Comamonas testosteroni (C. testosteroni) ATCC11996 is a gram negative bacterium which can use steroid as a carbon and energy source. 3,17β-hydroxysteroid dehydrogenase (3,17β-HSD) is a key enzyme for the degradation of steroid hormones in C. testosteroni. The LuxR regulation family is a group of regulatory proteins which play important role in gram negative bacterium. The luxr gene is located on 58 kb upstream of 3,17β-HSD gene with the opposite transcription orientation in the chromosomal DNA of C. testosteroni. An open reading frame of this putative luxr gene consists of 1125 bp and is translated into a protein containing 374 amino acids. The luxr gene was cloned into plasmid pK18 and plasmid pK-LuxR1 was obtained. E. coli HB101 was co-transformed by pK-LuxR1 and pUC912-10, pUC1128-5 or pUC3.2-4 (which contain βhsd gene and different length promoter, repeat sequences). The result of ELISA showed that LuxR protein is a negative regulator for 3,17β-HSD expression. The luxr gene in C. testosteroni was knock-out by homologous integration. 3,17β-HSD expression was increased in the mutant (C.T.-L-KO1) comparing to that in wild-type C. testosteroni (C.T.) after 0.5 mM testosterone induction. The mutant C.T.-L-KO1 and wild-type C. testosteroni were cultured at 27 °C and 37 °C. The result of growth curve proved that LuxR has also effect on the bacterial growth.
Keywords: 3,17β-Hydroxysteroid dehydrogenase; Comamonas testosteroni; Gene regulation; Knock-out mutant; LuxR repressor; Testosterone induction.
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