Mycobacterium smegmatis DNA gyrase: cloning and overexpression in Escherichia coli

Abstract

The cloning and characterization of DNA gyrase genes from Mycobacterium smegmatis is described. The DNA sequence of 5119 bp encoding both gyrB and gyrA genes was determined. The gene gyrB precedes gyrA with a short intergenic region of 29 nucleotides. The proteins encoded, GyrB and GyrA, exhibit 45-80% identity to gyrase polypeptides from other bacteria. The genes were further engineered for overexpression in Escherichia coli. Both genes were individually cloned into a phage T7 expression system and overexpressed. The expressed GyrB and GyrA proteins had molecular masses of 75 and 95 kDa, respectively, in agreement with that calculated from the ORFs. The extracts from the overexpressing clones were fractionated to enrich the subunits and assayed for enzyme activity. While the individual extracts showed no detectable activity, the combined extract exhibited a strong DNA supercoiling activity. This activity was ATP-dependent and novobiocin-sensitive. The identity of the genes was also confirmed by complementation analysis.