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Japan Marine Science and Technology Center Frontier Research System for Extremophiles further confirmed by northern hybridization. The increase of the transcription products of the eight genes also coincided with their gene products increased at alkaline pH conditions. The gene product increased at higher pH condition seems to be potentially important for growth in an alkaline environment. A functionally unknown kDa protein strongly induced in alkaline and high temperature conditions was named as ARP (alkaline response protein kDa). This gene was confirmed to be pH-dependent at mRNA level. On the other hand, the kDa protein ARP was found to be in the membrane fraction by the -DE analysis with the membrane fraction of B. halodurans C- in pH . culture (Fig.). The observation of the same protein in the membrane and cytoplasm suggested that APR was membrane associated but not an integral membrane protein. Flagellin, which is another protein induced depending on alkaline pH, was also found in either cytoplasm fraction or membrane fraction. ARP and flagellin gave a quickly response to alkaline when the cells of neutral culture was transferred to the alkaline medium. Generally, the membrane proteins or membrane associate proteins are thought to play the major role for keeping pH homeostasis in a cell. Therefore, these response proteins, may be involved in the pH homeostasis. For further understanding of pH response, studies will be under way to define more clearly the functions of these proteins responded to pH. And the protein-protein interaction analysis will be major functional analysis at the next stage. 1.7. Prediction of the function of Na + /Ca 2+ exchanger in the prokaryotic cells Ca + is a second messenger in the eukaryotic cells, playing an important role such as muscle contraction, neurotransmitter, and exocytosis of a hormone. The calcium transportation is manly performed by Ca + ion channel and Na + /Ca + exchanger. Especially, the Na + /Ca + exchanger has been well studied in the eukaryotic cells but poorly characterized in the prokaryotic cells. The homologous gene products to the Na + /Ca + exchanger previously identified in the eukaryotic cells were screened bioinformatically among all CDSs identified in the genomes of three Bacillus-related species whose complete genome sequences have been determined. Search of the protein database for motif similarity was performed using Pfam motif analysis tool. In this analysis, two candidate genes for Na + /Ca + exchanger in B. halodurans and one in O. iheyensis were identified, respectively in contrast to nothing in B. subtilis. It was also found that these genes showed significant similarity to those from Homo sapiens by Psi-Blast search analysis. On the other hand, it has been suggested by SOSUI program that these candidate genes are membrane protein with transmembrane segments. 2. Metabolism and Adaptation Research 2.1. Taxonomy and preservation of newly isolated deep-sea microorganisms We isolated and identified new genera of piezophilic bacteria, psychlophilic bacteria, useful enzyme produced bacteria and yeast from several deep-sea sediments. From these experiments, we isolated strains in genera. There were novel species of Shewanella, Cytopaga, Marinobacter and some new genera were observed. This year, a new genus of piezophilic bacteria has been identified which can now be described for publication in the journal (IJSEM) and deposited in the culturer collection (JCM and ATCC) as a novel piezophilic bacteria called Psychromonas hadaliensis. On yeast, Rhodotorula benthica sp. nov. and Rhodotorula calyptogenae sp. nov., novel yeast species from animals collected from the deep-sea floor, and Rhodotorula lysiniphila sp. nov., which is related phylogenetically, were described. Phylogenetic relationships of species within the Erythrobasidium clade, which included some yeast species originated from benthic animals such as tubeworms or giant white clams, were estimated based on sequeces of S 75
JAMSTEC 2002 Annual Report Frontier Research System for Extremophiles rDNA, internal transcribed spacer, .S rDNA, S rDNA and elongation facter -. Cryptococcus surugaensis sp. nov., a novel yeast species from sediment collected on the deep-sea floor of Suruga Bay was described. In , newly isolated microorganisms preserved in our facility included bacteria strains ( isolates and others) and yeast strains (from Japan Trench), and these strains are being stored in liquid nitrogen conditions. Twenty-two deep-sea sediment samples obtained by several research vessels in were also preserved in liquid nitrogen conditions. In total, we now have types of deep-sea sediment samples in the liquid nitrogen storage tank. 2.2. Microbial adaptation to high-pressure environments (a) Identification of the genes responsible for high-pressure growth in the yeast Saccharomyces cerevisiae The study aims to establish the molecular basis responsible for the properties of piezosensitive, piezotolerant or piezophilic growth in microorganisms and to identify certain piezosensor (s) of the cell. We have reported that the availability of tryptophan is primarily important for high-pressure growth in S. cerevisiae. During incubation of the wild-type cells at MPa (approximately atm), the tryptophan permase Tat is degraded leading to growth arrest. Overexpression of Tat confers cell growth at this pressure. Analysis of the high-pressure growth mutants yielded four linkage groups, that is, HPG1, HPG2, HPG3 and HPG4. The HPG1 mutation sites were located in the HECT-domain of the ubiquitin ligase Rsp. Fig. shows the mutation site within a predicted structure of the HECT-domain. Rsp is involved in the intracellular protein degradation including Tat. The Tat level was indeed enhanced in the HPG1 mutants at both . and MPa, although the level was decreased in the wild-type strain at MPa. The Rsp-binding protein Bul was revealed to be a negative regulator for Tat under high-pressure Fig. 4 The predicted structure of the HECT domain of Rsp5 ubiquitin ligase. The HPG1 and previously known mutation sites are shown in green. A probable pathway accessible of E2-bound ubiquitin is shown in orange. Red, α -helix; Blue, β -sheet. condition. We have also cloned the HPG2 gene. The HPG2 was allelic to TAT2 itself. The HPG2 mutation sites were located in the N- or the C-terminal domain of Tat and the Tat protein level was enhanced in the mutants. Taken all results together, we propose a model for the high-pressure sensing pathway, depicting that Rsp in combination with Bul regulates Tat through its N- or C-terminus for degradation in response to increasing hydrostatic pressure. This is the first case in which high-pressure response was molecularly investigated in eukaryotic cells. (b) Construction of transformants responding to high hydrostatic pressure in bacteria The gfp (green fluorescent protein) gene under the control of a high-pressure inducible-lac promoter was introduced to the cells of Escherichia coli. Green fluorescence was indeed detected when the transformant was grown at MPa (Fig. a). Next, we introduced the gfp gene under the control of glnA promoter of Shewanella violacea or cadA promoter of Moritella japonica to the cells of M. japonica. As a result, green fluorescence was detected when the transformant was 76
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