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沼津工業高等専門学校研究報告
第 56 号，2022 年 2 月㧗ᗘዲሷᛶྂ⣽⳦Haloarcula japonica ⏤᮶㉮Ẽᛶࢺࣛࣥࢫࢹ࣮ࣗࢧ࣮ࡢぶᰴ࡟࠾ࡅࡿⓎ⌧࡜ᶵ⬟ゎᯒ⏣ຊ㕲ᖹ*1࣭ᯇཎᝡ㧗*1࣭ஂಖ⏣ⰾᘯ*1࣭ᑠᆏ㈗ᖾ*1࣭ᑠ⃝Ꮥಇ*1࣭ඵἼ฼ᜨ*1࣭⚟ᒃಇ᫛*1࣭୰ᮧ⪽*1,2
Expression and Functional Analysis of Aerotaxis Transducers
from Extremely Halophilic Archaeon Haloarcula japonica
TADIKARA Teppei*1, MATSUBARA Toshitaka*1, KUBOTA Yoshihiro*1,
KOSAKA Takayuki*1, OZAWA Takatoshi*1,
YATSUNAMI Rie*1, FUKUI Toshiaki*1, NAKAMURA Satoshi*1,2Abstract: The open reading frames encoding aerotaxis transducers Htr8 and HemAT homologs,
named Htr8-Hj and HemAT-Hj respectively, were found on the genome of extremely halophilicarchaeon Haloarcula japonica. The htr8 and hemAT genes were cloned and successfully
expressed in Ha. japonica. Recombinant Htr8-Hj and HemAT-Hj were purified and characterized.
Absorption spectra showed that the Ha. japonica-produced Htr8-Hj and HemAT-Hj would beheme-containing proteins. The air bubble assays indicated that the aerotaxis response of Ha.
japonica cells would be accelerated by introducing the htr8 gene and decelerated by introducing
the hemAT gene.
Key Words: Aerotaxis, Transducer, Htr8, HemAT, Extremely halophilic archaeon䠍䠊䛿䛨䜑䛻࡭ࢇẟࢆࡶࡘᚤ⏕≀ࡢከࡃࡣࠊእ⏺࠿ࡽࡢ≀⌮ⓗ࣭໬ᏛⓗHaloarcula japonica ࡣ୕ゅᙧᖹᯈ≧࡜࠸࠺≉ᚩⓗ࡞ᙧែ่⃭ࢆཷࡅࠊࡑࡢ่⃭※࡟ྥ࠿࠺ࠊ࠶ࡿ࠸ࡣ㐲ࡊ࠿ࡿ࡜࠸࠺ࢆ᭷ࡍࡿ㧗ᗘዲሷᛶྂ⣽⳦࡛࠶ࡿࠋᮏ⳦ࡣ࡭ࢇẟࢆ᭷ࡋࠊ㐠ᣲືࢆ♧ࡍࠋࡇࡢࡼ࠺࡞ᣲືࡣ㉮ᛶ࡜ࡼࡤࢀࠊᚤ⏕≀ࡀ⮬㌟ືᛶࢆ♧ࡍࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ [1,2]ࠋࡋ࠿ࡋ࡞ࡀࡽࠊHa.

ࡢ⏕⫱࡟ࡼࡾዲࡲࡋ࠸⎔ቃ࡬࡜⛣ືࡍࡿࡓࡵࡢᡭẁ࡜⪃࠼ࡽjaponica ࡢ㉮ᛶ࡟㛵ࡍࡿ▱ぢࡣ࡯࡜ࢇ࡝ᚓࡽࢀ࡚࠸࡞࠸ࠋࡇࢀ࡚࠸ࡿࠋᚤ⏕≀ࡀ่⃭※࡟ྥ࠿࠺ࡼ࠺࡞㉮ᛶࡣṇࡢ㉮ᛶࠊࢀࡲ࡛࡟ࠊHa. japonica ࡼࡾ 3 ✀㢮ࡢࢺࣛࣥࢫࢹ࣮ࣗࢧ࣮

㏫࡟㐲ࡊ࠿ࡿሙྜࡣ㈇ࡢ㉮ᛶ࡜ࡼࡤࢀࡿࠋᚤ⏕≀ࡀ♧ࡍ㉮ᛶ㑇ఏᏊ࣍ࣔࣟࢢ hjtA㸪hjtB ࠾ࡼࡧ hjtC ࡢࢡ࣮ࣟࢽࣥࢢࡀ࡜ࡋ࡚ࡣࠊ࢔࣑ࣀ㓟ࡸ⢾ࡢࡼ࠺࡞໬Ꮫ≀㉁࡟ᑐࡍࡿ㉮ᛶ (㉮⾜ࢃࢀ࡚࠸ࡿࡀ [3,4] ࠊᙜヱࢺࣛࣥࢫࢹ࣮ࣗࢧ࣮ࡢᶵ⬟ゎ᫂໬ᛶ) ࠊ㓟⣲ࡢࡼ࠺࡞Ẽయ࡟ᑐࡍࡿ㉮ᛶ (㉮Ẽᛶ) ࠊග࡟ᑐࡍ࡟ࡣ⮳ࡗ࡚࠸࡞࠸ࠋࡿ㉮ᛶ (㉮ගᛶ) ࡞࡝ࡀ▱ࡽࢀ࡚࠸ࡿࠋ㉮ᛶࡢⓎ⌧࡟㝿ࡋ࡚㏆ᖺࠊHa. japonica ࡢ඲ࢤࣀ࣒㓄ิゎᯒࡀ⤊஢ࡋࡓ [5]ࠋࡣࠊእ⏺࠿ࡽࡢ่⃭ࡣఱࡽ࠿ࡢࢩࢢࢼࣝఏ㐩⣔ࢆ௓ࡋ࡚࡭ࢇ┦ྠᛶ᳨⣴ࡢ⤖ᯝࠊ㧗ᗘዲሷᛶྂ⣽⳦ Halobacteriumẟ࣮ࣔࢱ࣮࡬࡜ఏࢃࡿࠋࡉࡲࡊࡲ࡞ᚤ⏕≀࡟࠾࠸࡚ࠊ⣽⬊ෆsalinarum ࡟࠾࠸࡚ࡑࢀࡒࢀṇ࠾ࡼࡧ㈇ࡢ㉮Ẽᛶ࡬ࡢ㛵୚እࡢ่⃭ࢆឤ▱ࡋࠊ࡭ࢇẟ࣮ࣔࢱ࣮࡬ఏ㐩ࡍࡿࢱࣥࣃࢡ㉁ࡀ

ࡀ▱ࡽࢀ࡚࠸ࡿࢺࣛࣥࢫࢹ࣮ࣗࢧ࣮㑇ఏᏊ htr8 [6] ࠾ࡼࡧぢࡘ࠿ࡗ࡚࠾ࡾࠊࡑࢀࡽࡣ⥲ࡌ࡚ࢺࣛࣥࢫࢹ࣮ࣗࢧ࣮࡜ࡼࡤhemAT [7] ࡢ࣍ࣔࣟࢢࡀぢ࠸ࡔࡉࢀࡓࠋHa. japonica ࡢ㉮ࢀࡿࠋẼᛶ࡟ࡶࠊࡇࢀࡽࡢ࣍ࣔࣟࢢࡀࢥ࣮ࢻࡍࡿࢱࣥࣃࢡ㉁ Htr8-㹝㹝㹝㹝㹝㹝㹝㹝㹝㹝㹝㹝㹝㹝㹝㹝㹝㹝㹝㹝㹝㹝㹝㹝㹝㹝㹝Hj ࠾ࡼࡧ HemAT-Hj ࡀ㛵୚ࡋ࡚࠸ࡿྍ⬟ᛶࡀ㧗࠸ࠋࡇࢀ*1 ᮾிᕤᴗ኱Ꮫ ⏕࿨⌮ᕤᏛ㝔 School of Life Science andࡲ࡛࡟ࠊHemAT-Hj ࡢ኱⭠⳦࡟࠾ࡅࡿⓎ⌧ࡀ⾜ࢃࢀࠊ⤌᥮࠼

Technology, Tokyo Institute of Technology

HemAT-Hj ࡟࣒࣊ࡀྵࡲࢀࡿࡇ࡜ࢆ♧၀ࡍࡿ⤖ᯝࡀᚓࡽࢀ

*2 ἟ὠᕤᴗ㧗➼ᑓ㛛Ꮫᰯ National Institute of Technology࡚࠸ࡿࡀࠊࡑࢀࡽࡢᶵ⬟ࡣ౫↛࡜ࡋ࡚୙࡛᫂࠶ࡗࡓ [8]ࠋ(KOSEN), Numazu Collegeᮏ◊✲࡛ࡣࠊHa. japonica ࢤࣀ࣒ୖ࡟ぢ࠸ࡔࡉࢀࡓ htr82沼津工業高等専門学校研究報告

第 56 号

࠾ࡼࡧ hemAT 㑇ఏᏊ࣍ࣔࣟࢢࡢぶᰴ (Ha. japonica) ࡟࠾㑇ఏᏊᆺࡀ dam- ࠾ࡼࡧ dcm- ࡛࠶ࡿ኱⭠⳦ JM110 ᰴ࡟ࡅࡿ㐣๫Ⓨ⌧࡜⤌᥮࠼ࢱࣥࣃࢡ㉁ࡢᶵ⬟ゎᯒࢆ⾜ࡗࡓࠋᙧ㉁㌿᥮ࡋࠊࡇࡢᰴࡼࡾᅇ཰ࡋࡓࡶࡢࢆ౑⏝ࡋࡓࠋ࣓ࢳࣝ໬ࡉࢀ࡚࠸࡞࠸ࣉࣛࢫ࣑ࢻࢆ౑⏝ࡍࡿࡇ࡜࡟ࡼࡾࠊ㧗ᗘዲሷᛶྂ⣽⳦ࡢᙧ㉁㌿᥮ຠ⋡ࡀྥୖࡍࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ [13]ࠋ㻞䠊ᮦᩱ䛸᪉ἲ

Ha. japonica ࡢᙧ㉁㌿᥮ࡣ᪤ሗ [11] ࡟ᚑ࠸ࠊࢫࣇ࢙ࣟࣉࣛ2㸬1 ⳦ᰴࠊࣉࣛࢫ࣑ࢻ࠾ࡼࡧᇵᆅࢫࢺἲ࡟࡚⾜ࡗࡓࠋ DNA ౪୚య࡜ࡋ࡚ Ha. japonica (JCM 7785) ࢆ⏝࠸ࡓࠋ ኱⭠⳦࠾ࡼࡧ Ha. japonica ࠿ࡽࡢࣉࣛࢫ࣑ࢻ DNA ࡢ㑇ఏᏊࢡ࣮ࣟࢽࣥࢢࡢᐟ୺࡜ࡋ࡚ࡣ Escherichia coli (኱⭠ᢳฟࡣᡂ᭩ [12] ࡟ᚑ࠸ࠊ࢔ࣝ࢝ࣜ-SDS ἲ࡟࡚⾜ࡗࡓࠋ⳦) JM109 ᰴ࠾ࡼࡧ JM110 ᰴࢆࠊࡑࡋ࡚㑇ఏᏊⓎ⌧ࡢᐟ୺࡜ࡋ࡚ࡣ Ha. japonica ࢆ౑⏝ࡋࡓࠋ㑇ఏᏊࢡ࣮ࣟࢽࣥࢢ࡟ࡣ኱⭠⳦⏝࣋ࢡࢱ࣮ pUC119 ࢆ⏝࠸ࡓࠋpWL102 ࡣ㧗ᗘዲሷᛶྂ⣽⳦࠾ࡼࡧ኱⭠⳦ࡢࢩࣕࢺࣝ2㸬4 DNA ሷᇶ㓄ิゎᯒ DNA ሷᇶ㓄ิࡢゎᯒࡣࠊᮾிᕤᴗ኱Ꮫ࣮࢜ࣉࣥࣇ࢓ࢩࣜࢸ࢕ࢭࣥࢱ࣮࣭ࣂ࢖࢜㒊㛛࡟౫㢗ࡋ࡚⾜ࡗࡓࠋ࣋ࢡࢱ࣮࡛࠶ࡿ [9]ࠋHa. japonica ⏝Ⓨ⌧ᆺࣉࣛࢫ࣑ࢻࡢㄪ〇࡟⏝࠸ࡓ pJFZ33 ࡣࠊHa. japonica ⣽⬊⾲ᒙ⢾ࢱࣥࣃࢡ2㸬5 ⤌᥮࠼ࢱࣥࣃࢡ㉁ࡢ⢭〇㉁㑇ఏᏊ (csg 㑇ఏᏊ) ࣉ࣮ࣟࣔࢱ࣮࠾ࡼࡧ Ha. japonica

 Ⓨ⌧ᆺࣉࣛࢫ࣑ࢻࢆ᭷ࡍࡿ Ha. japonica ࢆᾮయᇵᆅ࡛⏤᮶ ftsZ2 ᵓ㐀㑇ఏᏊ඲㡿ᇦࢆ pWL102 ࡟㐃⤖ࡋࡓ⤌᥮

OD660 ࡀ 1.0 ๓ᚋ࡟࡞ࡿࡲ࡛ᇵ㣴ࡋࡓᚋࠊ㐲ᚰ (4°Cࠊ4,400

࠼ࣉࣛࢫ࣑ࢻ࡛࠶ࡿ [10]ࠋ

x gࠊ10 ศ㛫) ࡟ࡼࡾ㞟⳦ࡋࡓࠋᚓࡽࢀࡓ⳦యࢆ 2.0 M NaCl኱⭠⳦ࡢᇵ㣴࡟ࡣ LB ᇵᆅࢆ⏝࠸ࠊᚲせ࡟ᛂࡌ࡚⤊⃰ᗘ

ࢆྵࡴ 50 mM ࣜࣥ㓟ࣂࢵࣇ࢓࣮ (pH 8.0) ࡟ᠱ⃮ࡋࡓᚋࠊ

50 μg/ml ࡢ࢔ࣥࣆࢩࣜࣥ (ࢼ࢝ࣛ࢖ࢸࢫࢡ) ࢆῧຍࡋࡓࠋHa.

ࢯࢽࢣ࣮ࢱ࣮ (Bioruptor UCD-250ࠊࢥࢫ࣭ࣔࣂ࢖࢜) ࢆ⏝

japonica ࡢᇵ㣴࡟ࡣ 20% (w/vࠊ௨ୗ␎) NaClࠊ4% MgSO4࣭࠸࡚◚○ࡋࡓࠋ㐲ᚰ (4°Cࠊ20,400 x gࠊ10 ศ㛫) ࠾ࡼࡧ㉸㐲

7H2O ࢆྵࡴኳ↛ᇵᆅ [11] ࢆ⏝࠸ࠊᚲせ࡟ᛂࡌ࡚⤊⃰ᗘ 8ᚰ (4°Cࠊ100,000 x gࠊ1.5 ᫬㛫) ࡟ࡼࡾྍ⁐ᛶ⏬ศࢆᅇ཰ࡋࠊμg/ml ࡢࣉࣛࣂࢫࢱࢳࣥ (➨୍୕ඹᰴᘧ఍♫ࡼࡾ౪୚) ࠾ࡼ20 mM ࢖࣑ࢲࢰ࣮ࣝ࠾ࡼࡧ 2.0 M NaCl ࢆྵࡴ 50 mM

ࡧ 17 μg/ml ࡢ 5-࢔࣑ࣀࣞࣈࣜࣥ㓟ሷ㓟ሷ (ᐩኈࣇ࢖࣒ࣝࣜࣥ㓟ࣂࢵࣇ࢓࣮ (pH 8.0) ࡟ᑐࡋ࡚㏱ᯒࡋࡓࠋ࿴ග⣧⸆) ࢆῧຍࡋࡓࠋᅛయᇵᆅ࡟ࡣ 1.5% ᐮኳࢆຍ࠼ࡓࠋ ⤌᥮࠼ࢱࣥࣃࢡ㉁ࡢ࢔ࣇ࢕ࢽࢸ࢕࣮⢭〇࡟ࡣࠊÄKTA
FPLC ࢩࢫࢸ࣒ (GE ࣊ࣝࢫࢣ࢔) ࠾ࡼࡧࢽࢵࢣࣝ㸦Ni㸧ࢆ2㸬2 㑇ఏᏊᕤᏛⓗᡭἲᅛᐃ໬ࡋࡓ HiTrap ࣮࢟ࣞࢸ࢕ࣥࢢ࣒࢝ࣛ (GE ࣊ࣝࢫࢣ Ha. japonica ࢤࣀ࣒ DNA ࡢㄪ〇ࡣ᪤ሗ [11] ࡟ᚑࡗࡓࠋ࢔) ࢆ⏝࠸ࡓࠋ࣒࢝ࣛࢆ 20 mM ࢖࣑ࢲࢰ࣮ࣝ࠾ࡼࡧ 2.0 M

PCR ࣉࣛ࢖࣐࣮ࡣ࣮ࣘࣟࣇ࢕ࣥࢪ࢙ࣀ࣑ࢡࢫ࠾ࡼࡧࢢࣛNaCl ࢆྵࡴ 50 mM ࣜࣥ㓟ࣂࢵࣇ࢓࣮ (pH 8.0) ࡛ᖹ⾮໬࢖ࢼ࣮࣭ࢪࣕࣃࣥࡼࡾ㉎ධࡋࡓࠋࣉࣛ࢖࣐࣮࡟࠶ࡽ࠿ࡌࡵࣜࡋࡓᚋࠊୖ࡛ᚓࡽࢀࡓྍ⁐ᛶ⏬ศࢆ࢔ࣉࣛ࢖ࡋࡓࠋྠࣂࢵࣇࣥ㓟໬ࢆ᪋ࡋࡓࠋࡍ࡞ࢃࡕࠊࣜࣥ㓟໬ࡢ཯ᛂᾮ 20 μl ࡟ࡣྛ࢓࣮࡛࣒࢝ࣛࢆὙίᚋࠊ20-500 mM ࢖࣑ࢲࢰ࣮ࣝࡢࢢࣛࢪ15 pmol ࡢ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࠊ2 μl ࡢ Protruding End࢚ࣥࢺ࡟ࡼࡾ྾╔ࢱࣥࣃࢡ㉁ࢆ⁐ฟࡋࡓࠋ⤌᥮࠼ࢱࣥࣃࢡ㉁Kinase Buffer (ᮾὒ⣳)ࠊ2 μl ࡢ 20 mM ATP ࠾ࡼࡧ 10 Uࢆྵࡴ⏬ศࢆ㝈እℐ㐣⃰⦰ࡍࡿ࡜࡜ࡶ࡟ࠊࣂࢵࣇ࢓࣮ࢆ 2.0

ࡢ T4 ࣏ࣜࢾࢡࣞ࢜ࢳࢻ࢟ࢼ࣮ࢮ (ᮾὒ⣳) ࡀྵࡲࢀࡿࠋ཯M NaCl ࢆྵࡴ 50 mM ࣜࣥ㓟ࣂࢵࣇ࢓࣮ (pH 8.0) ࡬࡜ᛂᾮࢆ 37ºC ࡛ 30 ศ㛫࢖࣮ࣥ࢟ࣗ࣋ࢺࡋࠊࣜࣥ㓟໬ࣉࣛ࢖஺᥮ࡋࠊ⢭〇ᶆရ࡜ࡋࡓࠋ

࣐࣮ࢆᚓࡓࠋ⪏⇕ᛶ DNA ࣏࣓࣮ࣜࣛࢮ࡟ࡣ KOD -Plus(ᮾὒ⣳) ࢆ⏝࠸ࠊῧ௜ࡢࣉࣟࢺࢥ࣮ࣝ࡟ᚑ࠸ PCR ࢆ⾜ࡗࡓࠋ2㸬6 SDS-࣏ࣜ࢔ࢡࣜࣝ࢔࣑ࢻࢤࣝ㟁ẼὋື (SDS-PAGE)

ࢧ࣮࣐ࣝࢧ࢖ࢡ࣮ࣛࡣ iCycler (Bio-Rad) ࢆ౑⏝ࡋࡓࠋ

 SDS-PAGE ࡟ࡣ 12.5% ࣏ࣜ࢔ࢡࣜࣝ࢔࣑ࢻࢤࣝࢆ⏝࠸ࠊไ㝈㓝⣲ࡣᮾὒ⣳࠾ࡼࡧࢱ࢝ࣛࣂ࢖࢜ࡼࡾ㉎ධࡋࠊ࣓࣮࢝

Laemmli ࡢ᪉ἲ [14] ࡟ᚑࡗ࡚⾜ࡗࡓࠋὋືᚋࡢࢱࣥࣃࢡ㉁࣮ࡢᣦ♧࡟ᚑ࠸౑⏝ࡋࡓࠋDNA ࡢ㐃⤖࡟ࡣ Ligation Highࡣࢡ࣮࣐ࢩ࣮ࣈࣜࣜ࢔ࣥࢺࣈ࣮ࣝ R-250 (CBB) (ࢼ࢝ࣛ࢖ࢸ(ᮾὒ⣳) ࢆ⏝࠸ࠊῧ௜ࡢࣉࣟࢺࢥ࣮ࣝ࡟ᚑ࠸཯ᛂࢆ⾜ࡗࡓࠋࢫࢡ) ࡟ࡼࡿᰁⰍࢆ᪋ࡋࡓࠋ2㸬3 ᙧ㉁㌿᥮2㸬7 ⣸እ-ྍどࢫ࣌ࢡࢺࣝ ᐃ ኱⭠⳦ࡢᙧ㉁㌿᥮ࡣᡂ᭩ [12] ࡟ᚑ࠸ࠊሷ໬࢝ࣝࢩ࣒࢘ἲ ⣸እ-ྍどࢫ࣌ࢡࢺࣝ ᐃ࡟ࡣศගගᗘィ UV-1650PC ࡶࡲࡓࡣ࢚ࣞࢡࢺ࣏࣮ࣟࣞࢩࣙࣥἲ࡟࡚⾜ࡗࡓࠋࡋࡃࡣ UV-1800 (࠸ࡎࢀࡶᓥὠ) ࢆ⏝࠸ࡓࠋ ᐃἼ㛗ࡣ 250- Ha. japonica ࡢᙧ㉁㌿᥮࡟⏝࠸ࡿࣉࣛࢫ࣑ࢻ DNA ࡣࠊ700 nm ࡜ࡋࠊᐊ ୗࠊ㓟໬᮲௳ (✵Ẽ㓟໬) ࠾ࡼࡧ㑏ඖ᮲௳

高度好塩性古細菌 Haloarcula japonica 由来走気性トランスデューサーの親株における発現と機能解析[ளࢪࢳ࢜ࣥ㓟ࢼࢺ࣒ࣜ࢘ (ࢪࢳ࢜ࢼ࢖ࢺ) ࢆᚤ㔞ῧຍ] ࡟࠾࠸࡚ ᐃࢆ⾜ࡗࡓࠋ3࡚࠸ࡿࠋୖ࡛ᚓࡽࢀࡓ pht8JH1 ࢆ Nde I/BamH I ฎ⌮ࡋࠊhtr8
㑇ఏᏊࢆྵࡴ DNA ᩿∦ࢆྠࡌࡃ Nde I/BamH I ฎ⌮ࡋ

2㸬8 ㉮Ẽᛶ ᐃ

ࡓ pJFZ33 ࡜㐃⤖ࡍࡿࡇ࡜࡛ࠊHa. japonica ⏝ htr8 㑇ఏ㉮Ẽᛶ ᐃࡣ Brooun ࡽࡢ᪉ἲ [6] ࡟‽ࡌ࡚⾜ࡗࡓࠋࡍᏊⓎ⌧ᆺࣉࣛࢫ࣑ࢻ pht8JH3 ࢆྲྀᚓࡋࡓ (Fig. 1 ྑ)ࠋ

࡞ࢃࡕࠊⓎ⌧ᆺࣉࣛࢫ࣑ࢻࢆ᭷ࡍࡿ Ha. japonica ࢆࠊᾮయᇵpht8JH3 ࡟࠾࠸࡚ࡣࠊHa. japonica csg 㑇ఏᏊࣉ࣮ࣟࣔࢱ

ᆅࢆ⏝࠸࡚ OD660 ࡀ 1.0-1.2 ࡟࡞ࡿࡲ࡛ 37°C ࡛✜ࡸ࠿࡟࣮㡿ᇦࡢୗὶ࡟ htr8 ᵓ㐀㑇ఏᏊ࠾ࡼࡧ His ࢱࢢ㓄ิࡀ㐃᣺࡜࠺ᇵ㣴ࡋࡓࠋᇶᮏሷ⁐ᾮ (20% NaClࠊ4% MgSO4•7H2O)⤖ࡋ࡚࠸ࡿࠋ

࡛ 6-7 ಸᕼ㔘ࡋࡓᇵ㣴ᾮࢆࠊẟ⣽⟶⌧㇟࡟ࡼࡾ࣐࢖ࢡࣟࢫࣛ
࢖ࢻ (50 x 2.0 x 0.10 mmࠊWale Apparatus) ࡢ୰ኸ௜㏆ࡲ࡛ᑟධࡋࡓࠋ
࣐࢖ࢡࣟࢫࣛ࢖ࢻࡢ୧➃ࢆࣂ࣮ࢼ࣮࡟ࡼࡾ⁐⼥࣭ᑒ⟶ࡋࠊᬯᡤ࡛ 37°Cࠊ4 ᫬㛫࢖࣮ࣥ࢟ࣗ࣋ࢺࡋࡓࠋ఩┦ᕪ㢧ᚤ㙾࡟ࡼࡿほᐹࢆ⾜࠸ࠊẼᾮ⏺㠃௜㏆࡟࠾ࡅࡿ⳦యࡢᣲື࡟

Fig. 1 ࣉࣛࢫ࣑ࢻ pht8JH1㸦ᕥ㸧࠾ࡼࡧ pht8JH3㸦ྑ㸧ࡼࡾ㉮Ẽᛶࡢホ౯ࢆ⾜ࡗࡓࠋ
pht8JH3 ࢆᑟධࡋࡓ Ha. japonica ࡼࡾ⣽⬊㉁⏬ศࢆㄪ
2㸬9 ࢔ࢡࢭࢵࢩࣙࣥ␒ྕ〇ࡋࠊNi ࣮࢟ࣞࢺ࣒࢝ࣛࢆ⏝࠸ࡓ࢔ࣇ࢕ࢽࢸ࢕࣮ࢡ࣐ࣟࢺ

Ha. japonica ⏤᮶ htr8 ࠾ࡼࡧ hemAT 㑇ఏᏊࡢሷᇶ㓄ࢢࣛࣇ࢕࣮࡟౪ࡍࡿࡇ࡜࡛ࠊ⤌᥮࠼ Htr8-Hj ⢭〇ᶆရࢆྲྀิࡣ DDBJ/EMBL/GenBank ࢹ࣮ࢱ࣮࣋ࢫ࡟Ⓩ㘓ࡉࢀ࡚࠸ᚓࡋࡓࠋᚓࡽࢀࡓ⢭〇ᶆရࢆ SDS-PAGE ࡟౪ࡋࠊCBB ᰁࡿ (࢔ࢡࢭࢵࢩࣙࣥ␒ྕࡣࡑࢀࡒࢀ LC651378 ࠾ࡼࡧⰍࢆ⾜ࡗࡓ⤖ᯝࠊ⤌᥮࠼ Htr8-Hj ⢭〇ᶆရࡣ⣙ 120 kDa ࡢAB823705㸧ࠋ఩⨨࡟୺せ࡞ࢱࣥࣃࢡ㉁ࣂࣥࢻࢆ୚࠼ࡓ (Fig. 2 ᕥ)ࠋ࢔࣑ࣀ
㓟㓄ิࡼࡾ㢮᥎ࡋࡓ Htr8-Hj ࡢศᏊ㉁㔞ࡣ His ࢱࢢ㓄ิ㻟䠊⤖ᯝ䛸⪃ᐹࢆྵࡵ࡚⣙ 70 kDa ࡛࠶ࡾࠊSDS-PAGE ࡼࡾồࡵࡓศᏊ㉁㔞ࡣࡇࢀࡼࡾ኱ࡁࡃぢ✚ࡶࡽࢀ࡚࠸ࡿࠋࡇࢀࡣࠊHtr8-Hj ࡢ

3㸬1 Htr8-Hj ࡢ Ha. japonica ࡟࠾ࡅࡿ㐣๫Ⓨ⌧ࠊ⤌᥮࠼㓟ᛶ࢔࣑ࣀ㓟ྵ㔞ࡀ㧗࠸ (18%) ࡇ࡜࡟㉳ᅉࡍࡿ࡜ᛮࢃࢀࡿࠋ

Htr8-Hj ࡢ⢭〇࠾ࡼࡧᛶ㉁᳨ウ୍⯡࡟ࠊ㧗ᗘዲሷᛶྂ⣽⳦ࡀ⏕⏘ࡍࡿࢱࣥࣃࢡ㉁ࡣ㓟ᛶ࢔࣑htr8 ᵓ㐀㑇ఏᏊ඲㡿ᇦࡢ PCR ቑᖜ࡟ࡣࠊࢭࣥࢫࣉࣛ࢖ࣀ㓟ࢆከࡃྵࡴࡓࡵࠊSDS ࡜⤖ྜࡋ࡟ࡃ࠸ࡇ࡜ࡀ▱ࡽࢀ࡚࣐࣮ Htr8-KNm ࠾ࡼࡧ࢔ࣥࢳࢭࣥࢫࣉࣛ࢖࣐࣮ Htr8-࠸ࡿࠋࡑࡢࡓࡵ SDS-PAGE ࡟࠾ࡅࡿ⛣ືᗘࡀ㐜ࢀࠊ⤖ᯝ࡜

6HEBr ࢆ౑⏝ࡋࡓࠋHtr8-KNm ࡟ࡣไ㝈㓝⣲ Kpn I ࠾ࡼࡋ࡚ศᏊ㔞ࡀ኱ࡁࡃぢ✚ࡶࡽࢀࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ

ࡧ Nde I ࡢㄆ㆑㓄ิࢆࠊࡑࡋ࡚ Htr8-6HEBr ࡟ࡣ His ࢱ[15]ࠋࢢ㓄ิࠊ⤊Ṇࢥࢻࣥ࠾ࡼࡧไ㝈㓝⣲ BamH I ࡢㄆ㆑㓄ิࢆ௜ຍࡋࡓ (Table 1)ࠋࣉࣛ࢖࣐࣮࡟ࣜࣥ㓟໬ࢆ᪋ࡋࡓᚋࠊHa.

japonica ࢤࣀ࣒ DNA ࢆ㗪ᆺ࡜ࡍࡿ PCR ࢆ⾜ࡗࡓࠋPCR⏘≀ࡢ࢔࣮࢞ࣟࢫࢤࣝ㟁ẼὋືࢆ⾜࠸ࠊ⣙ 2.0 kb ࡢ DNA᩿∦ࢆࢤࣝ࠿ࡽᢳฟࡋࠊSma I ฎ⌮ࡋࡓ pUC119 ࡟㐃⤖ࡍࡿࡇ࡜࡛ࠊhtr8 㑇ఏᏊࢆྵࡴࣉࣛࢫ࣑ࢻ pht8JH1 ࢆྲྀᚓࡋࡓ (Fig. 1 ᕥ)ࠋᮏࣉࣛࢫ࣑ࢻ࡟࠾࠸࡚ࡣࠊHtr8-Hj ࡢୗὶ࡟ His ࢱࢢ㓄ิࡀ௜ຍࡋࡓ⤌᥮࠼ࢱࣥࣃࢡ㉁ࡀࢥ࣮ࢻࡉࢀ
Table 1 ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࣉࣛ࢖࣐࣮ࡢሷᇶ㓄ิ
Fig. 2 Ha. japonica ࡀ⏕⏘ࡋࡓ⤌᥮࠼ Htr8-Hj ࡢSDS-PAGE㸦ᕥ㸧࠾ࡼࡧ UV/VIS ࢫ࣌ࢡࢺࣝ㸦ྑ㸧⤌᥮࠼ Htr8-Hj ⢭〇ᶆရࡢ⣸እ-ྍどࢫ࣌ࢡࢺࣝ ᐃࢆ⾜ࡗࡓ⤖ᯝࠊ㓟໬ᆺ࡟࠾࠸࡚Ἴ㛗 410 nmࠊ㑏ඖᆺ࡟࠾࠸࡚࣎ࢵࢡࢫࡣ⤊ṆࢥࢻࣥࠋἼ㛗 420 nm ࡢࢯ࣮࣮ࣞᖏࡢ྾཰ࣆ࣮ࢡࡀほᐹࡉࢀࡓ4沼津工業高等専門学校研究報告

第 56 号(Fig. 2 ྑ)ࠋࡲࡓࠊ㓟໬ᆺ࡛ࡣ 595 nm ࡟ᙅ࠸ D ᖏࡢ྾཰ࣆ

࡜ྠᵝࠊHemAT-Hj ࡢ㓟ᛶ࢔࣑ࣀ㓟ྵ㔞ࡀ㧗ࡃ (24%)ࠊSDS

࣮ࢡࡀㄆࡵࡽࢀࡓࠋࡇࡢࢫ࣌ࢡࢺࣝࡣ࣒࣊ࢱࣥࣃࢡ㉁࡟ぢࡽࡀ⤖ྜࡋ࡟ࡃ࠸ࡇ࡜࡟㉳ᅉࡍࡿ࡜ᛮࢃࢀࡿࠋ

ࢀࡿࢫ࣌ࢡࢺࣝ࡜㢮ఝࡋ࡚࠸ࡓࡇ࡜࠿ࡽࠊ⤌᥮࠼ Htr8-Hj⤌᥮࠼ HemAT-Hj ⢭〇ᶆရࡢ⣸እ-ྍどࢫ࣌ࢡࢺࣝ ᐃࡣ࣒࣊ࢆྵࡴࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ୍᪉࡛ࠊ᪤ሗࡢ࣒࣊ࢱࣥࣃࢆ⾜ࡗࡓ⤖ᯝࠊ㓟໬ᆺ࡛Ἴ㛗 410 nmࠊ㑏ඖᆺ࡛Ἴ㛗 428 nm

ࢡ㉁ࡢࢫ࣌ࢡࢺࣝࣃࢱ࣮ࣥ࡜᏶඲࡟ࡣ୍⮴ࡏࡎࠊ⤌᥮࠼ࡢࢯ࣮࣮ࣞᖏࡢ྾཰ࣆ࣮ࢡࡀほᐹࡉࢀࡓ (Fig. 4 ྑ)ࠋࡲࡓࠊ

Htr8-Hj ࡣ᪂つ࡞࣒࣊ࢆྵࡴྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋྂ⣽⳦㓟໬ᆺ࡛ほᐹࡉࢀࡓ 545nm ࡢ E ᖏ࠾ࡼࡧ 580 nm ࡢ D࡟ࡣ⣽⳦ࡢ࣒࣊ a ࡸ࣒࣊ o ࡜ࡣ␗࡞ࡿ㦵᱁ࢆࡶࡘ࣒࣊ aᖏࡢ྾཰ࣆ࣮ࢡࡀࠊ㑏ඖᆺ࡛ࡣ 552 nm ࡢࣈ࣮ࣟࢻ࡞ࣆ࣮ࢡࡸ࣒࣊ o 㢮ఝయࡢᏑᅾࡀ☜ㄆࡉࢀ࡚࠸ࡿ [16]ࠋ⤌᥮࠼࡜࡞ࡗ࡚࠸ࡓࠋୖ㏙ࡢࡼ࠺࡞ࢫ࣌ࢡࢺࣝࡣࠊHemAT-Hs ࡢ྾

Htr8-Hj ࡣྂ⣽⳦≉᭷ࡢ࣒࣊⏕ྜᡂ㓝⣲࡟ࡼࡗ࡚ྜᡂࡉࢀ

཰ࢫ࣌ࢡࢺࣝ (㓟໬ᆺ࡛ࡣ 406ࠊ538ࠊ578 nm ࡢࣆ࣮ࢡࡀࠊࡿ᪂つ࡞࣒࣊ࢆྵࡴࡢ࠿ࡶࡋࢀ࡞࠸ࠋࡓࡔࡋࠊ௒ᅇᚓࡽࢀࡓ㑏ඖᆺ࡛ࡣ 425ࠊ555 nm ࡟ࢩࣇࢺ) [17] ࡸᯤⲡ⳦ (Bacillusࢫ࣌ࢡࢺࣝࡣ S/N ẚࡀపࡃࠊ⢭〇᫬࡟࣒࣊ࡀ⬺ⴠࡋࡓྍ⬟subtilis) ⏤᮶ HemAT-Bs (㓟໬ᆺ࡛ࡣ 410ࠊ542ࠊ580 nmᛶࡶ⪃࠼ࡽࢀࡿࡇ࡜࠿ࡽࠊ⢭〇㐣⛬ࡢぢ┤ࡋࡀᚲせ࡛࠶ࢁ࠺ࠋࡢࣆ࣮ࢡࡀࠊ㑏ඖᆺ࡛ࡣ 428ࠊ560 nm ࡟ࢩࣇࢺ) [18] ࡟㢮ఝࡋ࡚࠸ࡓࠋࡇࢀࡼࡾࠊHemAT-Hj ࡣࠊHemAT-Hs

ࡸ

3㸬2 HemAT-Hj ࡢ Ha. japonica ࡟࠾ࡅࡿ㐣๫Ⓨ⌧ࠊ⤌᥮

HemAT-Hs ࡜ྠᵝࠊ࣒࣊ b ࢆྵࡴࡇ࡜ࡀ♧၀ࡉࢀࡓࠋࡲࡓࠊ

࠼ HemAT-Hj ࡢ⢭〇࠾ࡼࡧᛶ㉁᳨ウHemAT-Hj ⢭〇ᶆရࢆ⏝࠸࡚࣒࣊ᰁⰍࢆ⾜ࡗࡓ࡜ࡇࢁࠊ㝧ᛶ

hemAT 㑇ఏᏊࡢࢡ࣮ࣟࢽࣥࢢࡣࠊhtr8 㑇ఏᏊࡢሙྜ࡜ྠᵝࡢ᪉ἲ࡛⾜ࡗࡓࠋࡍ࡞ࢃࡕࠊhemAT ᵓ㐀㑇ఏᏊ඲㡿ᇦࢆࣂࣥࢻࡀ☜ㄆࡉࢀ(ࢹ࣮ࢱ♧ࡉࡎ)ࠊHemAT-Hj ࡣ࣒࣊ࢆྵࡴࡇ࡜ࡀ☜࠿ࡵࡽࢀࡓࠋ

ቑᖜࡍࡿࡓࡵࡢࢭࣥࢫࣉࣛ࢖࣐࣮ HemAT-KNm ࠾ࡼࡧ࢔ࣥࢳࢭࣥࢫࣉࣛ࢖࣐࣮ HemAT-6HEBr (Table 1) ࢆࣜࣥ㓟໬ࡋࡓᚋࠊHa. japonica ࢤࣀ࣒ DNA ࢆ㗪ᆺ࡜ࡍࡿ PCR
ࢆ⾜ࡗࡓࠋ⣙ 1.5 kb ࡢ DNA ᩿∦ࢆࢤࣝ࠿ࡽᢳฟࡋࠊSmaI ฎ⌮ࡋࡓ pUC119 ࡟㐃⤖ࡍࡿࡇ࡜࡛ࠊhemAT 㑇ఏᏊࢆྵࡴࣉࣛࢫ࣑ࢻ phemJH1 ࢆྲྀᚓࡋࡓ (Fig. 3 ᕥ)ࠋᮏࣉࣛ
ࢫ࣑ࢻ࡟࠾࠸࡚ࡣࠊHemAT-Hj ࡢୗὶ࡟ His ࢱࢢ㓄ิࡀ௜ຍࡋࡓ⤌᥮࠼ࢱࣥࣃࢡ㉁ࡀࢥ࣮ࢻࡉࢀ࡚࠸ࡿࠋ
ୖ࡛ᚓࡽࢀࡓ phemJH1 ࢆ Nde I/BamH I ฎ⌮ࡋࠊhemAT 㑇 ఏᏊࢆ ྵࡴ DNA ᩿ ∦ ࢆ pJFZ33 ࡢ NdeI/BamH I ࢧ࢖ࢺ࡟ᤄධࡍࡿࡇ࡜࡛ࠊ Ha. japonica ⏝hemAT 㑇ఏᏊⓎ⌧ᆺࣉࣛࢫ࣑ࢻ phemJH3 ࢆྲྀᚓࡋࡓ(Fig. 3 ྑ)ࠋ

Fig. 4 Ha. japonica ࡀ⏕⏘ࡋࡓ⤌᥮࠼ HemAT-Hj ࡢSDS-PAGE㸦ᕥ㸧࠾ࡼࡧ UV/VIS ࢫ࣌ࢡࢺࣝ㸦ྑ㸧
3㸬3 Htr8-Hj ࠾ࡼࡧ HemAT-Hj 㐣๫Ⓨ⌧ᰴࡢ㉮Ẽᛶゎᯒ
Htr8-Hj ࠾ࡼࡧ HemAT-Hj ࡢᶵ⬟ࡢゎ᫂ࢆ┠ⓗ࡜ࡋ࡚ࠊHa. japonica ࡟ྛ✀Ⓨ⌧ᆺࣉࣛࢫ࣑ࢻࢆᑟධࡋࡓ Htr8-Hj
Fig. 3 ࣉࣛࢫ࣑ࢻ phemJH1㸦ᕥ㸧࠾ࡼࡧ phemJH3㸦ྑ㸧࠾ࡼࡧ HemAT-Hj 㐣๫๫Ⓨ⌧ᰴ࡟ࡘ࠸࡚㉮Ẽᛶゎᯒࢆ⾜ࡗࡓࠋ

phemJH3 ࢆᑟධࡋࡓ Ha. japonica ࡼࡾ⣽⬊㉁⏬ศࢆㄪ

Ha. japonica ᇵ㣴ᾮ࡜✵Ẽࢆᑟධࡋࡓ࣐࢖ࢡࣟࢫࣛ࢖ࢻ

〇ࡋࠊNi ࣮࢟ࣞࢺ࣒࢝ࣛࢆ⏝࠸ࡓ࢔ࣇ࢕ࢽࢸ࢕࣮ࢡ࣐ࣟࢺ

ࡢ୧➃ࢆᐦ㛢ࡋࠊᬯᡤ࡛ 37°Cࠊ4 ᫬㛫࢖࣮ࣥ࢟ࣗ࣋ࢺࡍࡿ

ࢢࣛࣇ࢕࣮࡟౪ࡍࡿࡇ࡜࡟ࡼࡾࠊ⤌᥮࠼ HemAT-Hj ⢭〇ᶆࡇ࡜࡛ᇵ㣴ᾮ୰ࡢ㓟⣲ࡀ Ha. japonica ࡟ࡼࡾᾘ㈝ࡉࢀࠊẼရࢆྲྀᚓࡋࡓࠋᚓࡽࢀࡓ⢭〇ᶆရࢆ SDS-PAGE ࡟౪ࡋࠊᾮ⏺㠃㏆ഐ࡟㓟⣲ࡢ⃰ᗘ໙㓄ࡀᙧᡂࡉࢀࡿࠋࡶࡋ⣽⬊ࡀ㓟⣲

CBB ᰁⰍࢆ⾜ࡗࡓ⤖ᯝࠊSDS-PAGE ࡟࠾࠸࡚⣙ 90 kDa ࡢ࡟ᑐࡋ࡚ṇࡢ㉮ᛶࢆ♧ࡍࡢ࡛࠶ࢀࡤࠊ⣽⬊ࡣᇵ㣴ᾮ୰࡛᭱ࡶ

఩⨨࡟୺せ࡞ࢱࣥࣃࢡ㉁ࣂࣥࢻࢆ୚࠼ࡓ (Fig. 4 ᕥ)ࠋ࢔࣑ࣀ

㓟⣲⃰ᗘࡢ㧗࠸Ẽᾮ⏺㠃࡟ᒁᅾࡋࠊ཯ᑐ࡟㓟⣲࡟ᑐࡋ࡚㈇ࡢ

㓟㓄ิࡼࡾ㢮᥎ࡋࡓ HemAT-Hj ࡢศᏊ㉁㔞ࡣ His ࢱࢢ㓄㉮ᛶࢆ♧ࡍࡢ࡛࠶ࢀࡤࠊ⣽⬊ࡣẼᾮ⏺㠃࠿ࡽᑡࡋ㞳ࢀࡓሙᡤิࢆྵࡵ࡚⣙ 55 kDa ࡛࠶ࡾࠊSDS-PAGE ࡼࡾồࡵࡓศᏊ࡟ᒁᅾࡍࡿࡇ࡜࡟࡞ࡿࠋࡼࡗ࡚ࠊ࢖࣮ࣥ࢟ࣗ࣋ࢩࣙࣥᚋࡢ࣐㉁㔞ࡣࡇࢀࡼࡾ኱ࡁࡃぢ✚ࡶࡽࢀ࡚࠸ࡿࠋࡇࢀࡣࠊHtr8-Hj࢖ࢡࣟࢫࣛ࢖ࢻ୰ࡢ Ha. japonica ⣽⬊ࡢᒁᅾࢆ఩┦ᕪ㢧ᚤ

高度好塩性古細菌 Haloarcula japonica 由来走気性トランスデューサーの親株における発現と機能解析

5㙾࡟ࡼࡗ࡚ほᐹࡍࡿࡇ࡜࡛ࠊ㉮Ẽᛶࢆホ౯ࡍࡿࡇ࡜ࡀ࡛ࡁࡿࠋ

japonica ࡀ⏕⏘ࡋࡓ⤌᥮࠼ Htr8-Hj ࡣ࣒࣊ࢱࣥࣃࢡ㉁࡛

ࣉࣛࢫ࣑ࢻࢆྵࡲ࡞࠸ Ha. japonica ࡢ㉮Ẽᛶゎᯒࡢ⤖ᯝࠊ࠶ࡾࠊ᪤ሗࡢ࣒࣊ࢱࣥࣃࢡ㉁࡜ࡣ␗࡞ࡿ᪂つ࡞࣒࣊ࢆྵࡴྍ4 ᫬㛫࢖࣮ࣥ࢟ࣗ࣋ࢩࣙࣥᚋࡢẼᾮ⏺㠃࡟⣽⬊ࡢᒁᅾࡀほ⬟ᛶࡀ♧၀ࡉࢀࡓࠋࡲࡓࠊHa. japonica ࡀ⏕⏘ࡋࡓ⤌᥮࠼ᐹࡉࢀࡓ (Fig. 5)ࠋࡇࡢࡇ࡜࠿ࡽࠊࣉࣛࢫ࣑ࢻࢆྵࡲ࡞࠸ Ha.

HemAT-Hj ࡣࠊ᪤ሗࡢ HemAT ࡜ྠᵝ࡞࣒࣊ࢆྵࡴࡇ࡜ࡀ

japonica ࡣṇࡢ㉮Ẽᛶࢆ♧ࡍࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ♧၀ࡉࢀࡓࠋ
✵࣋ࢡࢱ࣮ pWL102ࠊ htr8 ࠾ࡼࡧ hemAT 㑇ఏᏊⓎ⌧ᆺࣉࣛࢫ࣑ࢻࢆᑟධࡋࡓ Ha. japonica ࡟ࡘ࠸࡚㉮Ẽᛶゎᯒࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊHa. japonica ࡣṇࡢ㉮Ẽᛶࢆ♧ࡍࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋࡲࡓࠊHtr8-Hj ࡣ Ha. japonica ࡢṇࡢ㉮Ẽᛶࢆᙉ໬ࡍࡿᶵ⬟ࢆࡶࡘࡇ࡜ࠊࡑࡋ࡚ HemAT-Hj ࡣHa. japonica ࡢṇࡢ㉮Ẽᛶࢆᙅࡵࡿᶵ⬟ࢆࡶࡘࡇ࡜ࡀ♧၀
Fig. 5 ࣉࣛࢫ࣑ࢻࢆྵࡲ࡞࠸ Ha. japonica ࡢ㉮Ẽᛶゎᯒ

htr8 㑇ఏᏊࢆᑟධࡋࡓ Ha. japonica/pht8JH3 ᰴ࡛ࡣࠊ✵࣋ࢡࢱ࣮ࢆᑟධࡋࡓ Ha. japonica/pWL102 ᰴ࡜ẚ㍑ࡋ࡚

ࡉࢀࡓࠋㅰ㎡ṇࡢ㉮Ẽᛶࡀᙉ໬ࡉࢀ࡚࠸ࡓ (Fig. 6 ୖࠊ୰)ࠋࡇࢀࡽࡢࡇ࡜

ࣉࣛࣂࢫࢱࢳࣥࢆࡈ౪୚࠸ࡓࡔ࠸ࡓ➨୍୕ඹᰴᘧ఍♫ࠊ࡞

࠿ࡽࠊHtr8-Hj ࡣ Ha. japonica ࡟࠾࠸࡚ṇࡢ㉮Ẽᛶࢆᙉ໬ࡽࡧ࡟ DNA ሷᇶ㓄ิゎᯒ࡟ࡈ༠ຊ࠸ࡓࡔ࠸ࡓᮾிᕤᴗ኱Ꮫ

ࡍࡿᶵ⬟ࢆࡶࡘࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ

࣮࢜ࣉࣥࣇ࢓ࢩࣜࢸ࢕ࢭࣥࢱ࣮࣭ࣂ࢖࢜㒊㛛࡟ឤㅰࡍࡿࠋ୍ ᪉ ࡛ ࠊ hemAT 㑇 ఏ Ꮚ ࢆ ᑟ ධ ࡋ ࡓ Ha. japonica/phemJH3 ᰴ ࡛ ࡣ ࠊ ✵ ࣋ ࢡ ࢱ ࣮ ࢆ ᑟ ධ ࡋ ࡓHa.

japonica/pWL102 ᰴ࡜ẚ㍑ࡋ࡚ṇࡢ㉮Ẽᛶࡀᙅࡲࡗ࡚࠸ࡓ

ཧ⪃ᩥ⊩(Fig. 6 ୖࠊୗ)ࠋࡇࢀࡽࡢࡇ࡜࠿ࡽࠊHemAT-Hj ࡣ Ha.

[1] 㧗ရ▱඾, ᥀㉺≉Ṧ⎔ቃᚤ⏕≀ࣉࣟࢪ࢙ࢡࢺ◊✲ᴫせ㞟,japonica ࡟࠾࠸࡚ṇࡢ㉮Ẽᛶࢆᙅࡵࡿᶵ⬟ࢆࡶࡘࡇ࡜ࡀ♧pp. 25-38, ᪂ᢏ⾡㛤Ⓨ஦ᴗᅋ (1989).

၀ࡉࢀࡓࠋ[2] K. Horikoshi, S. Nakamura, et al.: Experientia, 49(1993) 497-502.
[3] ᑠ⃝Ꮥಇ, ಟኈㄽᩥ, ᮾிᕤᴗ኱Ꮫ (2002).
[4] T. Kosaka, T. Ozawa, et al.: Nucleic Acids Symp. Ser., 48(2004) 169-170.
[5] S. Nakamura, K. Nakasone, et al.: Extremophiles

Handbook [K. Horikoshi, G. Antranikian, et al. (eds.)], pp.
364-381, Springer (2011).
[6] A. Brooun, J. Bell, et al.: J. Bacteriol., 180 (1988) 16421646.
[7] S. Hou, R.W. Larsen, et al.: Nature, 403 (2000) 540-544.
[8] T. Tadikara, T. Matsubara, et al.: J. Jpn. Soc. Extr., 12(2013) 29-32.
[9] W. L. Lam, W. F. Doolittle: Proc. Natl. Acad. Sci. USA,Fig. 6 ✵࣋ࢡࢱ࣮㸦ୖ㸧ࠊhtr8㸦୰㸧࠾ࡼࡧ hemAT

86 (1989) 5478-5482.

㑇ఏᏊ㸦ୗ㸧ࢆᑟධࡋࡓ Ha. japonica ࡢ㉮Ẽᛶゎᯒ[10] ᑠ⃝୍㐨, ༤ኈㄽᩥ, ᮾிᕤᴗ኱Ꮫ (2005).
[11] S. DasSarma, E. M. Fleischmann (eds.): Archaea, A㻠䠊䛚䜟䜚䛻

Laboratory Manual: Halophiles, Cold Spring Harbor
Laboratory Press (1995).

Ha. japonica ࢤࣀ࣒ୖ࡟ぢ࠸ࡔࡉࢀࡓ htr8 ࠾ࡼࡧ[12] J. Sambrook, E. F. Fritsch, et al.: Molecular Cloning, AhemAT 㑇ఏᏊ࣍ࣔࣟࢢࡢぶᰴ (Ha. japonica) ࡟࠾ࡅࡿⓎ

Laboratory Manual, Cold Spring Harbor Laboratory Press⌧ࢆ⾜ࡗࡓࠋ⢭〇ᶆရࢆ⏝࠸ࡓศගᏛⓗᛶ㉁᳨ウࡢ⤖ᯝࠊHa.

(1989).

6

沼津工業高等専門学校研究報告

第 56 号[13] S. A. Sundberg, M. Alam, et al.: J. Bacteriol., 172 (1990)[16] M. Lübben, K. Morand: J. Biol. Chem., 269 (1994)

2328-2335.

21473-21479.

[14] U. K. Laemmli: Nature, 227 (1970) 680-685.

[17] M. Piatibratov, S. Hou, et al.: Biochim. Biophys. Acta,[15] H. Wakai, S. Nakamura, et al.: Extremophiles, 1 (1997)

1524 (2000) 149-154.

29-35.

[18] S. Hou, T. Freitas, et al.: Proc. Natl. Acad. Sci. USA, 98(2001) 9353–9358.