Molecular Basis of Gyrase Poisoning by the Addiction Toxin CcdB

Dao‐Thi, Minh‐Hoa; Melderen, Laurence Van; Genst, Erwin De; Afif, Hassan; Buts, Lieven; Wyns, Lode; Loris, Remy

doi:10.1016/j.jmb.2005.03.049

Cited by 130 publications

(155 citation statements)

References 65 publications

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“…The crystal structure of CcdB in complex with a fragment of DNA gyrase has been determined. The active-site residues of CcdB are defined as those that are involved in direct interaction with DNA gyrase, as determined by Ala and Asp scanning mutagenesis (10) and confirmed by X-ray crystallography of the CcdB:GyrA14 fragment complex (11). These comprise residues Ile24, Ile25, Asn95, Phe98, Trp99, and Ile101 (Fig.…”

Section: Resultsmentioning

confidence: 94%

Additional role for the ccd operon of F-plasmid as a transmissible persistence factor

Tripathi

Dewan

Barua

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Toxin-antitoxin (TA) systems are found on both bacterial plasmids and chromosomes, but in most cases their functional role is unclear. Gene knockouts often yield limited insights into functions of individual TA systems because of their redundancy. The well-characterized F-plasmid–based CcdAB TA system is important for F-plasmid maintenance. We have isolated several point mutants of the toxin CcdB that fail to bind to its cellular target, DNA gyrase, but retain binding to the antitoxin, CcdA. Expression of such mutants is shown to result in release of the WT toxin from a functional preexisting TA complex as well as derepression of the TA operon. One such inactive, active-site mutant of CcdB was used to demonstrate the contribution of CcdB to antibiotic persistence. Transient activation of WT CcdB either by coexpression of the mutant or by antibiotic/heat stress was shown to enhance the generation of drug-tolerant persisters in a process dependent on Lon protease and RecA. An F-plasmid containing a ccd locus can, therefore, function as a transmissible persistence factor.

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Section: Resultsmentioning

confidence: 94%

Additional role for the ccd operon of F-plasmid as a transmissible persistence factor

Tripathi

Dewan

Barua

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…(B) CcdB F -gyrase A14 bound crystal structure (PDB no. 1X75) (40). The key region of the interaction between CcdB F and gyrase A14 is circled.…”

Section: Discussionmentioning

confidence: 99%

Contribution of the Chromosomal ccdAB Operon to Bacterial Drug Tolerance

et al. 2017

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One of the first identified and best-studied toxin-antitoxin (TA) systems in Escherichia coli is the F-plasmid-based CcdAB system. This system is involved in plasmid maintenance through postsegregational killing. More recently, ccdAB homologs have been found on the chromosome, including in pathogenic strains of E. coli and other bacteria. However, the functional role of chromosomal ccdAB genes, if any, has remained unclear. We show that both the native ccd operon of the E. coli O157 strain (ccd O157 ) and the ccd operon from the F plasmid (ccd F ), when inserted on the E. coli chromosome, lead to protection from cell death under multiple antibiotic stress conditions through formation of persisters, with the O157 operon showing higher protection. While the plasmid-encoded CcdB toxin is a potent gyrase inhibitor and leads to bacterial cell death even under fully repressed conditions, the chromosomally encoded toxin leads to growth inhibition, except at high expression levels, where some cell death is seen. This was further confirmed by transiently activating the chromosomal ccd operon through overexpression of an active-site inactive mutant of F-plasmid-encoded CcdB. Both the ccd F and ccd O157 operons may share common mechanisms for activation under stress conditions, eventually leading to multidrug-tolerant persister cells. This study clearly demonstrates an important role for chromosomal ccd systems in bacterial persistence.IMPORTANCE A large number of free-living and pathogenic bacteria are known to harbor multiple toxin-antitoxin systems, on plasmids as well as on chromosomes. The F-plasmid CcdAB system has been extensively studied and is known to be involved in plasmid maintenance. However, little is known about the function of its chromosomal counterpart, found in several pathogenic E. coli strains. We show that the native chromosomal ccd operon of the E. coli O157 strain is involved in drug tolerance and confers protection from cell death under multiple antibiotic stress conditions. This has implications for generation of potential therapeutics that target these TA systems and has clinical significance because the presence of persisters in an antibiotic-treated population can lead to resuscitation of chronic infection and may contribute to failure of antibiotic treatment.KEYWORDS toxin-antitoxin, persistence I n the early 1940s it was reported that a small fraction of staphylococcal cells, about one in a million, survived prolonged penicillin treatment (1). These cells were termed persisters. Later it was established that persisters are phenotypic variants in a given population that show antibiotic tolerance due to probable differences in their physiology under a given set of conditions. Upon subsequent culturing, they give rise to a population that retains antibiotic sensitivity. They are different from resistant cells in which resistance is genetically encoded and is passed on from one generation to the next. In contrast, persisters are transient and rare, ranging from 10 Ϫ2 to 10 Ϫ6 in frequenc...

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“…In addition, like in MazF proteins, the loop ␤1-␤2 of CcdB is highly flexible in the free state of the protein (45,47,48) and becomes ordered upon antitoxin binding. The latter loop in CcdB does not, however, seem to fold in a unique conformation when gyrase is bound (49), although correct folding of the equivalent loop in EcMazF is required for substrate binding and catalysis. Together, these strong parallels strongly favor common ancestor origins for the MazF and CcdB proteins, and possibly also for the neutralization domains of their corresponding antitoxins.…”

Section: Structural Similarities Suggest That the Functional Divergenmentioning

confidence: 99%

Substrate Recognition and Activity Regulation of the Escherichia coli mRNA Endonuclease MazF

Zorzini

Mernik

Lah

et al. 2016

Journal of Biological Chemistry

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Escherichia coli MazF (EcMazF) is the archetype of a large family of ribonucleases involved in bacterial stress response. The crystal structure of EcMazF in complex with a 7-nucleotide substrate mimic explains the relaxed substrate specificity of the E. coli enzyme relative to its Bacillus subtilis counterpart and provides a framework for rationalizing specificity in this enzyme family. In contrast to a conserved mode of substrate recognition and a conserved active site, regulation of enzymatic activity by the antitoxin EcMazE diverges from its B. subtilis homolog. Central in this regulation is an EcMazE-induced double conformational change as follows: a rearrangement of a crucial active site loop and a relative rotation of the two monomers in the EcMazF dimer. Both are induced by the C-terminal residues Asp-78 -Trp-82 of EcMazE, which are also responsible for strong negative cooperativity in EcMazE-EcMazF binding. This situation shows unexpected parallels to the regulation of the F-plasmid CcdB activity by CcdA and further supports a common ancestor despite the different activities of the MazF and CcdB toxins. In addition, we pinpoint the origin of the lack of activity of the E24A point mutant of EcMazF in its inability to support the substrate binding-competent conformation of EcMazF.

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Molecular Basis of Gyrase Poisoning by the Addiction Toxin CcdB

Cited by 130 publications

References 65 publications

Additional role for the ccd operon of F-plasmid as a transmissible persistence factor

Additional role for the ccd operon of F-plasmid as a transmissible persistence factor

Contribution of the Chromosomal ccdAB Operon to Bacterial Drug Tolerance

Substrate Recognition and Activity Regulation of the Escherichia coli mRNA Endonuclease MazF

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