Role of proP and proU in betaine uptake by Yersinia enterocolitica under cold and osmotic stress conditions

Abstract

Yersinia enterocolitica is a food-borne pathogen with the ability to grow at cold temperatures and tolerate high osmolarity. The bacterium tolerates osmotic stress by intracellular accumulation of osmolytes, such as betaine. The proP gene and proU operon of Y. enterocolitica were sequenced, and single (ProP(-) ProU(+) and ProP(+) ProU(-)) and double (ProP(-) ProU(-)) mutants were generated. Upon exposure to osmotic or chill stress, the single and double mutants demonstrated a reduction in betaine uptake compared to that in the wild type, suggesting that proP and proU play a role in betaine uptake during osmotic and chill stress responses of Y. enterocolitica.

FIG. 1.

Accumulation of betaine, in response to osmotic stress (2.5% sodium chloride) at 30°C, by Y. enterocolitica. Cell suspensions containing [¹⁴C]betaine (1 mM) were incubated for 0, 20, 40, and 60 min. At appropriate time points, 1 ml of cell suspension was centrifuged and the pellet was subjected to scintillation counting to calculate betaine accumulation. Betaine accumulation was normalized per mg of total cellular protein. Error bars reflect standard errors of the means. ♦, 9610 (ProP⁺ ProU⁺); ▴, 9610P (ProP⁻ ProU⁺); •, 9610U (ProP⁺ ProU⁻); ▪, 9610PU (ProP⁻ ProU⁻).

FIG. 2.

Accumulation of betaine, in response to chill stress at 4°C, by Y. enterocolitica. Cell suspensions containing [¹⁴C]betaine (1 mM) were incubated for 0, 30, 60, and 120 min. At appropriate time points, 1 ml of cell suspension was centrifuged and the pellet was subjected to scintillation counting to calculate betaine accumulation. Betaine accumulation was normalized per mg of total cellular protein. Error bars reflect standard errors of the means. ♦, 9610 (ProP⁺ ProU⁺); ▴, 9610P (ProP⁻ ProU⁺); •, 9610U (ProP⁺ ProU⁻); ▪, 9610PU (ProP⁻ ProU⁻).

FIG. 3.

Growth, measured by absorbance at an optical density of 600 nm, of wild-type and mutant strains of Y. enterocolitica at 30°C in MMA medium (A), in MMA medium containing 2.5% sodium chloride (B), and in MMA medium containing 2.5% sodium chloride and 1 mM betaine (C). Error bars reflect standard errors of the means. ♦, 9610 (ProP⁺ ProU⁺); ▴, 9610P (ProP⁻ ProU⁺); •, 9610U (ProP⁺ ProU⁻); ▪, 9610PU (ProP⁻ ProU⁻).

FIG. 4.

Growth, measured by absorbance at an optical density of 600 nm, of wild-type and mutant strains of Y. enterocolitica at 4°C in MMA medium (A), in MMA medium containing 1 mM betaine (B), in MMA medium containing 2.5% sodium chloride (C), and in MMA medium containing 2.5% sodium chloride and 1 mM betaine (D). Error bars reflect standard errors of the means. ♦, 9610 (ProP⁺ ProU⁺); ▴, 9610P (ProP⁻ ProU⁺); •, 9610U (ProP⁺ ProU⁻); ▪, 9610PU (ProP⁻ ProU⁻).