Additional role for the
            <i>ccd</i>
            operon of F-plasmid as a transmissible persistence factor

Tripathi, Arti; Dewan, Pooja C.; Barua, Bipasha; Varadarajan, Raghavan

doi:10.1073/pnas.1121217109

Cited by 57 publications

(71 citation statements)

References 29 publications

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“…The ccd operon of the F plasmid can function as a transmissible persistence factor (20). Under stress conditions, the antitoxins of multiple TA systems undergo degradation by cellular proteases such as Lon, leading to activation of multiple TA systems, which eventually leads to multidrug tolerance, as previously suggested (20).…”

Section: Discussionmentioning

confidence: 83%

“…Under stress conditions, the antitoxins of multiple TA systems undergo degradation by cellular proteases such as Lon, leading to activation of multiple TA systems, which eventually leads to multidrug tolerance, as previously suggested (20). CcdB poisons the gyrase-DNA complex, blocking the passage of polymerases and leading to double-strand breaks.…”

Section: Discussionmentioning

confidence: 99%

“…The chromosomal ccd operon cloned on a plasmid shows a lower degree of postsegregational killing than ccdAB F (32). When the ccd operon present on the F plasmid is activated by stress, such as heat or a sublethal dose of antibiotics, an increased frequency of drug-tolerant persisters is observed (20). A previous study of E. coli O157:H7 suggests that CcdB O157 is also active and that when (12), were aligned with the CcdB F sequence using the Clustal Omega multiple-sequence alignment tool.…”

Section: Discussionmentioning

confidence: 99%

“…It was recently shown that the ccd operon of the F plasmid can also function as a transmissible persistence factor (20). Hence, it becomes important to test whether the ccd operon in the chromosomal context has a role to play in bacterial drug tolerance that is similar to or enhanced compared to that of its F-plasmid counterpart.…”

Section: Abstract Toxin-antitoxin Persistencementioning

confidence: 99%

“…This requires that the CcdA O157 antitoxin either binds weakly or does not bind to the CcdB F toxin. To explore this further, we employed a previously described methodology (20) to specifically activate a given TA system in the background of others using an active-site inactive mutant toxin. We observed that overexpression of the active-site inactive mutant toxin, CcdB F G100T, is nontoxic in cells lacking WT CcdB but leads to cell death at very low levels of induction (0.002%) when transformed in cells having the ccd F operon inserted in the chromosome (Fig.…”

Section: Figmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 83%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Abstract Toxin-antitoxin Persistencementioning

confidence: 99%

Section: Figmentioning

confidence: 99%

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Contribution of the Chromosomal ccdAB Operon to Bacterial Drug Tolerance

et al. 2017

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Ter‐Seq: A high‐throughput method to stabilize transient ternary complexes and measure associated kinetics

et al. 2022

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Regulation of biological processes by proteins often involves the formation of transient, multimeric complexes whose characterization is mechanistically important but challenging. The bacterial toxin CcdB binds and poisons DNA Gyrase. The corresponding antitoxin CcdA extracts CcdB from its complex with Gyrase through the formation of a transient ternary complex, thus rejuvenating Gyrase. We describe a high throughput methodology called Ter-Seq to stabilize probable ternary complexes and measure associated kinetics using the CcdA-CcdB-GyrA14 ternary complex as a model system. The method involves screening a yeast surface display (YSD) saturation mutagenesis library of one partner (CcdB) for mutants that show enhanced ternary complex formation. We also isolated CcdB mutants that were either resistant or sensitive to rejuvenation, and used surface plasmon resonance (SPR) with purified proteins to validate the kinetics measured using the surface display. Positions, where CcdB mutations lead to slower rejuvenation rates, are largely involved in CcdA-binding, though there were several notable exceptions suggesting allostery. Mutations at these positions reduce the affinity towards CcdA, thereby slowing down the rejuvenation process. Mutations at GyrA14-interacting positions significantly enhanced rejuvenation rates, either due to reduced affinity or complete loss of CcdB binding to GyrA14. We examined the effect of different parameters (CcdA affinity, GyrA14 affinity, surface accessibilities, evolutionary conservation) on the rate of rejuvenation. Finally, we further validated the Ter-Seq results by monitoring the kinetics of ternary complex formation for individual CcdB mutants in solution by fluorescence resonance energy transfer (FRET) studies.

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Plasmid Genomes, Introduction to

Thomas¹,

Frost²

2018

Molecular Life Sciences

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

Cited by 57 publications

References 29 publications

Contribution of the Chromosomal ccdAB Operon to Bacterial Drug Tolerance

Contribution of the Chromosomal ccdAB Operon to Bacterial Drug Tolerance

Ter‐Seq: A high‐throughput method to stabilize transient ternary complexes and measure associated kinetics

Plasmid Genomes, Introduction to

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