Formation of linear plasmid multimers promoted by the phage lambda Red-system in lon mutants of Escherichia coli

We report here the formation of plasmid linear multimers promoted by the Red-system of phage lambda using a multicopy plasmid comprised of lambda red alpha and red beta genes, under the control of the lambda cI857 repressor. Our observations have revealed that the multimerization of plasmid DNA is dependent on the red beta and recA genes, suggesting a concerted role for these functions in the formation of plasmid multimers. The formation of multimers occurred in a recBCD+ sbcB+ xthA+ lon genetic background at a higher frequency than in the isogenic lon+ host cells. The multimers comprised tandem repeats of monomer plasmid DNA. Treatment of purified plasmid DNA with exonuclease III revealed the presence of free double-chain ends in the molecules. Determination of the size of multimeric DNA, by pulse field gel electrophoresis, revealed that the bulk of the DNA was in the range 50-240 kb, representing approximately 5-24 unit lengths of monomeric plasmid DNA. We provide a conceptual framework for Red-system-promoted formation and enhanced accumulation of plasmid linear multimers in lon mutants of E. coli.

Performance of Tuned Half-Wave-Length Power Transmission Lines

The basic concepts of tuned half-wave lines were covered by Hubert and Gent [1]. In this paper the problem of overvoltages during faults and the stability of the system incorporating such tuned lines are discussed. The type of tuning bank and the line arrangements that will be satisfactory from the point of view of stability are suggested. The behavior of a line tuned by distributed capacitor is analyzed, and its performance is compared with the other type of tuned line.

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

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.

MazF-induced Growth Inhibition and Persister Generation in Escherichia coli

Background: MazEF is a chromosomally encoded bacterial toxin-antitoxin system whose cellular role is controversial. Results: Expression of chromosomal MazF inhibits cell killing by multiple antibiotics in a Lon and ClpP dependent manner. Conclusion: MazF is involved in reversible growth inhibition and bacterial drug tolerance. Significance: Inactive, active-site toxin mutants yield functional insights by selectively activating the corresponding WT toxin in vivo. Toxin-antitoxin systems are ubiquitous in nature and present on the chromosomes of both bacteria and archaea. MazEF is a type II toxin-antitoxin system present on the chromosome of Escherichia coli and other bacteria. Whether MazEF is involved in programmed cell death or reversible growth inhibition and bacterial persistence is a matter of debate. In the present work the role of MazF in bacterial physiology was studied by using an inactive, active-site mutant of MazF, E24A, to activate WT MazF expression from its own promoter. The ectopic expression of E24A MazF in a strain containing WT mazEF resulted in reversible growth arrest. Normal growth resumed on inhibiting the expression of E24A MazF. MazF-mediated growth arrest resulted in an increase in survival of bacterial cells during antibiotic stress. This was studied by activation of mazEF either by overexpression of an inactive, active-site mutant or pre-exposure to a sublethal dose of antibiotic. The MazF-mediated persistence phenotype was found to be independent of RecA and dependent on the presence of the ClpP and Lon proteases. This study confirms the role of MazEF in reversible growth inhibition and persistence.