Study On The Function And Regulation Of TeiR Gene From Comamonas Testosteroni

Gram-negative bacterium Comamonas testosteroni has the ability to utilize various steriods as a sole carbon and energe source. Therefore it plays an important role in the bioremediation of these stable compounds in the environment. Insterestingly, the steriod metabolic enzymes were not constitutively expressed, but induced by their respective substrates. This limits its wide use in environmental bioremediation. Testosterone inducible regulator, teiR gene controls a wide variety of activities in various biological processes, some as transcriptional activators, but some as repressors. In the present work, we cloned teiR gene and studyed the function to reveal the relationship between the teiR gene and 3a-HSD/CR. Directionally control the Comamonas testosteroni on gene transcriptional regulation level to empower its degradation ability. The results benefit for applying genetically engineered bacteria of C. testosteroni to assimilate stable hormonally active compounds effectively. The results were as follows:1. teiR gene was cloned from genomic DNA of Comamonas testosteroni. Sequence analysis showed that teiR gene (GenBank accession No. FJ890932) shared highest similarity with teiR (GenBank accession No. AY363220) and tesR gene (GenBank accession No. AB186487), 99 % and 95 %, respectively. teiR gene encoding protein was a putative LuxR-type transcriptional regulator, its domain distribution as N-terminal autoinducer binding domain and C-terminal HTH DNA binding domain.2. Plasmid pET28a-teiR was constructed and performed in E. coli to overexpress TeiR protein. The recombinant protein was purified by its His-tag sequence using Ni-NTA Spin Column supplied by Qiagen. TeiR protein, as assessed by SDS-polyacrylamide gel electrophoresis, was used to prepare antibodies against rabbits. The antibodies reacted very well with the native recombinant protein, and the dilution could be 1:10000.3. Then the teiR gene was sucloned into plasmids pKtac2 and pK18 to yield plasmids pKtac2-teiR and pKteiR100. The recombinant plasmids were transformed into competent Escherichia coli HB101, respectively, or with plasmid p6 (containing 3a-HSD/CR gene) to detect the TeiR and 3a-HSD/CR expression levels by enzyme-linked immunosorbent assay (ELISA). Our results proved that tac promoter was much more efficient than the lacZ promoter and that teiR gene could act as an activator for 3a-HSD/CR expression. 4. Recombinant plasmid pKmut was integrated into Comamonas testosteroni chromosome DNA through homologous recombination to generate teiR gene knock-out mutant. Protein expression levels and its degradation ability were determined by enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography (HPLC), respectively. teiR-disrupted mutant inhibited the 3a-HSD/CR and TeiR expression, using the wild type of Comamonas testosteroni as a control. Finally, the mutant lost the ability to use testosterone as a carbon source. Our results confirmed that the teiR gene mediates the steroid metabolism in Comamonas testosteroni and was necessary for the 3a-HSD/CR expression.5. Recombinant plasmid pKtac2-teiR was integrated into Comamonas testosteroni chromosome DNA through homologous recombination to generate teiR gene overexpression mutant AMP8. AMP8 was already stored at China General Microbiological Culture Collection Center (CGMCC NO.2365), and was made a national patent application (Application No. 2008100724597)1) Real time fluorescence quantitative RT-PCR for detecting the expression of teiR and 3a-HSD/CR had been established, in which the wild type of Comamonas testosteroni treated as a control. The results showed that testosterone could increase this two genes expression in the wild type of Comamonas testosteroni as well as AMP8. AMP8 expressed more than that in the wild type of Comamonas testosteroni under the same conditions. Furthermore, the RT-PCR results indicated that AMP8 constitutively expressed 3a-HSD/CR, but also expressed the enzymes involved in the degradation of steroids, such as 4-hydroxy-2-oxovalerate aldolase and 2-hydroxypenta-2,4-dienoate hydratase.2) AMP8 was cultured in different conditions, and proteins were extracted to detect the TeiR and 3a-HSD/CR expression levels by ELISA using the wild type of Comamonas testosteroni as a control. The results showed that AMP8 could produce several folds of TeiR and 3a-HSD/CR compared to that of the wild type of Comamonas testosteroni, and the best was 5.8-folds of TeiR and 20-folds of 3a-HSD/CR compared to the control. Our results confirmed that AMP8 could widely increase TeiR expression, also increase 3a-HSD/CR expression.3) 8 of 15 differentially expressed proteins were identified by MALDI-TOF-MS and database searching, including 6 up-expressed proteins, 2 down-expressed proteins. The most of the identified proteins were founctioned in the catabolism of steroids.4) AMP8 was cultured for 50 days and total cell lysate was extracted per five days to detect the expression level of 3a-HSD/CR and TeiR using ELISA. The results indicated that TeiR and 3a-HSD/CR were in high and stable expression when inoculated with antibiotic in LB medium, but unstable without antibiotic.5) A HPLC and spectrophotometry assays were established to investigate the dagradation ability between AMP8 and the wild type of Comamonas testosteroni. The results showed that AMP8 had better degradation ability than the wild type of Comamonas testosteroni.