| Thermoanaerobacter ethanolicus is a Gram-positive thermophile that produces considerable amounts of ethanol from starch and hemicellulose at elevated temperatures. The goals of this work were to define how T. ethanolicus regulates the transport of maltose and xylose, the main products arising from starch and hemicellulose degradation. Growth on maltose was accompanied by the synthesis of a 44 kDa membrane-associated maltose binding protein encoded by the malE gene. The protein was post-translationally modified and bound maltose with high affinity (Kd = 270 nM), as well as maltotriose and alpha-trehalose. Since maltase activity was detected only in the cytosol, it appeared that the disaccharide was transported into the cell intact. An operon was isolated that included malE, genes encoding membrane-associated transport components (malF and malG), and an ATP binding protein (malK). Sequence analysis showed that MalK had 52% amino-acid identity and over 70% similarity with its homolog from the archeon Thermococcus litoralis , whilst the membrane components and binding protein exhibited less similarity with a range of other thermophilic eubacteria. RT-PCR showed that the malFGK was a cistron, and that this transcription was inhibited by growth on glucose. A portion of the T. ethanolicus xylose permease gene (xylH) was isolated and sequenced. Northern blotting showed that growth on xylose induced the expression of xylH . The expression levels of xylF (xylose binding protein) and xylH transcripts were compared 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 genes were expressed in glucose batch cultures. In continuous culture, expression seemed to be growth rate---rather than substrate-dependent. These unusual sugar utilization seem to be related to the basal level of xyl gene expression. These observations show that it may be possible to control gene expression and sugar utilization patterns by manipulation of growth rate. Overall, this work substantially extends previous knowledge concerning plant carbohydrate utilization in T. ethanolicus , and the results will be especially useful in the manipulation of substrate and fermentation conditions to optimize enzyme and chemical production from biomass. |
