Cloning and expression in Escherichia coli of an azoreductase gene from Clostridium perfringens and comparison with azoreductase genes from other bacteria

A genomic library of Clostridium perfringens ATCC 3626 was constructed in p hage lambda gt11 and screened with an antibody against the C. perfringens a zoreductase, which catalyzes the reduction of azo dyes to aromatic amines. A positive recombinant phage, containing a 3.8 kb DNA fragment insert was s elected and purified. Lytic and lysogenic Escherichia coli cultures infecte d with the recombinant phage had higher azoreductase activity than cultures infected only with the vector lambda gt11. The 3.8 kb DNA fragment was amp lified 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 azored uctase gene. The fragment also hybridized with DNA from other azoreductase- producing Clostridium species, a Eubacterium sp., Enterobacter cloacae, Cit robacter 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 azoreductase gene of C, perfringens is conserved in so me anaerobes and facultative anaerobes, but not in others, and that differe nt types of azoreductase genes must be found in other anaerobic bacteria.