Testosterone catabolic genes from Comamonas testosteroni are induced by TeiR and testosterone metabolite-Coenzyme A thioester

LINARES M; PRUNEDA-PAZ JL; GENTI DE RAIMONDI SUSANA

España.

Congreso; Bio Micro World 2005 ? International conference on Enmvironmental, Industrial and Applied Microbiology; 2005

Teir is a LuxR-Type transcriptional factor that positively regulates the transcription of genes involved in the initial enzymatic steps of steroid degradation in C. testosteroni (Pruneda-Paz, et al, J. Bacteriol, 2004, 186:1430). Western blot assays demonstrated TeiR density- and testosterone-dependent manner expression reaching peak levels at the stationary phase. Furthermore, the expression of a steroid-inducible transcriptional fusion could be prematurely activated in cells at early to mid-exponential phase by the addition of ethyl acetate extracts of spent C. testosteroni cultures grown in presence of testosterone. The gene loci teor, encoding acyl-coenzyme A -acyl-CoA- dehydrogenase homolog, tead encoding enoyl-CoA dehydratase homolog and tekt encoding a -ketothiolase homolog were localized on a DNA region upstream of the teiR transcriptional regulator. To prove the essential involvement of teor, tead and tekt genes in the catabolism of testosterone in C. testosteroni, these genes were inactivated separately by the insertion of omega elements. The corresponding mutant strains were not able to grow on testosterone as well as to activate a steroid-inducible transcriptional fusion. The teor mutant phenotype complemented with a plasmid encoding teiR gene, can be restored by a diffusible extracellular factor (DSF) produced by C. testosteroni wt or C.testosteroni teiR mutant grown in presence of testosterone. The properties of DSF suggest that it may be a testosterone derivative-CoA thioester. In conclusion, the data demonstrate the necessity of a beta-oxidation cycle for testosterone degradation suggesting the activation of a testosterone metabolite to the corresponding CoA ester. We propose a model for the DSF system, which represents a novel mechanism for regulating testosterone catabolism revealing a connection between the upper pathway and the TCA cycle intermediates.