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Journal of Bacteriology, February 2008, p. 905-914, Vol. 190, No. 3
0021-9193/08/$08.00+0     doi:10.1128/JB.01525-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Study of Anoxic and Oxic Cholesterol Metabolism by Sterolibacterium denitrificans{triangledown}

Yin-Ru Chiang,1,{dagger} Wael Ismail,1,{dagger} Dimitri Heintz,2 Christine Schaeffer,2 Alain Van Dorsselaer,2 and Georg Fuchs1*

Mikrobiologie, Fakultät Biologie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany,1 Laboratoire de spéctrometrie de masse Bio-Organique, CRNS, ECPM, Université Louis Pasteur, Strasbourg, France2

Received 21 September 2007/ Accepted 12 November 2007

The initial enzymes and genes involved in the anoxic metabolism of cholesterol were studied in the denitrifying bacterium Sterolibacterium denitrificans Chol-1ST. The second enzyme of the proposed pathway, cholest-4-en-3-one-{Delta}1-dehydrogenase (AcmB), was partially purified. Based on amino acid sequence analysis, a gene probe was derived to screen a cosmid library of chromosomal DNA for the acmB gene. A positive clone comprising a 43-kbp DNA insert was sequenced. In addition to the acmB gene, the DNA fragment harbored the acmA gene, which encodes the first enzyme of the pathway, cholesterol dehydrogenase/isomerase. The acmA gene was overexpressed, and the recombinant dehydrogenase/isomerase was purified. This enzyme catalyzes the predicted transformation of cholesterol to cholest-4-en-3-one. S. denitrificans cells grown aerobically with cholesterol exhibited the same pattern of soluble proteins and cell extracts formed the same 14C-labeled products from [14C]cholesterol as cells that were grown under anoxic, denitrifying conditions. This is especially remarkable for the late products that are formed by anaerobic hydroxylation of the cholesterol side chain with water as the oxygen donor. Hence, this facultative anaerobic bacterium may use the anoxic pathway lacking any oxygenase-dependent reaction also under oxic conditions. This confers metabolic flexibility to such facultative anaerobic bacteria.

* Corresponding author. Mailing address: Mikrobiologie, Insitut Biologie II, Schänzlestr. 1, D-79104 Freiburg, Germany. Phone: (49) 761-2032649. Fax: (49) 761-2032626. E-mail: georg.fuchs{at}biologie.uni-freiburg.de

{triangledown} Published ahead of print on 26 November 2007.

{dagger} Y.-R.C. and W.I. contributed equally to this study.

Journal of Bacteriology, February 2008, p. 905-914, Vol. 190, No. 3
0021-9193/08/$08.00+0     doi:10.1128/JB.01525-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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