acrB mutation located at carboxyl-terminal region of gyrase B subunit reduces DNA binding of DNA gyrase.
Mutations that exhibit susceptibility to acriflavine have been isolated and classified as acr mutations in Escherichia coli. We cloned the acrB gene, which has been identified as a mutation of the gyrB gene, and found a double point mutation altering two consecutive amino acids (S759R/R760C) in the COOH-terminal region of the gyrase B subunit. The mutant B subunit was found to associate with the A subunit to make the quaternary structure, and the reconstituted gyrase showed an 80-fold reduction of specific activity in DNA supercoiling assay; the sensitivity to acriflavine was not different in the same unit of wild-type and mutant gyrases. The mutant enzyme retained intrinsic ATPase activity, but DNA-dependent stimulation was observed infrequently. A gel shift assay showed that acriflavine inhibited the DNA binding of gyrase. The acrB mutation also reduced significantly the DNA binding of gyrase but did not change the sensitivity to acriflavine. These results revealed that the acrB mutation is related to the inhibitory mechanism of acriflavine; and the acriflavine sensitivity of the mutant, at least in vitro, is caused mainly by reduction of the enzyme activity. Further, our findings suggest that the COOH-terminal region of the B subunit is essential for the initial binding of gyrase to the substrate DNA.
Funatsuki
K
K
Aburahi Laboratories, Shionogi and Company, Ltd., Koka, Shiga 520-34, Japan.
Tanaka
R
R
Inagaki
S
S
Konno
H
H
Katoh
K
K
Nakamura
H
H
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D002352:Carrier Proteins; D003001:Cloning, Molecular; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009154:Mutation; D011485:Protein Binding; D016415:Sequence Alignment
D001426:Bacterial Proteins; D002352:Carrier Proteins; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C083496:AcrB protein, E coli