Analysis of strains lacking known osmolyte accumulation mechanisms reveals contributions of osmolytes and transporters to protection against abiotic stress

Abstract

Osmolyte accumulation and release can protect cells from abiotic stresses. In Escherichia coli, known mechanisms mediate osmotic stress-induced accumulation of K(+) glutamate, trehalose, or zwitterions like glycine betaine. Previous observations suggested that additional osmolyte accumulation mechanisms (OAMs) exist and their impacts may be abiotic stress specific. Derivatives of the uropathogenic strain CFT073 and the laboratory strain MG1655 lacking known OAMs were created. CFT073 grew without osmoprotectants in minimal medium with up to 0.9 M NaCl. CFT073 and its OAM-deficient derivative grew equally well in high- and low-osmolality urine pools. Urine-grown bacteria did not accumulate large amounts of known or novel osmolytes. Thus, CFT073 showed unusual osmotolerance and did not require osmolyte accumulation to grow in urine. Yeast extract and brain heart infusion stimulated growth of the OAM-deficient MG1655 derivative at high salinity. Neither known nor putative osmoprotectants did so. Glutamate and glutamine accumulated after growth with either organic mixture, and no novel osmolytes were detected. MG1655 derivatives retaining individual OAMs were created. Their abilities to mediate osmoprotection were compared at 15°C, 37°C without or with urea, and 42°C. Stress protection was not OAM specific, and variations in osmoprotectant effectiveness were similar under all conditions. Glycine betaine and dimethylsulfoniopropionate (DMSP) were the most effective. Trimethylamine-N-oxide (TMAO) was a weak osmoprotectant and a particularly effective urea protectant. The effectiveness of glycine betaine, TMAO, and proline as osmoprotectants correlated with their preferential exclusion from protein surfaces, not with their propensity to prevent protein denaturation. Thus, their effectiveness as stress protectants correlated with their ability to rehydrate the cytoplasm.

FIG 1

Growth of E. coli strains CFT073 and WG1331 in high-osmolality urine. Representative data are shown; a more extensive data set is shown in Fig. S2 in the supplemental material. Growth of strains CFT073 (wild-type pyelonephritis isolate, solid lines) and WG1331 (ΔotsA ΔbetT ΔproP ΔproU, dashed lines) was monitored for 18 h in a nephelometer (see Materials and Methods). The media included a high-osmolality urine pool (HOU, pH 6.3, 0.63 M urea, 1.00 mol/kg) and MOPS medium adjusted with NaCl and urea to attain the same urea content and osmolality (HOM, see Materials and Methods). Data are means with bars representing standard errors obtained from two separate experiments with nine replicate cultures per experiment (urine pools) or from four separate experiments with nine wells per experiment (MOPS medium). For clarity, an error bar is shown for only every 10th data point.

FIG 2

Compositions of cell extracts. Bacterial cultures were prepared and metabolites were extracted for analysis by ¹³C-NMR spectroscopy as described in Materials and Methods. Peak assignments based on the spectra of standard compounds analyzed under the same conditions by ¹³C 1-dimensional and ¹H-¹³C 2-dimensional NMR spectroscopy were as follows: 1, glutamate; 2, glutamine; 3, trehalose; 4, TMAO; 5, urea; 6, creatinine. (A) Metabolites were extracted from strains WG1246 and MG1655 after cultivation in 1 liter of MMA supplemented with 0.5 M NaCl and yeast extract (1 mg/ml), TMAO (10 mM), or nothing (No Supplement). (B) Metabolites were extracted from strains CFT073 and WG1331 after cultivation in 0.5 liter of the indicated urine pool (except that WG1331 was cultivated in 0.35 liter of the low-osmolality urine pool).

FIG 3

Bacterial growth at high salinity. Growth of strains MG1655 (wild type), WG1265 (ΔbetT ΔproP ΔproU), and WG1246 (ΔotsA ΔbetT ΔproP ΔproU) in MMA supplemented with 0.5 M NaCl was monitored for 18 h in a nephelometer (see Materials and Methods). The medium was unsupplemented (No Supplement) or supplemented with Casamino Acids (CAA), brain heart infusion (BHI), or yeast extract (YE) (1 mg/ml). Data are means with bars representing standard errors for two separate experiments with six replicate cultures per experiment. For clarity, an error bar is shown for only every 10th data point. Brain heart infusion and yeast extract (1 mg/ml) stimulated the growth of strains WG1228 (BetT⁺), WG1230 (ProP⁺), and WG1232 (ProU⁺), but Casamino Acids (1 mg/ml) did not (data not shown).

FIG 4

Contributions of transporters ProP and ProU to osmoprotection and urea protection. Growth of E. coli strains WG1230 (ΔotsA ΔbetT proP⁺ ΔproU) and WG1232 (ΔotsA ΔbetT ΔproP proU⁺) in MMA supplemented with 0.5 M NaCl and the indicated osmoprotectant (top, Osmoprotection) or in MMA supplemented with 0.3 M NaCl, 0.6 M urea, and the indicated osmoprotectant (bottom, Urea Tolerance) was monitored for 18 h in a nephelometer (see Materials and Methods). Osmoprotectants were provided at 1 mM except for TMAO, which was provided at 100 mM. Shown are the means with bars representing standard errors for data obtained from at least 2 separate experiments with 3 replicate cultures per experiment (Osmoprotection) or from 3 separate experiments with 4 replicate cultures per experiment (Urea Tolerance). For clarity, an error bar is shown for only every 10th data point. Dotted midlines are included to facilitate comparisons among plots. The data in the left panels show that strain WG1246 (ΔotsA ΔbetT ΔproP ΔproU) failed to grow in the presence of these compounds (data are global averages ± standard errors for determinations of the growth of strain WG1246 in the presence of each of the listed osmoprotectants).

FIG 5

Glycine betaine is a more powerful osmoprotectant than TMAO. Growth of E. coli strains WG1230 (ΔotsA ΔbetT proP⁺ ΔproU) and WG1232 (ΔotsA ΔbetT ΔproP proU⁺) in MMA supplemented with 0.5 M NaCl and no osmoprotectant or glycine betaine or TMAO was monitored for an 18-h period in a nephelometer (see Materials and Methods). Glycine betaine and TMAO were provided at 1 mM, 10 mM, or 100 mM. Shown are the means with bars representing standard errors for 2 experiments with at least 6 replicate cultures per experiment. For clarity, an error bar is shown for only every 10th data point.

FIG 6

Contributions of transporters ProP and ProU to high-temperature thermotolerance. Radial streak tests were performed as described in Materials and Methods. Petri plates containing MOPS medium supplemented with 0.5 M NaCl were inoculated radially with E. coli strains MG1655 (wild type [WT]), WG1230 (ΔotsA ΔbetT proP⁺ ΔproU), and WG1232 (ΔotsA ΔbetT ΔproP proU⁺). Osmolytes (20 μmol for TMAO, 2 μmol for the other compounds) were applied to the central filter disk. Mean zones of growth stimulation (cm) are shown (± standard error) for 3 replicate tests. Osmolytes γ-aminobutyrate, hypotaurine, myoinositol, sarcosine, sorbitol, taurine, thiaproline, and trigonelline were also tested but did not stimulate growth.

FIG 7

Contributions of transporters ProP and ProU to low-temperature thermotolerance. E. coli strains WG1230 (ΔotsA ΔbetT proP⁺ ΔproU), WG1232 (ΔotsA ΔbetT ΔproP proU⁺), and WG1246 (ΔotsA ΔbetT ΔproP ΔproU) were cultured at 15°C in microtiter plates containing MMA supplemented with 0.4 M NaCl as described in Materials and Methods. For strains WG1230 and WG1232, data are the means with bars representing standard errors for one representative experiment (of two) and 4 replicate cultures per experiment. For strain WG1246, data are the means with bars representing standard errors for one experiment with 8 replicate cultures per experiment. Nil, no supplement.