Cloning and expression inEscherichia coli of an azoreductase gene fromClostridium perfringens and comparison with azoreductase genes from other bacteria

Abstract

A genomic library of Clostridium perfringens ATCC 3626 was constructed in phage lambda gt11 and screened with an antibody against the C. perfringens azoreductase, which catalyzes the reduction of azo dyes to aromatic amines. A positive recombinant phage, containing a 3.8 kb DNA fragment insert was selected and purified. Lytic and lysogenic Escherichia coli cultures infected with the recombinant phage had higher azoreductase activity than cultures infected only with the vector lambda gt11. The 3.8 kb DNA fragment was amplified by PCR and found to hybridize with one band from C. perfringens DNA digested with EcoR1, indicating the presence of a single copy of the azoreductase gene. The fragment also hybridized with DNA from other azoreductase-producing Clostridium species, a Eubacterium sp., Enterobacter cloacae, Citrobacter amalonaticus and E. coli, but not with DNA from some other species of anaerobic bacteria capable of reducing azo dyes. The data indicate that the sequence of the azoreducatse gene of C. perfringens is conserved in some anaerobes and facultative anaerobes, but not in others, and that different types of azoreductase genes must be found in other anaerobic bacteria.