Biochemical and Genetic Investigation on Initial Reactions in Aerobic Degradation of the Bile Acid Cholate in Pseudomonas sp. Strain Chol1

University of Konstanz, Department of Biology, Microbial Ecology, Konstanz, Germany, University of Konstanz, Department of Chemistry, Konstanz, Germany, Eawag, Swiss Federal Institute of Aquatic Science and Technology, Duebendorf, Switzerland

^* To whom correspondence should be addressed. Email: bodo.philipp{at}uni-konstanz.de.

	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 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 -oxidation of the acyl-side chain with the C₁₉-steroid dihydroxyandrostadienedione as end product. A-ring oxidizing enzyme activities leading to ^1,4-3-ketocholyl-CoA were detected in cell-free extracts and confirmed by LC-MS/MS. Cholate activation with CoA was demonstrated in cell-free extracts and confirmed with an 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 7,12-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) by NMR spectroscopy. The interrupted gene was identified as encoding a putative acyl-CoA-dehydrogenase (ACAD). DHOPDC activation with CoA in cell-free 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 which had high similarity to genes in other steroid degrading bacteria.