Isolation and identification of two repressors, TetR AND LuxR, FOR 3,17β-hsd and 3α-hsd/cr gene regulation in Comamonas testosteroni
In the 1950s, the importance of microbial degradation was acknowledgedrealised with the emergence of bioinformatics approaches. Numerous reports have investigated the elimination of steroid hormones from contaminated ecosystems by microbial degradation. Due to its capability of degrading multiple toxic aromatic contaminants, Comamonas testosteroni (C. testosteroni) is widely recognised as a significant environmental bacterium for metabolising various toxic aromatic pollutants, and it has been proven to be a prospective biocatalyst for environmental pollution. Enzymatic biodegradation and biosynthesis are important intracellular processes. However, the expression of some enzymes could be inhibited directly by many environmental pollutants, thus interfered by endogenous active steroid hormone levels. The hydroxysteroid dehydrogenases (HSDs) are a set of steroidogenic enzymes with a major role in steroid biosynthesis and metabolism. Thus, research on the regulation of HSDs has been a recent trend. 3,17β-Hydroxysteroid dehydrogenase (3,17β-HSD) from C. testosteroni is a pivotal HSD enzyme in steroid degradation. To elucidate the complete molecular regulation of C. testosteroni, improving the understanding of the 3,17β-hsd gene induction mechanism is essential. Sequencing of the C. testosteroni ATCC11996 genome showed that there are two repeat sequences (RS; 16 bp), RS1 and RS2, located upstream of 3,17β-hsd. Between RS1 and RS2, there are 1,661 bp. In this work, a tetR repressor (522 bp) downstream of 3,17β-hsd was identified. A bioinformatics analysis showed that the TetR family of proteins plays a critical role in transcriptional repressors, which control the regulation by environmental signals in vivo. Results also shown thatAdditionally, as a result, C. testosteroni can metabolise steroid compounds by a group of enzymes involved in environmental responses. Here it is proposed that TetR might be a repression factor for inhibiting the expression of 3,17β-hsd in C. testosteroni. The overarching goal of this dissertation was to identify the tetR regulation mechanism and its pathway for 3,17β-hsd, so tetR was cloned into various plasmids with different fragments of the 3,17β-hsd gene, with and without the enhanced green fluorescent protein (egfp) as a reporter gene, to infer the functional mechanism and/or overexpression. The results demonstrated that TetR plays a repressor role in 3,17β-hsd gene expression. In addition, various steroids can induce 3,17β-hsd expression, especially testosterone. In order to further confirm that TetR acts as a repressor to inhibit the 3,17β-hsd expression, a tetR knockout mutant of wild type C. testosteroni was synthesised. The expression of 3,17β-hsd in the tetR knockout mutant was low, as in wild-type cells. Interestingly, testosterone induced a strong increase in the expression of 3,17β-hsd, especially in tetR knockout mutants. In principle, the results of tetR knockout mutants support the hypothesis that TetR is a repressor for the 3,17β-hsd expression, but the exact role of testosterone in this research remains unclear. Finally, 3α-hsd/cr is another gene in C. testosteroni that has been detected previously. Therefore, the relationship between tetR and 3α-hsd/cr was studied here in a similar way. The results indicated that tetR does also mediate the regulation of 3α-hsd/cr. However, besides tetR, some other factors seem to regulate 3,17β-hsd expression. The luxR gene, located 1,125 bp upstream of 3,17β-hsd was identified by a bioinformatics analysis to be also involved in the regulation of 3,17β-hsd. It is also a potent enzyme in steroid degradation and sensitive to environmental signals. Moreover, the LuxR protein plays a vital role in quorum sensing (QS), which coordinates various genes’ expression for cell densities, including genes encoding bioluminescence, virulence factors, sporulation, antibiotic biosynthesise, nitrogen fixation, biofilm formation and so on . The characteristics and roles of the LuxR family members, especially in C. testosteroni, which located 57,915 bp upstream of 3,17β-hsd was detected. To investigate luxR regulation, luxR was cloned into plasmids for functional characterisation. The data indicated that LuxR indeed acts as a repressor for 3,17β-hsd expression. Testosterone, in turn, which is known to induce 3,17β-hsd expression, could not resolve luxR repression, except with promoter 2. In summary, this dissertation adds important information to the understanding of regulation of steroid hormone degradation in bacteria.
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