Osmotolerance in Escherichia coli Is Improved by Activation of Copper Efflux Genes or Supplementation with Sulfur-Containing Amino Acids

Abstract

Improvement in the osmotolerance of Escherichia coli is essential for the production of high titers of various bioproducts. In this work, a cusS mutation that was identified in the previously constructed high-succinate-producing E. coli strain HX024 was investigated for its effect on osmotolerance. CusS is part of the two-component system CusSR that protects cells from Ag(I) and Cu(I) toxicity. Changing cusS from strain HX024 back to its original sequence led to a 24% decrease in cell mass and succinate titer under osmotic stress (12% glucose). When cultivated with a high initial glucose concentration (12%), introduction of the cusS mutation into parental strain Suc-T110 led to a 21% increase in cell mass and a 40% increase in succinate titer. When the medium was supplemented with 30 g/liter disodium succinate, the cusS mutation led to a 120% increase in cell mass and a 492% increase in succinate titer. Introducing the cusS mutation into the wild-type strain ATCC 8739 led to increases in cell mass of 87% with 20% glucose and 36% using 30 g/liter disodium succinate. The cusS mutation increased the expression of cusCFBA, and gene expression levels were found to be positively related to osmotolerance abilities. Because high osmotic stress has been associated with deleterious accumulation of Cu(I) in the periplasm, activation of CusCFBA may alleviate this effect by transporting Cu(I) out of the cells. This hypothesis was confirmed by supplementing sulfur-containing amino acids that can chelate Cu(I). Adding methionine or cysteine to the medium increased the osmotolerance of E. coli under anaerobic conditions.IMPORTANCE In this work, an activating Cus copper efflux system was found to increase the osmotolerance of E. coli In addition, new osmoprotectants were identified. Supplementation with methionine or cysteine led to an increase in osmotolerance of E. coli under anaerobic conditions. These new strategies for improving osmotolerance will be useful for improving the production of chemicals in industrial bioprocesses.

Keywords: Cu(I); CusS; Escherichia coli; copper efflux; methionine; osmotolerance.

FIG 1

Effects of the cusS mutation in strain HX024 on cell growth and succinate production under high osmotic stress. Fermentations of strain HX024 and its derivative, with the mutated cusS restored to its original sequence, were performed in NBS medium containing 12% (weight per volume [wt/vol]) glucose. The pH was maintained at 7.0 by the automatic addition of a base containing 2.4 M potassium carbonate and 1.2 M potassium hydroxide. The fermentations were performed three times, and the error bars represent standard deviations. (A) Cell growth curve. (B) Succinate titer at 96 h. Strain MX-210 had the restored cusS. Significant differences were determined by t test. The asterisk indicates a significant difference from the control (**, P value of <0.01).

FIG 2

Reverse metabolic engineering of mutated cusS in the parent strain Suc-T110. Fermentations of strain Suc-T110 and its derivative, with the mutated cusS restored to its original sequence, were performed in NBS medium containing 5% (wt/vol) glucose, 12% (wt/vol) glucose, and 5% (wt/vol) glucose supplemented with 30 g/liter disodium succinate. The pH was maintained at 7.0 by the automatic addition of a base containing 2.4 M potassium carbonate and 1.2 M potassium hydroxide. The fermentations were performed three times, and the error bars represent standard deviations. (A, B) Cell growth curve and succinate titer in 5% (wt/vol) glucose. (C, D) Cell growth curve and succinate titer in 12% (wt/vol) glucose. (E, F) Cell growth curve and succinate titer in 5% (wt/vol) glucose and 30 g/liter disodium succinate. Strain NZ-504 had the mutated cusS. Significant differences were determined by t test; the asterisks indicate a significant difference from the control (**, P value of <0.01; ***, P value of <0.001).

FIG 3

Reverse metabolic engineering of mutated cusS in wild-type strain ATCC 8739. Fermentations of strain ATCC 8739 and its derivative, with the mutated cusS restored to its original sequence, were performed in NBS medium containing 5% (wt/vol) glucose, 20% (wt/vol) glucose, and 5% (wt/vol) glucose supplemented with 30 g/liter disodium succinate. The pH was maintained at 7.0 by the automatic addition of a base containing 6 M potassium hydroxide. The fermentations were performed three times, and the error bars represent standard deviations. (A) 5% (wt/vol) glucose. (B) 20% (wt/vol) glucose. (C) 5% (wt/vol) glucose and 30 g/liter disodium succinate. Strain MX-204 had the mutated cusS.

FIG 4

Characterization of the osmotolerance mechanism of the mutated cusS. Fermentations of strain Suc-T110 and its derivatives were performed in NBS medium containing 12% (wt/vol) glucose. The pH was maintained at 7.0 by automatic addition of a base containing 2.4 M potassium carbonate and 1.2 M potassium hydroxide. The fermentations were performed three times, and the error bars represent standard deviation. (A) The relative expression levels of cusS and cusC in strain Suc-T110 and its derivative NZ-504, which had the cusS mutation, in 12% (wt/vol) glucose. (B, C) Cell growth curves and succinate titers of strains Suc-T110 and its derivatives NZ-521 and NZ-523, which had cusS and cusC deleted, respectively, in 12% (wt/vol) glucose. (D, E) Cell growth curves and succinate titers of strains Suc-T110 and its derivatives MX-217 and MX-222, which had cusC modulated, in 12% (wt/vol) glucose. (F) The relative expression levels of cusC in strain Suc-T110 and its derivatives MX-217 and MX-222 in 12% (wt/vol) glucose. Significant differences were determined by one-way ANOVA; the asterisks indicate a significant difference from the control (***, P value of <0.001; **, P value of <0.01).

FIG 5

Working model of the CopA and Cus system for copper homeostasis in parent strain Suc-T110 and the cusS mutant. (A) For strain Suc-T110 under normal conditions (5% glucose), copper exists mostly in the form of less-toxic Cu(II), and the CopA and Cus system was not induced. (B) For strain Suc-T110 under high osmotic pressure (12% glucose), increasing free Cu(I) in the periplasmic space activates the expression of copA, cusS, cusR, and cusCFAB to expel toxic Cu(I). (C) For strain NZ-504 with the mutated cusS under high osmotic pressure (12% glucose), expression levels of cusS and cusCFAB are increased to expel more Cu(I) from the periplasmic space. Closed circles represent toxic Cu(I), and open circles represent less-toxic Cu(II).

FIG 6

Effects of methionine or cysteine supplementation on cell growth and succinate production of strain Suc-T110. Fermentations were performed in NBS medium containing 5% (wt/vol) or 12% (wt/vol) glucose. The pH was maintained at 7.0 by the automatic addition of a base containing 2.4 M potassium carbonate and 1.2 M potassium hydroxide. The fermentations were performed three times, and the error bars represent standard deviations. (A, B) Cell growth curve and succinate titer at 96 h under 5% (wt/vol) glucose. (C, D) Cell growth curve and succinate titer at 96 h under 12% (wt/vol) glucose. Significant differences were determined by one-way ANOVA; the asterisks indicate a significant difference from the control (***, P value of <0.001).

FIG 7

Effects of methionine or cysteine supplementation on the growth of wild-type E. coli strains ATCC 8739 and MG1655. Fermentations were performed in NBS medium containing 20% (wt/vol) glucose or 5% (wt/vol) glucose supplemented with 30 g/liter disodium succinate. The pH was maintained at 7.0 by automatic addition of a base containing 6 M potassium hydroxide. The fermentations were performed three times, and the error bars represent standard deviations. (A) Cell growth of strain ATCC 8739 under 20% (wt/vol) glucose. (B) Cell growth of strain ATCC 8739 under 5% (wt/vol) glucose supplemented with 30 g/liter disodium succinate. (C) Cell growth of strain MG1655 under 20% (wt/vol) glucose. (D) Cell growth of strain MG1655 under 5% (wt/vol) glucose supplemented with 30 g/liter disodium succinate.