Cholesterol degradation by Gordonia cholesterolivorans

Abstract

This paper reports physiological and genetic data about the type strain Gordonia cholesterolivorans, a strain that is able to degrade steroid compounds containing a long carbon side chain such as cholesterol (C(27)), cholestenone (C(27)), ergosterol (C(28)), and stigmasterol (C(29)). The length of the carbon side chain appears to be of great importance for this bacterium, as the strain is unable to grow using steroids with a shorter or nonaliphatic carbon side chain such as cholic acid (C(24)), progesterone (C(21)), testosterone, androsterone, 4-androstene-3,17-dione (all C(19)), and further steroids. This study also demonstrates that the degradation of cholesterol is a quite common feature of the genus Gordonia by comparing Gordonia cholesterolivorans with some other species of this genus (e.g., G. sihwensis, G. hydrophobica, G. australis, and G. neofelifaecis). Pyrosequencing of the genome of G. cholesterolivorans led to the identification of two conventional cholesterol oxidase genes on an 8-kb and a 12.8-kb genomic fragment with genetic organizations that are quite unique as compared to the genomes of other cholesterol-degrading bacteria sequenced so far. The identified two putative cholesterol oxidases of G. cholesterolivorans are both intracellularly acting enzymes of the class I type. Whereas one of these two cholesterol oxidases (ChoOx-1) shows high identity with an oxidoreductase of the opportunistic pathogen G. bronchialis and is not transcribed during growth with cholesterol, the other one (ChoOx-2) appears phylogenetically closer to cholesterol oxidases from members of the genus Rhodococcus and is transcribed constitutively. By using targeted gene disruption, a G. cholesterolivorans ChoOx-2 gene mutant strain that was unable to grow with steroids was obtained.

Fig. 1.

(A) Time course of growth of G. cholesterolivorans in minimal medium with long carbon side chain-containing steroids as the only carbon source (cholesterol, 1.5 mM [•]; cholestenone, 1.5 mM [▪]; ergosterol, 1.5 mM [⧫]; stigmasterol, 1 mM [▴]). (B) Time course of growth of G. cholesterolivorans (•), G. sihwensis (▪), G. hydrophobica (⧫), G. australis (▴), G. neofelifaecis (▾), and Rhodococcus equi (□) in minimal medium with cholesterol (1.5 mM). The absorbance data are the means of the values from triplicate incubations. The standard deviation was always within ±5% of the mean values.

Fig. 2.

Time course of the degradation of cholesterol during growth of G. cholesterolivorans in minimal medium and formation of intermediate compounds identified by LC/MS. Symbols: •, cholesterol; ▪, cholestenone; ▴, 26-hydroxycholest-4-en-3-one; ⧫, 26-hydroxycholesta-1,4-dien-3-one; and ▾, cholest-4-en-3-one-26-oic acid.

Fig. 3.

Schemes of the genetic organizations of an 8-kb genomic region (accession number GU320250) with the putative cholesterol oxidase ChoOx-1 gene (A) and a 12.8-kb genomic region (accession number GU320251) with the putative cholesterol oxidase ChoOx-2 gene of G. cholesterolivorans (B), and comparison of both sequences with similar regions of other phylogenetically related Gram-positive bacteria. The locus tags of the genes within their corresponding genomes of the strains are as follows: (from left to right for panel A) Gbro_1725 to Gbro_1729, RHA1_ro06198 to RHA1_ro06203, ROP_62580 to ROP_62630, nfa8940 to nfa8980, and Mvan_1510 to Mvan_1517, and (from left to right for panel B) Gbro_3954 to Gbro_3944, RHA1_ro04694 to RHA1_ro04704, ROP_47930 to ROP_48030, nfa5330 to nfa5430, and Mvan_0359 to Mvan_0370. White arrows without further description are hypothetical proteins with functions that are unknown so far. The sizes of the genes of G. cholesterolivorans are given in Tables S1 and S2 in the supplemental material. SDR, short-chain dehydrogenase/reductase.

Fig. 4.

Growth of bacterial strains on cholesterol oxidase indicator test plates made with minimal medium containing 16.4 mM cyclodextrine and 1.5 mM cholesterol. (A) Gordonia cholesterolivorans strain Chol-3^T (CECT 7408). (B) Rhodococcus ruber strain Chol-4 (CECT 7469). (C) Rhodococcus erythropolis strain 3014 (CECT 3014).

Fig. 5.

Transcription studies on the two putative cholesterol oxidase ChoOx-1 (A) and ChoOx-2 (B) genes of G. cholesterolivorans. Specific RT-PCR products amplified from RNA isolated from cells grown in minimal medium with citrate (Ci; control), octanoic acid (C₈), and cholesterol (C₂₇). H₂O is the control without cDNA. The RT⁻ assays are controls to exclude any contamination with DNA and chrD is the assay with chromosomal DNA to verify the functionality of the PCR primers (see Table S3 in the supplemental material).

Fig. 6.

In silico study of the promoter region upstream of the ChoOx-2 gene of G. cholesterolivorans (Gchol) and comparison with a similar region of Rhodococcus equi (Requi) and Rhodococcus erythropolis strain SK121 (Reryt). Initiation codons and Shine-Dalgarno sequences as well as the start codon of the divergently transcribed tetR gene of G. cholesterolivorans are in bold and lowercase letters. Elements (−10 and −35 boxes, and +1 transcription sites) of putative promoters are boxed. Inverted repeats are underlined and in bold italics. Putative binding sites for the response regulator OmpR (in the Gchol sequence) and the transcription factor RpoD17 (in the Requi sequence) are double underlined.