RT Journal Article SR Electronic(1) A1 Madhusudan, K. A1 Nagaraja, V.YR 1995 T1 Mycobacterium smegmatis DNA gyrase: cloning and overexpression in Escherichia coli JF Microbiology, VO 141 IS 12 SP 3029 OP 3037 DO https://doi.org/10.1099/13500872-141-12-3029 PB Microbiology Society, SN 1465-2080, AB The cloning and characterization of DNA gyrase genes from Mycobacterium smegmatis is described. The DNA sequence of 5119 bp encoding both gyrB an 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 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., UL https://www.microbiologyresearch.org/content/journal/micro/10.1099/13500872-141-12-3029