Bacterial degradation of steroids is widespread, but the metabolic pathways have rarely been explored. Previous studies with Pseudomonas sp. strain Chol1 and the C 24 steroid cholate have shown that cholate degradation proceeds via oxidation of the A ring, followed by cleavage of the C 5 acyl side chain attached to C-17, with 7␣,12␤-dihydroxy-androsta-1,4-diene-3,17-dione (12␤-DHADD) as the product. In this study, the pathway for degradation of the acyl side chain of cholate was investigated in vitro with cell extracts of strain Chol1. For this, intermediates of cholate degradation were produced with mutants of strain Chol1 and submitted to enzymatic assays containing coenzyme A (CoA), ATP, and NAD ؉ as cosubstrates. When the C 24 steroid (22E)-7␣,12␣-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO) was used as the substrate, it was completely transformed to 12␣-DHADD and 7␣-hydroxy-androsta-1,4-diene-3,12,17-trione (HADT) as end products, indicating complete removal of the acyl side chain. The same products were formed with the C 22 steroid 7␣,12␣-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) as the substrate. The 12-keto compound HADT was transformed into 12␤-DHADD in an NADPH-dependent reaction. When NAD ؉ was omitted from assays with DHOCTO, a new product, identified as 7␣,12␣-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA), was formed. This aldehyde was transformed to DHOPDC and DHOPDC-CoA in the presence of NAD ؉ , CoA, and ATP. These results revealed that degradation of the C 5 acyl side chain of cholate does not proceed via classical ␤-oxidation but via a free aldehyde that is oxidized to the corresponding acid. The reaction leading to the aldehyde is presumably catalyzed by an aldolase encoded by the gene skt, which was previously predicted to be a ␤-ketothiolase.

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.

Pseudomonas sp. strain PPD and Alcaligenes sp. strain PPH isolated from soil by enrichment culture technique utilize 2-, 3- and 4-hydroxybenzoates as the sole source of carbon and energy. The degradation pathways were elucidated by performing whole-cell O(2) uptake, enzyme activity and induction studies. Depending on the mixture of carbon source and the preculture condition, strain PPH was found to degrade 2-hydroxybenzoate either via the catechol or gentisate route and has both salicylate 1-hydroxylase and salicylate 5-hydroxylase. Strain PPD utilizes 2-hydroxybenzoate via gentisate. Both strains degrade 3- and 4-hydroxybenzoate via gentisate and protocatechuate, respectively. Enzymes were induced by respective hydroxybenzoate. Growth pattern, O(2) uptake and enzyme activity profiles on the mixture of three hydroxybenzoates as a carbon source suggest coutilization by both strains. When 3- or 4-hydroxybenzoate grown culture was used as an inoculum, strain PPH failed to utilize 2-hydroxybenzoate via catechol, indicating the modulation of the metabolic pathways, thus generating metabolic diversity.