科学研究費助成事業　　研究成果報告書

様　式　Ｃ－１９、Ｆ－１９、Ｚ－１９ （共通）

機関番号：

研究種目：

課題番号：

研究課題名（和文）

研究代表者

研究課題名（英文）

交付決定額（研究期間全体）：（直接経費）

１４３０１

挑戦的萌芽研究

2015～2014

体腔形成細菌の新規な金属輸送機構の解明とその放射性物質・レアメタル回収への応用

A novel metal-import system in the pit-forming bacterium and its application for recovery of radioactive compounds and rare metals

３０２７３５１９研究者番号：

橋本　渉（Hashimoto, Wataru）

京都大学・（連合）農学研究科（研究院）・教授

研究期間：

２６６６００６２

平成 年 月 日現在２８ ６ ３

円 3,100,000

研究成果の概要（和文）：金属キレート能を示す多糖アルギン酸を細胞内に取り込むSphingomonas属細菌A1株について、その細胞表層とアルギン酸結合タンパク質Algp7の構造と機能を解析した。アルギン酸カルシウムゲルに接着した細胞は、表層構造が窪んだ体腔を形成する。その窪みの深さは100 nm程度であり、体腔形成部位はその周囲と比較すると高電位である。Algp7は、レアメタル（Sm3+とTl3+）と結合し、Lys68とLys69による正電荷クラスターでアルギン酸と結合することが示唆される。以上の結果から、体腔の高電位が負電荷のアルギン酸の濃縮に機能し、Algp7を用いてレアメタルを回収できる可能性が示された。

研究成果の概要（英文）：A gram-negative bacterium, Sphingomonas sp. A1, incorporates metal-chelating alginates. In this study, structure and function of the bacterial cell surface and alginate-binding protein Algp7 were analyzed by microscopy, differential scanning fluorimetry, or X-ray crystallography. The bacterial cells attached to alginate-calcium gels formed a pit on the cell surface. The pit showed about 100 nm in depth and high electrical potential. Algp7 exhibited a capability to bind rare metals such as Sm3+ and Tl3+, and to associate with alginate at the positively charged cluster formed by Lys68 and Lys69. These results suggest that the high electrical potential in the pit contributes to concentration of negatively charged alginate and Algp7 is applicable for recovery of rare metals.

研究分野： 応用微生物学

キーワード： 細菌　アルギン酸　レアメタル　輸送体　電子顕微鏡　原子間力顕微鏡　X線結晶構造解析　示差走査型蛍光定量法

１版

!! "! #$%&'($%&')$%&*+,-!

%./01234567 ! 89':;<=>?@ABCD=EFGHIJ'AIKL5MN=EFGOPQRSTU5VWLXY@Z[5\]^_G`abc:d=8eZfXgGhi'jkT8lmneco@?G`p='qrstuvwx5ABC yzP{|}O}=~G8����n5�����^?G`��'�� ABC yzP{|}O}e��T?8lmn*EfeU, EfeO, EfeB-5x��������=~���b^����'�5KL��� in silico��������@?G`a� ��¡¢£z¤¥=¦§Yh Fe2+�¨S©ªOPQRSEfeO X©ªY'EfeO X¡«¬­q®}¯EfeBX5°±²³=~� Fe2+� Fe3+=AB��'Fe3+�´lm; EfeU =~�a�S=lm��G EfeU, EfeO, EfeB5µ��L9��b¶·¥¸^¬¡sPe¹ºY'EfeO=»¼5\]©ª½¾}t*Site I¿III-�x�gG`Y�Y'À���Á5OPQRS5�Â�ÃÄEJKLÅÄƲ³=EgG/09Ç�@?G` ! À�È^='É/0Ê�9'v«ËPAeÌ�ÍIÎXY@:d^ÏGÐz¥ÑÒSphingomonas]ab A1Ó*ÔÕ'A1Ó-eÖ×='�5ØKLÙÚv«ËPA5����n5Û;eÜ��=Y@Ïh`a� 5v«ËPA9a�ÝÞ=¹º��G;ß*à-=áâ��hã'¨S©ªOPQRS*AlgQ1X AlgQ2-X ABCyzP{|}O}*AlgM1-AlgM2/AlgS-AlgS-5²³=~�äåa�S=���È�G`���È�hv«ËPA9'a�S^ÌÚ=È^K���G`;ß9'æ:ç�è¸é45ØKLçS����êë^_G`Èh'v«ËPA©ªÃeìga�ÝÞOPQRSAlgp7eíîY';ß=>ïGv«ËPAáâ=�ÃgGÀXeìYh`��'Algp798lm=EFG¨S©ªOPQRS EfeO XØ?ÄíÒeìY'�5��L¸¦=9 EfeU, EfeO, EfeB=µðÄíÒeìgOPQRS SPH729, SPH728, SPH727�A1Ó¶·¥=ñ}ò��@?G`óo@'Algp79\]*8-5lm=cEôgGÀX�õö��G` ÷./05ø� ! É/0Ê�9'A1 ÓùúØKLÙÚv«ËPA����n5ûØüýÅ�ÂX�Ãþÿ��5�Üeø!Y@?G*" 1- v«ËPA9µ#\]e­$}ygGhi'Algp7�v«ËPAX\]5%�5����=�ÃgGÀX�ì&��G`é�'A1 Ó�\]e­$}yYhv«ËPA¶«=å'Y'�5¶«eK�gG()�*�e+h`Èh'Algp75,;�Â-î=~�'\]©ª./*Site III-=>ïG0I©ª12y3}ReÜ��=Yh`É/0^9'v«ËPA¶«eáâgG;ß5¹º��'45=\]lm EfenOPQRS5�Ã*\]©ªXlm-þÿ��e'µ#6æ7��'X8©

9�Â��':5./p;�<;��=~�Ü��=gGÀXeø�XgG`

! "#$%" &'()*+,'-./012$ =./05�> (1) ;ß¹º A1Óa�56æ7�� ! A1 Ó9a�ÝÞ=;ße¹º�?G@'´�Âea�SABC§�?G`;ß¹º./=9\]e­$}yYhv«ËPA¶«*DE-����G`�À^' A1 Ó5a�ÝÞ�ÂeFGHIL6æ7=~���Yh`Èh'JLÅK6æ7��=~� A1 Ó5:a�=ÖY@';ß¹ºeLMY';ß5NO¤Xa�ÝPIQeRîYh` (2) Algp75\]©ª�� ! Algp75\]©ªÃe'JLSTK�=~�±Uh `Èh'Differential Scanning Fluorimetry*DSF>-=~G\]©ªÒcVWYh`XTYI*SYPRO Orange-x�Õ^'Algp7Xµ#\]XeZªY'\]©ª=~G Algp75[<ÒpÒe��Yh` (3) \]:5v«ËPAX©ªgG Algp75�Â�� ! Site III*His115xxGlu118-5\]©ª½¾}tec¼Algp75\]©ª=EFG�ÂfWeÜ��=gGhi'Algp75©9e±\Yh +��h©9�� X8]^r}Oe_`Y' ØK�Ã^5r}O_`9'SPring-85abTÛcde=@foh g^='\]�©ªY@?T?©9�Âe-îY@?G`#ð5\]e³?@'\]�©ªYh Algp75©9�Âe'KLëg>=~�-îYh` ! Algp79v«ËPA©ªÒeìghi'�5v«ËPA©ª./e'./p;�<;;e³?@Ü��=gG`

h./0ºi (1) A1Ó5;ß¹ºXpÒ ! ��bX A1 Ó5a�e³?@'j«qk¥^¶«BYhv«ËPA=ÖgG²³e±Uh`�5©i'A1 Ó5a�9':d=lo@'v«ËPAj«qk¥¶«*ämn3 mm-eK�Yh*" 2Õ- ��'��b^9v«ËPAj«qk¥¶«9K���o=pxYh*" 2¸'qr-

! 34()*+,5)'6789:;<=>?:

%" &>@A:()*+,5)B$ ! FGHIL6æ7��=~�'v«ËPAj«qk¥¶«=å'Yh A1 Óa�='ÝÞ�Â�s�t;ß5¹º�Mi��h*"3u- ��='JLÅK6æ7^��YhXÀv':a�=>?@c;ß�¹º��GÀXeLMY'�5s�5w�� 76¿147 nmxy^_GÀX�K�oh*" 3z-Èh';ß¹º./9'�5{|X}~gGXØI/�_GÀX��*" 3 �-'À5ØI/��IQe�5hv«ËPA5áâ=�ôY@?GXõö��G`

! C4%" &'DEFG8H:IJKLMNOPQ>R:SMTUNOPQ>V:LWQ>X@

A:DEB$ (2) Algp75\]*$v�O«-©ªÃ ! Algp75\]©ªÒ=¼?@'JLST{¡Ry«>^��Yh Algp7X Fe3+Èh9Cu2+XeZªY'��=~�\]O¬Pe��Yhã'Algp7X©ªgG\]O¬Peî�Yh �5©i'��ã5Algp75�K='Fe3+Èh9 Cu2+�pxgGÀXeLMgGÀX�^Ïh`Èh'Algp75\]©ª=~GOPQRS5[<Ò��e±Uh DSF>9'¢�Pòx�Õ�?9�x�Õ^'OPQRSXXTYI SYPRO OrangeXeZªY'�[gGÀX=~��5[<Ò5<B*���y Tm-eVWgGc5^_G`�

�='¢�PòX©ªYhOPQRS9'�©ªOPQRSX}~gGX'�Â�îB=~�Ø?���yeìg g^='Fe2+'Fe3+'Zn2+'Cu2+5x�Õ^'Algp75���y�¸�gGÀXeLMY@?G`�À^'µ#$v�O«*Pd2+'Sm3+'Pt2+'Tl3+TU-e 0.1 mMx�Õ^'Algp75[<Òe±Uh �5©i'Sm3+X Tl3+5x�Õ^'Algp75���y�¸�Yh`$v�O«�x�Õ^9'Tm�9 54y^_oh�'Sm3+x�Õ^9 67 y'Tl3+x�Õ^9'63 yX��=Tm ��¸�Yh`��=' Sm3+5áye<B�?@���yeRîYhXÀv'áywx�=���y5¸��Mi��'0.05 mMÔ¸^�î�=�Yh`��'��= Sm3+

X5©ª����h�5OPQRS XLY =¼?@í!5ÛcefohXÀv' XLY 9Algp7�U'Sm3+=~G���y5¸�eì�T�oh`À��5ÀX��'Algp79_G#5$v�O«X��=©ªgGÀX�ì&��h` (3) Algp7 5v«ËPA©ª=EFG�IQRz{O} ! v«ËPA9\]­$}y²³eìghi'\]lm Efen5�ºfI^_G Algp75v«ËPA©ªÃe�Â5������Yh`X8©9�Â��=~�'Algp75ØK�Ã*1.99Å-5,;�Âe-îYh*" 4-

! Y4%Z[\] '2^8H:_`+ab)>V:6McdLe8fA:gLehijklBB$

! ØK�Ã5,;�Âe³?@'v«ËPA¬¢ ÚX5ò2­PÐq¡¢$}q£Pef?'�5¤ª;½r«=¨¥?@'v«ËPA5©ª=EFG¦Vp¨e��Yh`���5p¨e./p;�<;=~� Ala=ëgYh<;; E194A'N221A':5K68A/K69A e²\Y'<;;5v«ËPA©ªÃe§ S_>=~���Yh`¨:HX<;;5µOPQRSX#ð5áy5v«ËPAXe©ª�?@'280nm=>ïGS_{¡Ry«<B�ev«ËPAáy=ÖY@£s2yY'«¬­8e²ºYh T>'Lys68 X Lys69 5{®=9§ S_eìgTrp187�¯ëY@?G*" 5-

! m4%Z[\] '()*+,nopW$ ! E194AX N221A=¼?@'¨:H Algp7X�°í±5 ©ª�²î³�´���h5=ÖY'K68A/K69A 9v«ËPAµáy^S_{¡Ry«5<Bc¶T·'«¬­8c+��T?ÀX��'��T*20¸Ô¸-©ªÃ5¹¶����h À5ÀX��'Lys68X Lys69 9v«ËPA©ª=EFGºfTp¨^_GÀX�ì&��h*" 5- (4) Algp75\]©ª=EFG�ÂfW ! Algp7X\]X5©ª=EgG�ÂpÒeÜ��=gGhi'Zn2+x�Õ^ Algp7*Algp7-Zn-5©9�Âe 2.7Å K�Ã^-îYh Algp7-Zn =>?@'Zn2+9'Glu79, Glu82, Asp96, Glu178 5»¼5AÒp¨X©ªY@?G*" 6- ¼½w?ÀX='À5©ª./9'Site III*His115xxGlu118-X¶Y²�@?G`

! q4%Z[\] 'rsnopW$ ¾©¿À ! IL6æ7^9ÁýÂÃÕ^a�e��gGhi'À�È^=:a�^5ÄÅe+GÀX�ÆÇ^_oh`É/0^9'JLÅK6æ7^ A1 Óa�e��gGÀX=~�':a�^5;ß¹ºeÈLMY'�5NO¤cÉRgGÀX�^Ïh`��='ÝPI/eRîgGÀX=~�';ßXv«ËPAX5ÊI�ÄƲ³=¼?@äå�TLËe+GÀX�^Ïh` ! v«ËPA©ªÃeìg Algp7 � Fe2+'Fe3+'Zn2+'Cu2+=�ö@'_G#5$v�O«X©ªgGÀX�Ü��=Toh`Èh'�Â��=~�'Algp75v«ËPAX Zn2+

5©ª./9��Ì��X�=�IY@>�'À��5KLÝPIQ9\]e­$}yYhv«ËPAX5©ª=ºf^_GXõö��G` ! Ô¸5É/0ºi��'A1 Óa�9�IQ5v«ËPAe�=�I Yh;ß=áâY'Algp7�\]O¬Pe­$}yYhv«ËPA��\]O¬PXv«ËPAXeK²Y'\]O¬Pe8lm Efen='v«ËPAe ABC OP|}O}n=K¯gGyztÍ2RñPys}z}XY@�ÃgGÀX�ì&��h`Èh'A1 Ó5a�ÝÞ=þÿgG Algp7e³?@'$v�O«eÎϵ#\]e]_^ÏGÐÃÒ�ì��h` ÑÒ³ÓÔÕ Ö Braun, V. and Hantke, K.: Recent

insights into iron import by bacteria. Curr. Opin. Chem. Biol., 15, 2011, 328-334

× Cao, J., Woodhall, M.R., Alvarez, J., Cartron, M.L., and Andrews, S.C.: EfeUOB (YcdNOB) is a tripartite, acid-induced and CpxAR-regulated, low-pH Fe2+ transporter that is cryptic in Escherichia coli K-12 but functional in E. coli O157:H7. Mol. Microbiol., 65, 2007, 857-875

Ø Rajasekaran, M.B., Nilapwar, S., Andrews, S.C., and Watson, K.A.: EfeO-cupredoxins: major new members of the cupredoxin superfamily with roles in bacterial iron transport. Biometals, 23, 2010, 1-17

Ù Momma, K., Mishima, Y., Hashimoto, W., Mikami, B., and Murata, K.: Direct evidence for Sphingomonas sp. A1 periplasmic proteins as macromolecule-binding proteins associated with the ABC transporter: Molecular insights into alginate transport in the periplasm. Biochemistry, 44, 2005, 5053-5064

Ú Aso, Y., Miyamoto, Y., Harada, K.M., Momma, K., Kawai, S., Hashimoto, W., Mikami, B., and Murata, K.: Engineered membrane superchannel improves bioremediation potential of dioxin-degrading bacteria. Nat.

Biotechnol., 24, 2006, 188-189 Û He, J., Ochiai, A., Fukuda, Y.,

Hashimoto, W., and Murata, K.: A putative lipoprotein of Sphingomonas sp. A1 binds alginate rather than a lipid moiety. FEMS Microbiol. Lett., 288, 2008, 221-226

Ü Niesen, F.H., Berglund, H., and Vedadi, M.: The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability, Nat.

Protoc., 2, 2007, 2212-2221 Ý Maruyama, Y., Ochiai, A., Mikami, B.,

Hashimoto, W., and Murata, K.: Crystal structure of bacterial cell-surface alginate-binding protein with an M75 peptidase motif. Biochem. Biophys. Res.

Commun., 405, 2011, 411-416 Þ.ßTþÝ¿Ó± àáâ¿Óã*É 5Ã- Ö Yukie Maruyama, Takafumi Itoh, Ai

Kaneko, Yu Nishitani, Bunzo Mikami, Wataru Hashimoto, and Kousaku Murata: Structure of a bacterial ABC transporter involved in the import of the acidic polysaccharide alginate. Structure, Gä�, 23, 2015, 1643-1654

doi: 10.1016/j.str.2015.06.021 × Yukie Maruyama, Masahiro Kobayashi,

Kousaku Murata, and Wataru Hashimoto: Formation of a single polar flagellum by lateral and polar bacterial flagellar gene sets in Sphingomonas sp. strain A1. Microbiology, Gä� , 161, 2015, 1552-1560

doi: 10.1099/mic.0.000119 Ø Kanate Temtrirath, Kousaku Murata,

and Wataru Hashimoto: Structural insights into alginate binding by bacterial cell-surface protein. Carbohydr. Res., Gä� , 404, 2015, 39-45

doi: 10.1016/j.carres.2014.11.008 Ù Ryuichi Takase, Bunzo Mikami,

Shigeyuki Kawai, Kousaku Murata, and Wataru Hashimoto: Structure-based conversion of the coenzyme requirement of a short-chain dehydrogenase/reductase involved in bacterial alginate metabolism. J. Biol.

Chem., Gä�, 289, 2014, 33198-33214 doi: 10.1074/jbc.M114.585661 Ú Chie Hayashi, Ryuichi Takase, Keiko

Momma, Yukie Maruyama, Kousaku Murata, and Wataru Hashimoto: Alginate-dependent gene expression mechanism in Sphingomonas sp. strain A1. J. Bacteriol., Gä� , 196, 2014, 2691-2700

doi: 10.1128/JB.01666-14 à�åþÝã*É 1Ã-

Ö ¸æ�ç'\L_?'èéêë'0¨ìí'

îïðñ'òó ô'»¸Ó»'õö÷²'

øÉ! ù: ab ABCyzP{|}O}5

ATP�0K�9 closedH5¨S©ªOP

QRSX5ÄƲ³=~o@úû��G .

üÉýþB�åEæÿ.!å*" 493]#

$å-'2016� 2% 6ü'&'��()å

**&'+&',-

à"-ã*É 0Ã- .3/Y à01203ã 4�567*É 0Ã- .3/Y 4�+67*É 0Ã- .3/Y à�5�ã .3/Y 8./09: (1)/0;ÝÊ ! øÉ! ù*HASHIMOTO, Wataru- &'��<��=ý�/0><?@

! /0ÊABC30273519 (2)/0KDÊ ! õö! ÷²*MURATA, Kousaku- EF��<GH�.<?@

! /0ÊABC 90142299