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

Abstract

Trehalose uptake at 65°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: K m ‫؍‬ 156 ؎ 11 M 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 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 (K m ‫؍‬ 0.11 ؎ 0.03 M 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 M and V max ‫؍‬ 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 M); 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.is a nonreducing disaccharide that is widespread in the three domains of life. This sugar often accumulates intracellularly and protects against a variety of stresses (4, 17). Given the ubiquitous distribution of trehalose in all types of biotopes, it is not surprising that efficient systems for uptake of this sugar evolved in many organisms. In particular, several mechanisms have been discovered in prokaryotes and studied to different extents. In mesophilic bacteria, like Escherichia coli, the uptake of trehalose is typically mediated by the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) (2). In contrast, the PTS has not been found in extreme thermophiles or hyperthermophiles, and the ATP-binding cassette (ABC) transport system is used for trehalose uptake in the few cases examined thus far (5, 9, 18, 24, 25). The high affinity of the ABC transporters seems to be crucial for utilization of the scarce carbon sources typical of the hostile environments where thermophiles and hyperthermophiles are found.Rhodothermus marinus is a thermophilic bacterium with an optimal growth temperature of 65°C, and it was isolated from marine hot springs in the Azores Islands (1). In response to heat or osmotic stress, R. marinus accumulates ...

Genetics and physiology of the thermophilic acetogenic bacteria Thermoanaerobacter kivui lacking key genes coding for electron re-cycling enzymes

“…Pyrococcus furiosus, a hyperthermophilic archaeon grows on mannitol (Fiala and Stetter, 1986) and the metabolically engineered strain is reported to produce 3-hydroxypropionate (3HP) from maltose (Hawkins et al, 2015). Trehalose utilization has been reported in an aerobic marine thermophilic bacterium, Rhodothermus marinus, where trehalose plays a minor role in osmo-or thermo-adaptation of this organism (Jorge et al, 2008). In addition to the transport system, trehalose and maltose hydrolase genes are also found in the T. kivui genome, annotated as kojibiose phosphorylase.…”