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

Abstract

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 α-linked glucosides, the glucose-binding activity showed a broader specificity. All maltose-and glucose-binding activity was found in the supernatant retrieved following centrifugation (100 000 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 maltosebinding-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 maltosebinding proteins, MalE1 and MalE2, encoded in the genome. The maltosebinding 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.

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

“…One of these gene clusters is an almost exact copy of the maltose/trehalose cluster of T. litoralis (21), and its presence was attributed to a lateral gene transfer phenomenon (13). In the genome of the hyperthermophilic bacterium T. maritima, two putative maltose-binding proteins sharing very high sequence similarity (around 80%) were found (31). A similar redundancy was found in Sinorhizobium meliloti, where two maltose/trehalose/sucrose ABC transporters were characterized (23,44).…”

“…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).…”

Whole-Genome Expression Profiling of Thermotoga maritima in Response to Growth on Sugars in a Chemostat

“…In many organisms catabolite transport is linked to transcriptional control of gene expression, but little is known about such regulation in T. maritima or other members of the Thermotogales. A close relative, Thermotoga neapolitana, prioritizes its carbon sources in a cyclic AMP (cAMP)-independent manner and regulates expression of some of its carbohydrases in response to its carbon source (16,18,30). The genome sequence of T. maritima shows no evidence of cAMPdependent regulatory systems, suggesting that this mechanism is absent from this lineage.…”

Substrate Specificities and Expression Patterns Reflect the Evolutionary Divergence of Maltose ABC Transporters in Thermotoga maritima

“…T. maritima MSB8 T (DSM 3109 T ) was obtained from the Deutsche Sammlung von Mikroorganismen and Zellkulturen, Braunschweig, Germany. Cells were grown on defined basal media as described previously (15). Sugars used as carbon and energy sources were filter sterilized and added separately to media at a final concentration of 5 g/liter.…”

“…Maltose transport has been the subject of previous investigations into the processes of sugar transport and its regulation in T. maritima. Maltose binding activities in periplasmic extracts of T. maritima cells grown on different sugars have been measured (15). MalE2, one of two putative periplasmic maltose binding proteins identified in the T. maritima genome sequence, has been shown to bind maltose, maltotriose, and trehalose (32).…”