Role of Periplasmic Trehalase in Uptake of Trehalose by the Thermophilic Bacterium Rhodothermus marinus

Abstract

Trehalose uptake at 65 degrees C in Rhodothermus marinus was characterized. The profile of trehalose uptake as a function of concentration showed two distinct types of saturation kinetics, and the analysis of the data was complicated by the activity of a periplasmic trehalase. The kinetic parameters of this enzyme determined in whole cells were as follows: Km = 156 +/- 11 microM and Vmax = 21.2 +/- 0.4 nmol/min/mg of total protein. Therefore, trehalose could be acted upon by this periplasmic activity, yielding glucose that subsequently entered the cell via the glucose uptake system, which was also characterized. To distinguish the several contributions in this intricate system, a mathematical model was developed that took into account the experimental kinetic parameters for trehalase, trehalose transport, glucose transport, competition data with trehalose, glucose, and palatinose, and measurements of glucose diffusion out of the periplasm. It was concluded that R. marinus has distinct transport systems for trehalose and glucose; moreover, the experimental data fit perfectly with a model considering a high-affinity, low-capacity transport system for trehalose (Km = 0.11 +/- 0.03 microM and Vmax = 0.39 +/- 0.02 nmol/min/mg of protein) and a glucose transporter with moderate affinity and capacity (Km = 46 +/- 3 microM and Vmax = 48 +/- 1 nmol/min/mg of protein). The contribution of the trehalose transporter is important only in trehalose-poor environments (trehalose concentrations up to 6 microM); at higher concentrations trehalose is assimilated primarily via trehalase and the glucose transport system. Trehalose uptake was constitutive, but the activity decreased 60% in response to osmotic stress. The nature of the trehalose transporter and the physiological relevance of these findings are discussed.