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

Staudenbauer, Walter L.; Orr, Elisha

doi:10.1093/nar/9.15.3589

Cited by 182 publications

(129 citation statements)

References 24 publications

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“…The isolation of gyrases was performed according to Staudenbauer and Orr [11] using a novobiocin-sepharose column. For enzymes from Streptomyeetes distinct modifications of the method were applied [12].…”

Section: Enzymesmentioning

confidence: 99%

Biochemical complementation studies in vitro of gyrase subunits from different species

1995

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To investigate the functional equivalence of DNA gyrase subunits from different bacterial sources hybrid enzymes were formed using purified A and B subunits from three species of Streptomycetes, E. coli and B. subtilis. The activity of gyrase hybrids composed of heterologous gyr A and gyr B proteins and of the gyrases containing homologous subunits was characterized by binding studies and a cleavage assay with two different DNA fragments. Likewise the enzyme activity was monitored by the super-coiling and relaxation assay with pBR322 DNA. We found that cleavage reactions are largely determined by the source of the gyr B subunits whereas DNA supercoiling and relaxation reactions of pBR322 catalyzed by DNA gyrase are limited to a combination of homologous A and B subunits or of heterologous A and B subunits from the taxonomically related bacteria Streptomycetes.

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Section: Enzymesmentioning

confidence: 99%

Biochemical complementation studies in vitro of gyrase subunits from different species

1995

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“…GyrB R and ParY R were first expressed in S. lividans TK24, using the Streptomyces expression vector pUWL201. Affinity chromatography on novobiocinSepharose (Staudenbauer & Orr, 1981;Thiara & Cundliffe, 1988) allowed the purification of the genuine aminocoumarin-sensitive GyrB protein from S. lividans, which could be reconstituted with GyrA from the same organism to yield active gyrase. However, the aminocoumarinresistant GyrB R and ParY R proteins did not bind to the affinity column and apparently eluted with the bulk of the proteins.…”

Section: Resultsmentioning

confidence: 99%

Identification of a topoisomerase IV in actinobacteria: purification and characterization of ParYR and GyrBR from the coumermycin A1 producer Streptomyces rishiriensis DSM 40489

Schmutz

Hennig

et al. 2004

Microbiology

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The biosynthetic gene clusters of the gyrase inhibitors coumermycin A 1 and clorobiocin contain two different resistance genes (gyrB R and parY R ). Both genes code for B subunits of type II topoisomerases. The authors have now overexpressed and purified the encoded proteins, as well as the corresponding A subunits GyrA and ParX. Expression was carried out in Streptomyces lividans in the form of hexahistidine fusion proteins, allowing purification by nickel affinity chromatography. The complex of GyrA and GyrB R was found to catalyse ATP-dependent supercoiling of DNA, i.e. to function as a gyrase, whereas the complex of ParX and ParY R catalysed ATP-dependent decatenation and relaxation, i.e. the functions of topoisomerase IV (topo IV). This is believed to represent the first topo IV identified in the class of actinobacteria, and the first demonstration of the formation of a topo IV as a resistance mechanism of an antibiotic producer.

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“…pPYGB was transformed into E. coli MlS(pREP4) (Qiagen) and single colonies were screened for the production of the GyrB fusion protein as instructed by the manufacturer (Qiagen) and analysed using 10 ' / o SDS-PAGE (Laemmli, 1970). facturer's instructions (Qiagen), and affinity chromatography on novobiocin-Sepharose as described previously (Orr & Staudenbauer, 1982;Staudenbauer & Orr, 1981).…”

Section: Methodsmentioning

confidence: 99%

Molecular analysis of the DNA gyrB gene from Myxococcus xanthus

et al. 1998

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DNA gyrase, an essential type II topoisomerase, mediates negative supercoiling of the bacterial chromosome, thereby affecting the processes of DNA replication, transcription, recombination and repair. The gyrB gene from the Gram-negative soil bacterium Myxococcus xanthus was sequenced. The sequence predicts a protein of 81 5 amino acid residues displaying significant homology to all known GyrB proteins. A 6-His-GyrB fusion protein was overexpressed in Escherichia coli and purified to near homogeneity using affinity chromatography on Ni-nitrilotriacetic acid-agarose and novobiocin-Sepharose columns. The fusion protein bound novobiocin and cross-reacted with anti-€. coli GyrB antibodies, indicating structural and functional similarities to the E. coli DNA GyrB. The gene was mapped to the region of the origin of replication (oriC) of M. xanthus.

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

Cited by 182 publications

References 24 publications

Biochemical complementation studies in vitro of gyrase subunits from different species

Biochemical complementation studies in vitro of gyrase subunits from different species

Identification of a topoisomerase IV in actinobacteria: purification and characterization of ParYR and GyrBR from the coumermycin A1 producer Streptomyces rishiriensis DSM 40489

Molecular analysis of the DNA gyrB gene from Myxococcus xanthus

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