ABSTRACT
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 investigated on the biochemical and genetic level in strain Chol1. These reactions comprised A-ring oxidation, activation with coenzyme A (CoA), and beta-oxidation of the acyl side chain with the C(19)-steroid dihydroxyandrostadienedione as the end product. A-ring oxidizing enzyme activities leading to Delta(1,4)-3-ketocholyl-CoA were detected in cell extracts and confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cholate activation with CoA was demonstrated in cell extracts and confirmed with a chemically synthesized standard by LC-MS/MS. A transposon mutant with a block in oxidation of the acyl side chain accumulated a steroid compound in culture supernatants which was identified as 7alpha,12alpha-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) by nuclear magnetic resonance spectroscopy. The interrupted gene was identified as encoding a putative acyl-CoA-dehydrogenase (ACAD). DHOPDC activation with CoA in cell extracts of strain Chol1 was detected by LC-MS/MS. The growth defect of the transposon mutant could be complemented by the wild-type ACAD gene located on the plasmid pBBR1MCS-5. Based on these results, the initiating reactions of the cholate degradation pathway leading from cholate to dihydroxyandrostadienedione could be reconstructed. In addition, the first bacterial gene encoding an enzyme for a specific reaction step in side chain degradation of steroid compounds was identified, and it showed a high degree of similarity to genes in other steroid-degrading bacteria.
Subject(s)
Cholates/metabolism , Metabolic Networks and Pathways/genetics , Metabolic Networks and Pathways/physiology , Pseudomonas/enzymology , Pseudomonas/genetics , Acyl-CoA Dehydrogenase/genetics , Aerobiosis , Amino Acid Sequence , Coenzyme A/metabolism , DNA Transposable Elements , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Gene Deletion , Genetic Complementation Test , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Mutagenesis, Insertional , Oxidation-Reduction , Pseudomonas/chemistry , Pseudomonas/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Tandem Mass SpectrometryABSTRACT
A facultative anaerobic bacterium, Pseudomonas sp. strain Chol1, degrading cholate and other bile acids was isolated from soil. We investigated how strain Chol1 grew with cholate and whether growth was affected by the toxicity of this compound. Under anoxic conditions with nitrate as electron acceptor, strain Chol1 grew by transformation of cholate to 7,12-dihydroxy-1,4-androstadiene-3,17-dione (DHADD) as end product. Under oxic conditions, strain Chol1 grew by transformation of cholate to 3,7,12-trihydroxy-9,10-seco-1,3,5(10)-androstatriene-9,17-dione (THSATD), which accumulated in the culture supernatant before its further oxidation to CO(2). Strain Chol1 converted DHADD into THSATD by an oxygenase-dependent reaction. Addition of cholate (> or =10 mM) to cell suspensions of strain Chol1 caused a decrease of optical density and viable counts but aerobic growth with these toxic cholate concentrations was possible. Addition of CCCP or EDTA strongly increased the sensitivity of the cells to 10 mM cholate. EDTA also increased the sensitivity of the cells to DHADD and THSATD (< or =1.7 mM). The toxicity of cholate and its degradation intermediates with a steroid structure indicates that strain Chol1 requires a strategy to minimize these toxic effects during growth with cholate. Apparently, the proton motive force and the outer membrane are necessary for protection against these toxic effects.
