BackgroundBacterial populations contain persisters, phenotypic variants that constitute approximately 1% of cells in stationary phase and biofilm cultures. Multidrug tolerance of persisters is largely responsible for the inability of antibiotics to completely eradicate infections. Recent progress in understanding persisters is encouraging, but the main obstacle in understanding their nature was our inability to isolate these elusive cells from a wild-type population since their discovery in 1944.ResultsWe hypothesized that persisters are dormant cells with a low level of translation, and used this to physically sort dim E. coli cells which do not contain sufficient amounts of unstable GFP expressed from a promoter whose activity depends on the growth rate. The dim cells were tolerant to antibiotics and exhibited a gene expression profile distinctly different from those observed for cells in exponential or stationary phases. Genes coding for toxin-antitoxin module proteins were expressed in persisters and are likely contributors to this condition.ConclusionWe report a method for persister isolation and conclude that these cells represent a distinct state of bacterial physiology.

Toxin-antitoxin (TA) systems are plasmid-or chromosome-encoded protein complexes composed of a stable toxin and a short-lived inhibitor of the toxin. In cultures of Escherichia coli, transcription of toxin-antitoxin genes was induced in a nondividing subpopulation of bacteria that was tolerant to bactericidal antibiotics. Along with transcription of known toxin-antitoxin operons, transcription of mqsR and ygiT, two adjacent genes with multiple TA-like features, was induced in this cell population. Here we show that mqsR and ygiT encode a toxin-antitoxin system belonging to a completely new family which is represented in several groups of bacteria. The mqsR gene encodes a toxin, and ectopic expression of this gene inhibits growth and induces rapid shutdown of protein synthesis in vivo. ygiT encodes an antitoxin, which protects cells from the effects of MqsR. These two genes constitute a single operon which is transcriptionally repressed by the product of ygiT. We confirmed that transcription of this operon is induced in the ampicillin-tolerant fraction of a growing population of E. coli and in response to activation of the HipA toxin. Expression of the MqsR toxin does not kill bacteria but causes reversible growth inhibition and elongation of cells.Bacterial toxin-antitoxin (TA) systems (for reviews, see references 16 and 54) are complexes consisting of a stable toxin component and a short-lived antitoxin. Toxins of TA systems are autotoxic; they target vital functions of the producing bacterium itself. TA complexes were discovered because they are plasmid-stabilizing entities. When a bacterium carries a plasmid which encodes a toxin and an antitoxin, both molecules are produced continuously and have no effect on the activities of the cell. When the plasmid is lost during cell division, the toxin is released and kills or inhibits the cell, because the unstable antitoxin is degraded faster. Chromosomal TA systems were found later (36), and comparative studies determined that they are widespread in free-living bacteria (35, 41).There are two different types of bacterial TA systems, which depend on the nature of the antitoxin. In type I TA systems, the antitoxin is a small regulatory RNA (15, 18). In type II TA systems, which are relevant to this study, both the toxin and the antitoxin are proteins. Protein antitoxins neutralize toxins by direct interaction, forming catalytically inactive complexes. All known TA genes are organized as operons. The antitoxin is usually encoded by the first gene and always acts as a transcriptional autorepressor of the operon either alone or in a complex with the toxin molecule. Thus, antitoxins control toxin activity in two ways: through direct binding and through transcriptional regulation (17).The toxins of type II systems attack essential functions of a bacterial cell, either protein synthesis through cleavage of free or ribosome-bound mRNA (e.g., RelE, MazF, HigB, and HicA) (10,11,25,43) or the replication and integrity of DNA through interference with DNA gyrase (e.g., CcdB and ...