Three enzymes for trehalose synthesis in Bradyrhizobium cultured bacteria and in bacteroids from soybean nodules

Abstract

alpha,alpha-Trehalose is a disaccharide accumulated by many microorganisms, including rhizobia, and a common role for trehalose is protection of membrane and protein structure during periods of stress, such as desiccation. Cultured Bradyrhizobium japonicum and B. elkanii were found to have three enzymes for trehalose synthesis: trehalose synthase (TS), maltooligosyltrehalose synthase (MOTS), and trehalose-6-phosphate synthetase. The activity level of the latter enzyme was much higher than those of the other two in cultured bacteria, but the reverse was true in bacteroids from nodules. Although TS was the dominant enzyme in bacteroids, the source of maltose, the substrate for TS, is not clear; i.e., the maltose concentration in nodules was very low and no maltose was formed by bacteroid protein preparations from maltooligosaccharides. Because bacteroid protein preparations contained high trehalase activity, it was imperative to inhibit this enzyme in studies of TS and MOTS in bacteroids. Validamycin A, a commonly used trehalase inhibitor, was found to also inhibit TS and MOTS, and other trehalase inhibitors, such as trehazolin, must be used in studies of these enzymes in nodules. The results of a survey of five other species of rhizobia indicated that most species sampled had only one major mechanism for trehalose synthesis. The presence of three totally independent mechanisms for the synthesis of trehalose by Bradyrhizobium species suggests that this disaccharide is important in the function of this organism both in the free-living state and in symbiosis.

FIG. 1.

(A) Effect of validamycin A on trehalase activity from the cytosol of soybean nodules. Note that the scale for validamycin A concentration is not linear. (B) Effect of trehazolin concentration on trehalase activity from the cytosol of soybean nodules. Note that the scale for enzyme activity is logarithmic and that the scale for trehazolin concentration is not linear. Enzyme activity with a trehazolin concentration of 280 nM was undetectable.

FIG. 2.

Growth of B. japonicum USDA 110 and B. elkanii USDA 324 in medium with AG as carbon sources or maltose as the sole carbon source.