Study On Related Proteins In Comamonas Testosteroni Induced By Progesterone

In recent years,environmental hormone pollution which is difficult to degrade in nature has become more and more serious.Therefore,the degradation mechanism of environmental hormone pollutants by microorganisms and the regulation effect of enzyme genes and regulatory genes have been widely concerned by many researchers,and are being explored and studied continuously.Comamonas testosteroni ATCC11996(C.testosteroni for short)is a kind of Gram-negative bacteria that can degrade steroid hormones.Many enzymes are involved in its metabolic process,but the specific metabolic network is not fully understood.Through the analysis of the transcriptome of C.testosteroni induced by progesterone and testosterone stored in the laboratory,five differential genes with high expression under progesterone induction and low or even down-regulated expression under testosterone induction were screened out,and their physiological activities of degrading steroid hormones were investigated by gene knockout and high performance liquid chromatography(HPLC).The regulation of nicotinate dehydrogenase subunit A(nicA)gene with the highest expression in progesterone transcriptome was investigated by means of recombinant plasmid construction,whole plasmid amplification,plasmid co-transformation and Western-blot.The experimental results showed that 5 different genes with high expression in progesterone transcriptome and low or even down-regulated expression in testosterone transcriptome included:nicA,nicinate dehydrogenase subunit B(nic B),cytochrome C(cyt.C),ABC transporter substrate binding protein(t.pro),hemin receptor(hemin).Their expression levels in the progesterone transcriptome were 2^7.17?2^7.02?2^6.79?2^5.35?2^4.99,and in the testosterone transcriptome were 2^0.29?2^0.5?2^-0.16?2^0.02?2^0.47,respectively.Knockout strains(referred to as KCT)of the five genes mentioned above are successfully constructed,including KCT-nicA,KCT-nic B,KCT-cyt.c,KCT-t.pro,and KCT-hemin.HPLC method verifies that these five genes influence the degradation of progesterone by C.testosteroni,which is compared with the wild strains:KCT-nicA progesterone degradation rate fell by 72.31%,KCT-nic B progesterone degradation rate fell by66.08%,KCT-cyt.c progesterone degradation rate fell by 53.76%,KCT-t.pro progesterone degradation rate fell by 55.66%,KCT-hemin progesterone degradation rate fell by 58.92%,knock out a wild strain degradation strains of progesterone degradation rate shows the five different genes are involved in the C.testosteroni effect on the degradation of progesterone and nicA gene relative maximum;The co-transformation results showed that Lux R,Lys R,Tet R,Tet R and Ara C up-regulated nicA by 53.02%,63.16%,5.53%,69.35% and 27.69% respectively,indicating that regulatory proteins Lux R,Lys R,Ic IR and Ara C were involved in the regulation of nicA gene to varying degrees,while regulatory protein Tet R was almost not involved in the regulation of nicA gene.The results of this study provide experimental basis for the biodegradation and gene regulation of C.testosteroni in nature.