GyrB Proteins Research Articles

Mutations in DNA gyrase result in novobiocin resistance in halophilic archaebacteria

We have developed a cloning vector for use in halophilic archaebacteria which has a novobiocin resistance determinant as a selectable marker. The resistance determinant, which was derived from the genome of a resistant mutant strain, was mapped to a site within a 6.7-kb DNA clone by using a recombination assay and was sequenced. An open reading frame of 1.920 nucleotides (640 amino acids) was identified, with the predicted protein being highly homologous to the DNA gyrase B subunit (i.e., GyrB) of eubacteria. Three mutations were identified in the GyrB protein of the resistant mutant compared with the wild type (at amino acids 82, 122, and 137) which together enable Haloferax cells to grow in concentrations of novobiocin some 1,000 times higher than that possible for cells carrying only the wild-type enzyme. One base beyond the stop codon of gyrB was the start of gyrA, coding for the gyrase A subunit.

Cloning and simplified purification of Escherichia coli DNA gyrase A and B proteins.

We have transferred the Escherichia coli gyrA and gyrB genes onto plasmids that allow the overproduction of the DNA gyrase A and B proteins and have designed relatively simple purification procedures for both proteins. The pure proteins are obtained in good yield; from 2 liters of culture (12 g of cells), one can recover 25 mg of GyrA or 3 mg of GyrB protein.

Bacillus subtilis DNA gyrase: purification of subunits and reconstitution of supercoiling activity.

DNA gyrase from Bacillus subtilis 168 was purified by affinity chromatography on novobiocin-Sepharose and shown to consist of two subunits, A and B, with molecular weights of 100,000 and 85,000, respectively. The B subunits, which contains novobiocin-sensitive. ATPase activity, could complement the gyrA protein of Escherichia coli. No complementation was detected between the A subunit and the E. coli gyrB protein.

DNA gyrase: affinity chromatography on novobiocin-Sepharose and catalytic properties.

Novobiocin-Sepharose was prepared by coupling of novobiocin to Epoxy-activated Sepharose 6B and used as an affinity adsorbent. Four novobiocin-binding proteins were isolated from crude extracts of Escherichia coli with molecular weights of 105, 92, 85 and 40 kdal. The two larger proteins were identified as the A subunit (gyrA protein) and the B subunit (gyrB protein) of DNA gyrase topoisomerase II). By this method the two gyrase components can be easily separated and purified in high yield. Although both proteins are involved in the ATP-dependent supercoiling of relaxed plasmid DNA, only the gyrB protein is required for catalyzing the cleavage of ATP. The gyrB protein ATPase activity is competitively inhibited by novobiocin and related coumarin antibiotics. ATP hydrolysis is unaffected by the addition of either gyrA protein or DNA but stimulated in the presence of both.

An Escherichia coli mutant thermosensitive in the B subunit of DNA gyrase: Effect on the structure and replication of the colicin E1 plasmid in vitro

An E. coli strain which carries a mutation conferring clorobiocin resistance and temperature sensitivity for growth has recently been described and evidence has been presented suggesting that the mutation is located in the gyrB gene (Orr et al. 1979). The replication of the ColE1 plasmid was analysed in cell-free extracts from this thermosensitive strain. These extracts were totally deficient in the replication of exogenous plasmid DNA and were unable to maintain the superhelical structure of the plasmid DNA. Both defects could be fully complemented by addition of purified gyrB protein.