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Periplasmic maltose- and glucose-binding protein activities in cell-free extracts of Thermotoga maritima

Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269-3125, USA¹

Author for correspondence: Kenneth M. Noll. Tel: +1 860 486 4688. Fax: +1 860 486 4331. e-mail: noll{at}uconnvm.uconn.edu

In this study, high-affinity maltose- and glucose-binding activities in cell-free extracts of Thermotoga maritima were detected; these activities were distinct and specific. At the gross level, the expression of binding-protein activities was repressed by growth of T. maritima in the presence of the cognate sugar. Growth of the organism in the presence of maltose reduced maltose-binding activity but not glucose-binding activity, while growth in the presence of glucose reduced glucose-binding activity but not maltose-binding activity. In competition assays, these binding activities showed distinct patterns of substrate specificity: whereas the maltose-binding activity showed specificity for -linked glucosides, the glucose-binding activity showed a broader specificity. All maltose- and glucose-binding activity was found in the supernatant retrieved following centrifugation (100000 g) of the cell-free extracts prepared by French-pressure-cell treatment; no activity was found in an octyl-glucoside-treated extract of the membrane fraction. The maltose-binding-protein activity was recovered from the periplasmic fraction by selective release of the periplasmic contents of T. maritima cells using a newly developed freeze–thaw procedure. Annotation of the complete genome sequence of T. maritima suggests that there may be at least two maltose-binding proteins, MalE1 and MalE2, encoded in the genome. The maltose-binding activity corresponded to a protein of 43 kDa, which was consistent in size with either of the putative proteins. These data demonstrate that the hyperthermophilic bacterium T. maritima possesses separate maltose- and glucose-binding-protein activities that are freely soluble in its periplasm, in contrast to the membrane-bound sugar-binding proteins found in archaeal hyperthermophiles.

Keywords: hyperthermophile, sugar transport, ABC transporters, transporter evolution

Abbreviations: ABC, ATP-binding cassette

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