of cholesterol- and deoxycholate-degrading bacteria from soil samples: evidence of a common pathway

SummaryThe distribution and the metabolic pathways of bacteria degrading steroid compounds released by eukaryotic organisms were investigated using the bile salt cholate as model substrate. Cholatedegrading bacteria could be readily isolated from freshwater environments. All isolated strains transiently released steroid degradation intermediates into culture supernatants before their further degradation. Cholate degradation could be initiated via two different reaction sequences. Most strains degraded cholate via a reaction sequence known from the model organism Pseudomonas sp. strain Chol1 releasing intermediates with a 3-keto-Δ 1,4 -diene structure of the steroid skeleton. The actinobacterium Dietzia sp. strain Chol2 degraded cholate via a different and yet unexplored reaction sequence releasing intermediates with a 3-keto-Δ 4,6 -diene-7-deoxy structure of the steroid skeleton such as 3,12-dioxo-4,6-choldienoic acid (DOCDA). Using DOCDA as substrate, two Alphaproteobacteria, strains Chol10-11, were isolated that produced the same cholate degradation intermediates as strain Chol2. With DOCDA as substrate for Pseudomonas sp. strain Chol1 only the side chain was degraded while the ring system was transformed into novel steroid compounds accumulating as dead-end metabolites. These metabolites could be degraded by the DOCDA-producing strains Chol10-11. These results indicate that bacteria with potentially different pathways for cholate degradation coexist in natural habitats and may interact via interspecies cross-feeding.

Section: Resultsmentioning

confidence: 80%

Section: Introductionmentioning

confidence: 97%

Evidence of distinct pathways for bacterial degradation of the steroid compound cholate suggests the potential for metabolic interactions by interspecies cross‐feeding

Holert

Yücel

Suvekbala

et al. 2014

“…We examined the catabolic pathway(s) required for the degradation of cholate, deoxycholate, lithocholate, ursodeoxycholate, chenodeoxycholate, androstanolone, AD and testosterone ( Fig. 1) by the strain DOC21 (Merino et al, 2013). To identify steroid degradation (std) genes we used the Tn5 to generate a library of transposon mutants (Selvaraj and Iyer, 1983;Herrero et al, 1990), from which we isolated 54 mutants (Fig.…”

Section: Identification Of Genes Involved In Steroid Degradation In Pmentioning

confidence: 99%

“…A variety of Gram-negative bacteria are able to catabolize different families of steroids, including bile acids, testosterone and their close structural analogues (or derivatives). Although most of the studies have been done with Comamonas and Pseudomonas strains, none of these have been reported to degrade sterols, suggesting that the presence of a terminal carboxylate on the C-17 side chain is required for the catabolism of these compounds (Merino et al, 2013). Studies of steroiddegrading Gram-negative bacteria have been aimed mainly at: (i) analysing the enzymes involved in the catabolism of the C-17 side chain and (ii) determining the chemical steps leading to catabolism of rings A and B of the steroid nucleus (see Fig.…”

Section: Introductionmentioning

confidence: 99%

Functional analyses of three acyl‐CoA synthetases involved in bile acid degradation in Pseudomonas putida DOC21

Barrientos

Merino

Casabon

et al. 2014

Pseudomonas putida DOC21, a soil-dwelling proteobacterium, catabolizes a variety of steroids and bile acids. Transposon mutagenesis and bioinformatics analyses identified four clusters of steroid degradation (std) genes encoding a single catabolic pathway. The latter includes three predicted acyl-CoA synthetases encoded by stdA1, stdA2 and stdA3 respectively. The ΔstdA1 and ΔstdA2 deletion mutants were unable to assimilate cholate or other bile acids but grew well on testosterone or 4-androstene-3,17-dione (AD). In contrast, a ΔstdA3 mutant grew poorly in media containing either testosterone or AD. When cells were grown with succinate in the presence of cholate, ΔstdA1 accumulated Δ(1/4) -3-ketocholate and Δ(1,4) -3-ketocholate, whereas ΔstdA2 only accumulated 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC). When incubated with testosterone or bile acids, ΔstdA3 accumulated 3aα-H-4α(3'propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP) or the corresponding hydroxylated derivative. Biochemical analyses revealed that StdA1 converted cholate, 3-ketocholate, Δ(1/4) -3-ketocholate, and Δ(1,4) -3-ketocholate to their CoA thioesters, while StdA2 transformed DHOPDC to DHOPDC-CoA. In contrast, purified StdA3 catalysed the CoA thioesterification of HIP and its hydroxylated derivatives. Overall, StdA1, StdA2 and StdA3 are acyl-CoA synthetases required for the complete degradation of bile acids: StdA1 and StdA2 are involved in degrading the C-17 acyl chain, whereas StdA3 initiates degradation of the last two steroid rings. The study highlights differences in steroid catabolism between Proteobacteria and Actinobacteria.

“…Steroids are naturally occurring hydrophobic molecules that present a structure core formed by four fused alicyclic rings named gonane. Steroid compounds are frequently found in the biosphere and can be used as carbon and energy sources by different bacteria (e.g., Mycobacterium, Pseudomonas, Sterolibacterium, Sphingomonas, Novosphingobium and Comamonas) (Fujii et al, 2002;Tarlera and Denner, 2003;Horinouchi et al, 2003a;Philipp et al, 2006;van der Geize et al, 2007;Roh and Chu, 2010;Leu et al, 2011;Merino et al, 2013). Most bacteria capable of degrading aerobically sterols (e.g., cholesterol and phytosterols), which are one of the most abundant steroid compounds in nature, belong to the phylum Actinobacteria (e.g., Mycobacterium, Rhodococcus and Gordonia) (van der Geize et al, 2007;Kendall et al, 2007;Drzyzga et al, 2009;Uhía et al, 2012;Fern andez de las Heras et al, 2013;Li et al, 2014;Bergstrand et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

Unravelling a new catabolic pathway of C‐19 steroids in Mycobacterium smegmatis

Fernández-Cabezón

Galán

Garcı́a

2018

In this work, we have characterized the C-19+ gene cluster (MSMEG_2851 to MSMEG_2901) of Mycobacterium smegmatis. By in silico analysis, we have identified the genes encoding enzymes involved in the modification of the A/B steroid rings during the catabolism of C-19 steroids in certain M. smegmatis mutants mapped in the PadR-like regulator (MSMEG_2868), that constitutively express the C-19+ gene cluster. By using gene complementation assays, resting-cell biotransformations and deletion mutants, we have characterized the most critical genes of the cluster, that is, kstD2, kstD3, kshA2, kshB2, hsaA2, hsaC2 and hsaD2. These results have allowed us to propose a new catabolic route named C-19+ pathway for the mineralization of C-19 steroids in M. smegmatis. Our data suggest that the deletion of the C-19+ gene cluster may be useful to engineer more robust and efficient M. smegmatis strains to produce C-19 steroids from sterols. Moreover, the new KshA2, KshB2, KstD2 and KstD3 isoenzymes may be useful to design new microbial cell factories for the 9α-hydroxylation and/or Δ1-dehydrogenation of 3-ketosteroids.