Effects of DNA gyrase inhibitors in Escherichia coli topoisomerase I mutants.

ABSTRACTAnl-glutamine-overproducing mutant of anEscherichia coliK-12-derived strain was selected from randomly mutagenized cells in the course ofl-alanyl-l-glutamine strain development. Genome-wide mutation analysis unveiled a novel mechanism forl-glutamine overproduction in this mutant. Three mutations were identified that are related to thel-glutamine overproduction phenotype, namely, an intergenic mutation in the 5′-flanking region ofyeiGand two nonsynonymous mutations ingyrA(Gly821Ser and Asp830Asn). Expression ofyeiG, which encodes a putative esterase, was enhanced by the intergenic mutation. The nonsynonymous mutations ingyrA, a gene that encodes the DNA gyrase α subunit, affected the DNA topology of the cells. Gyrase is a type II topoisomerase that adds negative supercoils to double-stranded DNA. When the opposing DNA-relaxing activity was enhanced by overexpressing topoisomerase I (topA) and topoisomerase IV (parCandparE), an increase inl-glutamine production was observed. These results indicate that a reduction of chromosomal DNA supercoils in the mutant caused an increase inl-glutamine accumulation. The mechanism underlying this finding is discussed in this paper. We also constructed anl-glutamine-hyperproducing strain by attenuating cellularl-glutamine degradation activity. Although the reconstituted mutant (withyeiGtogether withgyrA) produced 200 mMl-glutamine, metabolic engineering finally enabled construction of a mutant that accumulated more than 500 mMl-glutamine.

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Structural Maintenance of Chromosomes Protein of Bacillussubtilis Affects Supercoiling In Vivo

Journal of Bacteriology ◽

10.1128/jb.184.19.5317-5322.2002 ◽

2002 ◽

Vol 184 (19) ◽

pp. 5317-5322 ◽

Cited By ~ 43

Author(s):

Janet C. Lindow ◽

Robert A. Britton ◽

Alan D. Grossman

Keyword(s):

Topoisomerase I ◽

Dna Gyrase ◽

Dna Supercoiling ◽

Null Mutant ◽

Wild Type ◽

Mutant Plasmid ◽

Chromatid Cohesion ◽

Structural Maintenance Of Chromosomes ◽

Gyrase Inhibitors

ABSTRACT Structural maintenance of chromosomes (SMC) proteins are found in nearly all organisms. Members of this protein family are involved in chromosome condensation and sister chromatid cohesion. Bacillus subtilis SMC protein (BsSMC) plays a role in chromosome organization and partitioning. To better understand the function of BsSMC, we studied the effects of an smc null mutation on DNA supercoiling in vivo. We found that an smc null mutant was hypersensitive to the DNA gyrase inhibitors coumermycin A1 and norfloxacin. Furthermore, depleting cells of topoisomerase I substantially suppressed the partitioning defect of an smc null mutant. Plasmid DNA isolated from an smc null mutant was more negatively supercoiled than that from wild-type cells. In vivo cross-linking experiments indicated that BsSMC was bound to the plasmid. Our results indicate that BsSMC affects supercoiling in vivo, most likely by constraining positive supercoils, an activity which contributes to chromosome compaction and organization.

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Escherichia coli DNA Topoisomerase I and Suppression of Killing by Tn5 Transposase Overproduction: Topoisomerase I Modulates Tn5 Transposition

Journal of Bacteriology ◽

10.1128/jb.180.22.5866-5874.1998 ◽

1998 ◽

Vol 180 (22) ◽

pp. 5866-5874 ◽

Cited By ~ 5

Author(s):

Hesna Yigit ◽

William S. Reznikoff

Keyword(s):

Escherichia Coli ◽

Chromosome Segregation ◽

Topoisomerase I ◽

Dna Gyrase ◽

Wild Type ◽

Dna Topoisomerase ◽

Dna Relaxation ◽

Tn5 Transposase ◽

Lines Of Evidence

ABSTRACT Tn5 transposase (Tnp) overproduction is lethal toEscherichia coli. The overproduction causes cell filamentation and abnormal chromosome segregation. Here we present three lines of evidence strongly suggesting that Tnp overproduction killing is due to titration of topoisomerase I. First, a suppressor mutation of transposase overproduction killing, stkD10, is localized in topA (the gene for topoisomerase I). ThestkD10 mutant has the following characteristics: first, it has an increased abundance of topoisomerase I protein, the topoisomerase I is defective for the DNA relaxation activity, and DNA gyrase activity is reduced; second, the suppressor phenotype of a second mutation localized in rpoH, stkA14 (H. Yigit and W. S. Reznikoff, J. Bacteriol. 179:1704–1713, 1997), can be explained by an increase in topA expression; and third, overexpression of wild-type topA partially suppresses the killing. Finally, topoisomerase I was found to enhance Tn5 transposition up to 30-fold in vivo.

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Transcriptional regulation of Mn-superoxide dismutase gene (sodA) of Escherichia coli is stimulated by DNA gyrase inhibitors

Archives of Biochemistry and Biophysics ◽

10.1016/0003-9861(92)90261-t ◽

1992 ◽

Vol 299 (1) ◽

pp. 185-192 ◽

Cited By ~ 7

Author(s):

Laura W. Schrum ◽

Hosni M. Hassan

Keyword(s):

Escherichia Coli ◽

Superoxide Dismutase ◽

Transcriptional Regulation ◽

Dna Gyrase ◽

Superoxide Dismutase Gene ◽

Gyrase Inhibitors

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Interaction of the Plasmid-Encoded Quinolone Resistance Protein Qnr with Escherichia coli DNA Gyrase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.1.118-125.2005 ◽

2005 ◽

Vol 49 (1) ◽

pp. 118-125 ◽

Cited By ~ 179

Author(s):

John H. Tran ◽

George A. Jacoby ◽

David C. Hooper

Keyword(s):

Escherichia Coli ◽

Dna Gyrase ◽

Quinolone Resistance ◽

Catalytic Cycle ◽

Repeat Family ◽

Biochemical Basis ◽

Chromosomal Genes ◽

Resistance Protein ◽

Cleavage Complex ◽

Gyrase Inhibitors

ABSTRACT Quinolone resistance normally arises by mutations in the chromosomal genes for type II topoisomerases and by changes in the expression of proteins that control the accumulation of quinolones inside bacteria. A novel mechanism of plasmid-mediated quinolone resistance was recently reported that involves DNA gyrase protection by a pentapeptide repeat family member called Qnr. This family includes two other members, McbG and MfpA, that are also involved in resistance to gyrase inhibitors. Purified Qnr-His6 was shown to protect Escherichia coli DNA gyrase directly from inhibition by ciprofloxacin. Here we have provided a biochemical basis for the mechanism of quinolone resistance. We have shown that Qnr can bind to the gyrase holoenzyme and its respective subunits, GyrA and GyrB. The binding of Qnr to gyrase does not require the presence of the complex of enzyme, DNA, and quinolone, since binding occurred in the absence of relaxed DNA, ciprofloxacin, or ATP. We hypothesize that the formation of Qnr-gyrase complex occurs before the formation of the cleavage complex. Furthermore, there was a decrease in DNA binding by gyrase when the enzyme interacted with Qnr. Therefore, it is possible that the reaction intermediate recognized by Qnr is one early in the gyrase catalytic cycle, in which gyrase has just begun to interact with DNA. Quinolones bind later in the catalytic cycle and stabilize a ternary complex consisting of the drug, gyrase, and DNA. By lowering gyrase binding to DNA, Qnr may reduce the amount of holoenzyme-DNA targets for quinolone inhibition.

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