GlpD and PlsB participate in persister cell formation in Escherichia coli - PubMed (original) (raw)

GlpD and PlsB participate in persister cell formation in Escherichia coli

Amy L Spoering et al. J Bacteriol. 2006 Jul.

Abstract

Bacterial populations produce dormant persister cells that are resistant to killing by all antibiotics currently in use, a phenomenon known as multidrug tolerance (MDT). Persisters are phenotypic variants of the wild type and are largely responsible for MDT of biofilms and stationary populations. We recently showed that a hipBA toxin/antitoxin locus is part of the MDT mechanism in Escherichia coli. In an effort to find additional MDT genes, an E. coli expression library was selected for increased survival to ampicillin. A clone with increased persister production was isolated and was found to overexpress the gene for the conserved aerobic sn-glycerol-3-phosphate dehydrogenase GlpD. The GlpD overexpression strain showed increased tolerance to ampicillin and ofloxacin, while a strain with glpD deleted had a decreased level of persisters in the stationary state. This suggests that GlpD is a component of the MDT mechanism. Further genetic studies of mutants affected in pathways involved in sn-glycerol-3-phosphate metabolism have led to the identification of two additional multidrug tolerance loci, glpABC, the anaerobic sn-glycerol-3-phosphate dehydrogenase, and plsB, an sn-glycerol-3-phosphate acyltransferase.

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Figures

FIG. 1.

FIG. 1.

Selection of an E. coli overexpression clone library for increased persister production. Ampicillin (100 μg/ml) was added to exponential-phase cells in a growth medium, and after 3 h of incubation surviving cells were collected, washed, and regrown for a subsequent round of selection. The percent survival is normalized to the amount of killing of the negative control on each day. Black bars, negative control (KLE401); white bars, positive control (KLE401) and high-persistence mutant HM22 added at 1:10,000 before round 1; hatched bars, expression library. The level of persisters for the positive control was not determined on day 4; this is indicated by a star.

FIG. 2.

FIG. 2.

Levels of persistence to ampicillin or ofloxacin in late exponential phase. Cells were diluted 1:1,000 and induced with 0.2% arabinose at time zero. After 5 h; samples were removed and tested for their multidrug tolerance to 100 μg/ml ampicillin or 5 μg/ml ofloxacin. The starting cell number for each was 1 × 109 CFU/ml. Black bars, wild-type control (KLE421); white bars, overexpression of GlpD (KLE414); hatched bars, overexpression of inactive GlpD (KLE443). These data are the averages of three independent experiments, and the error bars correspond to the standard deviations of the means.

FIG. 3.

FIG. 3.

Survival of mutants affected in putative persister genes. Cells were grown to stationary phase and diluted 1:10 in fresh medium (final concentration, ∼3 × 108 CFU/ml) containing 5 μg/ml ciprofloxacin. After 6 h of exposure, cells were washed, diluted, and spot plated. The wild type is EMG2, and all mutations were made in this background. These data are the averages of the log percent survival of at least three independent experiments, and the error bars correspond to the standard deviations of the means.

FIG. 4.

FIG. 4.

Role of glycerol-3-phosphate in E. coli metabolism. Enzyme names in bold indicate involvement in persistence. Underlined enzyme names indicate there was no detectable change in persistence in a deletion mutant.

FIG. 5.

FIG. 5.

Intracellular G3P concentration in stationary phase in strains involved in G3P metabolism. These data are the averages of at least four independent experiments, and the error bars correspond to the standard deviations of the means.

FIG. 6.

FIG. 6.

Changes in G3P following dilution of cells in fresh medium. (A) G3P concentration in the wild type; (B) G3P concentration in the glpD deletion strain, KLE416. These data are the averages of three independent experiments, and the error bars correspond to the standard deviations of the means.

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