Sequence analysis, purification, and study of inhibition by 4-quinolones of the DNA gyrase from Mycobacterium smegmatis

Revel-Viravau, V.; Truong, Que Chi; Moreau, N.; Jarlier, Vincent; Sougakoff, Wladimir

doi:10.1128/aac.40.9.2054

Cited by 24 publications

(15 citation statements)

References 51 publications

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“…DNA gyrase from M. smegmatis was purified by novobiocin‐Sepharose column, as described [21] with the following modifications. During purification, TGEM buffer was used and the purified protein fractions were stored in TGEM buffer with 20% glycerol.…”

Section: Methodsmentioning

confidence: 99%

Monoclonal antibodies to mycobacterial DNA gyrase A inhibit DNA supercoiling activity

Manjunatha¹,

Mahadevan²,

Visweswariah³

et al. 2001

European Journal of Biochemistry

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DNA gyrase is an essential type II topoisomerase found in bacteria. We have previously characterized DNA gyrase from Mycobacterium tuberculosis and Mycobacterium smegmatis. In this study, several monoclonal antibodies were generated against the gyrase A subunit (GyrA) of M. smegmatis. Three, MsGyrA:C3, MsGyrA:H11 and MsGyrA:E9, were further analyzed for their interaction with the enzyme. The monoclonal antibodies showed high degree of crossreactivity with both fast-growing and slow-growing mycobacteria. In contrast, none recognized Escherichia coli GyrA. All the three monoclonal antibodies were of IgG 1 isotype falling into two distinct types with respect to epitope recognition and interaction with the enzyme. MsGyrA:C3and MsGyrA:H11 IgG, and their respective Fab fragments, inhibited the DNA supercoiling activity catalyzed by mycobacterial DNA gyrase. The epitope for the neutralizing monoclonal antibodies appeared to involve the region towards the N-terminus (residues 351±415) of the enzyme in a conformation-dependent manner. These monoclonal antibodies would serve as valuable tools for structure± function analysis and immunocytological studies of mycobacterial DNA gyrase. In addition, they would be useful for designing peptide inhibitors against DNA gyrase.Keywords: DNA gyrase; enzyme inhibition; epitope mapping; monoclonal antibody; mycobacterium.The topological state of DNA plays an important role in biological activity and is maintained by a group of enzymes called topoisomerases [1,2]. These enzymes change the topology of DNA by passing one segment through another. The type I enzymes catalyze DNA-strand passage by transiently breaking one strand at a time and type II enzymes create transient breaks in both strands of a DNA segment for the enzyme-mediated passage of the second segment [1±3]. Type II topoisomerases are essential cellular enzymes that function in the segregation of newly replicated chromosome pairs, chromosome condensation and in altering DNA superhelicity [4]. DNA gyrase is a type II topoisomerase exclusively found in prokaryotes. It is the only enzyme that can introduce negative supercoiling, in a reaction that depends on ATP hydrolysis. The enzyme also catalyzes a number of other topological interconversions such as knotting±unknotting and catenation±decatenation [3].DNA gyrase from Escherichia coli is composed of two subunits, A (GyrA) and B (GyrB). The active enzyme is a tetramer of two subunits each of GyrA and GyrB [5,6]. The GyrA subunit has the active site for DNA binding, cleavage and resealing, whereas GyrB powers the reaction by catalyzing ATP hydrolysis [7]. DNA gyrase is the target of two distinct classes of inhibitors, coumarins and quinolones. Coumarins bind to GyrB and are competitive inhibitors with respect to ATP. In contrast, quinolones bind DNA gyrase when the enzyme is complexed with DNA and trap the enzyme in an abortive ternary complex [8].Of the 50 species in the genus mycobacterium, about 20 are of medical importance. Apart from well-known human mycobacterial pathogen...

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Section: Methodsmentioning

confidence: 99%

Monoclonal antibodies to mycobacterial DNA gyrase A inhibit DNA supercoiling activity

Manjunatha¹,

Mahadevan²,

Visweswariah³

et al. 2001

European Journal of Biochemistry

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“…In this isolate, amino acid 95 is threonine [23], which makes its GyrA QRDR identical to that of M. smegmatis (Genbank accession numbers X94224 and L27512). The M. smegmatis isolate encoded a threonine at position 507 of the GyrB protein rather than the arginine reported elsewhere [24].…”

Section: Bacterial Strains and Growth Conditionsmentioning

confidence: 96%

Selection of Antibiotic‐Resistant Bacterial Mutants: Allelic Diversity among Fluoroquinolone‐Resistant Mutations

Zhou¹,

Dong²,

Zhao³

et al. 2000

J INFECT DIS

131

107

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To obtain a general framework for understanding selection of antibiotic-resistant mutants, allelic diversity was examined with about 600 fluoroquinolone-resistant mutants of mycobacteria. Selection at low fluoroquinolone concentration produced many low-level resistance mutants. Some of these contained mutations that conferred unselected antibiotic resistance; none contained alterations in the quinolone-resistance-determining region of the GyrA protein, the principal drug target. As selection pressure increased, a variety of GyrA variants became prevalent. High concentrations of antibiotic reduced the variety to a few types, and eventually a concentration was reached at which no mutant was recovered. That concentration defined a threshold for preventing the selection of resistance. The pattern of variants selected, which was also strongly influenced by antibiotic structure, readily explained the variants present in clinical isolates. Thus, resistance arises from selection of mutants whose identity depends on drug concentration and structure, both of which can be manipulated to restrict selection.

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“…Table 1 shows that the dormant bacteria were killed by metronidazole whereas the actively growing aerobic bacteria were not affected by the drug. Table 1 also shows that dormant M. smegmatis culture was resistant against the anti‐mycobacterial gyrase inhibitor ofloxacin [8], which killed the actively growing culture. These data suggest that the dormant M. smegmatis culture has a similar antibiotic susceptibility pattern as dormant tubercle bacilli.…”

Section: Resultsmentioning

confidence: 99%

Oxygen depletion induced dormancy inMycobacterium smegmatis

Dick

Lee

Murugasu-Oei

1998

FEMS Microbiology Letters

135

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We report here that the physiological behaviour of the fast growing saprophytic Mycobacterium smegmatis under in vitro oxygen-depletion and reactivation conditions is strikingly similar to the characteristics shown by the slow growing pathogenic M. tuberculosis. M. smegmatis died rapidly when shifted abruptly from aerobic to anaerobic conditions. In contrast to the lethal shock of abrupt oxygen depletion, the slow depletion through a self generated oxygen gradient permitted an adaptation to a persistent state which showed increased resistance against the bactericidal effects of anaerobiosis. The anaerobic persistent culture did not synthesise DNA and showed synchronised division upon reactivation in oxygen rich medium, indicating that the persistent bacilli are uniformly arrested at a defined stage of the cell cycle. Upon reactivation the persistent culture started synthesising DNA only after the first cell division, suggesting that the persistent cells contain two chromosomes. Furthermore, the persistent culture developed sensitivity to metronidazole and resistance against ofloxacin. These results suggest that M. smegmatis might be useful as a fast growing non-pathogenic model for comparative molecular analyses of mycobacterial dormancy.

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Sequence analysis, purification, and study of inhibition by 4-quinolones of the DNA gyrase from Mycobacterium smegmatis

Cited by 24 publications

References 51 publications

Monoclonal antibodies to mycobacterial DNA gyrase A inhibit DNA supercoiling activity

Monoclonal antibodies to mycobacterial DNA gyrase A inhibit DNA supercoiling activity

Selection of Antibiotic‐Resistant Bacterial Mutants: Allelic Diversity among Fluoroquinolone‐Resistant Mutations

Oxygen depletion induced dormancy inMycobacterium smegmatis

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