| Budget Amount *help |
¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1996: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
The hyperthermophilic bacteria living undersea hydrothermal vents have the remarkable property of growing optimally at temperatures near and even above 100ﾟC.Some of their proteins have been described to have abilities to operate at their growing extreme temperature, however, little is known about the membrane related function. Our recent studies have shown that the alanine transporter protein from moderate thermophilic bacterium PS3 is driven by an electrochemical gradient of either Na^+ or H^+, and probably contains 10 to 12 membrane spanning domain in the molecule which is one of typical features among various transpoter proteins. The present works are to study the structure-function relationships of amino acid transporter in the hyperthermophile. In order to clone the gene encoding the alanine-glycine transporter, it is necessary to establish Escherichia coli strain lacking the activity. Using the E.coli alanine-glycine transporter gene (EdagA) which have been cloned previously, we
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established the alanine-glycine transporter negative strain AK430 by homologous recombination. Then, we carried out the expression cloning from the genome library of hyperthermophilic Thermococcus sp. KS-1 which have been donated from Dr.Hoaki of Marine Biotechnology Laboratory, Mishima. Among a few colonies grown on a minimal medium containing D-alanine as a sole carbon source, we obtained two strains prominently enhancing the glycine transport. We then obtained the plasmid from one of the stain and carried out the DNA sequencing, and found an open reading frame encoding 232 amino acids (ORF232) in the insert. The ORF232 is hydrophobic and probably consisting of 5 to 6 membrane spanning domains in the molecule. A homology search revealed high homologous scores with some E.coli membrane proteins, however, there was no homology with known transporters. It is thus proposed that the ORF232 itself is a novel amino acid transporter which works as a homodimeric form or a heteromer with other unknown tranport related proteins. Less
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