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Isolation of cholesterol- and deoxycholate-degrading bacteria from soil samples: evidence of a common pathway

  • Environmental biotechnology
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Abstract

Nineteen different steroid-degrading bacteria were isolated from soil samples by using selective media containing either cholesterol or deoxycholate as sole carbon source. Strains that assimilated cholesterol (17 COL strains) were gram-positive, belonging to the genera Gordonia, Tsukamurella, and Rhodococcus, and grew on media containing other steroids but were unable to use deoxycholate as sole carbon source. Surprisingly, some of the COL strains unable to grow using deoxycholate as sole carbon source were able to catabolize other bile salts (e.g., cholate). Conversely, strains able to grow using deoxycholate as the sole carbon source (two DOC isolates) were gram-negative, belonging to the genus Pseudomonas, and were unable to catabolize cholesterol and other sterols. COL and DOC were included into the corresponding taxonomic groups based on their morphology (cells and colonies), metabolic properties (kind of substrates that support bacterial growth), and genetic sequences (16S rDNA and rpoB). Additionally, different DOC21 Tn5 insertion mutants have been obtained. These mutants have been classified into two different groups: (1) those affected in the catabolism of bile salts but that, as wild type, can grow in other steroids and (2) those unable to grow in media containing any of the steroids tested. The identification of the insertion point of Tn5 in one of the mutants belonging to the second group (DOC21 Mut1) revealed that the gene knocked-out encodes an A-ring meta-cleavage dioxygenase needed for steroid catabolism.

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Acknowledgments

This work has been supported by grants BFU2009-11545-C03-01 from Ministerio de Ciencia e Innovación (Spain) and LE246A11-2 from Junta de Castilla y León (Consejería de Educación, Spain). JR, AB, and EM are recipient of fellowships given by the Ministerio de Ciencia e Innovación (JR and AB) and by the Ministerio de Educación y Ciencia (EM).

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Authors and Affiliations

  1. Departamento de Biología Molecular (Área Bioquímica), Universidad de León, Campus de Vegazana s/n, 24071, León, Spain

    E. Merino, A. Barrientos, J. Rodríguez, J. M. Luengo & E. R. Olivera

  2. Departamento de Sanidad Animal (Área de Microbiología), Universidad de León, 24071, León, Spain

    G. Naharro

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  1. E. Merino
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  2. A. Barrientos
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  3. J. Rodríguez
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  4. G. Naharro
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  5. J. M. Luengo
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  6. E. R. Olivera
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Correspondence to E. R. Olivera.

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E. Merino and A. Barrientos contributed equally to this article.

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Suppl. Figure 1

Structure of the steroid compounds tested in this work. 1. Cholesterol; 2. Ergosterol; 3. β-Sitosterol; 4. Stigmasterol; 5. 5β-Cholanate; 6. Lithocholate; 7. Chenodeoxycholate; 8. Ursodeoxycholate; 9. Deoxycholate; 10. Cholate; 11. Dehydrocholate; 12. Progesterone; 13. 5-Pregnen-3β-ol-20-one; 14. Testosterone; 15. 17α-Methyltestosterone; 16. 1-Dehydro-17α-Methyltestosterone; 17. Prednisone; 18. Androstanolone; 19. β-Estradiol; 20. trans-dehydroandrosterone; 21. Estrone; 22. trans-androsterone; 23. 4-Androstene-3,17-dione. Carbon atoms are numbered over colesterol structure (1). (JPEG 1.36 MB)

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Suppl. Figure 2

Morphology of the colonies (a) and cells (b) of the COL isolates. Strains numbered 1–8 belong to the genus Gordonia: COL11 (1), COL17 (2), COL19 (3), COL20 (4), COL21 (5), COL23 (6), COL26 (7), and COL33 (8); strains numbered 9–11 belong to the genus Tsukamurella: COL14 (9), COL16 (10), and COL18 (11); and strains numbered 12–17 belong to the genus Rhodococcus: COL22 (12), COL25 (13), COL27 (14), COL28 (15). COL29 (16), and COL30 (17). (a) optical microscopy images, bar indicates 1 mm; (b) scanning microscopy images, bar indicates 1 μm. (JPEG 162 kb)

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Suppl. Figure 3

(A and C) Optical microscopy images of the colonies and (B and D) scanning microscopy images of the cells of strains DOC19 (A and B) and DOC21 (C and D). In the optical microscopy images (A and C), the bar indicates 1 mm; in the scanning microscopy photographs (B and D), the bar indicates 1 μm. (JPEG 34 kb)

High resolution image file (TIFF 1855 kb)

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Merino, E., Barrientos, A., Rodríguez, J. et al. Isolation of cholesterol- and deoxycholate-degrading bacteria from soil samples: evidence of a common pathway. Appl Microbiol Biotechnol 97, 891–904 (2013). https://doi.org/10.1007/s00253-012-3966-7

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