Effects of temperature, salinity, and medium composition on compatible solute accumulation by thermococcus spp

Abstract

The effects of salinity and growth temperature on the accumulation of intracellular organic solutes were examined by nuclear magnetic resonance spectroscopy (NMR) in Thermococcus litoralis, Thermococcus celer, Thermococcus stetteri, and Thermococcus zilligii (strain AN1). In addition, the effects of growth stage and composition of the medium were studied in T. litoralis. A novel compound identified as beta-galactopyranosyl-5-hydroxylysine was detected in T. litoralis grown on peptone-containing medium. Besides this newly discovered compound, T. litoralis accumulated mannosylglycerate, aspartate, alpha-glutamate, di-myo-inositol-1,1'(3,3')-phosphate, hydroxyproline, and trehalose. The hydroxyproline and beta-galactopyranosyl-5-hydroxylysine were probably derived from peptone, while the trehalose was derived from yeast extract; none of these three compounds was detected in the other Thermococcus strains examined. Di-myo-inositol-1,1'(3,3')-phosphate, aspartate, and mannosylglycerate were detected in T. celer and T. stetteri, and the latter organism also accumulated alpha-glutamate. The only nonmarine species studied, T. zilligii, accumulated very low levels of alpha-glutamate and aspartate. The levels of mannosylglycerate and aspartate increased in T. litoralis, T. celer, and T. stetteri in response to salt stress, while di-myo-inositol-1,1'(3,3')-phosphate was the major intracellular solute at supraoptimal growth temperatures. The phase of growth had a strong influence on the types and levels of compatible solutes in T. litoralis; mannosylglycerate and aspartate were the major solutes during exponential growth, while di-myo-inositol-1,1'(3,3')-phosphate was the predominant organic solute during the stationary phase of growth. This work revealed an unexpected ability of T. litoralis to scavenge suitable components from the medium and to use them as compatible solutes.

FIG. 1

Proton decoupled ¹³C-NMR spectrum of an ethanol extract of T. litoralis grown in Bacto Marine Broth containing 4% NaCl at 85°C. Resonances due to aspartate (peaks a), MG (peaks b), DIP (peaks d), glutamate (peaks g), GalHL (peaks h), and trehalose (peaks t) are indicated.

FIG. 2

¹³C-¹H correlation spectrum through one bond coupling (heteronuclear multiple quantum correlation) of the novel compound GalHL. (Inset) Schematic representation of GalHL.

FIG. 3

Effect of the growth temperature, at the optimum salinity, on the accumulation of the following compatible solutes by T. litoralis (A), T. celer (B), and T. stetteri (C): DIP (░⃞), MG (▨), glutamate (), aspartate (□), trehalose(▩), GalHL (), and hydroxyproline ().

FIG. 4

Effect of the NaCl concentration of the medium, at the optimum growth temperature, on the accumulation of the following compatible solutes by T. litoralis (A), T. celer (B), and T. stetteri (C): DIP (░⃞), MG (▨), glutamate (), aspartate (□), trehalose (▩), GalHL (), and hydroxyproline ().

FIG. 5

Correlation between growth phase and accumulation of organic solutes by T. litoralis. The culture was grown in medium containing 4.0% NaCl at 85°C (■). The intracellular concentrations of the following solutes were determined at different phases of growth: DIP (░⃞), MG (▨), glutamate (), aspartate (□), trehalose (▩), GalHL (), and hydroxyproline ().

FIG. 6

Effect of medium composition on the accumulation of organic solutes by T. litoralis grown at 85°C with 3.0% NaCl. The media contained peptone plus yeast extract (bar A), peptone plus maltose (bar B), Casitone plus yeast extract (bar C), Casitone plus maltose (bar D), tryptone plus yeast extract (bar E), and tryptone plus maltose (bar F). The intracellular concentrations of the following solutes were determined: DIP (░⃞), MG (▨), glutamate (), aspartate (□), trehalose (▩), GalHL (), and hydroxyproline ().