Characterization and regulation of the NADP-linked 7α-hydroxysteroid dehydrogenase gene from Clostridium sordellii

Abstract

A bile acid-inducible NADP-linked 7α-hydroxysteroid dehydrogenase (7α- HSDH) from Clostridium sordellii ATCC 9714 was purified 310-fold by ion- exchange, gel filtration, and dye-ligand affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified enzyme showed one predominant peptide band (30,000 Da). The N-terminal sequence was determined, and the corresponding oligonucleotides were synthesized and used to screen EcoRI and HindIII genomic digests of C. sordellii. Two separate fragments (4,500 bp, EcoRI; 3,200 bp, HindIII) were subsequently cloned by ligation to pUC19 and transformation into Escherichia coli DH5α-MCR. The EcoRI fragment was shown to contain a truncated 7α-HSDH gene, while the HindIII fragment contained the entire coding region. E. coli clones containing the HindIII insert expressed high levels of an NADP-linked 7α-HSDH. Nucleotide sequence analyses suggest that the 7α-HSDH is encoded by a monocistronic transcriptional unit, with DNA sequence elements resembling rho-independent terminators located in both the upstream and downstream flanking regions. The transcriptional start site was located by primer extension analysis. Northern (RNA) blot analysis indicated that induction is mediated at the transcriptional level in response to the presence of bile acid in the growth medium. In addition, growth-phase- dependent expression is observed in uninduced cultures. Analysis of the predicted protein sequence indicates that the enzyme can be classified in the short-chain dehydrogenase group.