No 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.

Thermoanaerobacter ethanolicus is a Gram-positive thermophile that converts xylose to ethanol. A portion of the T. ethanolicus xylose transport permease gene ( xylH) was cloned, and the deduced protein exhibited greater than 60% similarity to homologs in enterobacteria. Xylose-binding protein ( xylF) and ( xylH) transcripts were quantitated and compared from cells grown in batch or continuous cultures grown on xylose, glucose, or a mixture of both sugars. In contrast to the strong repression of xyl operons by glucose in other bacteria, both xylF and xylH expression were detected in the presence of this hexose sugar. Expression of xylF and xylH generally increased with dilution rate (3- and 1.5-fold, respectively) and seemed to be growth rate rather than substrate dependent. Overall, these unusual sugar utilization patterns in batch and continuous culture seem to result from a basal expression level of xyl genes in the absence of xylose. T. ethanolicus is unique in possessing a triumvirate of xylose transport and catabolism operons and, given its extensive hemicellulolytic capabilities, may have evolved to constitutively express xyl genes.

Thermoanaerobacter ethanolicus strain 39E is a Gram-positive thermophile that converts sugars resulting from plant carbohydrate polymer degradation into ethanol. A putative maltose ATP-binding cassette (ABC) transport operon was isolated with genes encoding for the integral membrane components (malF and malG); the ATP-binding protein (malK); and a partial gene for the maltose-binding protein (malE). This operon is unlike most other maltose transport operons, which do not contain a contiguous malK gene. Sequence analysis showed that the individual genes in the putative operon possessed a considerable range of similarities to their respective homologs in other eubacteria and archaea. MalK had 52% amino-acid identity and over 70% similarity with its homolog from the archaeon Thermococcus litoralis, while the membrane components and binding protein exhibited much less similarity with a range of other thermophilic eubacteria. Transcript was not detected in maltose-, glucose-, or xylose-grown cells using Northern blotting, but RT-PCR showed that malFGK were expressed in cells grown on maltose or xylose. Based on these results, the strain 39E maltose operon may be subject to glucose catabolite repression.

Thermoanaerobacter ethanolicus is a xylose-utilizing thermophilic anaerobe that produces considerable amounts of ethanol. A protein in xylose-growing cells was solubilized from cell membranes by extraction with octyl-beta-glucoside. Internal peptide sequencing revealed that the protein was the product of a gene, xylF, encoding a putative D-xylose-binding protein. Metabolic labeling with 14C palmitic acid suggested that this is a lipoprotein that is anchored to the cell membrane via a cysteine residue. Binding was highly specific for xylose as evident by the lack of competition by sugars with structures similar to xylose. The apparent Kd of the protein for xylose was approximately 1.5 &mgr;M, and this value was very similar to the affinity constant determined for xylose transport by whole cells at low substrate concentrations. Uptake experiments with cells also suggested the presence of a separate low-affinity system. Binding activity varied less than 20% over a pH range of 4-8, and the level of activity was virtually unaffected when temperature was varied between 40 degreesC and 80 degreesC. This is the first biochemical characterization of a D-xylose-binding protein from a thermophilic organism.