MazF-induced growth inhibition and persister generation in Escherichia coli.

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.

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.

Antimicrobial action of prototypic amphipathic cationic decapeptides and their branched dimers.

Toward delineation of antimicrobial action, a prototypic amphipathic, cationic decapeptide Ac-G-X-R-K-X-H-K-X-W-A-NH(2) was designed and peptides for which X was didehydrophenylalanine (DeltaFm), alpha-aminoisobutyric acid (Um), or phenylalanine (Fm) were synthesized. A growth kinetics experiment indicated that the bacteriostatic effects were nil (Um), mild and transient (Fm), and strong and persistent (DeltaFm) respectively. Though at par in binding to lipopolysaccharide, DeltaFm and Fm, but not Um, caused outer membrane permeabilization. Inner membrane permeabilization was attenuated and membrane architecture rehabilitated with DeltaFm but not Fm. Reverse phase high-performance liquid chromatography revealed that DeltaFm was translocated into Escherichia coli, while Um and fragments of Fm were detected in the medium. Among these monomers, only DeltaFm was modestly antibiotic [minimum inhibitory concentrations (MICs) of 110 microM (E. coli) and 450 microM (Staphylococcus aureus)]. Interestingly, a linear dimer of DeltaFm, viz. (DeltaFm)(2), turned out to be highly potent against E. coli [MIC of 2 microM and minimum bactericidal concentration (MBC) of 2 microM] and modestly potent against S. aureus (MIC of 20 microM and MBC of 20 microM). In contrast, a lysine-based branched dimer of DeltaFm, viz. DeltaFd, was found to be a potent antimicrobial against both E. coli (MIC of 2.5 microM) and S. aureus (MIC of 5 microM). Studies with analogous branched dimers of Fm and Um have indicated that dimerization represents a scaffold for potentiation of antimicrobial peptides and that the presence of DeltaF confers potent activity against both E. coli and S. aureus. De novo design has identified DeltaFd as a potent, noncytotoxic, bacterial cell-permeabilizing and -penetrating antimicrobial peptide, more protease resistant than its monomeric counterpart. We report that in comparison to the subdued and sequential "membrane followed by cell interior" mode of action of the monomeric DeltaFm, the strong and simultaneous "membrane along with cell interior" targeting by the dimeric DeltaFd potentiates and broadens its antibiotic action across the Gram-negative-Gram-positive divide.

Structural correlates of the temperature sensitive phenotype derived from saturation mutagenesis studies of CcdB.

Temperature sensitive (ts) mutants are widely used to reversibly modulate protein function in vivo and to understand functions of essential genes. Despite this, little is known about the protein structural features and mechanisms responsible for generating a ts phenotype. Also, such mutants are often difficult to isolate, limiting their use. In this study, a library consisting of 75% of all possible single-site mutants of the 101-residue, homodimeric Escherichia coli toxin CcdB was constructed. Mutants were characterized in terms of their activity at two different temperatures and at six different expression levels. Of the total of 1430 single-site mutants that were screened, 231 (16%) mutants showed a ts phenotype. The bulk of these consisted of 120 ts mutants found at all 22 buried sites and 34 ts mutants at all seven active site residues involved in binding DNA gyrase. Of the remaining ts mutants, 16 were found at residues in van der Waals contact with active site residues, 36 were at partially buried residues, and 30 resulted from introduction of Pro. Thus virtually all ts mutants could be rationalized in terms of the structure of the native protein and without knowledge of folding pathways. Data were analyzed to obtain insights into molecular features responsible for the ts phenotype and to outline structure- and sequence-based criteria for designing ts mutants of any globular protein. The criteria were validated by successful prediction of ts mutants of three other unrelated proteins, TBP, T4 lysozyme, and Gal4.