Cloning and physical characterization of katE and katF required for catalase HPII expression in Escherichia coli

Abstract

Two genes, katE and katF, affecting the synthesis of catalase HPII in Escherichia coli, have been cloned. The multistep cloning protocol involved: screening for the tet gene in a transposon interrupting the genes, selecting DNA adjacent to the transposon, and using it to probe a library of wild-type DNA to select clones from which katE and katF were subcloned into pAT153. The clones were physically characterized and the presence of the genes confirmed by complementation of their respective mutations. The location of the transposon insertions in the two genes was determined by Southern blotting of genomic digests to further confirm the identity of the cloned genes. A 93-kDa protein, the same size as the subunit of HPII, was encoded by the katE plasmid, indicating that katE was the structural gene for HPII. A 44-kDa protein was encoded by the katF plasmid.