GyrA Protein Research Articles

Quinolone resistance-determining region in the DNA gyrase gyrA gene of Escherichia coli

Nucleotide sequence analysis of the gyrA genes of 10 spontaneous quinolone-resistant gyrA mutants of Escherichia coli KL16, including four mutants examined previously, disclosed that quinolone resistance was caused by a point mutation within the region between amino acids 67 and 106, especially in the vicinity of amino acid 83, of the GyrA protein.

The ParD‐ mutant of Escherichia coli also carries Aam mutation. The complete sequence of gyrA

The phenotype of a recently-described mutant (OV6), conditionally defective in chromosome partitioning and septal positioning, was originally thought to be due to a new gene (parD) mapping at 88.4 min. We have now shown that, in addition to the parD mutation, OV6 carries a gyrAam mutation and that this mutation is probably responsible for the gross phenotype of the mutant. We have cloned the gyrA gene, identified the GyrA protein, sequenced the gyrA gene and flanking genes, cloned and sequenced the gyrAam mutation, and identified its truncated product. In addition, we have identified the transcriptional start point of the gyrA gene. The E. coli GyrA protein has extensive homologies with Gyrase proteins of other organisms and weak sequence homologies with some eukaryotic cytoskeletal proteins.

Genetic and physical organization of the cloned gyrA and gyrB genes of Bacillus subtilis.

An 8-kilobase fragment already known to contain the gyrA gene of Bacillus subtilis was shown to encode the gyrB gene as well. Plasmids containing this fragment can rescue both B. subtilis gyrA and gyrB mutants and complement Escherichia coli gyrA mutants. Deletion analysis has indicated the gene locations on the cloned fragment. Under low-stringency conditions the cloned E. coli gyrA and gyrB genes each hybridized to the appropriate subfragments, confirming the assignment of the gene locations on the cloned DNA. In E. coli maxicells, proteins of 67,000 (gyrA) and 77,000 (gyrB) Mr were synthesized. Analysis of proteins encoded by various subfragments indicated the direction of transcription. Although the gyrA and gyrB genes are located adjacent to each other on the chromosome, they may be transcribed independently since expression of gyrA protein is not dependent upon the gyrB gene in maxicells.

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.