Archaeal Binding Protein-Dependent ABC Transporter: Molecular and Biochemical Analysis of the Trehalose/Maltose Transport System of the Hyperthermophilic Archaeon Thermococcus litoralis

Abstract

We report the cloning and sequencing of a gene cluster encoding a maltose/trehalose transport system of the hyperthermophilic archaeonThermococcus litoralis that is homologous to themalEFG cluster encoding the Escherichia colimaltose transport system. The deduced amino acid sequence of themalE product, the trehalose/maltose-binding protein (TMBP), shows at its N terminus a signal sequence typical for bacterial secreted proteins containing a glyceride lipid modification at the N-terminal cysteine. The T. litoralis malE gene was expressed in E. coli under control of an inducible promoter with and without its natural signal sequence. In addition, in one construct the endogenous signal sequence was replaced by the E. coli MalE signal sequence. The secreted, soluble recombinant protein was analyzed for its binding activity towards trehalose and maltose. The protein bound both sugars at 85°C with aKd of 0.16 μM. Antibodies raised against the recombinant soluble TMBP recognized the detergent-soluble TMBP isolated from T. litoralis membranes as well as the products from all other DNA constructs expressed in E. coli. Transmembrane segments 1 and 2 as well as the N-terminal portion of the large periplasmic loop of the E. coli MalF protein are missing in the T. litoralis MalF. MalG is homologous throughout the entire sequence, including the six transmembrane segments. The conserved EAA loop is present in both proteins. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the binding protein-dependent ABC transport systems in these two phylogenetic branches.

Maltodextrin‐binding proteins from diverse bacteria and archaea are potent solubility enhancers

“…Moreover, although a statistical model [10] predicted that four of the six MBPs were likely to be insoluble in E. coli, we found that all of them were highly soluble at 37 ‡C. Tli MBP does not bind to amylose resin [11], but all of the other MBPs were quantitatively retained on an amylose column, indicating that they were properly folded (data not shown).…”

Sugar transport in Sulfolobus solfataricus is mediated by two families of binding protein‐dependent ABC transporters

“…Binding of [ 14 C]-maltose was strongly inhibited by a 10-fold excess of unlabelled maltose (76%), weakly by maltotriose (44%) and not at all by trehalose (0%). This binding activity is therefore specific for maltose/maltodextrin, and it differs from the maltose/trehalose-binding protein found in many other organisms (Xavier et al, 1996;Horlacher et al, 1998;Wassenberg et al, 2000). As S. solfataricus can grow on trehalose as sole carbon and energy source, and [ 14 C]trehalose binds to all examined ConA fractions ( Fig.…”

“…Such systems have also been found for the uptake of sugars in other archaea as, for instance, the uptake of maltose and trehalose in the hyperthermophilic Thermococcus litoralis (Xavier et al, 1996). The genes encoding this system have been cloned and sequenced and shown to be homologous to the maltose transporter of Escherichia coli, MalEFGK (Horlacher et al, 1998). Recently, three ABC transporters of Haloferax volcanii have been functionally characterized and shown to be essential for nitrate respiration (Wanner and Soppa, 1999).…”

Compatible solutes of organisms that live in hot saline environments

“…Low level accumulation of trehalose in Pyrococcus horikoshii and P. furiosus, and high levels of trehalose in Thermococcus litoralis, appears to derive exclusively from yeast extract in the medium. In these organisms, trehalose appears to be taken up from yeast extract through a high affinity ABC maltose/trehalose transporter (Horlacher et al, 1998;Koning et al, 2002). The hyperthermophilic crenarchaeote Pyrobaculum aerophilum, accumulates trehalose exclusively at its optimum growth temperature and salinity, unlike other hyperthermophilic archaea.…”