Ectopic Overexpression of Wild-Type and Mutant hipA Genes in Escherichia coli : Effects on Macromolecular Synthesis and Persister Formation

Abstract

Persistence is an epigenetic trait that allows a small fraction of bacteria, approximately one in a million, to survive prolonged exposure to antibiotics. In Escherichia coli an increased frequency of persisters, called "high persistence," is conferred by mutations in the hipA gene, which encodes the toxin entity of the toxin-antitoxin module hipBA. The high-persistence allele hipA7 was originally identified because of its ability to confer high persistence, but little is known about the physiological role of the wild-type hipA gene. We report here that the expression of wild-type hipA in excess of hipB inhibits protein, RNA, and DNA synthesis in vivo. However, unlike the RelE and MazF toxins, HipA had no effect on protein synthesis in an in vitro translation system. Moreover, the expression of wild-type hipA conferred a transient dormant state (persistence) to a sizable fraction of cells, whereas the rest of the cells remained in a prolonged dormant state that, under appropriate conditions, could be fully reversed by expression of the cognate antitoxin gene hipB. In contrast, expression of the mutant hipA7 gene in excess of hipB did not markedly inhibit protein synthesis as did wild-type hipA and yet still conferred persistence to ca. 10% of cells. We propose that wild-type HipA, upon release from HipB, is able to inhibit macromolecular synthesis and induces a bacteriostatic state that can be reversed by expression of the hipB gene. However, the ability of the wild-type hipA gene to generate a high frequency of persisters, equal to that conferred by the hipA7 allele, may be distinct from the ability to block macromolecular synthesis.

Regulation of phenotypic variability by a threshold-based mechanism underlies bacterial persistence

“…In contrast, in a hipB þ strain, cells are fully rescued from the toxic effect of HipA expression (Fig. 1B), in agreement with previous observations (23). But whereas the number of colonies was not affected by HipA in the presence of HipB, their growth dynamics were, and some of the colonies appeared over an extended period of time, compared to colonies that did not express HipA, as apparent also in ref.…”

“…We induced HipA expression in cells for 3 h and plated them in conditions in which the HipA expression was repressed. We found that, without HipB, even leakage levels of expression of HipA interrupts cell growth and prevents the subsequent appearance of colonies, as previously observed (23,24). In contrast, in a hipB þ strain, cells are fully rescued from the toxic effect of HipA expression (Fig.…”

“…1A). Overexpression of the toxin was demonstrated to lead to a growth arrest that could be reversed by the expression of the antitoxin (23)(24)(25)(26). These findings confirm that the hipBA TA module plays a key role in determining bacterial persistence.…”

“…But whereas the number of colonies was not affected by HipA in the presence of HipB, their growth dynamics were, and some of the colonies appeared over an extended period of time, compared to colonies that did not express HipA, as apparent also in ref. 23.…”

Role for dnaK locus in tolerance of multiple stresses in Staphylococcus aureus

“…The persister cells do not contain genetic traits that confer resistance to antibiotics, and, as a result, the progeny of these persisters are just as sensitive to the antibiotic used to treat the parental cells (Korch & Hill, 2006;Stewart, 2002). Although oxacillin tolerance was reduced in the dnaK mutant of meticillin-resistant S. aureus strain COL, no such reduction in the oxacillin MIC was observed in the dnaK mutant of meticillin-susceptible S. aureus strain SH1000.…”