Physiological consequences of multiple-target regulation by the small RNA SgrS in Escherichia coli

Abstract

Cells use complex mechanisms to regulate glucose transport and metabolism to achieve optimal energy and biomass production while avoiding accumulation of toxic metabolites. Glucose transport and glycolytic metabolism carry the risk of the buildup of phosphosugars, which can inhibit growth at high concentrations. Many enteric bacteria cope with phosphosugar accumulation and associated stress (i.e., sugar-phosphate stress) by producing a small RNA (sRNA) regulator, SgrS, which decreases phosphosugar accumulation in part by repressing translation of sugar transporter mRNAs (ptsG and manXYZ) and enhancing translation of a sugar phosphatase mRNA (yigL). Despite a molecular understanding of individual target regulation by SgrS, previously little was known about how coordinated regulation of these multiple targets contributes to the rescue of cell growth during sugar-phosphate stress. This study examines how SgrS regulation of different targets impacts growth under different nutritional conditions when sugar-phosphate stress is induced. The severity of stress-associated growth inhibition depended on nutrient availability. Stress in nutrient-rich media necessitated SgrS regulation of only sugar transporter mRNAs (ptsG or manXYZ). However, repression of transporter mRNAs was insufficient for growth rescue during stress in nutrient-poor media; here SgrS regulation of the phosphatase (yigL) and as-yet-undefined targets also contributed to growth rescue. The results of this study imply that regulation of only a subset of an sRNA's targets may be important in a given environment. Further, the results suggest that SgrS and perhaps other sRNAs are flexible regulators that modulate expression of multigene regulons to allow cells to adapt to an array of stress conditions.

Fig 1

Model for the SgrS-mediated glucose-phosphate (GP) stress response. During glucose-phosphate stress, SgrR activates transcription of sgrS. SgrS associates with Hfq and negatively regulates the ptsG and manXYZ mRNAs, which encode major PTS sugar transporters. In addition, SgrS positively regulates the yigL mRNA, encoding a phosphatase. Dephosphorylation of sugars is a prerequisite for their efflux through unknown transporters. The regulation of these target mRNAs by SgrS, in turn, helps cellular recovery from GP stress. aMG, αMG; P, phosphate group.

Fig 2

SgrS mutant alleles differentially regulate expression of ptsG, manX, and yigL. (A) Base pairing between SgrS and the three targets, the ptsG, manX, and yigL mRNAs, are indicated by vertical lines. The sequence directly above SgrS and allele names (SgrS1, SgrS26, and SgrS28) indicate the mutated bases and their positions in different SgrS mutants. (B) The ΔsgrS strains with ptsG′-′lacZ, manX′-′lacZ, or yigL′-′lacZ carrying an empty vector, P_lac-sgrS, P_lac-sgrS1, P_lac-sgrS26, or P_lac-sgrS28 were grown to early log phase and exposed to 0.1 mM IPTG. Samples were collected 60 min after IPTG addition and assayed for β-galactosidase activity. Specific activities were normalized to the levels in the strains carrying the empty vector to yield relative activity (fold). Three independent experiments were performed; results reported are averages plus standard deviations (error bars). (C) Strains were grown to early log phase and exposed to 0.5% αMG for 10 min. Rifampin (Rif) (250 μg/ml) was then added to all cultures, and RNA samples were harvested at the indicated time points and subjected to Northern blot analysis. Blots shown are representative of three independent experiments. WT, wild type.

Fig 3

Regulation of ptsG by SgrS is crucial for recovery from αMG-induced stress. Strains were grown in LB medium overnight and then subcultured 1:200 in fresh medium, both in the presence of 0.1 mM IPTG. Cells were harvested at an optical density at 600 nm of ∼0.1 by filtration, washed, and resuspended in fresh medium with 0.5% αMG and in the absence (A) or presence (B) of 0.1 mM IPTG. Growth of all cultures was monitored by OD₆₀₀ throughout the whole procedure, but only the measurements following resuspension of cells are reported in the graphs. Results shown are representative of at least three independent trials.

Fig 4

SgrS-mediated regulation of multiple targets, including ptsG, yigL, and additional targets, is required for recovery from αMG-induced stress. (A and B) Strains were grown in minimal MOPS medium supplemented with 0.4% glycerol in the presence of 25 ng/ml aTc to an OD₆₀₀ of ∼0.1 and then exposed to 0.5% αMG. (C) Strains were grown overnight in minimal MOPS medium supplemented with 0.4% glycerol and 25 μg/ml kanamycin and then subcultured 1:200 in fresh medium, both in the presence of 0.1 mM IPTG. aTc (25 ng/ml) was also present in all the subcultures. Cells were harvested at an OD₆₀₀ of ∼0.1 by filtration, washed, and resuspended in fresh medium with 0.5% αMG and 25 ng/ml aTc. Growth of all cultures was monitored by OD₆₀₀ throughout the whole procedure, but only the measurements following resuspension of cells were reported in the graphs. (D) Strains were grown in minimal MOPS medium supplemented with 0.2% fructose in the presence of 25 ng/ml aTc to an OD₆₀₀ of ∼0.1 and then exposed to 0.5% αMG. All results are representative of at least three independent experimental trials.

Fig 5

Supplementation with Casamino Acids improves growth during glucose-phosphate stress. Strains were grown in minimal MOPS medium supplemented with 0.2% fructose (A and B) or 0.4% glycerol (C and D). In addition, 0.5% αMG and/or 0.1% Casamino Acids (CAA) were present in the media as indicated. Results shown are representative of at least three independent experimental trials.