The role of periplasmic trehalase in the uptake of trehalose by the thermophilic bacterium Rhodothermus marinus

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande 6, Apartado 127, 2780-156 Oeiras, Portugal; Department of Biology, University of Konstanz, D-78434 Konstanz, Germany

^* To whom correspondence should be addressed. Email: santos{at}itqb.unl.pt.

	Abstract

Trehalose uptake at 65°C was characterised in Rhodothermus marinus. The profile of trehalose uptake as a function of concentration showed two distinct 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: K_m=156±11 µM and V_max=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 characterised. To discriminate the several contributions in this intricate system, a mathematical model was developed taking into account the experimental kinetic parameters of 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 are perfectly fitted with a model considering a high affinity/low capacity transport system for trehalose (K_m=0.11±0.03 µM and V_max=0.39±0.02 nmol/min/mg of protein), and a glucose transporter with moderate affinity and capacity (K_m=46±3 µM and V_max=48±1 nmol/min/mg of protein). The contribution of the trehalose transporter is important only in trehalose poor environments (up to 6 µM concentration); at higher concentration trehalose is primarily assimilated via trehalase and the glucose transport system. Trehalose uptake was constitutive, but the activity decreased by 60% in response to osmotic stress. The nature of the trehalose transporter as well as the physiological relevance of these findings are discussed.