Biochemical and genetic investigation of initial reactions in aerobic degradation of the bile acid cholate in Pseudomonas sp. strain Chol1

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.

FIG. 1.

Proposed degradation pathway for cholate (compound I) in Pseudomonas sp. strain Chol1. The following compounds have been experimentally detected in this study: II, 3-ketocholate; III, Δ⁴-3-ketocholate (P1); IV, Δ^1,4-3-ketocholate (P2); V, cholyl-CoA (P5); VI, 3-ketocholyl-CoA; VII, Δ⁴-3-ketocholyl-CoA; VIII, Δ^1,4-3-ketocholyl-CoA; XII, DHOPDC-CoA (P6); and XVII, DHOPDC (P4). As the position of the double bond in the A-ring has not yet been identified, compounds III and VII could also be Δ¹-3-ketocholate and Δ¹-3-ketocholyl-CoA, respectively. Compounds XV (DHADD) and XVI (THSATD) were identified in a previous study (28). The compounds indicated by numbers in brackets (IX, X, XI, XIII, and XIV) have not been detected so far. DHOPDC (XVII) is a dead-end metabolite in the mutant strain R1.

FIG. 2.

HPLC chromatogram of a culture supernatant of Pseudomonas sp. strain R1 grown with succinate in the presence of cholate. The products P4, P3, P2, and P1 were identified as DHOPDC, its Δ¹ or Δ⁴ monoene derivative, Δ^1,4-3-ketocholate, and its Δ¹ or Δ⁴ monoene derivative, respectively.

FIG. 3.

Analysis of bile acid CoA ester formation. (a) Overlaid HPLC chromatograms of a sample from a cholate-CoA ligase assay (dotted trace) and of chemically synthesized cholyl-CoA (solid trace). P5 was identified as cholyl-CoA. P1 represents the monoene derivative of Δ^1,4-3-ketocholate (Fig. 2). (b) Mass spectrum and chemical structure of ([M+H]⁺) of cholyl-CoA (P5). (c) Overlaid chromatograms of a DHOPDC-CoA ligase assay (dotted trace) and a control assay in which cell extract had been omitted (solid trace). P6 was identified as DHOPDC-CoA. P4 represents DHOPDC (Fig. 2). (d) Mass spectrum and chemical structure of ([M+H]⁺) of DHOPDC-CoA (P6).

FIG. 4.

Cell suspension experiment with Pseudomonas sp. strain R1 in culture supernatant of strain R1 after growth with succinate in the presence of cholate. Conversion of Δ¹/Δ⁴-3-ketocholate (▵), Δ^1,4-3-ketocholate (▴), and the monoene derivative of DHOPDC (□) into DHOPDC (▪). AU, arbitrary units.

FIG. 5.

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.

FIG. 6.

Growth of Pseudomonas sp. strains Chol1 (×), R1 pBBR (triangles), and R1 pBBR(ACAD) (diamonds) with 2 mM cholate. Strains R1 pBBR and R1 pBBR(ACAD) were incubated in the presence of kanamycin without (closed symbols) or with gentamicin (open symbols). Open triangles overlap with closed triangles.