PhoU is a persistence switch involved in persister formation and tolerance to multiple antibiotics and stresses in Escherichia coli

Abstract

When a bactericidal antibiotic is added to a growing bacterial culture, the great majority of the bacterial population is killed but a small number of metabolically quiescent bacteria called persisters survive antibiotic treatment. The mechanism of this bacterial persistence is poorly understood. In Escherichia coli, we identified a new persistence gene, phoU, whose inactivation leads to a generalized higher susceptibility than that of the parent strain to a diverse range of antibiotics, including ampicillin, norfloxacin, and gentamicin, and stresses, such as starvation, acid pH, heat, peroxide, weak acids, and energy inhibitors, especially in stationary phase. The PhoU mutant phenotype could be complemented by a functional phoU gene. Mutation in PhoU leads to a metabolically hyperactive status of the cell, as shown by an increased expression of energy production genes, flagella, and chemotaxis genes and a defect in persister formation. PhoU, whose expression is regulated by environmental changes like nutrient availability and age of culture, is a global negative regulator beyond its role in phosphate metabolism and facilitates persister formation by the suppression of many important cellular metabolic processes. A new model of persister formation based on PhoU as a persister switch is proposed. PhoU may be an ideal drug target for designing new drugs that kill persister bacteria for more effective control of bacterial infections.

FIG. 1.

Killing curve of the PhoU mutant and the wild-type strain W3110 upon ampicillin treatment (A) and the effect of complementation with the functional phoU gene (B). Log phase cultures of the E. coli PhoU mutant and wild-type W3110 and the PhoU mutant transformed with the phoU gene and the vector control were exposed to ampicillin 100 μg/ml for various times up to 3 h. The viability of the bacterial cultures was determined by CFU counts on LB plates. Error bars indicate standard deviations.

FIG. 2.

Susceptibilities of the PhoU mutant JHU-313 and E. coli wild-type strain W3110 to stresses and energy inhibitors. More experimental details are described in Materials and Methods. (A) Susceptibilities to starvation in saline. (B) Susceptibilities to acid at pH 4.0 in LB. (C) Susceptibilities to energy inhibitors, 1 mM CCCP and 5 mM DCCD in MOPS minimal medium at pH 5.0. Error bars indicate standard deviations.

FIG. 3.

Susceptibilities of the log phase and stationary-phase PhoU mutant and the wild-type strain W3110 to pyrazinamide (2 mg/ml) exposure in MOPS minimal medium at pH 5.0. Error bars indicate standard deviations.

FIG. 4.

Expression of PhoU in E. coli wild-type strain W3110 in response to nutrient availability by Western blot analysis. Bacterial extracts were loaded onto a 14% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel. After electrophoresis and electrotransfer, the nitrocellulose membrane was stained with polyclonal antiserum against E. coli PhoU peptides to monitor PhoU expression. Lane 1, 27-kDa molecular mass marker; lane 2, W3110 grown overnight in MOPS minimal medium with 2 mM K2HPO4; lane 3, log phase growth of W3110 grown in rich LB medium; lane 4, stationary-phase growth of W3110 grown in LB medium.