Reinhold Horlacher

Evidence of recent lateral gene transfer among hyperthermophilic Archaea

A total of 153 nucleotide differences were found over a contiguous 16 kb region between two hyperthermophilic Archaea, Pyrococcus furiosus and Thermococcus litoralis. The 16 kb region in P. furiosus is flanked by insertion sequence (IS) elements with inverted and direct repeats. Both IS elements contain a single open reading frame (ORF) encoding a putative protein of 233 amino acids identified as a transposase. This 16 kb region has the features of a typical bacterial composite transposon and represents a possible mechanism for lateral gene transfer between Archaea or possibly between Archaea and Bacteria. A total of 23 homologous IS elements was found in the genome sequence of P. furiosus, whereas no full‐length IS elements were identified in the genomes of Pyrococcus abyssi and Pyrococcus horikoshii. Only one IS element was found in T. litoralis. In P. furiosus and T. litoralis, the 16 kb region contains an ABC transport system for maltose and trehalose that was characterized biochemically for T. litoralis. Regulation of expression studies showed that the malE gene, located on the transposon, and the encoded trehalose/maltose‐binding protein (TMBP) are induced in the presence of maltose and trehalose in both P. furiosus and T. litoralis. The implications of transposition as a mechanism for lateral gene transfer among Archaea are discussed.

Characterization of TreR, the Major Regulator of the Escherichia coli Trehalose System

The pathway of trehalose utilization inEscherichia coli is different at low and high osmolarity. The low osmolarity system takes up trehalose as trehalose 6-phosphate which is hydrolyzed to glucose and glucose 6-phosphate.treB and treC, the genes for the enzymes involved, form an operon that is controlled by TreR (encoded bytreR), the repressor of the system, for which trehalose 6-phosphate is the inducer. We have cloned and sequencedtreR. The protein contains 315 amino acids with a molecular weight of 34,508. TreR was purified and shown to bind as a dimer trehalose 6-phosphate and trehalose with a K d of 10 and 280 μm, respectively. The conformations of the protein differ from each other with either one or the other substrate-bound. Protease treatment removed the DNA-binding domain from the intact protein leaving the dimerization domain (a 29-kDa carboxyl-terminal fragment) intact. Nuclease protection experiments revealed a palindromic sequence located directly upstream of the −35 promoter sequence of treB that functions as the operator of the system.

An internally modulated, thermostable, pH sensitive Cys-loop receptor from the hydrothermal vent worm Alvinella pompejana

Background: Cys loop receptors from the hydrothermal vent worm Alvinella pompejana were studied. Results: A Cys loop receptor opens at low pH, is chloride-specific, is modulated by its N-terminal extension, and is modestly thermostable. Conclusion: Cys loop receptors from worms living in extreme and secluded habitats are more closely related to eukaryotic than to prokaryotic family members. Significance: We studied the first known Cys loop receptor from a hydrothermal vent worm. Cys loop receptors (CLRs) are commonly known as ligand-gated channels that transiently open upon binding of neurotransmitters to modify the membrane potential. However, a class of cation-selective bacterial homologues of CLRs have been found to open upon a sudden pH drop, suggesting further ligands and more functions of the homologues in prokaryotes. Here we report an anion-selective CLR from the hydrothermal vent annelid worm Alvinella pompejana that opens at low pH. A. pompejana expressed sequence tag databases were explored by us, and two full-length CLR sequences were identified, synthesized, cloned, expressed in Xenopus oocytes, and studied by two-electrode voltage clamp. One channel, named Alv-a1-pHCl, yielded functional receptors and opened upon a sudden pH drop but not by other known agonists. Sequence comparison showed that both CLR proteins share conserved characteristics with eukaryotic CLRs, such as an N-terminal helix, a cysteine loop motif, and an intracellular loop intermediate in length between the long loops of other eukaryotic CLRs and those of prokaryotic CLRs. Both full-length Alv-a1-pHCl and a truncated form, termed tAlv-a1-pHCl, lacking 37 amino-terminal residues that precede the N-terminal helix, formed functional channels in oocytes. After pH activation, tAlv-a1-pHCl showed desensitization and was not modulated by ivermectin. In contrast, pH-activated, full-length Alv-a1-pHCl showed a marked rebound current and was modulated significantly by ivermectin. A thermostability assay indicated that purified tAlv-a1-pHCl expressed in Sf9 cells denatured at a higher temperature than the nicotinic acetylcholine receptor from Torpedo californica.