MYCOBACTERIUM-SMEGMATIS DNA GYRASE - CLONING AND OVEREXPRESSION IN ESCHERICHIA-COLI

The cloning and characterization of DNA gyrase genes from Mycobacteriu m smegmatis is described. The DNA sequence of 5119 bp encoding both gy rB and gyrA genes was determined. The gene gyrB precedes gyrA with a s hort intergenic region of 29 nucleotides. The proteins encoded, GyrB a nd GyrA, exhibit 45-80% identity to gyrase polypeptides from other bac teria. The genes were further engineered for overexpression in Escheri chia coli. Both genes were individually cloned into a phage T7 express ion system and overexpressed. The expressed GyrB and GyrA proteins had molecular masses of 75 and 95 kDa, respectively, in agreement with th at calculated from the ORFs. The extracts from the overexpressing clon es were fractionated to enrich the subunits and assayed for enzyme act ivity. While the individual extracts showed no detectable activity, th e combined extract exhibited a strong DNA supercoiling activity. This activity was ATP-dependent and novobiocin-sensitive. The identity of t he genes was also confirmed by complementation analysis.