High-Affinity Maltose Binding and Transport by the Thermophilic Anaerobe Thermoanaerobacter ethanolicus 39E

Abstract

Thermoanaerobacter ethanolicus is a gram-positive thermophile that produces considerable amounts of ethanol from soluble sugars and polymeric substrates, including starch. Growth on maltose, a product of starch hydrolysis, was associated with the production of a prominent membrane-associated protein that had an apparent molecular weight of 43,800 and was not detected in cells grown on xylose or glucose. Filter-binding assays revealed that cell membranes bound maltose with high affinity. Metabolic labeling of T. ethanolicus maltose-grown cells with [ 14 C]palmitic acid showed that this protein was posttranslationally acylated. A maltose-binding protein was purified by using an amylose resin affinity column, and the binding constant was 270 nM. Since maltase activity was found only in the cytosol of fractionated cells and unlabeled glucose did not compete with radiolabeled maltose for uptake in whole cells, it appeared that maltose was transported intact. In whole-cell transport assays, the affinity for maltose was approximately 40 nM. Maltotriose and ␣-trehalose competitively inhibited maltose uptake in transport assays, whereas glucose, cellobiose, and a range of disaccharides had little effect. Based on these results, it appears that T. ethanolicus possesses a high-affinity, ABC type transport system that is specific for maltose, maltotriose, and ␣-trehalose.Thermoanaerobacter ethanolicus is a gram-positive anaerobic thermophile (11) that produces considerable amounts of ethanol from a wide range of polymeric and soluble carbohydrates (15,24,25). The physiology of T. ethanolicus type strain JW200 and strain 39E has been studied in some detail, and the high specific rate of ethanol production makes this species an attractive candidate for use in bioconversion processes (7-9). Starch is a potentially useful substrate for biomass conversion because of its availability and relatively low cost (26). The physiology (24) and enzymology (7) of starch breakdown in T. ethanolicus have been studied previously. However, there are still many gaps in our knowledge concerning fundamental processes, such as substrate transport. T. ethanolicus grows more slowly on maltose, cellobiose, or starch than on glucose (16,23,25) and apparently lacks disaccharide phosphorylase activities possessed by other thermophiles (16); it was therefore hypothesized that in this organism a glucose permease is responsible for uptake of monosaccharides derived from extracellularly degraded maltose and starch (7). However, there has been no systematic comparison of the substrate transport systems of T. ethanolicus cultures grown on glucose, maltose, or starch, and an alternative hypothesis is that the differences in growth rates observed are due to discrete sugar transport systems for mono-and disaccharides.

Maltose and Maltodextrin Transport in the Thermoacidophilic Gram-Positive Bacterium Alicyclobacillus acidocaldarius Is Mediated by a High-Affinity Transport System That Includes a Maltose Binding Protein Tolerant to Low pH

“…Uptake of maltose is mediated by a high-affinity binding-protein-dependent ABC transport system that is specific for maltose and maltodextrins. Both, the kinetic parameters of transport and the chemical nature of inhibiting sugars clearly suggest that the transporter is more closely related to that of enterobacteria than to the maltose-trehalose transport system found in the archaeon Thermococcus litoralis (61) or to a transporter in the thermophilic anaerobic bacterium Thermoanaerobacter ethanolicus that accepts maltose, maltotriose, and trehalose (31). This notion is further supported by the properties of the purified binding protein, such as a dissociation constant for maltose in the low micromolar range and its sensitivity to the same competing sugars.…”

“…This notion is further supported by the properties of the purified binding protein, such as a dissociation constant for maltose in the low micromolar range and its sensitivity to the same competing sugars. Here, the protein also differs from maltose binding proteins of other thermophilic and hyperthermophilic microorganisms, which often display K d values in the nanomolar range (1,28,31,58).…”

The High-Affinity Maltose/Trehalose ABC Transporter in the Extremely Thermophilic Bacterium Thermus thermophilus HB27 Also Recognizes Sucrose and Palatinose

“…Sugar transport mediated by binding protein-dependent ABC transporters has been characterized to different extents only in a few (hyper)thermophiles: in the hyperthermophilic archaeon Sulfolobus solfataricus (2,26); in the hyperthermophilic bacterium Thermotoga maritima (31,42); and in the thermophilic bacteria Thermoanaerobacterium thermosulfuri-genes (33), Alicyclobacillus acidocaldarius (22), and Thermoanaerobacter ethanolicus (24,25).…”

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

“…In fact, the occurrence of PTS in thermophiles and hyperthermophiles is extremely rare; the only exception is the thermophilic bacterium Bacillus stearothermophilus, which possesses PTSs for mannitol and cellobiose (8,12). On the other hand, all thermophilic and hyperthermophilic organisms examined thus far transport trehalose via ABC transport systems (5,9,11,18,24,25). The strong reduction in trehalose transport induced by arsenate in R. marinus is consistent with this type of system.…”

“…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.…”