Characterization of a bifunctional specific regulator for genes involved in maltodextrin transport and metabolism in Thermococcus litoralis

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The hyperthermophilic archaeon Thermococcus litoralis contains a high affinity and binding protein-dependent transport system for trehalose and maltose that is specifically induced by trehalose in the medium. The proposal aims at the identification and characterization of the specific gene regulator of this system. The proposed strategy for the identification is based on several findings: i, the structure of MalK, the ATP-binding subunit of the transporter, reveals a conserved domain found also in the mesophilic homologue in Escherichia coli whose only function is to interact with its specific gene regulator. Thus, MalK of Thermococcus litoralis will be used in a biochemical approach to identify the gene regulator that is expected to interact with it. ii, the gene cluster encoding the entire ABC transporter is contained in a 16kb DNA fragment in Thermococcus litoralis that has been moved into Pyrococcus furiosus by horizontal gene transfer. The necessary regulatory elements must be contained on this 16 kb fragment since the system exhibits the same inducibility in the new host. Thus, we will express genes in E. coli contained in this fragment and test their products for the ability to interact with MalK and to act as transcriptional regulators.