ISSN 2186-9936

Mar. 2019$

Journal of Japanese Society for Extremophiles





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1Journal of Japanese Society for Extremophiles (2018) Vol.17

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3

Journal of Japanese Society for Extremophiles (2018) Vol.17

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4

Journal of Japanese Society for Extremophiles (2018) Vol.17

Journal of Japanese Society for Extremophiles (2018) Vol.17, 5-11

/ !# --!#

Shigi N

Characterization of the biosynthesis of 2-thiouridine in thermophile tRNAs
which confers the thermostability to translation systemՐȵǘͫэkУͫŘ>g tRNA ζ՘ĀՈƾƷQ;ƁɯL̈ЭQѴʽƩϒβώԍΗ̰× Ϳ˽ɸџЋƁβώɳ ŇѕƷΠβώӮԋՕ ΢̆
Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science andTechnology (AIST), 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan
Corresponding AuthorխNaoki Shigi, naoki-shigi@aist.go.jp
Phone: +81-3-3599-8265, Fax: +81-3-3599-8494
Received: Sep. 14, 2018 / Accepted: Sep. 14, 2018
1. R=_OΗѮ<D#8h\K¬xµĀՈϭRΧ˴;ͯQ

ρR͆ӇģЇě;e1 1974 ȬOΗѮ:hDՐȵǘ]OǣƮ>gL:hJ2D1"˟βώOdfΧ̎Ϲ1)

эKR=_JBQǣƮkη҉<D#Χ˴;ͯQ¬

Q;Ɓɯ̈̀QѴʽkўGJ2D#ՐȵǘͫΧ̎ϹxµĀՈϭQұ͏Rũ˴;ͯQζ՘ŘƁͯQ;Ɓ

э Thermus thermophilus Qөæǡʠóϭkʢс<"ζɯϭO+g8LkѥÜ6g`QK+g#ͫэµzҭƁɯϭkУͫŘ>gζ՘ĀՈƾƷ՘ĀՈƾƷQ;ƁɯөæǡМkƃǭ<D#8hOd
fĀՈ̉ʜ˳RՐ͇ɦŴɚkκ>8LkѮĳ<"x ¸ ´ ¸  խ tRNA, thiouridine, thermophile,tRNA Qζ՘ŘOdgПѿϭQͫǨǭŘ1ǘͫэQ

post-transcriptional

Ր͇¾KQ;ЪOɓԸK+g8LkҀʽ<D#

modification

modification,

post-translational\D tRNA ζ՘ĀՈűɕk҂ՍϠĨKĪ̀ɯ<"ζ՘ŘӴ϶ TtuA RӶ϶ɦŴɚQԀζ՘z¯¸k2ա ζ՘ĀՈƾƷ 2-uq°µQ̈Э̷ɚOɓѬL<"\Dζ՘xª°n¸µzҭ mRNA ½Q~µLn¦ӶkϾVÜ6g tRNA

TtuB OϾƁ<D L-pµ΂˥Q̷ɚŘ:hD

ORǊʬQҼĭɉĀՈ1+f"N0K`ζ՘ĀՈƾζ՘k RNA OҼφ>g"L,-Ͱɐkʽe0O<

ƷR~µ҉қ (34 ë) aϒð̀ӗQǨǭŘ (54

D#ǊʬQ;Ɓɯƣǡ1ԏÀ<"Ӷ϶ɦŴɚK`+ë) NMQɄņkʂGJ,g [Ƨ 1A]#BQD_"

GDD_"Ѵ˨ϭQϒF½7RƥԧK+GD1"

tRNA Oζ՘kȁğ>g̈̀R;ƎOLGJɓԸK

¦µϜQÛQ;ðζ՘ŘƁͯQ;Ɓɯ̈̀O`Ĥ

+g1"BQ҆ϹRԊȬ¿ʽK+GD#

ӕ>g"űɕɚQՐ,̷ɚŘζ՘kxª°n¸µ q°µQ 2 ëQӶ϶ũǡ1ζ՘ũǡOИʚ:hzҭOϾƁ:@JԮͰΌΛNűɕkԑ,K,g"DŘǥ̀ӗk`H 2-uq°µ (s2U) R tRNA Q

L,-<4]k+2e0O>g8L1K2D#\Dnµ~µL T-±¸O]ehg [Ƨ 1A, B]#nƃ́Qϭ1Χ˴;ͯO`þǣ:hJ,g8L1Ĺʽµ~µ 1 ʯǢΡ (34 ë) QĀՈR~µǼƁk<"8QϭQәŘΛNŶ:LӻѬɚ1κ:hD#ӦĵOĽɌ<"̎ηKŎͷΛNµzҭƁɯOL:eOζ՘xª°n¸µzҭK+g TtuB R"

GJӻѬK+g#Z[ĠJQ;ͯQ{±¦µӶ"

BQ¬xµOéD̀ӗL̈̀Odf"ǘͫэĨ{±¦µ"°µQ tRNA Q 34 ëQq°µR"

KǊʬQ̂ΛµzҭOĤ˗ϾƁ<J,g8Lk

5-¨±-2-uq°µҋȁð (xm5s2U) OĀՈ:5

Journal of Japanese Society for Extremophiles (2018) Vol.17

hg 2) ՞ƾƷQ 5 ë`ĀՈ:hJ/f"BQ̀ӗR

 \D T-±¸Q 54 ëRĠJQ tRNA ψK 5-¨;ͯψOdfΌNg՟#2-uq°µR"0:Ր,±q°µ՞m5U"°£¦µ՞rT՟L`,-՟ƾƷQ 2-uƷL°£¸Q 2’-̢ӶƷQϒðԢǴ

K+g1"ǘͫэ՞Χ̎Ϲэ Thermus thermophilus"

3)

Odf C3’-endo ƲkLfa>4Ng [Ƨ 1B ¾̙]#Aquifex aeolicus"ŶϹэ Pyrococcus furiosus NM՟K

8QD_°£¸§½Knµ~µQĀՈ:hD

R¹Ӯ1ζ՘Ř:h"5-¨±-2-uq°µ

U R~µ 3 ʯǢΡQ A·G LQǼƁkȾ_"ĺQ

՞m5s2U"\DR s2T՟LNGJ,g 1) [Ƨ 1A, B]#n¦Ӷkʉǭ>g C·U LǼƁ>gQkԑ3"2-

m5s2U ` C3’-endo Ʋ1ǨǭK+gQK"T-±¸L~µ£zQ̎ηNѴҐ1ӡɯ:hg#ǰԡĀ

D-±¸1åƁ>g tRNA QШԺƴQ 2 ˟Ԉ̀ӗ1

Ոk̉ʜ>gL"Ǽɕ>g~µK°£¸§1ĉǨǭŘ:hgD_"Ͼ˫ΛO tRNA Ġð1ͫǨǭŘ

͓<"ПѿÄQµzҭQϒð̀ӗɀɯ1-\4:hg 5) [Ƨ 1C]#dfՐ͇KƶՉ>gLζ՘Ř:h,0?ıԤð1ёϋ<J"ϹЬO²kÀ.g

g tRNA QņƁ1ǁ."\Dζ՘Ř:hD tRNA R

4)

Ր͇KQµzҭƁɯ̷ɚ1Ր,##Ƨ 1. tRNA Qζ՘ĀՈ (54 ë) RՐ͇ŘKQ;ЪOɓԸK+g
A. ζ՘ĀՈƾƷQ tRNA ½KQëИ#
B. q°µ (½̙ț) Lζ՘ĀՈƾƷ 2-uq°µ (½̙Ž) Q̀ӗ#2-uq°µR C2’-endo Ʋ (¾̙ț) df` C3’-endo Ʋ (¾̙Ž) kLfa>,#
C. 54 ëQ 2-uq°µR T-±¸L D-±¸0eNg tRNA QШԺƴQ 2 ˟Ԉ̀ӗkǨǭŘ>g#
D. 2-uq°µ̉ʜ˳RՐ͇ɦŴɚkκ>#

6

Journal of Japanese Society for Extremophiles (2018) Vol.17ա ǘͫэζ՘ĀՈƾƷQ;Ɓɯ̈̀QѴʽ
3ա

ғѨ<J"ζ՘Řűɕk҂ՍϠĨKĪ̀ɯ<"űɕ ½ӍQd-OǘͫэKR"tRNA Q 54 ëQq°̈̀kҭӽĴ˨ѤИaʤǿɚ¯
7,8,9)±<DƷҭϜk`[Ƨ 2A]#ζ՘xª°n¸µƾƷ1ζ՘Ř:hg8LOdfУͫŘ:h"Ր͇

F,J҆ϹOѴ˨<D

KQµzҭƁɯ1źЭONgLС.ehJ,D#µz TtuB Qv±£x˞ϕQv±£x±ƷρRǘͫɚΧ̎Ϲэ Thermus thermophilus k©±

(TtuB-COOH) 1"TtuC Odfn±Ř:hJ̷

O΀,"өæǡʠóϭkʢс·Ռõ<Jζ՘ĀՈƾɚŘ:hg#B8O L-Cysteine Qζ՘ũǡ1ƷQ;ƁɯөæǡМ (iscS, sufS, ttuA, ttuB, ttuC, ttuD)pµвζӴ϶ (IscS/SufS) Odf̷ɚŘ:h"TtuD

kƃǭ>g8LOɯŉ<D

6,7,8,9)#ɉONGJj0G

kÙ<Juv±£xðkɀɯ>g(TtuB-COSH)#

D1"ǊʬQ;Ɓɯƣǡ1ԏÀ<0HӶ϶1̷ɚk

8Qζ՘1ζ՘ŘӴ϶ TtuA Odf tRNA Oȁğ:hԒǴ>gD_"ӕȧQζ՘Ř̷ɚϭQ̀ϣLƀψv

g#ͥӶ϶¾KQ;ŘǥǰՍL̀ӗѴ˨0e"ζ՘¯§OdgϹЬʁĳ̾0eQζ՘ŘӴ϶QϫѨR-ŘӴ϶ TtuA RӶ϶ɦŴɚQԀζ՘z¯¸k̷

\4,0N0GD#B8K tRNA OϾƁ>gµ

ɚOɓѬL>gʵǑNӴ϶K+g8Lk+2e0OzҭkǘͫэϹЬʁĳ̾0e͜В<"ҭӽĴ˨ѤИ

<D 10,11) [Ƨ 3]#8QǘͫэQϭRũ˴;ͯQ 34 ë

Odfζ՘ŘӴ϶ TtuA Lζ՘xª°n¸µzQζ՘ĀՈƾƷQ;Ɓɯϭ 12)LRΌNf"ɤǉO`

ҭ TtuB kƃǭ>g8L1K2D#;ƁɯƣǡМQ

Χ˴;ͯQ 34 ëQ;Ɓɯϭ [Ƨ 2B]LՂé<J,Dөæǡεǂ˳Rζ՘ĀՈƾƷk̉ʜ<D1"8he

13)Q˳RՐ͇ɦŴɚkκ< 7,8) [Ƨ 1D]"tRNA Qζ՘Āµ҉қOɓԸK+g)#8heQѴ˨0e RNA ζ՘ՈƾƷOdgПѿϭQͫǨǭŘ1ǘͫэQՐ͇KQĀՈƾƷQ;ƁɯϭR 2 ψՂ+g1"BQMFe`;ЪOɓԸK+g8Lk΢ʕΛOҀʽ>g8L1K

űɕɚQՐ,̷ɚŘζ՘kxª°n¸µzҭO

2D#

ϾƁ:@JԮͰΌΛNűɕkԑ,K,g"L,-Ĥ H,K;ƁɯµzҭМQϼʚ.µzҭk

ӕQͰɐkѮĳ<D#(½Ӎ<Dd-O 34 ëQζ՘ĀՈRĠ;ͯK~Ƨ 2. 2-uq°µQ;Ɓɯϭ
A. ǘͫэQ 2-uq°µ (54 ë) Q;Ɓɯϭ#
B. Χ˴;ͯϹЬҭQ 2-uq°µ (34 ë) Q;Ɓɯϭ#

7

Journal of Japanese Society for Extremophiles (2018) Vol.17

Ƨ 3. Ӷ϶ɦŴɚԀζ՘z¯¸k˗>gζ՘ŘӴ϶ TtuA
A. ͥӶ϶ǰՍѤИ
B. TtuA RԀζ՘z¯¸QD_щтkƉ>g#C. ζ՘ŘӴ϶ TtuA Lζ՘xª°n¸µzҭ TtuB QѩƁð̀ӗ#
D. TtuA Q̷ɚÄɒQ̀ӗ#ζ՘kӝlK4g TtuB Q C ˞ϕ"ATP n³{"[4Fe-4S] z¯¸1ӊʕ<J,g#E. TtuA Qʖǭűɕ̈̀#8

Journal of Japanese Society for Extremophiles (2018) Vol.17

4. ʵ ѯµzҭПѿɉĀՈQΗѮ

 \DΧ˴;ͯKR̂Λµzҭ0e¬xµ βώQӟχKɤǉNΗѮ`+GD#ǘͫɚΧ̎ϹkĵfԦ>"вĀՈӴ϶`̂ΛQ̈ЭĽɌOӻѬNэ T. thermophilus Qζ՘xª°n¸µzҭ TtuB

Ĕ2kʂGJ,g1"BQ¢©³{`ǘͫэOǣƮ

LBQn±ŘӴ϶ TtuC R¬xµLBQ̷

<J,D 14)#8hRәŘQӟχK"ζ՘ŘƁͯQ;

ɚŘӴ϶ (E1) OéD̀ӗkLf"¬xµŘLƁɯ̈̀1ПѿɉĀՈ̈̀XL̈ЭǇŘ<D8LkՂé<DűɕOdfǊʬQ̂ΛµzҭOĤ˗ϾκƔ>gǌǇнƌ̀,ήѮK+g#ζ՘ŘƁͯQ;

Ɓ<J,D

14)

ƁɯQD_O̷ɚŘ (n±Ř) :hD¬x[Ƨ 4A]#8h\K¬xµϭ [Ƨ4B]RΧ˴;ͯO<0N,LС.ehJ,DQE1"µՂéĴǡR"+gL2ČΗΛOƋӇQµzҭŶϹэKQƻƊ (SAMP) 15) OЅ2"Χ̎ϹэO`+

Lűɕ<Ĥ˗ϾƁ<J<\GD"LС.ehg#8g8Lkĸ_Jκ<D#¬xµa¬xµՂ

h1ζ՘xª°n¸0eПѿɉĀՈƣǡXQ̈ЭǇ

éĴǡ (NEDD8 a SUMO NM) RΧ˴;ͯOþǣŘQ¤pµK+GDQEi-#ZLlMQũ˴;:hDПѿɉĀՈµzҭK"̂ΛLNgµͯ}§O¬xµ"E1"в¬xµӴ϶OՂzҭQ̈Эkғϡ>g#̂ΛµzҭQĴѴaȇ

é<Dөæǡ1+gQK"8heRũ˴;ͯQʵѯƮkĽɌ<"ϹЬƋ˛Qәўa{±æӡNMǊ

NПѿĀՈͺҟOdgµzҭ̈ЭQĽɌ̈̀L

́NՐ̊;Ǝ̈ЭOԏÀ>g#ǘͫэO/6ĝΛ<JĔ,J,gźЭɚ1С.eh"ØɉQβώQә

µzҭQЈКΛƃǭOH,JR\EәўÄK+Ȍ1˛ɇ:hg#g1"ζ՘ŘĀՈϭQ TtuA (ζ՘ŘӴ϶) L TtuC

 N/¬xµ"E1 Ӵ϶"E2 Ӵ϶"E3 Ӵ϶"/

(TtuB ̷ɚŘӴ϶) 1 TtuB ŘĀՈ:hJ,g8Lk

dVв¬xµӴ϶өæǡu¡³µ1nw±Ѯĳ<J,g

14)#:eO TtuA Q̷ɚÄɒӊčQ 3

ŶϹэNMQΧ˴;ͯQ΢ʕQνěLС.ehgŶ

HQ Lys ̗Ʒ1 TtuB OdfПѿɉĀՈ:hJ/f"ϹэМQ}§OR+f"ǰԡOBheQөæǡͿ

8hOdf RNA ζ՘Řűɕ1ĽɌ:hJ,g8L

ͯRПѿɉĀՈ̷ɚk`H8L1κ:hJ/f"Χ

1κƔ:hD#

˴;ͯQѩԥN¬xµ§QũƲR>KOŶϹэO+GDLС.ehJ,g 16,17)#

Ƨ 4. ǘͫэ TtuB RПѿɉĀՈƣǡK`+g
A. (½̙) ǘͫэQ 54 ëQ 2-uq°µQ;Ɓɯϭ#(¾̙) ПѿɉĀՈƣǡL<J`Ĕ4#
B. Χ˴;ͯQ¬xµĀՈϭ#

9Journal of Japanese Society for Extremophiles (2018) Vol.17

5. Ø ɉQȌ˚

position of the anticodon. Proc. Natl. Acad. Sci.

 ½Ӎ<Dd-Oζ՘ĀՈƾƷRǘͫэÞǉQ;ͯ

USA 82:4905-4909.

KRnµ~µդǢΡ (34 ë) OˈӞΛOǣƮ<"4) Nedialkova D.D., Leidel S.A., 2015. Optimization~µ҉қOɓԸK"8Qζ՘ĀՈQ̉ʜRK

of Codon Translation Rates via tRNA Modifications

RΖa¦~µ°nΑQũƣONg#8QƾƷQ

Maintains Proteome Integrity. Cell 161:1606-1618.

;ƁɯűɕRǘͫэϭLƃ́K+f"Qζ՘Ř5) Watanabe K., Shinma M., Oshima T., Nishimura S.,ĀՈӴ϶`¬xµOՂé<DĀՈk-6g#ǘ

1976. Heat-induced stability of tRNA from anͫɚΧ̎ϹэaŶϹэk©±O<J"8Qζ՘Ř

extreme thermophile, Thermus thermophilus.

ĀՈűɕQĽɌ̈̀kѴʽ>g8LOdf"ζ՘Ā

Biochem. Biophys. Res. Commun. 72:1137-1144.
6) Shigi, N., Suzuki T., Terada T., Shirouzu M.,ՈƾƷΌȧOdgΐΑQũƣѴʽ"̬Ε̰QԍΗO
HN7D,#\D¬xµOՂé<D8QПѿɉYokoyama S., Watanabe K., 2006.

ĀՈR"ũ˴;ͯK¹пΛNĽɌ̈̀QźЭɚ1+

Temperature-dependent biosynthesis ofg#Αũэa˗΀ͯҭ;ͿэkѴ˨<"ĀՈ̈̀Q

2-thioribothymidine of Thermus thermophilus tRNA.

ԒǴaüәk҂]"ɼ;ńQԍΗ"ͯҭ;ͿэQʢ

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сXQɕ΀`ՑňΛN̊QҒԾK+g#

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6. ҚӅ

required for cell growth at extremely high ˕ɉO˟βώkǚ_gǓ̈kÀ.J¾:f"\Dtemperatures. J. Biol. Chem. 281:14296-14306.
8) Shigi, N., Sakaguchi Y., Asai S., Suzuki T.,ԊȬO͆f9ʉȁ·9԰ʟkҫf\<D"˦Öǌǥʦ ͆ӇģЇ Ƅ҇ʩʐOŨ4Ɍλ΄<½7\>#

Watanabe K., 2008. Common thiolation mechanism

RNA QҭӽĴ˨kR=_βώĠпOH2\<J9in the biosynthesis of tRNA thiouridine andʩʐ·9ʉȁԵ2\<D˦Öǌǥ ӿ˜ Ő ʩʐO`

sulphur-containing cofactors. EMBO J.

ɦҚм<\>#\DͼŘǥβώɳ ̅ȏшÈ ½Ȥβ27:3267-3278.

ώƒ"˦řǌǥ ΁ÄсƏ ʩʐ"ƃ ՚і ̎΅ ʩʐ9) Shigi, N., Asai S., Watanabe K., 2016. Identification

kR=_L>gĤƃβώТQʶ$O`ɦҚ΄<½7

of a rhodanese-like protein involved in thiouridine\>#\D"́$N9ŋѶ·9ŋňkԵ,D"Րsbiosynthesis in Thermus thermophilus tRNA. FEBS

±y¸ŊӖƟβώ̈̀ ŝ΁ýƑ ʩʐ"͆Ӈ·ӿ

Lett. 590:4628-4637.
10) Nakagawa H., Kuratani M., Goto-Ito S., Ito T.,

˜βώǲQ¨µ¸QΜ́O`ɦҚ΄<½7\>#\D"˟βώQ¹ӮRʯӮσǥΥσβҧ (KAKENHI

Katsura K., Terada T., Shirouzu M., Sekine S., Shigi,

19770096, 22770140, 24570173, 16K07311)"Ĩіѻɘ

N., Yokoyama S., 2013. Crystallographic andσǥʌнҡƤ"ā΁ѻɘʺϒσǥɸџҡƤ"̏΁σ

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ǥʌнҡƤ"ϫǷ͈ǭɸџʌнҡƤ"/dVȂϘũ11) Chen M., Asai S., Narai S., Nambu S., Omura N.,σǥɸџʌнҡƤQŋɯOdfӛў>g8L1K2

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K., Yao M., Shigi, N., Tanaka Y., 2017.
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Sugahara J., Kazama H., Chee G.J., Hattori M.,

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Journal of Japanese Society for Extremophiles (2018) Vol.17/ !# --!#

Journal of Japanese Society for Extremophiles (2018) Vol.17, 12-18

Minegishi H

Isolation and Phylogenetic Analysis of Novel halophilic Archaeon, andCharacterization of Halophilic Enzymes.
ʵѯǘƾɚŶϹэQĴԦ·ϭЂѴ˨/dVǘƾɚӴ϶Qβώ
˦̵ǌǥͼȚǥӮɕ΀ŘǥσȔȓ Ǫʽ
Faculty of Science and Engineering, Toyo University, 2100 Kujirai, Kawagoe-shi, Saitama 350-8585, Japan
Corresponding AuthorխHiroaki Minegishi, minehiro@toyo.jp
Phone: +81-49-239-1390, Fax: ++81-49-234-250
Received: Sep. 22, 2018 / Accepted: Sep. 22, 2018
1. R=_O+f"Ǌ́ɚQȂ:,ϭЂМK+f"ʺ˟ƩĨO/Ɏ;ͯRBQǁ̘˕Ӧ NaCl ͜ȵOdGJĴՂ:h6gǘƾɚŶϹэQĴԦöRԮȧOȃN4"BQĴ

g#ZLlMQƭǃɎ;ͯ1Ԯǘƾэ՞ǁ̘˕ӦԦƻƊöR"Haloarcula japonica /dV Natrialba

NaCl ͜ȵխ0հ0.2 M՟OƇ\h"̵̼ϹэQǊ4R

asiatica Qj?0 2 ƻK+GD#ʺ˟ķȕR̵̼OƦìȵǘƾэ՞0.2հ0.5 M՟OƇ\hg#ԮǘƾэQ\hJR,g1"ƩĨORՐƾ͜ȵͽǀRǣƮ<NÄORУƾэ`Ƈ\h"10՝ Þ½QƾÄK`ǁ̘ź

0GDD_LС.ehg#<0<N1e"2002 ȬÞ

ЭK+g#Äȵǘƾэ՞0.5հ2.5 M՟QǊ4R":

Ԕ"ƾQңǆй΂ŘOdf"ƩĨͿQǍʺƾkR=\;\NƇƾ҂ʳ0eǊʬĴԦ:hg#B<J"Ր_ӃğȒƾ·Ǎʺƾ·͊ƾNM1ƾȠƼOŊjf"ȵǘƾэ՞2.5 Mծ՟Rƾ΁"Ǎʺƾ"Ȓƾap¯ʺ˟ƩĨO/,J`ǘƾэQĴԦ͏K+gǍʺƾ"s±Q̼̔NMQƾ͊L,GDՐ͜ȵƾͽǀÄ0eȒƾa͊ƾNMkǵʾOğɴ>g8L1źЭLNG

Ĵ Ԧ : h " B Q Z L l M R Euryarchaeota ԋD#ͺƮ"ʺ˟QƾӃğӽRÁΈ¹K+f"ƀƩ0

Halobacteria ЇQǘƾɚŶϹэOĴՂ:hg#ӊȬ"eƾkӃğ<J,gE6KN4"ƩĨO/,J`Ǎ

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g1"˟ωKRͰOʽѻQͥ,ԕf Halobacteria Ї

K+gƌƞ"ӷ̫"͕ͯNMR8QƾkǊӽOƇlkǘƾɚŶϹэL>g#

K/f"ԮǘƾэOdgдʨkԑ5LĤO"Уƾэ ǘƾɚŶϹэR¹ӮQöǉkԗ2ǘ̡ɚ{¯§ԙaǘƾэOdgԕǭĴѴOdfͲͰQՄƌkӸ<ĳɚQŘǥƁɯɊȍ˰Չ;ͯK+f"ǸǍƶƯ½OҮ

>#ƾRʺ˟ͲйQΗӴͿ˽OLGJǌǇӻѬK̉,~³¸kɀɯ>g#BQϹЬɀɧR˸э"ͻэ"

0@N,`QK+f"ǘƾэRʺ˟×OLGJǌǇ\DR¼ѳɀaơѳɀkƇ^ȫ˧ͱQэ1ήehJ

ҹӊNɎ;ͯK+g#88KR"ʺ˟KğɴźЭN,g#8heQǌӮĴRлӦǁ̘͇ȵ1 40ÜӊQȠңƾkĴԦ͏L<DʵѯǘƾɚŶϹэQĴԦöLÄ͇э"ǁ̘лӦ pH RÄɚ\DRn±v°ɚK+

Bhe1;Ϳ>gǘƾɚnw¯¸OH,JϺÙ<f"ǘƾɚL,-8Lkԗ6SBQɚҭR¹пϹэ

D,#

Lǌ24ǇjeN,#Ƶϛˆ՞2018 Ȭ 9 ˖՟O/,J"Halobacteria ЇR 3 σ 60 ȍ 249 ψ1ģȹOѻ

x ¸ ´ ¸  խ Haloarchaea, Halophilic enzyme,

ҿ:hJ,g1"10 ȬZMł\KR"j?0O 1 Ρ

Phylogenetic Analysis

՞Halobacteriales՟1 σ՞Halobacteriaceae՟10 ȍK

12Journal of Japanese Society for Extremophiles (2018) Vol.17ա ǘӶɚǘƾɚŶϹэQĴԦ2ա ˟βώKR"Ƞңƾk΀,JǘӶɚǘƾɚQĴԦ ǘƾɚŶϹэRp¯s±·®±µOʕ>g̔k҂]D#ʵDOǘӶɚǘƾɚŶϹэĴԦ΀OғѨ

̼"n¨°vƁѝƩ¬șQ{²¸·±͊"<D̾ðƶƯ՞pH 4.5՟OȠңǍͧƾkɮ͛<"37n¯nŞȕřѫӮOëИ>g̼̔a|nQ¥w

KԭИԤϋƶՉ<D#ĴԦ˳Q 16S rRNA өæǡOo͊"ÄƩǔƯaՐũO+gƾ͊0e"

ƷI4ϭЂѴ˨/dVǁ̘лӦ pH Qη҉kўGD

Ǌ4QʵȍʵψQĴԦƻƊ:hJ,g#\D"ϯ 4

Ͼ˫"27 ψՂQƾ0e 400 ˳Þ½QǘӶɚQǘƾɚ

Ė 1900 »ȬłQŶ;Ý±±Ϯ0eϯ 180 »ȬłŶϹэQąѧ˳1ųɋK2D#8heĴԦ˳Qǁ̘

Q˒ʵÁr¸¯˛\KQƯҭȬÝQȒƾԂȱ0e

pH R 4.5-7.0"ǁ̘лӦ pH R 4.5-6.0 QϢƦĨK+`ʬǊ4ΗѮ:h":eO¨x~Q}²³{³f"16S rRNA өæǡQƾƷӲķkη҉<DϾ˫"ʹƾ΁NMÁΈƀƯQƾ΁"ƾђՆƐ0e`ĴԦ:hήψK+g Halobacterium noricense L 92%Þ¾QΣ

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D MH1-52-1 ˳R:l9Qƾ0eĴԦ:hD#:Ӧ pH RÄɚ\DRn±v°ɚK+g1"ǁ̘лӦl9QƾRu¸¯°n0eQӃğǍʺƾO:pH 1ӶɚK+g˳QƻƊRΜͥK+GD#Šѫu¸l9Ϧ˞kŊ.D`QK+f"BQ 25%͒̾R pH

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9.0 K+GD#MH1-52-1 ˳R pH 4.2-4.8 Kǁ̘<"

Ʈ>g1"ǘӶɚǘƾɚŶϹэQĴԦƻƊRN,#

BQлӦǁ̘ pH R 4.4 K+f"pH 4.0-5.8 kҳ.gӶɚˤáK`ǁ̘>gǘƾɚŶϹэL<JRˤáKRм̔ΛK+g8L1η҉K2D՞Fig. 1՟#Halococcus hamelinensis /dV Halococcus

8Q MH1-52-1 ˳kƷ͐ψL<J Halarchaeum

qingdaonensis 1ƻƊ:hJ,g1"BQǁ̘ϢƦ pH

acidiphilum QʙƖOлGD 1-2)#ͺƮ\KQǘӶɚǘ

R 4.0-9.0 K+f"ǁ̘лӦ pH R 6.0 QÄɚэK+ƾɚŶϹэQĴԦƻƊR>YJ Halarchaeum ȍK+g#f"5 ψ1Ηѡ:hJ,g 2-6)#

Fig. 1. Halarchaeum acidiphilum MH1-52-1 ˳Qƀ pH ˤáOdgǁ̘ˑЎ
3ա ǘƾɚŶϹэQĴǡϭЂǥΛѴ˨ӊȬQɎ;ͯĴՂO/,JӻѬNɡƻQ¹HLNG

 ͺƮ"́$N;ͯψO/,J"BQϭЂǥΛëИ

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Proposed Minimal Standards for Description of New

8heQƾƷӲķRBQþǣɚ1Ր4"әŘϭЂ1

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rRNA өæǡƾƷӲķQՂéȵ"DNA-DNA p

1 97%Þ¾K+hSĺψK+gLʖǭK2gNM"°p¸­µQΣƃć1ӻѬNɡƻLNGJ,
13

Journal of Japanese Society for Extremophiles (2018) Vol.17

g#<0<N1e"ǘƾɚŶϹэO/,J"BQ 16S

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N Q 0 Ĺ ʴ 1 H 0 N , # B 8 K ɰ $ R 14 ψ Q:hJ,g#N0K` Halosimplex carlsbadense R

Haloarchaea }§Q¸k҆ϹOғY"16S rRNA

rrnA"rrnB /dV rrnC Q 3 HQ 16S rRNA өæǡkөæǡ՞2-3 Ă+gƼƁRBQĨ¹H՟Q½̹OŜ}§½Oþʈ<J/f"BheQΣƃɚRì4"

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RNA ¤°¨¯¸¬ Bөæǡ(rpoB')O

f"pyrD-23S Q]KǁȨ>g 26 ȍ 89 ψ

dgϭЂѴ˨kўGD#BQϾ˫"rpoB'өæǡ/d՞subgroup1՟\DR lpdA-23S Q]KǁȨ>VBQn¦ӶӲķOƷI4ϭЂ̆R 16S rRNA ө

g 12 ȍ 43 ψ՞subgroup 2՟QեHQ{±¸OǩĠæǡOdgBhLՂé<J/f"ǘƾɚŶϹэRȃO Ĵ 6 e h D ՞ Fig. 2՟# ͺ Ʈ " Halobacteria Ї R

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Halobacteriaceae σ/dV Haloferacaceae σ1Ƈ\hFig. 2. ƀ¯p¥¸Odg orthologous 16S rRNA өæǡQǁȨϾ˫

14

Journal of Japanese Society for Extremophiles (2018) Vol.17

lpdA-23S Q]KǁȨ>g subgroup 2՞clade՟

4. ǘ ƾɚŶϹэ1;Ϳ>gӴ϶Oԏ>gβώ

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hg8L1ʽηK+GD#rRNA u¡³µQ½̹OQ-1,4 ϾƁӮëk҉қ<ĵʴ>g#nw¯¸R+g nonhistone chromosomal protein՞MC1՟өæǡ0nw³¸Ԫ̴̡őɉQnw³¸ʴͬÄ0e

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՞Fig. 3՟#  Fig. 3. 197A ˳Q~³¸LǸǍĴѴʲ ǘƾɚnw¯¸;ͿэR JCM No.169 ƶƯkõ197A ˳O̲Ρ<JўGD#197A ˳kǭȧ˛\Kƶ΀<JĴԦ:hD 62E ˳/dV 197A ˳Q 16S rRNA

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DNA-DNA  p  °  p  ¸  ­ µ Q Ͼ ˫ "˫"˟Ӵ϶Runw³Ϭk;ɯ>g8L0e-

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15

Journal of Japanese Society for Extremophiles (2018) Vol.17

80K 60 ĴԎQŊͫĲͼɉ`̷ɚ1ì¾@?"95

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Fig. 5. Aga-HC QĴѴ́ȹ

16

Journal of Japanese Society for Extremophiles (2018) Vol.17

Fig. 6. Aga-HC QͫǨǭɚ

5. Ø ɉQȌ˚

ɰ1ėOʯŷ`,j?Ü2ƁGJ4hD˦̵ǌǥ ǘƾɚŶϹэR"ǘƾɚQÛOǘӶɚ"ǘn±vҳɉ ӁʪŢǄ"̵̼βώԍΗ̈̀ ȗ˵ ȷȻŢǄO°ɚ"ϸǉЎУɚNMQɚҭk˗>g8L0eɦҚ΄<½7\>#\D"́$N9ŋѶ·9ŋňk

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 ¹ʶK"ǘƾɚӴ϶RУͫɚa˗̈͒ǞУɚk˗

fJŨ4Ɍλ΄<½7\>#>g8L1ήehJ/f"BQɚҭkѴʽ>g8L
RǘƾɚӴ϶QȚ˽Λɕ΀QźЭɚkκ>LL`O"Ⱥ΀ʯʹǘƾɚ"Уͫɚa˗̈͒ǞУɚL,GDµzҭ1)Minegishi H, Mizuki T, Echigo A, Fukushima T,

Q;ŘǥΛͼѴk̀_gɤƌK`нƌ̀,#:eO"

Kamekura M, Usami R. 2008. Acidophilic

8h\KǘƾɚŶϹэLѶ.S"Halobacterium ЇQhaloarchaeal strains are isolated from various solar

Ðk:<J,D1"ӊȬ8h\KLRĠ4ΌNgĴ

salts. Saline Systems. 4:16.
2)

ՂМQ³n¸xnQǣƮ1ƻƊ:hD#8h

Minegishi H, Echigo A, Nagaoka S, Kamekura M,e`Ƈ_"%ǘƾɚ&L,-x¸´¸kÄɒL<J

Usami R. 2010. Halarchaeum acidiphilum gen.

Øɉ`βώkә_J,2D,#

nov., sp. nov., a moderately acidophilic
haloarchaeon isolated from commercial solar salt.
Int. J. Syst. Evol. Microbiol. 60:2513-2516.

6. ҚӅ
3)

 ˕ɉO"˟βώkә_gOȿDf9ʉȁ·9԰ʟ

Yamauchi, Y., Minegishi, H., Echigo, A., Shimane,kҫf\<Dǘƾэβώɳ Ìā ̎ŢŢǄ"ϝ̱ǌ

Y., Shimoshige, H., Kamekura, M., Itoh, T.,ǥ ̇˟ Ɠ΅ʩʐ"OŨ4Ɍλ΄<½7\>#ρQ

Doukyu, N., Inoue, A., Usami, R. 2013.

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Journal of Japanese Society for Extremophiles (2018) Vol.17

Halarchaeum salinum sp. nov., a moderately

4)7)

acidophilic haloarchaeon isolated from commercial

Usami, R., and Hashimoto, T. 2010. Further

sea salt. Int. J. Syst. Evol. Microbiol.

refinement of the phylogeny of the

63:1138-1142.

Halobacteriaceae based on the full-length RNA

Yamauchi, Y., Minegishi, H., Echigo, A., Shimane,polymerase subunit B' (rpoB') gene. Int. J. Syst.

Y., Kamekura, M., Itoh, T., Ohkuma, M., Doukyu,

Evol. Microbiol. 60:2398-2408.

N., Inoue, A., Usami, R. 2013. Halarchaeum

5)8)

Minegishi, H., Kamekura, M., Kitajima-Ihara, T.,rubridurum sp. nov., a moderately acidophilic

Nakasone, K., Echigo, A., Shimane, Y., Usami, R.,

haloarchaeon isolated from commercial sea salt

Itoh, T., and Ihara, K. 2012. Gene orders in thesamples. Int. J. Syst. Evol. Microbiol.

upstream of 16S rRNA genes divide genera of the

63:3143-3147.

family Halobacteriaceae into two groups. Int. J.

Minegishi, H., Yamauchi, Y., Echigo, A., Shimane,

Syst. Evol. Microbiol. 62:188-195.

Y., Kamekura, M., Itoh, T., Ohkuma, M., Usami, R.

6)

Minegishi, H., Kamekura, M., Itoh, T., Echigo, A.,9)

Minegishi H, Echigo A, Shimane Y, Kamekura M,

2013. Halarchaeum nitratireducens sp. nov., a

Itoh T, Ohkuma M, Usami R. 2015. Halococcus

moderately acidophilic haloarchaeon isolated from

agarilyticus sp. nov., an agar-degrading

commercial sea salt. Int. J. Syst. Evol. Microbiol.

haloarchaeon isolated from commercial salt. Int. J.

63:4202-4206.

Syst. Evol. Microbiol. 65:1634-1639.

Shimane, Y., Minegishi, H., Echigo, A., Kamekura,

10)

Minegishi, H., Shimane, Y., Echigo, A., Ohta, Y.,

M., Itoh, T., Ohkuma, M., Tsubouchi, T., Usui, K.,

Hatada, Y., Kamekura, M., Maruyama, T., andMaruyama, T., Usami, R., Hatada, Y. 2015.

Usami, R. 2013. Thermophilic and halophilic

Halarchaeum grantii sp. nov., a moderately

b-agarase from a halophilic archaeon Halococcus

acidophilic haloarchaeon isolated from asp. 197A. Extremophiles. 17:931-939.

commercial salt sample. Int. J. Syst. Evol.
Microbiol. 65:3830-3835.

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Journal of Japanese Society for Extremophiles (2018) Vol.17

Journal of Japanese Society for Extremophiles (2018) Vol.17, 19-30

ORIGINAL PAPER

Sakagoshi Ya and Takada Yb a

Isocitratet ddehydrogenaseh dfrom a Gram-positive psychrotrophic bacterium,
strain G-4aBiosystems Science Course, Graduate school of Life Science, Hokkaido University, Kita 10-jo Nishi 8-chome, Kita-ku,

Sapporo, Hokkaido 060-0810, Japanb

Depertment of Biological Sciences, Faculty of Science, Hokkaido University, Kita 10-jo Nishi 8-chome, Kita-ku,

Sapporo, Hokkaido 060-0810, Japan
Corresponding author: Yasuhiro Takada, ytaka@sci.hokudai.ac.jp
Phone & Fax: +81-11-706-2742
Received: Mar. 28, 2018 / Revised: Apr. 12, 2018 / Accepted: Apr. 17, 2018
By western blot analysis with antibodies

Abstract

Introduction
NADP+-dependent isocitrate dehydrogenase (IDH; EC

against monomeric and dimeric isocitrate dehydrogenases(IDHs),aGram-positive

1.1.1.42)

psychrotrophic

catalyzes

thereduction

of

NADP+

bacterium, strain G-4, was confirmed to possess only

concomitantly with the oxidative decarboxylation ofmonomeric IDH. The gene encoding this enzyme

D-isocitrate to 2-oxoglutarate in TCA cycle. Bacterial(G-4IDH) was cloned and sequenced. The open reading

IDHs are divided into two types; dimer type constituted

frame of this gene was 2,220 bp in length, correspondingby same two subunits with molecular weights of 40,000

to a polypeptide composed of 739 amino acids. The

to 45,000 and monomer type with a molecular weight oftranscriptional initiation site for the gene was guanine,

80,000 to 100,00014). Many bacteria only have either

located at 695 bases upstream of the translational start

type of IDH. IDHs of Escherichia coli1) and Thermus

codon. The deduced amino acid sequence showed highthermophilus2) have been reported to be dimeric, while

identities with those of the monomeric IDHs from

Azotobacter vinelandii20), Corynebacterium glutamicum3)

Gram-negative bacteria such as Colwellia maris andand Vibrio parahaemolyticus4) have monomeric IDH.

(62%

identity,

However, the homology of amino acid sequence is very

respectively) and, particularly, the putative IDHs oflow between the two type IDHs nevertheless theyGram-positive

Azotobacter

vinelandii

and

64%

and

catalyze the same reaction. Furthermore, immunological

Paenarthrobacter aurescens (91% and 81% identity,

cross-reactivity is different from each other8). On therespectively). The His-tagged G-4IDH was overproduced

other hand, several bacteria such as a psychrophilicin the Escherichia coli cells and was purified andbacterium, Colwellia maris16), and a psychrotrophic

characterized. The G-4IDH showed the highest activity

bacterium, Pseudomonas psychrophila15), are known toat 45°C, indicating that it is mesophilic. However, thepossess both monomeric and dimeric IDHs. The

residual activity after incubation for 10 min at 35°C wasmonomeric IDH of C. maris (IDH-II) shows the

37%, and G-4IDH was more thermolabile than a typicalmaximum activity at 20°C and about 50% of the activity

mesophilic IDH of A. vinelandii.

is lost by incubation for 10 min at 30°C, indicating that itbacteria,

Arthrobacter

alpinusis a typical cold-adapted enzyme16). In contrast, theKey words: Psychrotrophic bacterium strain G-4,

optimum temperature for the activity of the dimeric IDH

Gram-positive bacterium, Isocitrate dehydrogenase, gene

(IDH-I) is 40°C and it retains the activity completely

cloning

after incubation for 10 min at 40°C. Therefore, thisenzyme is mesophilic. These two IDH genes are located19Journal of Japanese Society for Extremophiles (2018) Vol.17

in tandem on the chromosomal DNA9), but their genewith intervals for 2 min on ice. The cell lysate wasexpressions are controlled independently; the IDH-I genecentrifuged at 17,700 × g for 20 min at 4°C, andisissupernatant was stored at -80°C and used as crude

cold-inducible . Furthermore, a nucleotide sequence

extract. Protein concentration was determined by the“CCAAT” has been reported to play as a promoter of themethod of Lowry et al.13) using bovine serum albumin as

cold-inducible expression of IDH-II gene19). On the othera standard.

acetate-inducible

while

the

IDH-II

gene

25)

hand, the two type IDHs of P. psychrophila has beenreported to be mesophilic15).

Western blot analysis

A psychrotrophic bacterium, strain G-4, was isolated

Sodium22)

dodecylsulfate-polyacrylamide

gelfrom a cold room of a food package center . It was aelectrophoresis (SDS-PAGE) was carried out by theGram-positive rod-shaped bacterium, and its optimum

method of Leammli12) with 10% acrylamide gel at 120V.

temperature for growth was about 25°C, but it could

After electrophoresis, protein bands on the gel weregrow at 0°C and 30°C. A mesophilic Gram-positivetransferredbacterium, Bacillus subtilis, has been found to have only

(Immobilon-P Transfer Membrane, Millipore) with 2

23)

to

polyvinylidene

fluoride

membranedimeric IDH , but IDHs from psychrophilic or

mA/cm2 for 90 min at 4°C. Western blot analysis was

psychrotrophic Gram-positive bacteria have been hardly

done as reported previously24) except for using rabbit

characterized. In this study, the gene for IDH of strain

antibodies against the purified dimeric IDH-I of C.

G-4 (G-4IDH) was cloned, and several properties of thismaris8) and the purified monomeric IDH of Vibrio

enzyme were examined.

parahaemolyticus4) as primary antibodies diluted to
1/30,000 and 1/5,000, respectively, and anti-rabbit IgG,
HRP-linked whole antibody (GE Healthcare) or goat

Materials and Methods

anti-rabbit IgG (H+L), HRP conjugate, cross adsorbed(Life technologies) as secondary antibody diluted to

Bacterial strains, plasmids and growth media

1/50,000 or 1/20,000, respectively.

The psychrotrophic bacterium strain G-4 was grown at
its optimum temperature for growth, 25°C, with vigorous
shaking in a nutrient medium (pH 7.0) consisting of 1%

Cloning of G-4IDH gene(w/v) meat extract and 1% (w/v) peptone. A mutant of

Genomic DNA of strain G-4 was prepared as27)

Escherichia coli defective in IDH, strain DEK2004 ,described previously10). To obtain a probe for genomic

having a glutamate auxotrophic phenotype, was used as aSouthern hybridization, PCR was done with Veriti 96

host for expression of the IDH gene and grown inwell Thermal cycler (Applied Biosystems) in the reaction

21)

28)

or Super broth medium .

mixture (50 µl) containing approximately 90 ng of

If necessary, ampicillin and tetracycline were added togenomic DNA, 15 pmol each of forward and reverse

the culture media at final concentrations of 50 µg/ml andprimers, CpM-s and CpM-as, respectively14) (Table 1),

15 µg/ml, respectively. Plasmids pBluescript SK(+)

designed from two highly conservative sequences(pBS; Stratagene) and pTrcHisB (Invitrogen) were used

between the genes for monomeric IDH of A. vinelandii

Luria-Bertani (LB) medium

as vectors for the construction of a genomic DNA library(AvIDH) and IDH-II of C. maris (CmIDH), and 1U ofof strain G-4 and the subcloning of the IDH gene and for

KOD-Plus-Neo DNA polymerase (Toyobo) with buffer

conferring the N-terminal His-tag on the expressed

system prepared by the manufacturer. Cycling conditions

proteins, respectively. All transformation in this study

after predenaturation for 2 min at 94°C were as follows:21)

was done by a conventional calcium chloride method .

denaturation for 10 s at 98°C, annealing for 30 s at 50°C,
extension for 10 s at 68°C, for each of 30 cycles. The
PCR product was purified and ligated into the EcoRV

Preparation of crude extract
Crude extract of the strain G-4 was prepared assite of pBS for propagation. The nucleotide sequence of24)

reported previously . Bacterial cells suspended in a

the insert DNA in pBS was determined in both direction

sonication buffer (20 mM potassium phosphate (pH 7.5)

by appropriate primers and a BigDye Terminator v3.1

containing 0.15 M NaCl, 2 mM MgCl2 and 10 mM

Cycle Sequencing Kit (Applied Biosystems) with a2-mercaptoethanol) were ten times sonicated for 2 minsequencer (3130 genetic Analyzer, Applied Biosystems).

20

Journal of Japanese Society for Extremophiles (2018) Vol.17

Table 1. Oligonucleotides used in this study.
Primer nameNucleotide sequence (5’→3’)

Cloning of IDH gene
CpM-s14)
CpM-asAAYATCATCAAGYTGCCRAAYATY

14)TGGCGCRCGRCGRTCRGAGTTRCCTTC

G-4 698H

TCCTCACCGCACAGGTTGCCCGCGCC

G-4 R110T

AGATTTCTTTCCCTCCCAATCTACCCG

G-4R-invH1

GCCCACCGGCTCCCGCTAACATCGTG

Determination of transcriptional initiation site
' G-4siteC1-R' ' ' ' ' ' ' ' ' ' ' ' CAGGGTGTCGGCTGCGCGTTGCTCTTC' G-4BssHIIsite-R

CTTCACAGAAAGGGGCGCGCGGCGGT

poly-C

CCCCCCCCCCCCCCC

poly-GGGGGGGGGGGGGGGG

Construction of pHisG-4IDH
G-4-F pTrcHisB

gcgcggatccgGCCAAGATTATCTACACC

G-4-R pTrcHisB

gcgcgagctcTTAGGACAGCGTGTCGATG

G-4-F c1487g

GACATTCGAGCTGGGCGAAGCCGGCAC

G-4-R c1487g

GTGCCGGCTTCGCCCAGCTCGAATGTC

Small letters indicate additional bases for introducing the digestion sites for BamHI and SacI (underlined anddouble-underlined letters, respectively).
Letters in gray boxes indicate bases substituted to delete the SacI site by site-directed mutagenesis.
For genomic Southern hybridization21), the genomic

inverse PCR18) was carried out. The genomic DNA

DNA was digested with EcoRI. After the electrophoresis

fragments of strain G-4 digested with SacI wereof the digested DNA, the DNA fragments on gels wereself-ligated with Ligation-Convenience Kit (NIPPON+

transferred to a membrane (Hybond-N , GE Healthcare).

GENE) and used as a template for the PCR.

Hybridization was done with AlkPhos Direct Labelling

Amplification was performed for 30 cycles in a reaction

and Detection system with CDP-Star (GE Healthcare).

mixture (50 µl) containing 100 ng of the self-ligated

The DNA fragments hybridized with the probe were

DNA, 15 pmol each of G-4 R110T and G-4 698H (Table

detected with Luminescent Image Analyzer LAS-3000

1) as forward and reverse primers, respectively, and 1 U(FUJIFILM). About 3.4-kbp DNA fragments digested

KOD-plus-Neo DNA polymerase in a buffer systemwith EcoRI were purified and ligated to pBS digested

prepared by the manufacturer. Cycle conditions after thewith EcoRI. Then, colony hybridization5) was carried out

predenaturation for 2 min at 94°C were the same as those+with Hybond-N membrane and the probe for genomic

described above except for the extension for 2 min at

Southern hybridization to select the plasmid harboring

68°C. Nucleotide sequence of the obtained PCR product,

DNA fragment which contained the G-4IDH gene

G-4R-inv (Fig. 1), was determined by a direct sequencing(named pBSG-4IDH).

with a primer, G-4R-invH1 (Table 1), as described

Since a sufficient length of upstream region was notabove.

contained in the DNA fragment of the obtained plasmid,

21

Journal of Japanese Society for Extremophiles (2018) Vol.17

Fig. 1. Restriction and physical map of the genomic DNA fragments containing the G-4IDH gene. Arrow and box
represent the ORF of G-4IDH and the position of the probe for genomic Southern hybridization, respectively.
Determination of transcriptional initiation site
The transcriptional initiation site of the G-4IDH gene

Construction of His-tagged G-4IDH genes

was determined by the method of 5’-rapid amplification

The G-4IDH gene was amplified by PCR to introduce

21)

of cDNA ends (5’-RACE) . Total RNA of the strain

the restriction sites for BamHI and SacI at the 5’- andG-4 (4 µg) prepared with High pure RNA isolation kit

3’-terminals of its ORF, respectively. Furthermore, since(Roche), a primer, G-4BssHIIsite-R (Table 1), anda SacI site is present in the ORF, it was deleted without

PrimeScript Transcriptase (TaKaRa) were used tothe change of amino acid residues in the translatedsynthesize the template strand of cDNA. After poly-C orprotein by site-directed mutagenesis using PCR7) with

poly-G was added to the 3’-terminal of the cDNA withthe primers for the deletion of SacI site, G-4-F c1487g

terminal deoxynucleotidyl transferase (TaKaRa), theand G-4-R c1487g and those for introducing themodified cDNA was used as a template for the following

restriction sites at both terminals of the ORF, G-4-F

PCR. Amplification was performed for 30 cycles in apTrcHisB and G-4-R pTrcHisB (Table 1). Forward andreaction mixture (50 µl) containing the template, 15

reverse primers were G-4-F pTrcHisB and G-4-R c1487g

pmol each of forward primer, poly-C or poly-G, andfor first PCR, and G-4-F c1487g and G-4-R pTrcHisB

reverseUfor second one, respectively. These amplifications were

KOD-plus-Neo DNA polymerase in a buffer system

performed for 30 cycles in a reaction mixture (50 µl)

one,

G-4siteC1-R(Table

1),

and

1

prepared by the manufacturer. Cycling condition after

containing 100 ng of pBSG-4IDH as a template, 15 pmol

predenaturation for 2 min at 94°C was as follows:each of the forward and reverse primers and 1 U

denaturation for 10 s at 98°C, annealing for 30 s at 54°C,

KOD-plus-Neo DNA polymerase in a buffer system

extension for 75 s at 68°C. For accurate determination of

prepared by the manufacturer. Cycling conditions after

the initiation site, the PCR products were used aspredenaturaion at 94°C for 2 min were as follows:

templates for semi-nested PCR29) with poly-C or poly-G

denaturation for 10 s at 98°C, annealing for 30 s at 57°C,

and G-4R110T (Table 1) as primers. Cycling condition

extension for 1 min at 68°C. Final PCR was carried out

after the predenaturation for 2 min at 94°C was asin the same reaction mixture as the first and second

follows: denaturation for 10 s at 98°C, annealing for 30 s

PCRs except for the use of G-4-F pTrcHisB and G-4-R

at 54°C, extension for 45 s at 68°C. The resultant PCR

pTrcHisB as primers and the first and second PCR

products were ligated into the EcoRV site of pBS, andproducts (75 ng and 65 ng, respectively) as templates.

their nucleotide sequences were determined as described

Predenaturation and cycling conditions were the same asabove.

the two PCRs except for the extension for 75 s. Resultant
22

Journal of Japanese Society for Extremophiles (2018) Vol.17

PCR product was then digested with BamHI and SacI

His-tagged

G-4IDH

activitywas

assayed,NaCl

and ligated to the BamHI-SacI site of pTrcHisB to obtain

concentration in the reaction mixture was changed toa plasmid pHisG-4WT.

0.125 M. One unit of enzyme activity was defined as theamount capable of catalyzing the reduction of 1 µmol of
NADP+ per min.

Overexpression and purification of His-tagged IDHs
E. coli DEK2004 transformed with pHisG-4WT was

To examine the thermostability for IDH activity, thegrown at 37°C in Super broth medium until OD600 of thepurified G-4IDH with His-tag was dialyzed overnight at

culture reached 1.1. The culture was then cooled for 30

4°C against 20 mM sodium phosphate (pH 8.0),

min on ice. For the overexpression of the IDH protein,containing 2 mM MgCl2, 0.3 M NaCl, 1 mM

the culture was incubated at 15°C overnight after thedithiothreitol and 10% (v/v) glycerol. The dialyzed

addition of 1 mM isopropyl-β-D-thiogalactopyranoside.

enzymes were incubated for 10 min at various

Then, cells were harvested at 4°C and were suspended intemperatures and were then immediately cooled on ice

30 ml of 0.05 M sodium phosphate buffer (pH8.0),

for 10 min.

containing 2 mM MgCl2, 0.3 M NaCl, 10 mM imidazole,
10% (v/v) glycerol and 10 mM 2-mercaptoethanol. A

Results

final concentration of 2 mg/ml lysozyme from egg white
was added to the cell suspension, and the mixture wasWestern blot analysis of the G-4IDH

6)gently shaken for 1 h at 4°C. As described previously ,To confirm what type(s) of IDH are present in thethe overproduced IDH was then purified by Ni-NTA

strain G-4 cells, western blot analysis was carried out asagarose (Qiagen) column chromatography, and thedescribed in Materials and Methods (Fig. 2). While noelutant containing IDH activity was concentrated,

protein cross-reacting with the antibody against thedialyzed and stored until use.

dimeric IDH-I of C. maris was detected in the crude
extract of strain G-4, a cross-reactive protein with theantibody

Enzyme assay

againstthemonomeric

IDH

of

V.

The IDH activity was measured as describedparahaemolyticus was existent. Its molecular mass waspreviously30). Unless otherwise noted, 2 ml of reaction

estimated to be about 80 kDa, coincident with that of the

mixture consisted of 33 mM Tris-HCl (pH 8.0), 0.67 mM

purified monomeric IDH-II of C. maris. These results+

MnCl2, 0.05 M NaCl, 0.12 mM NADP , 2 mM sodium

suggest that this bacterium possesses only monomericisocitrate and an appropriate amount of enzyme, and the

IDH.

assay was carried out at 40°C. When the purified

Fig. 2. Western blot analysis of G-4IDH. Rabbit antibodies against the dimeric IDH of C. maris (left) and themonomeric IDH of V. parahaemolyticus (right), were used. Lanes M, marker proteins. Lanes 1 and 4, the purified
dimeric IDH-I of C. maris (0.3 µg protein). Lanes 2 and 5, the purified monomeric IDH-II of C. maris (0.3 µg protein).
Lanes 3 and 6, crude extract of strain G-4 (80 µg protein).
23

Journal of Japanese Society for Extremophiles (2018) Vol.17

Cloning of the G-4IDH gene and its nucleotide andobtained. By nucleotide sequencing of the product,

deduced amino acid sequences

guanine

The product of genomic PCR described in Materials

locatedtranslational

start

at

695

bases

upstream

of

the

codon

was

found

be

the

to

and Methods was 209 bp in length. The deduced aminotranscriptional initiation site for the G-4IDH gene (star inacid sequence of this probe was analogous to those of theFig. 3). Furthermore, promoter motifs at -10 and -35 for

identity,

the G-4IDH gene were deduced to be GATAGC

respectively). In genomic Southern hybridization, an(nucleotides between 121 and 126) and TTCCGA

about 3.4-kbp DNA fragment digested with EcoRI(nucleotides between 96 and 101), respectively (thehybridized with this probe. Then, plasmid harboring theunderlined sequences in Fig. 3).

AvIDH

and

CmIDH

(84%

and

67%

G-4IDH gene was selected by colony hybridization andnamed pBSG-4IDH. The restriction map of the DNA

Properties of His-tagged G-4IDH

fragment inserted in this plasmid is shown in Fig. 1. The

The G-4IDH gene was introduced into the expression

fragment contained the complete ORF of the G-4IDH

vector, pTrcHisB, as described in Materials and Methods.

gene and its 5’- and 3’-flanking regions (up to 157 bp

The His-tagged G-4IDH was overproduced in the E. coli

upstream from the translational start codon and 1,022 bp

cells and purified by Ni-agarose column chromatography.

downstream from the stop codon, respectively) (Figs. 1

SDS-PAGE of the final elutant in the chromatography

and 3). The ORF was 2,220 bp in length and encoded a

revealed that the G-4IDH was almost homogeneously

polypeptide of 739 amino acids with a calculated

purified and its molecular mass was about 80 kDa (Fig.

molecular mass of 78,986.56 Da. A putative ribosome

5). Since the purified enzyme showed high IDH activity,

binding site, AGGAG, was present 12-16 bases upstream

the cloned gene was demonstrated to encode the IDHof the ATG codon. On the other hand, sequence of aprotein as described above. The activity of the purified

terminator-like stem-loop structure, composed of a 15-bp

sample of His-tagged G-4IDH was assayed at various

stem with a 6-nucleotide loop, was found downstream ofpHs (Fig. 6, left) and in the presence of various

the ORF. The nucleotide sequence of the G-4IDH geneconcentrations of NaCl (Fig. 6, right). The His-tagged

has been deposited in the DDBJ database under

G-4IDH showed the highest activity at pH 8.0 and 0.125

accession no. LC376051. The deduced amino acid

M NaCl. On the other hand, optimum temperature for its

sequence of G-4IDH showed high degrees of homology

activity was 45°C and the enzyme retained 37% of

to monomeric IDHs from other bacteria; for example,

activity after incubation for 10 min at 35°C (Fig. 7). It

64% and 62% identity to the IDHs of Gram-negative

was reported that a typical mesophilic AvIDH withbacteria, A. vinelandii and C. maris, respectively (Fig. 4).

His-tag maintains completely the activity even after

Especially, the identities to the putative IDHs fromincubation for 10 min at 40°C and its optimum

Gram-positiveandtemperature for activity is 47.5°C28). Therefore, although

Paenarthrobacter aurescens (database accession no.

G-4IDH was more thermolabile than AvIDH, the former

CP013200 and no. CP000474), were 91% and 81%,

IDH was judged to be mesophilic enzyme. The IDH in

respectively. Furthermore, the amino acid residues

the crude extract of the strain G-4 showed the maximum

involved in the binding of isocitrate, metal ion andactivity at 50°C, and the remaining activity after

NADP+ were completely conserved among G-4IDH,

incubation for 10 min at 35°C was about 60%. Therefore,

AvIDH and CmIDH (Fig. 4). These results imply that thisthe optimum temperature and thermostability for activity

ORF encodes the monomeric IDH of strain G-4.

of the purified G-4IDH with His-tag resembled those ofAnalysis of 5’-terminal region of the G-4IDH mRNA

N-terminal of G-4IDH does not significantly affect their

To elucidate the transcriptional initiation site of thethermal properties as reported in the monomeric IDHs of

G-4IDH gene by the method of 5’-RACE, the upstream

several bacteria14, 15, 28). The values of Km for isocitrate,

bacteria,

Arthrobacter

alpinusthe native G-4IDH, indicating that His-tagging at theregion of the ORF was further subcloned by inverse PCR,

kcat and kcat/Km of G-4IDH at 20°C were 9.70±0.33 µM,

and the nucleotide sequence of the PCR product was

50.46±0.98 s-1 and 52.04×105 s-1M-1, respectively.

determined (Figs. 1 and 3). When poly-G (Table 1) wereused as a primer for the 5’-RACE, a PCR product was24

Journal of Japanese Society for Extremophiles (2018) Vol.17

Fig. 3. Nucleotide and deduced amino acid sequences of the G-4IDH gene. The boxed nucleotides are the translational
start codon. The double underlined sequence is the putative ribosome binding site (RBS). The probable stem-loop
structure located downstream of the stop codon is shown by two opposite arrows. The underlined sequences indicate theputative -35 and -10 sequences of the promoter. Star shows the transcriptional initiation site.
25

Journal of Japanese Society for Extremophiles (2018) Vol.17

Fig. 4. Alignment of amino acid sequences of monomeric IDHs from several bacteria.

The deduced amino acidsequence of the G-4IDH is aligned with those of AvIDH (database accession no. D73443) and CmIDH (no. BAA03134).
The amino acid residues involved in the binding of isocitrate, metal ion and NADP+ are indicated by asterisks, closed
circles and closed triangles, respectively.

Fig. 5. SDS-PAGE of purified G-4IDH. Lane M, marker proteins. Lane 1, the purified G-4IDH (3 µg protein).

26

Journal of Japanese Society for Extremophiles (2018) Vol.17

Fig. 6. Effects of pH (left) and NaCl (right) on the His-tagged G-4IDH activity. The activities are represented aspercentages of the maximum activity. In left, 33 mM Tris-HCl () and 33 mM MES-NaOH (!) were used for pH

range of 7-8.5 and 5.5-7, respectively. The data are the means ±SD of at least four individual measurements.

Fig. 7. Effect of temperature on the His-tagged G-4IDH activity (left) and its thermostability (right). In right, residual
activities assayed at 40°C after incubation for 10 min at the indicated temperatures are represented as percentages ofthat without the incubation. The data are the means ±SD of at least four individual measurements.
The corresponding values of AvIDH at 20°C have beenclarified. By western blot analysis, it was found that only

reported to be 12.58±0.32 µM, 159.48±2.39 s-1 anda IDH protein cross-reactive with antibody against the

5

-1

-1

6)

126.9×10 s M , respectively . The Km for isocitrate ofmonomeric IDH, but not dimeric IDH, was present in the

G-4IDH was comparable to that of AvIDH, but the kcat

crude extract of strain G-4 (Fig. 2). When the primers

and kcat/Km values of the latter were about 3-fold higher

designed from highly conservative sequences between

than those of the former.

the dimeric IDH genes of C. maris and E. coli were usedin the genomic PCR, no product was obtained. These
results indicate that this bacterium possesses only

Discussionamonomeric IDH. The G-4IDH showed higher degrees ofpsychrotrophic bacterium, strain G-4, was cloned and

In

this

study,

monomeric

IDH

gene

of

amino acid sequential homology to the putative IDHs ofsequenced. This gene was expressed in the E. coli cells,

Gram-positive bacteria, A. alpinus and P. aurescensand several properties of the purified enzyme werewhich is a bacterium belonging to the same family

27

Journal of Japanese Society for Extremophiles (2018) Vol.17(Micrococcaceae) as the genus Arthrobacter, than thoseconfirmed to be comparable with that of the cold-adaptedof Gram-negative bacteria such as AvIDH and CmIDH.

CmIDH. On the other hand, the specific activities of IDH

On the other hand, 16S ribosomal RNA gene of strain

at 10°C in the crude extracts of strain G-4 and C. maris

G-4 showed high sequential identities (86-89%) to thosegrown on a nutrient medium consisting of 1% peptone

of

al.,

and 1% meat extract were 0.38 U/mg protein and 0.23

unpublished data), implying that this bacterium belongs,

U/mg protein16), while the growth rate of strain G-4 at

or is closely related, to the genus Arthrobacter. The

10°C on the nutrient medium was lower than that of C.

bacteria

ofArthrobacter(Sakagoshi

etIDHs of Gram-positive bacteria have been lessmaris on the same medium supplemented by 0.4 M NaClcharacterized than those of Gram-negative ones. To ourfor the optimum growth22, 26). These reveal that theknowledge, many Gram-positive bacteria have only

catalytic ability of G-4IDH is enough to sustain growth

dimeric IDH or its putative gene, while monomeric IDHof strain G-4 at low temperatures.

gene is presumed to be present in only a few
Gram-positive bacteria from their genomic sequences.

References

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Inactivationisocitrateofisocitrate

dehydrogenasebyphosphorylation is mediated by the negative charge

psychrophilicof the phosphate. J. Biol. Chem. 262: 10422-10425.

bacterium, Vibrio sp. strain ABE-1. J. Gen. Appl.

28) Watanabe, S., Yasutake, Y., Tanaka, I., and Takada,

Microbiol. 30: 479-487.

Y. 2005. Elucidation of stability determinants of18) Ochman, H., Gerber, A. S., and Hartl, D. L. 1998.

cold-adapted monomeric isocitrate dehydrogenase

Genetic applications of an inverse polymerase chain

from a psychrophilic bacterium, Colwellia maris, byreaction. Genetics 120: 621-623.

construction of chimeric enzymes. Microbiology 151:

19) Sahara, T., Suzuki, M., Tsuruha, J., Takada, Y., and1083-1094.

Fukunaga, N. 1999. cis-Acting elements responsible29) Walker, J. M., and Raply, R. 2009. Molecular

for low-temperature-inducible expression of the gene

Biology and Biotechnology. 5th ed. The Royalcoding for the thermolabile isocitrate dehydrogenase

Society of Chemistry, Cambridge, UK.

29

Journal of Japanese Society for Extremophiles (2018) Vol.1730) Yasuda, W., Kobayashi, M., and Takada, Y. 2013.

protein monomerization by a domain duplication.

Analysis of amino acid residues involved in cold

Structure 10: 1637-1648.

activity of monomeric isocitrate dehydrogenase from32) Yasutake, Y., Watanabe, S., Yao, M., Takada, Y.,

psychrophilic bacteria, Colwellia maris and Colwellia

Fukunaga, N., and Tanaka, I. 2003. Crystal structure

psychrerythraea. J. Biosci. Bioeng. 116: 567-572.

of the monomeric isocitrate dehydrogenase in the31) Yasutake, Y., Watanabe, S., Yao, M., Takada, Y.,

presenceof36897-36904.

Fukunaga, N., and Tanaka, I. 2002. Structure of themonomeric isocitrate dehydrogenase: evidence of a30

NADP+.

J.

Biol.

Chem.

278:

Journal of Japanese Society for Extremophiles (2018) Vol.17

Journal of Japanese Society for Extremophiles (2018) Vol.17, 31-43

ORIGINAL PAPER

Matsumoto Ya, Aoshima Tb, Endou Hb and Takada Yc

Gene cloning and thermal properties of isocitrate dehydrogenase isozymes froma psychrotrophic bacterium, Psychrobacter okhotskensisaDepertment of Biological Sciences, School of Science, Hokkaido University, Kita 10-jo Nishi 8-chome, Kita-ku,
Sapporo, Hokkaido 060-0810, Japanb
Biosystems Science Course, Graduate school of Life Science, Hokkaido University, Kita 10-jo Nishi 8-chome, Kita-ku,
Sapporo, Hokkaido 060-0810, Japanc
Depertment of Biological Sciences, Faculty of Science, Hokkaido University, Kita 10-jo Nishi 8-chome, Kita-ku,
Sapporo, Hokkaido 060-0810, Japan
Corresponding author: Yasuhiro Takada, ytaka@sci.hokudai.ac.jp
Phone & Fax: +81-11-706-2742
Received: Dec. 19, 2018 / Revised: Jan. 17, 2019 / Accepted: Jan. 31, 2019andKey words: psychrotrophic bacterium, Psychrobacter

dimer-type isocitrate dehydrogenase (EC 1.1.1.42)

okhotskensis, isocitrate dehydrogenase, isozymes, geneisozymescloning

Abstract

The
from

genes
a

encoding

Gram-negative

monomer-

psychrotrophic

bacterium, Psychrobacter okhotkensis, (PoIDH-M andPoIDH-D, respectively) were cloned and sequenced. The

Introduction

open reading frames of the genes encoding PoIDH-M

Isocitrate dehydrogenase (IDH; EC 1.1.1.41(42))

and PoIDH-D were 2,217 and 1,257 bp in length andcatalyzes the oxidative decarboxylation of D-isocitrate tocorresponded to polypeptides composed of 739 and 419

2-oxoglutarate and CO2 with a concomitant reduction ofamino acids, respectively. Their deduced amino acid

NAD(P)+ in the central metabolic pathway, TCA cycle,

sequences showed high degrees of homology with thoseand is present ubiquitously in a variety of organisms.

of the respective type IDHs from Colwellia maris (61This enzyme is NADP+-dependent (EC 1.1.1.42) in mostand 71% identity, respectively) and, particularly, thebacteria13). Bacterial IDHs are classified into two types;

putativegenus

homodimer-type constituted by 40−45 kDa subunits andPsychrobacter (more than 87% identity). The two genes

monomer-type, which is a single polypeptide with aexisted facing each other with a spacer of 322 bases inmolecular mass of 80−100 kDa. Many bacteria have only

the order of the PoIDH-M and PoIDH-D genes on theone of either type IDH. The former type IDH has beenchromosomal DNA. The His-tagged PoIDH-M andreported in many bacteria including Escherichia coli1, 2, 4,

PoIDH-D overproduced in Escherichia coli cells showed

9)

the highest activities at 30 and 50°C, respectively. Theirglutamicum5) and Vibrio parahaemolyticus6) possess theresidual activities after incubation for 10 min at 45°C

later one. Despite catalyzing the same reaction in the twowere 0 and about 80%, respectively, and the latter IDH

type IDHs, homology of amino acid sequence is very

activity was lost completely by incubation at 50°C. Fromlowthese results, PoIDH-M and PoIDH-D were concluded tocross-reactivities are different from each other6, 10, 11). On

be cold-adapted and mesophilic enzymes, respectively.

the other hand, the coexistence of the two type IDHs

IDHs

from

other

bacteriaof, while Azotobacter vinelandii23), Corynebacterium

between

them,

and

their

immunologicalhave been found in several bacteria such as psychrophilic
bacteria,

Colwellia

pschrerythaea
31

17, 26)maris19,

20)

andColwellia, and a psychrotrophic bacterium,

Journal of Japanese Society for Extremophiles (2018) Vol.17

Pseudomonas psychrophila18). The monomer-type IDH

were done by an electroporation with Gene Pulser IIof C. maris (CmIDH-II) shows the maximum activity at(Bio-Rad).

20°C and about 50% of the activity is lost by incubationfor 10 min at 30°C, indicating that it is a typicalcold-adapted

enzyme19,temperature

for

20)

.

In

of

the

activity

contrast,

optimum

dimer-type

IDH

Preparation of crude extract and sodium dodecyl
sulfate-polyacrylamidegelelectrophoresis

(SDS-

PAGE)

(CmIDH-I) is 40°C and it completely retains the activity

The P. okhotskensis cells suspended in a sonication

after incubation for 10 min at 40°C. These indicate thatbuffer (50 mM potassium phosphate (pH 7.5), containing

this enzyme is mesophilic. These two IDH genes were

0.5 M NaCl and 2 mM MgCl2) were disrupted by fivereported to be located in tandem in the same direction ontimes sonication for 1 min with intervals of 2 min on ice.

11)

The cell lysate was centrifuged at 16,000 × g for 20 minthechromosomal

DNA .

However,

their

gene

expressions are independently controlled, and theat 4°C, and the supernatant was stored at -30°C and used

expressions of CmIDH-I and CmIDH-II genes are

as crude extract. Protein concentration was determined

induced by acetate and low temperature, respectively25).

by the method of Lowry et al.16) using bovine serum

On the other hand, both of the two type IDHs from P.

albumin as a standard. SDS-PAGE was carried out by

18)

the method of Leammli15) with 7.5% polyacrylamide gel.

psychrophila have been reported to be mesophilic .
Furthermore, no remarkable induction of their gene
expressions by acetate and/or low temperature wasobserved.

Cloning of PoIDH isozyme genes
Genomic DNA of P. okhotskensis was prepared as

Psychrobacter

described previously12, 31). To obtain a probe for genomic

okhotskensis, isolated from the Monbetsu coast of theSouthern hybridization, genomic PCR was performed for

Okhotsk Sea in Hokkaido, Japan, is a Gram-negative

30 cycles in a Veriti 96-well Thermal cycler (AppliedA

psychrotrophic

bacterium,29)

coccobacillus and hydrolyzes lipids at 5°C . It can grow

Biosystems). As the forward and reverse primers for the

between 0 and 35°C and its optimum temperature formonomer- and dimer-type PoIDH (PoIDH-M andgrowth is 25°C. In this study, the genes coding for IDH

PoIDH-D, respectively) genes, those for the cloning of

isozymes from P. okhotskensis (PoIDHs) were cloned,

IDH isozyme genes from C. psychrerythraea NRC1004

and thermal properties of these enzymes were examined.

reported previously17) (CpM-s and CpM-as, and CpD-s

Materials and methods

mixture (50 µl) contained 100 ng genomic DNA as

and CpD-as, respectively) were used (Table 1). Reaction
template, 10 pmol each of the forward and reverse
primers and 1U KOD-Plus-Neo DNA polymerase

Bacterial strains, plasmids and growth media
The

psychrotrophic

bacterium,

Psychrobacter(Toyobo)inabuffersystempreparedbytheokhotskensis29), was grown for 16 h at 25°C with

manufacturer. Cycling conditions after predenaturation at

vigorous shaking in Marine broth 2116 medium (Difco).

94°C for 3 min were as follows: denaturation at 94°C for27)

A mutant of E. coli defective in IDH, strain DEK2004 ,30 s, annealing at 55°C for 30 s, extension at 68°C for 30

was used as a host for expression of the IDH isozymes. The PCR products were purified and ligated into thegenes and grown in Luria-Bertani (LB) medium24) or

SmaI site of pBS for propagation and were used for

30)

Super broth medium . If necessary, ampicillin andgenomic Southern hybridization and the following

tetracycline were added to the culture media at final

colony hybridization. The nucleotide sequences of theconcentrations of 100 µg/ml and 15 µg/ml, respectively.

PCR products were determined in both directions by

Plasmids pBluescript SK(+) (pBS; Stratagene) andappropriate primers and a BigDye Terminator v1.1 Cycle

pTrcHisB (Invitrogen) were used as vectors for the

Sequencing Kit (Applied Biosystems) with a sequencer,

construction

ABI 3130 Genetic Analyzer.

of agenomic

DNAlibraryofP.

okhotskensis and the subcloning of the IDH genes andGenomic

Southernhybridization

andcolonyfor conferring the N-terminal His-tag on the expressedhybridization24) were carried out with a nylon membraneproteins, respectively. All transformations in this study(Hybond-N+, GE Healthcare) and the probes labeled with
AlkPhos Direct

32

Labeling and

Detection system with

Journal of Japanese Society for Extremophiles (2018) Vol.17

Table 1. Oligonucleotides used in this study
Primer nameNucleotide sequence (5'→3')

Cloning of IDH genes
CpM-s

AAYATCATCAAGYTGCCRAAYATY

CpM-asTGGCGCRCGRCGRTCRGAGTTRCCTTC

CpD-s

ATGAAGTTYACYGAAGGMGCKTTTCpD-asACCKGCATATTTMGGWGCRGTWCC

inverse-M1

TCTTGTTCTTCGTCGGTTTTTGGATC

inverse-M2

AATTCGTCAGCGCTATGGTAAAAGTC

inverse-D1

TTGCTGCAAAAAGGCATCAGCAATG

inverse-D2

ATTCTGATGCGTCCTGCCGATTATTC

genome-direct-dimer

CCAGCGAACGGGACGTTGGCATACATAG

Construction of gene encoding His-tagged IDHs
monomer-F-BamHIplus

gcgcggatccgTCAAAAATTATTTATAC

monomer-R-SacIplus

gcgcgagctcTTACAACGATGCCAAC

dimer-F-BamHIplus

gcgcggatccgTCATATGATAAGGTTATC

dimer-R-SacIplus

gcgcgagctcCTAGGCATCCATGTGCTTA

Small letters indicate additional bases for introducing the digestion sites for BamHI and SacI (underlined anddouble-underlined letters, respectively).
CDP-Star (GE Healthcare). The DNA fragments anddetermined as described above except for the use of acolonies hybridized with the probes were detected with a

BigDye Terminator v3.1 Cycle Sequencing Kit (Applied

Luminescent Image Analyzer, LAS-3000 (FUJIFILM).

Biosystems).

For further cloning of the PoIDH isozyme genes, inverse
21)

Nucleotide sequence of 5’-terminal region of thewas carried out. Approximately 5-kbp genomic

PoIDH-D gene, which was not contained in the inverse

DNA fragments digested with PstI (0.2 µg) were

PCR product, was determined by genome direct

PCR

self-ligated with Ligation-Convenience Kit (NIPPON

sequencing by using 200 ng genomic DNA digested withGENE) and then used as the template for inverse PCR.

ClaI as template and the BigDye Terminator v3.1 Cycle

The primers (inverse-M1 and inverse-M2 for PoIDH-M,

Sequencing Kit (Fig. 1). For this purpose, the primer,and inverse-D1 and inverse-D2 for PoIDH-D; Table 1)

genome-direct-dimer (Table 1), was designed from thewere designed from the determined nucleotide sequences

determined sequence of the inverse PCR product.

of the probes for their cloning described above.
Amplification was performed for 30 cycles in a reaction

Construction of His-tagged PoIDH isozyme genes

mixture (50 µl) containing about 200 ng the self-ligated

The PoIDH-M and PoIDH-D genes were amplified by

DNA, 10 pmol each of the primers and 1 U

PCR to introduce restriction sites for BamHI and SacI at

KOD-plus-Neo DNA polymerase in a buffer system

the 5’- and 3’-terminals of their ORFs, respectively.

prepared by the manufacturer. Cycle conditions after

Therefore,monomer-F-BamHIplus

predenaturation at 94°C for 3 min were as follows:

R-SacIplus

for

the

PoIDH-Mandgenemonomerand

dimer-

denaturation at 94°C for 30 s, annealing at 60°C for 30 s,

F-BamHIplus and dimer-R-SacIplus for the PoIDH-D

extension at 68°C for 3 min. The PCR products weregene were used as forward and reverse primers,

purified and ligated into the EcoRV site of pBS.

respectively (Table 1). Amplification was carried out for

Nucleotide sequences of the DNA fragments selected by

35 cycles in a reaction mixture (50 µl) containing 80 ng

colony hybridization and the inverse PCR products were

genomic DNA of P. okhotskensis as template,

33

10 pmolJournal of Japanese Society for Extremophiles (2018) Vol.17

Fig. 1. Restriction and physical map of the genomic DNA fragments containing the PoIDH isozyme genes. Arrows andboxes represent the ORFs of PoIDH genes and the position of the probes for genomic Southern hybridization,
respectively.
each of the forward and reverse primers and 1 U

elutants containing IDH activity were concentrated,

KOD-plus-Neo DNA polymerase in a buffer system

dialyzed and stored at -30°C until use.

prepared by the manufacturer. Cycling conditions after
predenaturaion at 94°C for 3 min were as follows:denaturation at 94°C for 30 s, annealing at 50°C for 30 s,

Enzyme assay
The

IDH

activitywas

assayedasdescribed

19)extension at 68°C for 90 s except for extension at 68°C

previously . Unless otherwise noted, 2 ml of reaction

for 60 s for the PoIDH-D gene. Resultant PCR products

mixture consisted of 33 mM Tris-HCl (pH 8.0), 0.67 mM

were then digested with BamHI and SacI and ligated to

MnCl2, 0.12 mM NADP+, 2 mM sodium isocitrate, andthe BamHI-SacI site of pTrcHisB to obtain plasmids

an appropriate amount of enzyme. One unit of enzyme

pHisPoIDH-M and pHisPoIDH-D, containing the ORFs

activity was defined as the amount capable of catalyzingof the PoIDH-M and PoIDH-D genes, respectively.

the reduction of 1 µmol of NADP+ per min.

Overexpression and purification of His-tagged IDHs

purified PoIDH isozymes with His-tags were dialyzed

To examine thermostability for the IDH activity, theE. coli DEK2004 transformed with pHisPoIDH-M,

overnight at 4°C against 20 mM sodium phosphate (pHpHisPoIDH-D, or pTrcHisB carrying the gene for IDH8.0), containing 2 mM MgCl2, 0.3 M NaCl, 1 mM

30)

of Azotobacter vinelandii (AvIDH), pHisAvWT , wasdithiothreitol and 10% (v/v) glycerol. The dialyzed

grown at 37°C in 1 l of Super broth medium until OD600

enzymes were incubated for 10 min at variousof the culture reached 0.6-0.8. The culture was thentemperatures and were then immediately cooled on ice

cooled for 30 min on ice. For the overexpression of thefor 10 min. The remaining activities of PoIDH-M andIDH proteins, the culture was further incubated for 19 h

PoIDH-D were assayed at 30°C and 40°C, respectively.

at

15°C

after

the

addition

of

1

mM

isopropyl-β-D-thiogalactopyranoside. Then, cells were

Results

harvested at 4°C and were suspended in 30 ml of 50 mM
sodium phosphate buffer (pH 8.0), containing 2 mM

Cloning of the PoIDH isozyme genes and their

MgCl2, 0.3 M NaCl, 10 mM imidazole, 10% (v/v)

nucleotide and deduced amino acid sequences

glycerol and 10 mM 2-mercaptoethanol. A final

As probes used for the cloning of the PoIDH-M andconcentration of 2 mg/ml lysozyme from egg white was

PoIDH-D genes, 210 and 342 bp of each DNA fragment

added to the cell suspension, and the mixture was gently

was obtained by genomic PCR described in “Materials

shaken for 1 h at 4°C. As described previously8), theand Methods”. The deduced amino acid sequences ofoverproduced IDHs were then purified by Ni-NTA

these probes were analogous

agarose (Qiagen) column chromatography, and the34

to those of the respective

Journal of Japanese Society for Extremophiles (2018) Vol.17

Fig. 2. Nucleotide and deduced amino acid sequences of the PoIDH isozyme genes. The underlined nucleotides are thetranslational start codon. The double underlined sequences are the putative ribosome binding sites. The probablestem-loop structures located downstream of the stop codons are shown by two opposite arrows.
type IDH genes from other bacteria. By genomic

them, only a 3.0-kbp genomic DNA fragment digested

Southern blot analysis, it was revealed that about 3.0 andwith HindIII was selected by the following colony

4.5 kbp of single fragment of the P. okhotskensis

hybridization by using these probes, but it did not

genomic DNA digested with HindIII and PstI and about

contain the complete IDH isozyme genes (Fig. 1).

1.5 and 4.5 kbp of those hybridize with the probes for the

Therefore, inverse PCR and genome direct sequencing

PoIDH-M and PoIDH-D genes, respectively. Amongdescribed in “Materials and Methods”

35

were performed,

Journal of Japanese Society for Extremophiles (2018) Vol.17

Fig. 3. Alignment of amino acid sequences of monomer-type IDHs from several bacteria. The deduced amino acidsequence of PoIDH-M (P.o) is aligned with those of monomer-type IDH from P. cryohalolentis K5 (P.c; databaseaccession no. CP000323), CmIDH-II (C.m; no. D14047) and AvIDH (A.v; no. D73443). The amino acid residues
involved in the binding of isocitrate and metal ion and of NADP+ are indicated in gray and white boxes, respectively.
and the two IDH genes were completely cloned. Theirbacteria (Figs. 3 and 4). In particular, the identities ofnucleotide sequences are shown in Fig. 2. The two IDH

PoIDH-M to the putative monomer-type IDHs ofisozyme genes were found to exist facing each other with

Psychrobactera spacer of 322 bases in the order of the PoIDH-M andimmobilis and Psychrobacter cryohalolentis K5, were 92,

PoIDH-D genes on the chromosomal DNA (Figs. 1 and89 and 89%, respectively. On the other hand, the2). The ORF of PoIDH-M gene was 2,217 bp in length

identities to AvIDH and CmIDH-II were 62 and 61%

and encoded a polypeptide of 739 amino acids with arespectively. In the case of PoIDH-D, the identities to thecalculated molecular mass of 83,101.42 Da. A putative

putative dimer-type IDHs of the above three bacteria ofribosome binding site, AGG, was present 15-17 bases

the genus Psychrobacter were 96, 96 and 87%,

upstream of the ATG codon. Furthermore, sequence of arespectively, while those to the E. coli IDH andterminator-like stem-loop structure, composed of a 7-bp

CmIDH-I were 72 and 71%, respectively. Furthermore,stem with a 9-nucleotide loop, was found downstream of

the amino acid residues involved in the binding of

the ORF. On the other hand, the ORF of PoIDH-D geneisocitrate, metal ion and NADP+28) were completely

was 1,257 bp in length and encoded a polypeptide of 419

conserved among these IDHs (Figs. 3 and 4). These

amino acids with a calculated molecular mass of

results suggest that the cloned genes encode the45,772.61 Da. A putative ribosome binding site,monomer- and dimer-type IDH isozymes of P.

AGGAG, was present 9-13 bases upstream of the ATG

okhotskensis.

aquaticus

CMS

56,

Psychrobactercodon. Sequence of a terminator-like stem-loop structure,composed of a 5-bp stem with a 11-nucleotide loop, was

Thermal properties of His-tagged PoIDH isozymes

also found downstream of the ORF. The nucleotide

The PoIDH-M and PoIDH-D genes were introduced

sequences of the PoIDH isozyme genes have been

into the expression vector, pTrcHisB, to confer adeposited in the DDBJ database under accession no.

(His)6-tag on the N-terminal of the translated proteins.

LC431291. The deduced amino acid sequences of

The

PoIDH-M and PoIDH-D showed high degrees ofoverproduced in the E. coli cells and purified byhomology to the respective type IDHs from otherNi-agarose36

His-tagged

PoIDH-Mcolumnandchromatography

PoIDH-Dasweredescribed

Journal of Japanese Society for Extremophiles (2018) Vol.17

Fig. 4. Alignment of amino acid sequences of dimer-type IDHs from several bacteria.
The deduced amino acidsequence of the PoIDH-D (P.o) is aligned with those of dimer-type IDH from P. cryohalolentis K5 (P.c; databaseaccession no. CP000323), CmIDH-I (C.m; no. D14047) and IDH form E. coli (E.c; no. J02799). The amino acidresidues involved in the binding of isocitrate and metal ion and of NADP+ are indicated in gray and white boxes,
respectively.
“Materials and Methods”. SDS-PAGE of the final elutant(Fig. 6). In the IDHs of several bacteria, His-tagging atin the chromatography revealed that the two His-tagged

their N-terminals was found to not significantly affect

IDHs were almost homogeneously purified (Fig. 5).

their thermal properties such as optimum temperature

Since the purified enzymes showed high IDH activities,

and thermostability for activity7, 17, 18, 26). The His-tagged

the cloned genes were demonstrated to encode IDH

PoIDH-M showed the highest activity (27.66  0.83

proteins as described above. Activities of the purified

U/mg protein) at 30°C and completely lost it at 60°C. On

PoIDH isozymes were assayed at various temperatures

the other hand, optimum temperature for the PoIDH-D

37

Journal of Japanese Society for Extremophiles (2018) Vol.17

Fig. 5. SDS-PAGE of crude extract of P. okhotskensis and the purified PoIDH isozymes. Lane M, marker proteins;.lane
1, the purified CmIDH-II (1 µg protein); lane 2, the purified CmIDH-I (1 µg protein); lane 3, crude extract of P.
okhotskensis (85 µg protein); lane 4, the purified PoIDH-M (3 µg protein); lane 5, the purified PoIDH-D (3 µg protein)

Fig. 6. Effect of temperature on the His-tagged PoIDH activities. Activities of monomer- and dimer-type PoIDHs areindicated by  and ◯, respectively. Values indicate the means ±SD of at least duplicate assays from two independent
experiments.

38

Journal of Japanese Society for Extremophiles (2018) Vol.17

Fig. 7. Thermostability for activities of the His-tagged PoIDHs. Residual activities of monomer- () and dimer-type (◯)
PoIDHs assayed at 30°C and 40°C, respectively, after incubation for 10 min at the indicated temperatures are
represented as percentages of those without the incubation. Values indicate the means ±SD of at least duplicate assays
from two independent experiments.
activity was 50°C, and the enzyme retained 34% of thetemperature and thermostability for activity, indicating

maximum activity at 60°C. The maximum activity ofthat this enzyme is cold-adapted. Several kinetic

PoIDH-D (128.610.5 U/mg protein) was much higher

parameters of PoIDH-M and AvIDH at 30°C are

than that of PoIDH-M. Thermostability of the PoIDH-M

summarized in Table 2. The Km value for isocitrate ofand PoIDH-D activities is shown in Fig. 7. The former

PoIDH-M was about 3-fold higher than that of AvIDH,

IDH activity completely lost after incubation for 10 min

whereas the kcat value of the later IDH was about 4-foldat 45°C and retained 807 and 302% of activity after

higher than that of the former IDH. Therefore, theincubation at 30 and 40°C, respectively. In contrast, thecatalytic efficiency, kcat/Km, value of the latter was muchlatter IDH retained completely the activity even after

higher than that of the former.

incubation at 40°C, but no activity was detected after
incubation at 50°C. It was reported that a typical

Discussion

mesophilic AvIDH with His-tag retains completely the

In this study, the monomer- and dimer-type IDH isozyme

activity after incubation for 10 min at 40°C and its

genes of P. okhotskensis were cloned and sequenced.

optimum temperature for activity is 47.5°C30). Therefore,

These genes were found to exist facing each other on the

PoIDH-D is judged to be a mesophilic enzyme. On thechromosomal DNA of this bacterium (Figs. 1 and 2).

otherThis arrangement of thehand,

PoIDH-M

showedlower

optimumP. okhotskensis IDH isozyme

Table 2 Kinetic parameters of AvIDH and PoIDH-M at 30°C
kcat (s-1)

Km for isocitrate (μM)

kcat/Km×105 (s-1·μM-1)

AvIDH

315.05±13.99

23.15±4.83

140.97

PoIDH-M

78.91±3.41

63.06±7.71

12.64

39

Journal of Japanese Society for Extremophiles (2018) Vol.17

Fig. 8. Location of IDH isozyme genes on the chromosomal DNA in several bacteria. Relative configurations of IDH
genes of C. maris, C. psychrerythraea strains NRC1004 and 34H, Pseudomonas fluorescens strain Pf0-1, P.
psychrophila, P. okhotskensis, and P. cryohalolentis strain K5 (database accession no. CP000323) on theirchromosomal DNAs are shown. Locational data for IDH genes of these bacteria except for P. cryohalolentis strain K5
were obtained from previous reports18, 26).
genes is different from those of C. maris, C.
andthose of the two bacteria. These imply that the

Pseudomonas

coexistence of monomer- and dimer-type IDH isozyme

fluorescens (Fig. 8). Bacteria of the genus Psychrobacter

genes and the arrangement on the chromosomal DNA areare closely related to those of the genus Pseudomonas,

not necessarily correlated to phylogenetic relationshipand the two genera belong to the same order,

betweenpschrerythaea,P.

psychrophilabacteria

of

Amino

acid

sequence

and

genus

immunological cross-reactivity of the two type IDHs are

Colwellia, belonging to order Alteromonadales, are also

different from each other, but similar within each typecomparatively allied to those of the two genera. One theIDHs6, 10, 11). On the other hand, the monomer-type IDH

other hand, from the genome sequence of Burkholderia

has been suggested to originate from partial genecenocepacia, belonging to class Betaproteobacteria, theduplication of the dimer-type one27). Therefore, it isputative monomer- and dimer-type IDH isozyme genes

assumed that the coexistence of the two type IDHs arose

are expected to be present in this bacterium. Similar to C.

dispersedly in several bacteria after appearance of themaris and C. pschrerythaea, the putative two IDH genes

monomer-type IDH.

Pseudomonadales.

Furthermore,bacteria.

of B. cenocepacia are located tandem in the same

The PoIDH-M and PoIDH-D genes were expressed indirection, but the order of the genes is different fromthe E. coli cells, and several thermal properties of the40

Journal of Japanese Society for Extremophiles (2018) Vol.17

purified enzymes were clarified in this study. The

further study is required to clarify the regulation of the

genome sequence has been determined in several

PoIDH-M and PoIDH-D gene expressions.

bacteria of the genus Psychrobacter. However, to ourknowledge, this is the first report on the characterization

Acknowledgementsof IDHs from bacteria of this genus. From the elucidated

We sincerely thank Dr. Isao Yumoto of the National

thermal properties of PoIDH-M and PoIDH-D (Figs. 6

Institute of Advanced Industrial Science and Technology

and 7), these enzymes were confirmed to be cold-adaptedof kind donation of P. okhotskensis.

and mesophilic, respectively, like IDH isozymes from C.
maris and C. pschrerythaea17, 19, 26). In the case of C.

References

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Burke, W. F., Johanson, R. A., and Reeves, H. C.

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Chung, A. E., and Braginski, J. E. 1972. Isocitrateof PoIDH-M was lower at all temperatures tested thandehydrogenase from Rhodopseudomonas sphe-that of PoIDH-D (Fig. 6). Similar results were reported

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Chung, A. E., and Franzen, J. S. 1969. Oxidized
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genetype IDHs may be required for its growth at low

biochemical

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Fukunaga, N., Yoshida, S., Ishii, A., Imagawa, S.,

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Takada, Y., and Sasaki, S. 1988. Isolation andto be a cis-element responsible for its cold-induciblecharacterization of a mutant defective in one of twogene expression 22, 25). This sequence was also foundisozymes of the isocitrate dehydrogenase from a

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psychrophilic bacterium, Psychromonas marina.
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okhotskensis sp. nov., a lipase-producing facultativefrom a psychrophilic bacterium, Colwellia maris bypsychrophile isolated from the coast of the Okhotsk

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4331) Wilson, K. 1987. Current protocols in molecularbiology. Wiley, N Y.

Journal of Japanese Society for Extremophiles (2018) Vol.17

Journal of Japanese Society for Extremophiles (2018) Vol.17, 44-57

ORIGINAL PAPER

Hamana Ka, Hayashi Ha, Kobayashi Mb, Furuchi Tb, Niitsu Mb,
Itoh Tc and Ohkuma Mc

Polyamine analysis of the new members of euryarchaeal methanogens,euryarchaeal and crenarchaeal thermophiles, and thaumarchaeal
ammonia-oxidizers-Polyamine catalogues of bacterial (eubacterial) and archaeal (archaebacterial)
extremophiles IXa

Faculty of Engineering, Maebashi Institute of Technology, Maebashi, Gunma 371-0816, Japan.
Faculty of Pharmacy and Pharmaceutical Sciences, Josai University, Sakado, Saitama 350-0295, Japan.
cJapan Collection of Microorganisms (JCM), RIKEN, BioResource Center, Tsukuba, Ibaraki 305-0074, Japan.
bCorresponding author: Koei Hamana, koeihamana@gmail.com
Phone: +81-27-234-4611, Fax: +81-27-234-4611
Received: Nov. 8, 2018 / Revised: Dec. 25, 2018 / Accepted: Dec. 27, 2018
Cellular polyamines were acid-extracted

containedfrom the newly validated 59 strains of euryarchaeal

Mesophilic

methanogens, euryarchaeal and crenarchaeal thermo-

belonging to the order Methanomassiliicoccales of the

Abstract

philes,andthaumarchaeal

ammonia-oxidizers

analyzed by HPLC and HPGC.

andSpermidine andputrescine,bacterium and Methanothermobacter of the order

Thermoplasmatales

A high spermine level was foundthermophilic

Methanothermobacter

species.

In

spermine.
luminyensis

class Thermoplasmata contained spermidine, homospermidine and spermine.
moderately

in an alkaliphilic Methanobacterium species and threeandMethanomassiliicoccus

spermine were distributed in the genera MethanoMethanobacteriales.

spermidine

thermophilic

In euryarchaea, slightly/
acidophiles

contained

belongingspermidine

to

(plus

spermine) whereas extreme thermophiles belonging toandThermococcales

Archaeoglobales

ubiquitously

4

contained N -bis(aminopropyl)spermidine (plus N4-bis

Methanococcales, Methanococcus contained spermidine(aminopropyl)norspermidine).

and agmatine whereas hyperthermophilic Methano-

caldopentamine and thermopentamine, and a linear

caldococcus

contained

spermineandabranched

4hexa-amine,caldohexamine,

Linearwere

penta-amines,
detectedinpenta-amine, N -bis(aminopropyl)spermidine, in addition

crenarchaeal extremely/hyper-thermophilic Desulfuro-

to the two polyamines.

Distribution of homospermidine

coccales but not in the extreme/hyper thermophiles of

was limited in Methanoculleus (Methanomicrobiales),Acidilobales, Sulfolobales, Fervidicoccales and Thermo-

Methanoplanus and Methanocella (Methanocellales),proteales.

and Methanosaeta, Methanosarcina, Methanolobus,

Nitrosopumilus belonging to the third archaeal phylum

Methanomethylovorans and Methanosalsum (Methano-

Thaumarchaeota

sarcinales), but was heterogeneous within the orders andspermidine alone.

genera.

Low spermidine and spermine levels inconcentrations depend on their growth temperature, pH

halophilic Methanohalophilus (Methanosarcinales) andand salt conditions in addition to their phylogenetic

high spermine and agmatine levels in alkaliphilic

locations.

Methanosalsum

zhilinae

observed.

Thermo-haloalkaliphilic

Methanonatronarchaeum

werethermo-philum belonging to the class Methanonatronarchaeia

44

Key

Ammonia-oxidizing Nitrososphaera andwords:

contained

putrescine

and

homo-

Archaeal polyamine profiles andArchaea,methanogen,thermophile, ammonia-oxidizing archaea

polyamine,Journal of Japanese Society for Extremophiles (2018) Vol.17

Using the combination of high-performance liquid

Introduction
By our extensive cellular polyamine analyses ofarchaea (of the domain Archaea) and bacteria (of thechromatography(HPLC),

high-performancegaschromatography (HPGC) and HPGC-mass spectrometry

domain Bacteria), it has been suggested that cellular(HPGC-MS) developed in our laboratory, additional

polyamineapolyamine distribution catalogues for 37 new archaeal

chemotaxonomic marker, are related to their optimum

methanogens including alkaliphilic, halophilic andgrowth temperature, pH and salt concentration, inthermophilic species, 18 new archaeal thermophiles

addition to their phylogenetically classified locations

including acidothermophiles, and four new ammonia-

11,21,25)

Ionic interaction of polycationic polyamines tooxidizing archaea (nitrite archaea) were presented here to

nucleic acids and other acidic components in the cells issearch for unique polyamine profiles in the domain.

distribution

profiles,such

important for their growth environments

as37,45).

LinearArchaea.

Nine difficult-to-culture methanogens wereand branched penta-amines and hexa-amines found in

re-cultured and newly analyzed in the present study

various bacterial and archaeal thermophiles grown in(shown as reanalysis in Table 1) to confirm and correcthigh thermal environments are possibly associated with

their polyamine profiles.

stabilizing cellular nucleic acid structure as a major
function of the high basic polyamines14-20,22-24,26,27).
These polyamine levels were salt dependent in some23)

archaeal thermophiles .

Materials and Methods
Archaeal strains were provided by JCM of the RIKEN

Archaeal extreme halophiles

BRC through the National Bio-Resource Project of thegrown in high salt media showed low cellular

MEXT, Japan29) and purchased from NBRC of theconcentrations

Biotechnology Center, National Institute of Technologyofspermidine,

spermine

and/oragmatine13,18,22,43).
An

and Evaluation, Japan34) and were cultivated in thearchaebacterial

Euryarchaeota,

aerobic or anaerobic liquid culture media (100-1000ml)

encompasses diverse lineages including the six previous

designated in JCM Catalogue 29) or NBRC Catalogue 34),

orders of methanogens (Methanobacteriales of the classat the temperature, pH and NaCl concentration listed in

Methanobacteria,
Methanoccoci,

phylum,Methanococcales

Methanomicrobiales,

of

the

class

Methanocellales

and Methanosarcinales of the class Methanomicrobia,

Table 1.

Cells in the stationary phase were harvested

and polyamines were extracted completely from the cells
by 0.5M perchloric acid (PCA) three times.

Theand Methanopyrales of the class Methanopyri), two

PCA-extract was subjected to a Dowex 50WX8 column

orders(1cm I.D. x 3cm) and then the column was washed withofthermophiles
one

Thermococcales),(Thermoplasmatales),(Archaeoglobales

order
and

one

of

and

acidothermphiles

order

35)

of

extreme

halophiles(Halobacteriales) .

However,mesophilic

methanogens

to

1M HCl.

Polyamines were eluted with 6M HCl fromthe column.

The concentrated polyamines weretheanalyzed by HPLC on a Hitachi L6000 using a column

orderof cation-exchange resin, Hitachi 2619F (=Hitachi 2720)

Methanomassiliicoccales are located in the class(4mm I.D. x 50mm) and determined by post-labeled

Thermoplasmata
extremely

4).

halophilic

belongingthe

A novel slightly thermophilic,fluorometry(haloalkaliphilic)

Spectrophotometermethanogen,onaHitachi
after

F1050
being

Fluorescence
heatedwith

Methanonatronarchaeum thermophilum, belongs to theo-phthalaldehyde 12,19,20).

new class Methanonatronarchaeia41).

Another phylum,of the concentrated polyamine samples, HPGC on a

Crenarchaeota is comprised of the four thermophilic

SHIMADZU GC-17A and HPGC-MS on a JEOL

orders Acidilobales, Desulfurococcales, Sulfolobales and

1MS (0.32mm I.D. x 30m) (GL Sciences) were

Fervidicoccales35). Ammonia-oxidizing Nitrososphaera

performed, and polyamines were determined by a flame

and Nitrosopumilus species were published as the firstionization detector (FID) 12,19,20).

pure

polyamine peaks was performed by peak high of theculture

of

a

the

new

third

thermophilic

JMS-700, equipped with a capillary column of Inert Cap

order,

Thermoproteales

andAfter heptafluorobutyrizationarchaeal

phylumThaumarchaeota and belong together to the class
Nitrososphaeria

1,28,43).

45

polyamine standards.

Quantification ofJournal of Japanese Society for Extremophiles (2018) Vol.17

Table 1. Cellular distribution and concentration of polyamines in the phyla Euryarchaeota, Crenarchaeota and Thaumarchaeota

46

Journal of Japanese Society for Extremophiles (2018) Vol.17

47

Journal of Japanese Society for Extremophiles (2018) Vol.17

Numeric codes of polyamines abbreviated as number of methylene (CH2) groups between amino (NH2) or imino (NH)groups are used. 3, 1,3-diaminopropane; 4, putrescine; 5, cadaverine; 6, 1,6-diaminohexane; 33, norspermidine; 34,
spermidine; 35, aminopropylcadaverine; 44, homospermidine; 333, norspermine; 3(3)3, N4-aminopropylnorspermidine;
3(3)4, N4-aminopropylspermidine; 343, spermine; 334, thermospermine; 344, aminopropyhomospermidine; 434,
canavalmine; 3333, caldopentamine; 3343, thermopentamine; 3334, homocaldopentamine; 3(3)(3)3,N4-bis(aminopropyl)norspermidine; 3(3)(3)4, N4-bis(aminopropyl)spermidine; Agm, agmatine; *, not calculated; -, notdetected (<0.005); T, type strain; °C, culture temperature; pH, medium pH; NaCl, NaCl concentration (%) in media;
JCM, Japan Collection of Microorganisms, RIKEN, Wako, Saitama, Japan; NBRC, Biological Resource Center,
National Institute of Technology and Evaluation, Kisarazu, Chiba, Japan; IFO, Institute for Fermentation Osaka, Japan(IFO strains have been transferred to NBRC); ATCC, American Type Culture Collection, Manassas, Virginia, USA.
Ref. are shown in the References (published year of Hamana et al.) of the present report (59 blanks are the data of thisstudy in 2019). #, the muss culture of the strains was difficult so that the polyamine data of the strains in the present
study are insufficient. Agmatine level of P. horikoshi and P. woesei (not determined in the previous reports)
recalculated from the previous HPLC data.
France.

werePyrococcus glycovorans AL585 is deposited in IFREMER, Plouzane,

Former names are shown in parentheses.

Molar concentrations of cellular polyamines per gramof wet archaeal cell pellet (μmol/g wet wt. cell)
estimated from the HPLC and HPGC analyses (Figs. 1

profilesinmanydifficult-to-culturearchaealextremophiles.
Numeric codes of polyamines abbreviated as numberand 2) are shown in Table 1 and are compared with thoseof methylene (CH2) groups between amino (NH2) orof our previous data on phylogenetically related archaea.

imino (NH) groups are used in Table 1, Fig. 1 and Fig. 2

Our analytical data without statistical analysis would beof the present study.

validated in reference to cellular polyamine distribution48

Journal of Japanese Society for Extremophiles (2018) Vol.17

Fig. 1. HPLC chromatogramof the polyamines of Methanimicrococcus blatticola JCM

31471 (A),Methanomassiliicoccus lumenyensis JCM 31472 (B), Cuniculiplasma divulgatum JCM 30642 (C), Nitrosphaera
gargensis JCM 31473 (D), Thermococcus kodakarensis JCM 12380 grown at 85°C, 1.25% NaCl (Ea) and
Thermococcus kodakarensis JCM 12380 grown at 65°C, 2.5% NaCl (Eb). Three tetra-amines 343, 334 and 3(3)4 wereco-eluted in the HPLC, however, analyzed separately in HPGC. Abbreviations for polyamines are given in Table 1.
alcaliphilum grown under an alkaline medium condition

Results and Discussion

at pH 8.4, contained a high concentrations of spermine
Phylum Euryarchaeota(343),

Order Methanobacteriales (class Methanobacteria)

accumulation

occurredmethanogens.

In this order, the spermine (343) level

The newly available five species of Methanobacterium
analyzed

as

well

as

16

other

speciesofsuggestingthat

cellularspermine

also

the

in

(343)

alkaliphilic

was lower in the mesophilic Methanobrevibacter andMethanobacterium previously analyzed showed theMethanosphaera16,17,22)

ubiquitous occurrence of spermidine (34) and spermine

thermophiles belonging to Methanothermobacter grown(343) (Table 1).

at 55-65°C and Methanothermus grown at 80-85°C as

In spite of mesophilic methanogens, a

than

in

the

methanogenic

high spermine level was found in eight species among

previously reported17,24).

the 22 species analyzed.

species were heterogeneous in the polyamine distribution

Two strains of alkaliphilic M.

49

Five Methanobrevibacter

Journal of Japanese Society for Extremophiles (2018) Vol.17

Fig. 2.

HPGC chromatogram of the polyamines of Saccharolobus solfataricus 98/2 JCM 30930 (A), Acidianus sp.

RZ1 JCM 30227 (B), Thermococcus kodakarensis JCM 12380 grown at 85°C, 1.25% NaCl (Ca) and Thermococcus
kodakarensis JCM 12380 grown at 65°C, 2.5% NaCl (Cb).

Chromatograms by SHIMADZU GC-17A are shown.

It

has been known that 3(3)(3)4 is converted to 3(3)3 and 3(3)4 at a ratio of 3:1, and a major part of agmatine is destroyed
during HPGC after heptafluorobutyrization.

The peak high of 3(3)3 x 1/3 might be equal nearly to the peak high of

3(3)4 converted from 3(3)(3)4, so that the peak high of 3(3)4 found on the chromatogram minus the former peak high of
3(3)3 x 1/3 is roughly calculated as original 3(3)4 in the sample before HPGC (peak high of 3(3)3 and 3(3)4 have same
quantity).

Abbreviations for polyamines are given in Table 1.

profiles, as shown in Table 1.

M. arboriphilus and M.

ruminantium including reanalyzed samples contained
homospermidine (44) and spermine (343).

Newly

analyzed M. boviskoreani and M. smithii contained50

spermidine (34) and spermine (343).

Putrescine (4) andspermidine (34) were detected in M. oralis.

Journal of Japanese Society for Extremophiles (2018) Vol.17

same

Order Methanococcales (class Methanoccoci)
Extremely thermophilic Methanocaldococcus species

strainNBRC

101202

and

three

other

Methanoculleus species, indicating miss polyamine

previously analyzed, grown at 80-85°C, contained

assignment in 2002.

4

N -bis(aminopropyl)spermidine (3(3)(3)4) as a major
polyamine18,20).

Since

the

quaternary

branched

Order Methanocellales of the class Methanomicrobia

penta-amine have been found in extremely thermophilic

Newly analyzed Methanocella conradii JCM 17849

Methanotorris grown at 70-80°C and has not been

as well as Methanocella arvoryzae and Methanocelladetected in mesophilic Methanococcus grown at 25-37°C

paludicola previously analyzed contained putrescine (4)and moderately thermophilic Methanothermococcus

and homospermidine (44) as the major polyamines

16)

grown at 60°C belonging to this order , the occurrence(Table 1), suggesting ubiquitous occurrence of putrescineof the branched penta-amine seems to correlate to their(4) and homospermidine (44) in the genus Methanocella.

extreme thermophily.

It has been shown that putrescinesynthesis

via

proceeds

L-arginine

decarboxylation

Order Methanosarcinales of the class Methano-

pathway and spermidine (34) and spermine (343) are
synthesizedbyaminopropyltransferase

microbia

in

Methanogens belonging to this order are known to10)

Methanococcus species .

contribute to methanogenesis from acetate and methyl

A unique iron-corroding methanogen, Methanococcus
49)

compounds(methanol

andmethylamine).

isolated from

Methanosaeta is a specialist that uses acetate specifically.

a crude-oil storage tank in Japan contained putrescine (4),Other methanogens belonging to the other five orders use

spermidine (34), spermine (343) and agmatine (Agm),

H2 and formate for their methanogenesis.

maripaludis strain KA1 (NBRC 102054)

Including newly analyzed species, Methanosarcinaand had same polyamine profile in other strains of M.
maripaludis including the type strain JJ (Table 1).

ubiquitously

contained

Recently, hydrogenase mediating iron corrosion inDistribution of homospermidine (44) is conflicting

another iron-corroding strain OS7 (NBRC 103642) ofwithin the species of genera Methanolobus andMethanococcus maripaludis was reported48).

Methanomethylovorans.

Methanosalsum

homospermidine

and

(44)

homospermidine(44).

contained

spermine(343).

Methanococcoides and Methanimicrococcus contained

Order Methanobacteriales (class Methanobacteria)
In this mesophilic order, polyamine distribution

spermidine (34) and spermine (343).

profiles varied within the six genera Methanomicrobium

Spermidine (34) (and spermine (343)) in addition to(spermidine (34)), Methanogenium (putrescine (4),putrescine (4) was found in the re-cultured (reanalysis)

spermidine (34), homospermidine (44) and spermine

samples of Methanomethylovorans hollandica and(343)), Methanocalculus (putrescine (4), spermidine (34)

Methanomethylovorans uponensis but spermidine (34)and homospermidine (44)), Methanoregula (putrescineand spermine (343) had not been detected in their(4) and spermidine (34)), Methanospirillum (putrescineprevious cultures (Table 1).

(4) and spermidine (34)) and Methanoplanus (putrescine

putrescine (4), spermidine (34) and spermine (343) are(4) and homospermidine (44)) as analyzed in the present

varied in cultures of them.

and previous studies16,17,21).

thermophilic

Methanogenium cariaci inwhich spermidine (34), homospermidine (44) andCellular concentrations ofOn the other hand,

Methanomethylovorans

thermophila

contained homospermine (44) as the major polyamine.

spermine (343) were not detected in 2002 contained

Alkaliphilic Methanosalsum species grown at pH 9.0

them in the reanalyzed sample in the present study.

in the presence of 1.6-4% NaCl contained homospermine

Two Methanolinea species and four Methanoculleus(44) and/or spermine (343) as the major polyamine

species had conflicting results in regard to the occurrence

whereas halophilic Methanohalophilus species grown atof

and/orpH 7.2 in the presence of 7% NaCl and extremely

spermidine(34),homospermidine

spermine (343) (Table 1).

(44)

Although Methanoculleus

halophilic Methanohalobium species grown at pH 7.4 inchikugoensis JCM 10825 contained spermidine (34) andthe presence of 25% NaCl lacked major polyamines.

spermine (343) in 2002, homospermidine (44) was found

Their polyamine profiles are related to their alkaliphilic

as the major polyamine of the re-culture as well as theand halophilic growth conditions.
51

Journal of Japanese Society for Extremophiles (2018) Vol.17

Methanosaeta thermophila NBRC 101360 grown at
55°C

contained

Mesophiles,

4

N -bis(aminopropyl)spermidine

slightthermophiles

andmoderate

thermophiles were distributed in the acidophilic archaeal

22)(3(3)(3)4) , but the branched tetra-amine was not

order

Thermoplasmatales

grown

at

pH

1.0-3.0.

9)detected in another strain, NBRC 109048, grown at 60°C.

Mesophilic Cuniculiplasma divulgatum analyzed in the

A novel thermophilic methoxydotrophic methanogen,present study contained spermidine (34) as the major

Methermicoccus shengliensis producing methane frompolyamine.

32)

Acidophilic properties of the archaea

andwould not be affected to their major polyamine profiles,

contained spermidine (34) alone, as shown in the present

thus long polyamines have not been found in the order.

study.

ofThe lower limit of optimum growth temperature for theslight/moderate thermophily (45-60°C) for the cellularoccurrence of the significant amount of a tetra-amine,polyamine levels of the thermophiles of this order was

spermine (343), was estimated to be around at 60°C

coal and grown at 55°C is located in this orderWithout

statisticalanalyses,

affectnot clear, however, the occurrence of the branchedwithintetra-amine seems to be advantageous for a high growth

Ferroplasma,

thegeneraCuniculiplasma,

Thermoplasma,

Acidiplasma,

Picrophilus,

Thermo-16,20,22,26,27).

temperature.

gymnomonas of this acidophilic order

Order Methanopyrales (class Methanopyri)

Order Archaeoglobales (class Archaeoglobata)
Five extremely thermophilic Archaeoglobus species

The absence of the long polyamines in Methanopyrus
species grown at 95-100°C

22)

is unique, indicating its

grown

at

70-85°C

ubiquitously

N4-biscontained(3(3)(3)4)

N4-bisprimitive polyamine synthetic ability within the domain(aminopropyl)spermidine

andArchaea.

(aminopropyl)norspermidine (3(3)(3)3) (Table 1).

Theoccurrence of the two-branched penta-amines as the
majorClass Methanonatronarchaeia

polyamines

in

the

genus

of

the

order

Methanogens inhabiting hypersaline environments

Archaeoglobales is unique among the domain Archaea.

have been identified within the order Methanosarcinales.

Relatively lower penta-amine levels in A. infectus15,18), A.

Extremely halophilic, methyl-reducing Methanonatron-

sulfaticallidus20) and A. veneficus, a newly analyzed

archaeum thermophilum was isolated from a hypersaline

Archaeoglobus species, might be due to their low growthlake and its lineage, the class Methanonatronarchaeia, istemperature at 70-75°C.

most closely related to the class Halobacteria in
Archaea42).

Extremely halophilic methanogen grown

Order Thermococcales (class Thermococci)

under slightly thermophilic (at 45°C) and slightly

N4-bis(aminopropyl)spermidine (3(3)(3)4) was foundalkaline (pH 9.0) conditions, likely in halobacteria,

as a major polyamine in the three new marine

contained putrescine (4), spermidine (34) and spermine

Thermococcus species, T. prieurii, T. nautili and T.

(343).

The polyamine component and concentration

eurythermalis, a new marine Pyrococcus species, P.

were similar to those of the extreme-haloarhaea

abyssi, and a new marine species of Palaerococcus, P.

(belonging to the order Halobacteriales) polyamine

pacifica grown at 80°C.

profiles.

(aminopropyl)spermidine (3(3)(3)4) and agmatine (Agm)in

OrdersMethanomassiliicoccales

andneutrophilic

order

and

Thermococcus,

studies15,17,20,23,24,27).

Methanomassiliicoccus

Methanomassiliicoccales

thermophilicorder Thermococcales has been reported in previous

luminyensis4), a novel archaeal methanogen, belongs totheextremely

Pyrococcus and Palaeococcus species belonging to the

Thermo-plasmatales of the class Thermoplasmata
Mesophilic,other

The occurrence of N4-bis

Canavalmine

containedatranticus

20)

(434)

detectedpreviouslyinT.

was also found in four of the newly

homospermidine (44) and spermine (343) as the main

analyzed five species.

The branched penta-amine level

polyamines and spermidine (34) as a minor polyamine.

decreased under a lower salt concentration (1.25% NaCl)

In another order Thermoplasmatales of the class,or a lower growth temperature (65°C) in T. kodakarensis

homospermidine (44) has not been detected.

(Fig. 1, 2 and Table 1), as previously shown in someother Thermococcus species23).
52

Putrescine (4) andJournal of Japanese Society for Extremophiles (2018) Vol.17

agmatine (Agm) productions and a dual biosynthetic

tengchongensis, NBRC 109472 grown at 70°C in thepathway for spermidine (34) from putrescine (4) andpresent study.

This is the first report on the cellularagmatine (Agm) were found in some Thermococcus andoccurrenceaminopropylcadaverine

Pyrococcus species

3,7,8,33)

.

of

in

Archaea.

Since aminopropyltransferases have been characterized
to produce linear triamines and tetra-amines in a

Phylum Crenarchaeota

Sulfolobus species2), aminopropylcadaverine (35) can be

Order Acidilobales of the class Thermoprotei

produced from cadaverine (5).

1,6-Diaminohexane (6)was detected as a minor polyamine in Saccharolobus

Norspermidine (33) and spermidine (34) were foundas the major polyamine, and norspermine (333) andcaldissimus (Table 1).

spermine (343) (and/or thermospermine (334)) were

occurrence of the diamine in archaea.

detected as a minor polyamine in the newly validated

agmatine/cadaverine

extreme thermophile, Acidilobus saccharovorans growncharacterized for polyamine biosynthesis from an

at 80°C, as well as Acidilobus aceticus and Caldisphaera

archaeon Pyrococcus furiosus belonging to the order

lagunensis

22,27)(Table 1.).

The acidothermophiles did

aminopropyl

cellular

Thermococcales,

not produce penta-amines.

This is the first report on the
Althoughtransferase

occurrence

of

was

amino-

propylcadaverine (35) has not been in the archaeon.
Penta-amines

The new strain of archaea belonging to this order was
not available in the present study.

have

not

been

detectedintheacidothermophiles of the order including extreme

Order Desulfurococcales of the class Thermoprotei

thermophiles/hyperthermophiles grown at 80-90°C.

Linear hexa-amines

and/or penta-amines have been found in Aeropyrum,

Order Fervidicoccales of the class Thermoprote

Staphylo-

Fervidicoccus fontis belongs to the acidothermophilic

thermus, Stetteria and Thermodiscus species, but not inorder Fervidicoccales newly validated as the fifth

Desulfurococcus,

Sulfophobococcus,

crenarchaeal order38).

Thermosphaera,

Thermogladius,

Ignicoccus,

Hyperthermus,

species

Caldococcus

Pyrodictium,

in

Ignisphaera,

Pyrolobus

the

order

spermidine

and

other

present

Metallosphaera

study,

sedulaAcidianus

HO615,sp.

RZ1,

IC-006

therefore,

fourordersAcidilobales

andSulfolobales,

Desulforococcales,

Thermoproteales

containing

Metallosphaera
wereOrder Thermoproteales of the class Thermoprotei

available.

Polyamine profiles in the six genera Caldivirga,

Saccharolobus, Sulfodiicoccus and Sulfurisphaera were

Pyrobaculum, Thermocladium, Thermofilum, Thermo-

proposed 39,40,47 ) as a new genus in the order.

Four

proteus and Vulcanisaeta, analyzed in our previous

former Sulfolobus species have been reclassified.

The

reports14,17,22,24,27) and the present study are shown in

Sulfolobus

sp.

(343),

spermidine (34) and spermine (343).

tengchongensis NBRC 109472, Sulfolobus solfataricus
98/2,

spermine

norspermidine (33) and norspermine (333) in addition toOrder Sulfolobales of the class Thermoprotei
the

and

distinguished from other thermophiles belonging to the

Desulfuro-

coccales14,17,22,23), as cited in the Table 1.

In

(34)

The acidothermophile contained

eight new strains ubiquitously contained norspermidine

Table 1.

(33), spermidine (34), and norspermine (333) as thethe present study, the mass culture of the four strains was

major polyamines (Table 1).

difficult and their growth was poor, so that theMetallosphaera,

Therefore, Acidianus,

Saccharolobus,

Although four strains were newly analyzed inpolyamine data shown as a reference in Table 1 were

Sulfurisphaera,

Sulfolobus, Sulfodiicoccus and Stygiolobus species

insufficient.

grown at 65-90°C have similar polyamine profiles (Table

norspermidine (33), spermidine (34), norspermine (333),

1).

spermine

Thermospermine (334) was found as a major

polyamine in Acidianus sp. RZ1.

Spermine (343) was

1,3-Diaminopropane (3), putrescine (4)

(343)

and

widespread in the order.

thermospermine

(334)

were

However, a major occurrence

found as a major polyamine in some Metallosphaera andof long polyamines is impossible in the extremely/hyper-

Sulfurisphaera species.

thermophilic order.

Among them, cadaverine (5) and aminopropylcadaverine (35) were detected in Metallosphaera
53

Journal of Japanese Society for Extremophiles (2018) Vol.17

Phylum Thaumarchaeota

Pyrobaculum belonging to the order Thermoproteales

Orders Nitrososphaerales and Nitrosopumilales of thehas been reported31).
Ammonia-oxidizingclass Nitrososphaeria

Nitrosopumilus belonging to the third archaeal phylum

Slightly thermophilic ammonia-oxidizing Nitrososphaera viennensis42) and Nitrososphaera gargensis28),

Thaumarchaeota

were isolated from garden soil from Austria and a hothomospermidine (44) alone.

spring in Austria, respectively, and grown at optimum

thaumarchaeal extremophiles is in waiting.

42-45°C.

and

Nitrososphaeracontained

putrescine

(4)

and

Pure culture of otherAmmonia-oxidizing Nitrosopumilus adria-

Strictly halophilic thermophile and strictly alkaliphilic

ticus and Nitrosopumilus piranensis growing at 30°C

thermophile have never been isolated in archaea.

were isolated from coastal surface water sampled off

Anaerobic methanotrophic (methane-oxidizing) archaea

1)

Piran, Slovenia .

They contained putrescine (4) andbelonging

to

the

order

Methanomicrobiales

or

46)

and

obligateorsymbiotic

homospermidine (44) alone by the HPLC (Fig. 1) andMethanosarcinales

HPGC analyses.

uncultivated archaea located in the phyla Nanoarchaeota,

Usual triamine, spermidine (34) andtetra-amines were not detected in the nitrite archaea.

Korarchaeota,

Aigarchaeota,

andLokiarchaeota

5,35)

have never been

Same polyamine profile has been found in somebelonging to five different lineages

mesophilic methanogens belonging to the genera

available as pure culture for our polyamine studies.

Methanolinea, Methanoclleus, Methanoplanes, Methanocella, Methanococcus and Methanomethylovorans within

Author contributions

Archaea (Table 1). Nitrosarchaeum koreense, anThis study was carried out as the joint research project

30)“Polyamine distribution profiles as a chemotaxonomic

ammonia-oxidizing thaumarchaeon, isolated in Korea
has never been available to our study.

marker in Bacteria and Archaea” between Maebashi
Institute of Technology (Koei Hamana and Hidenori
Hayashi)Conclusion and FutureIneuryarchaea,branched

penta-amines

were

and

JCM, RIKEN

BioResource(Takashi Itoh and Moriya Ohkuma).

Center

Masaki Kobayashi,ubiquitously distributed within the thermophilic orders

Takemitsu Furuchi and Masaru Niitsu of Josa University

Archaeoglobales and Thermococcales and extremely

participated in the HPGC and HPGC-MS analyses ofthermophilic methanogenic genera Methanocaldococcus

polyamines.

and Methanotorris of the order Methanococcales but notinthehyperthermophilic

methanogenic

genus

Acknowledgements

Methanopyrus of the order Methanopyrales, three

We thank Dr. Koji Mori and Dr. Yoshihito Uchino ofmesophilic/slightly thermophilic methanogenic orders

NBRC for their advices for the culture of NBRC strains.

andWe thank also Dr. Gael Erauso of Universite de Bretagne

Methanomicrobiales,

Methanocellales

Methanosarcinales and the acidophilic (mesophilic/

Occidentale,

Plouzane,France(thedepositorof

6)

moderately thermophilic) order Thermoplasmatales.

Pyrococcus abyssi JCM 18155 ), Dr. Wagner, D. of The

Methanogens are distributed in several diverse lineages

Institute for Polar and Marine Research, Potsdam,

after evolution in Archaea under various growth

Germany (the depositor of Methanobacterium movilense

environments and have been shown in various polyamine

JCM 1847041)), and Dr. Blum, P. of University of

distribution types.

Nebraska-Lincoln, USA (the depositor of Sulfolobus
solfataricus 98/2 JCM 30930).

In crenarchaea, branched penta-amines have not been
detected in the two thermophilic orders Thermoproteales
and Desulfurococcales and in the two thermoacidophilic

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43) Stieglmeier, M., Klingl,A., Alves, R.J., Rittmann,

Identification, chemical synthesis, and biological

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Polyamine analysis of extremely halophilic archae-anaerobic, thermophilic crenarchaeote from terrestrial

bacteria and methanogenic archaebacteria. Ann.

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and Oshima, T. 2005. Stabilization of nucleic acids

39) Sakai, H.D. and Kurosawa, N. 2017. Sulfodiicoccus

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archaeon isolated from Indonesian hot spring, andInt. J. Syst. Evol. Microbiol. 68: 1271-1278.

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W., Hellebrand-Voiculesco, A.,and Wagner, D. 2014.
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Uchiyama, T., Iino, T., Hosoyama, A., Ataku, H.,from the anoxic sediment of a subsurface lake. Int. J.

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Harayama, S. 2010. Iron-corroding methanogensaline lakes comprising a new euryarchaeal classisolated from a crude-oil storage tank. App. Environ.

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Journal of Japanese Society for Extremophiles (2018) Vol.17

MINI REVIEWJournal of Japanese Society for Extremophiles (2018) Vol.17, 58-61

Nunoura T a, Chikaraishi Yb and Atomi Hc

A primordial and reversible TCA cycle in a thermophilic bacteriumǘ ͫϹэ0eQźӒΛNǚũΛ TCA ƢҷQΗѮ̵̼βώԍΗ̈̀·̵̼;ƎͼȚǥβώԍΗµ¸ař̼Ӡǌǥì͇σǥβώɳ bÖӱǌǥȚǥӮ cȡ̻ ʄӭ a·ňΰ Ɯ× b·ҶѮ ˊȭ caResearch Center for Bioscience and Nanoscience (CeBN) , Japan Agency for Marine-Earth Science & Technology(JAMSTEC), b. Hokkaido University, c. Kyoto University
Corresponding author: Takuro Nunoura, takuron@jamstec.go.jp
Phone: +81-46-867-9707, FAX +81-46-867-9715
Received: Jan. 8, 2019 / Revised: Feb. 18, 2019 / Accepted: Feb. 18, 2019
TCA Ƣҷ(tricarboxylic acid Ƣҷ"+g,RzsµӶƢҷ)QǊ́ɚ TCA ƢҷORĦƲöLͼѴ:hJ2DӶŘΛ
TCA Ƣҷ՞ƧդA՟QÛ"ĴȐƲ՞ƧդB՟"͢ӶƨǭÖЭ>gӫęƲ՞ƧդC՟"B<J¹Ӯ1̉ʜ>g¿ǩĠNϽҷapϽҷ`ήehg#\D"Ƣҷkʂ-űɕQ̕MORұ͏QΌNgѩʬQӴ϶
1ǣƮ>g#H\f"TCA ƢҷOR̕MQ;ͯOñ
e0QɀK̈Э>gՐ,þǣɚQ¹ʶK"BhLΣ
ű>gǊ́ɚ"ˮһɚ1Ѯehg¬¸zN
§K+g#8Q TCA ƢҷQɄņL<JӶŘΛƢҷK
Rs±y¸;Ϳ1"ӫęΛ TCA ƢҷKR͢Ӷƨǭ

1Ⱦғ:hg1"ĠJQƲOĤӕ>gɄņL<J%n¦Ӷa˴ӶϜQłՌðLNgÄԎÝҚͯQ;Ϳ&1ʋ7ehg#B<J"8QĤӕ<DɄņL§L<JQՐ,þǣɚ1ʦO TCA ƢҷQұ͏RŘǥәŘQˆÝO\KӥgźЭɚ1ҜҖ:h 3՟"\D"ӊȬ"Ԯ;ͯǥΛO TCA Ƣҷ1;=-g8L1Σ̊,KƻƊ:hJ,g 5,8՟#ÄK`"ӫęΛ TCA Ƣҷ՞ƧդC՟R"¨µэaӳӶэ1˗>gWood-Ljungdahl Ͻҷ՞n± CoA Ͻҷ՟LÃV"˕`ǚũΛN͢϶ƨǭϽҷK+gLС.ehJ2D1)#Ƨ 1 ʹήQǩĠƲ TCA Ƣҷ՞ӶŘƲ-A·ĴȐƲ-B·ӫęƲ-C՟L Thermosulfidibacter takaii 0eѮĳ:hD
ĴȐƲ TCA Ƣҷ-D#
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Journal of Japanese Society for Extremophiles (2018) Vol.17

 ¹ʶ"ͫňǥQѰͣ0e TCA ƢҷQǊ́ɚkΨ
_gL"˕`ǨǭNÚϼ]R"d4ήehgӶŘΛ
TCA Ƣҷ՞ƧդA՟KRN4"йΗΛNűɕ՞Ηs±µűɕ՟Q]0eNgĴȐƲ TCA ƢҷQ]K+g՞ƧդB՟#ӶŘΛ TCA Ƣҷ`"˾ϕNԮйΗΛűɕ՞ƈs±µűɕ՟K+g°µӶв̢϶Ӵ϶
՞dG0’ = +30 kJ՟kƇ]"ӫęΛƢҷKR"±µӶr²xµӶŘӫęӴ϶՞±µӶƁɯӴ϶՟ŮV 2-ux{±±Ӷ(-|{±±Ӷ)r²xµӶŘӫęӴ϶՞2-ux{±±ӶƁɯӴ϶՟՞,?h`dG0’ = +19 kJ՟1ƈs±µűɕK̈Э>g 2՟#\D"ӫęΛ TCA ƢҷkͰɐÜ6
gL:hJ2D ATP zsµӶ°n¸՞+g,R°± CoA ƁɯӴ϶L°± CoA °n¸Qϼ]Ɓj@՟՞dG0’ = +4 kJ՟QǣƮ`"ͫňǥΛѰͣ0eҏʽ:hJ2D#NANe"ӶŘΛ TCA ƢҷaĴȐƲ TCA ƢҷO/6gzsµӶƁɯӴ϶Odg
zsµӶƁɯűɕR˾_JйΗΛNűɕ՞δG0 =-37.6 kJ՟4՟K+gD_"Ӓűɕ1;ðĨKұ2gL
RС.ehJ/e?"ǰԡO"ʹήQӫęΛ TCA ƢҷKR ATP Qs±y¸kĻ΀>g8heQÚϼ]1Ѯĳ:hJ,D0eK+g#ѣűɕ՞zsµӶƁɯӴ϶QӒűɕ՟Q̷̟ɚ՞ux³ӳӶ;Ϳ̷ɚL<J͈ǭ՟1ʹήQӫęΛ
TCA Ƣҷk˗>g Aquificae ԋϹэQ ATP øǣΛzsµӶԍѣűɕQ̷̟ɚLZ[ƃϜK+g8L"¹
ʶ ATP ԮøǣΛNzsµӶԍѣűɕ1 ATP ̓ŊO
dGJüә:hN,8L1κ:hD#B<J"ӶŘʶƅKQ̷ɚ͈ǭKR"ȧOԮȧOȾ,zsµӶƁɯӴ϶̷ɚ1ǣƮ>g8L1ʽe0LNGD#8Q
8LR"ӶŘΛ TCA ƢҷaĴȐƲ TCA Ƣҷ1ʹή
Q͢϶ƨǭϽҷLĤǣ<JzsµӶƁɯӴ϶1zsµӶƁɯÖЭ>gLßǭ<DƼƁ"zsµӶƁɯӴ϶L͢϶ƨǭϽҷOƇ\hgn± CoA kƷҭL>gӴ϶՞ö.S"v±£µӶ/ 4-³x
±ӶƢҷKR±µӶr²xµӶŘӫęӴ϶"3-³x³uµӶ/4-³x
±ӶƢҷKRn± CoA v±£x¯¸՟QԎKƷҭϖƁ1;=g1"Ⱦ,zsµӶƁɯӴ϶̷ɚQǣƮR"n± CoA kÄԎÝҚͯL>gʹήQ͢϶ƨǭϽҷQ̈ЭkԒǴ>gźЭɚ1˾_JՐ,8Lkκ>#8he¹ӘQϾ˫R"Ɋ˥QȧқL
ű<"zsµӶƁɯӴ϶QӒűɕOdg ATP ԮøǣΛNzsµӶԍѣűɕkƇ^ӫęΛ TCA Ƣҷ1 T.
takaii O/,J̈Э<J,gźЭɚ1Ր,8Lkɤ
ƌ<J,g#<0<N1e"Ă$QӴ϶̷ɚ͈ǭaөæǡΗͺQη҉Q]KR"BhChQƶՉˤáO/6g TCA ƢҷQƢҼʶƅķǭI6g8LRĳ
˥N,# B8Kɰ$RʵDOԍΗ<DÝҚѴ˨ɴ̰kȁğ<"BhChQƶՉˤáO/6g TCA ƢҷQƢҼʶƅQѴʽk҂]D#8Qʵɴ̰R 109 ϹЬO͎DN,ȃӽQϹЬKǰʷźЭK+f"Ɋ˥Q NMR a
HPLC ϜOdgÝҚѴ˨KRӦ΀K2N0GDǁ̘ͷQì,ͽǀɎ;ͯO`ÝҚѴ˨1Ӧ΀źЭLNg#
N/"ǨǭƃëðƷҭk΀,gƶՉϜ"BQǰՍQ
ǌ\0N̹hR¾ѻQӕf"ʹǣQÝҚѴ˨ɴ̰Lƃ́K+f"TCA ƢҷOdf;Ϳ:hDÄԎÝҚͯ
kłՌðL>gn¦ӶQĴǡĨ̂қkқĺ>g8
LOdf"TCA ƢҷQűɕʶƅkηǭ:@g#8Q
ɴ̰kͰɐÜ6gQRʵ<4ԍΗ:hDqrn MassWorks QĻ΀K+g#(1)Ǩǭƃëð̂қ:hDӻ͢Ӷ+g,R˗̈ӶkƷҭL<DƶՉ(2)ĠэðљΙҭQŊ̢ĴѴLn¦ӶQҋȁðŘ(3)GC/MS OdgĴ˨(4)qrn MassWorks Odgƃëð̂қͷQϟĳ(5)TCA ƢҷQűɕʶƅQηǭ Thermosulfidibacter takaii O / 6 g ͢ ϶ ƨ ǭ ϽҷQʔϷ ǚũΛN Bacteria ϭЂМ Aquificae ԋOӊБN
Thermosulfidibacter takaii R"̢϶ӶŘζ՘ӫęOd
gs±y¸ͶɋkɓԸL>gǠ̡ɚǘͫэ՞лӦǁ̘͇ȵ 70C՟K+f"Ͳϒ˰Չˤá¾E6KN4
Ӵ̝sxa˗̈Ӷk͢϶͏L>ǵƁ˰ՉƶƯÄK`сǘNǁ̘kκ> 9)#Aquificae ԋͲϒ˰ՉϹэLƃ́"˟эRͲϒ˰Չǁ̘ˆORӫęΛ TCA Ƣҷ
k΀,g8L1Îɣ:hJ,D#<0<"}§Ѵ
˨QϾ˫R"˟э1ǩĠN TCA ƢҷԏӘөæǡk˗
>¹ʶ"ӫęΛ TCA ƢҷO¿ź̉L:hJ2D ATP
zsµӶ°n¸aʹήQÝ˔̈̀՞°± CoAƁɯӴ϶·°± CoA °n¸Q 2 ̙ԝűɕ՟k~¸>gөæǡkþʈ<N,8L1ʽe0LNG
D#\D"ӫęΛ TCA ƢҷÞǉQʹή͢ӶƨǭϽҷk˗>gźЭɚ`˾_Jì,8L`κ:hD#B8
K"T. takaii 1ʹήQ ATP zsµӶ°n¸LRΌ
Ng̈̀KzsµӶԍѣűɕkў,"ӫęΛ TCA Ƣҷk̈Э:@J,gźЭɚk˼Ҁ>gD_"ϹЬʁĳ̾QӴ϶̷ɚ͈ǭ"¯µz°¸§"³u¸§Ѵ˨kǰʷ<D#8he¹ӘQѴ˨Ͼ˫R"Ͳϒ˰Չ"́Ɓ˰ՉQñhO/,J`ɜȧΛO TCAƢҷ1Ηͺ>g8LkκƔ<D#ͰOӫęʶƅKQӴ϶̷ɚ͈ǭO/,J"ATP ԮøǣΛNzsµӶԍ

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Journal of Japanese Society for Extremophiles (2018) Vol.17

Ƨե Thermosulfidibacter takaii O/6gǨǭƃëð̂қƷҭk΀,DÝҚѴ˨Qǰԡ#ҮQͣЎR"³u¸§KQΗͺ1η҉:hJ,g1"ÝҚѴ˨KRźѰŘ:hN0GDϽҷ#ԬQͣЎR"³u¸§KQΗͺ1η҉:hJ,g1"ͼҖΛO˟ÝҚѴ˨0eRη҉K2N,Ͻҷ#ˤáKR"Ʒҭ̞OͰΌΛNĴȐƲ TCA Ƣҷ1̈Э>g"(c)~zӶLӳӶQÂʶk̓Ŋ<DˤáKR"
TCA Ƣҷ
ҷRn³²ozϽҷkƇ_JӶŘΛÖЭ>g8Lkʽe0O<D(Ƨե)#\D"Ӵ̝sxQ]k˗̈ͯL<J̓Ŋ<DˤáK`"
TCA ƢҷRӶŘΛÖЭ<"~zӶLӳӶQÂʶk̓Ŋ<DƼƁLƃ́"͢ӶƨǭkўjN,8L1

 ˟βώKR"±µӶ"ux³ӳӶ"ux{±±ӶQĴǡĨƃëð̂қkѴ˨>gD_"T.
takaii Q}§ɡƻ0eƃǭ<Dn¦Ӷ;ƁɯϽҷOƷI2"BhChn¯µ"n¯yµӶ"{±¦µӶQƃëð̂қͷkѴ˨<D#BQϾ˫"(a)Ͳϒ˰Չǁ̘ˆO/,JӫęΛ TCA Ƣҷ1̈Э>g"(b)~zӶ+g,RӳӶk̓Ŋ<D́Ɓ˰Չ
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Journal of Japanese Society for Extremophiles (2018) Vol.17

κ:hD# ˒O˟βώKR"T. takaii QzsµӶƁɯӴ϶kǌжэKΗͺ<JBQӴ϶ǥΛɚͱkѴ˨<D#B
QϾ˫"ÝҚѴ˨ϜQǰՍϾ˫1κ>ӕf"ʹήQ
ATP zsµӶ°n¸Lƃ́OzsµӶOǼ<JՐ,ƷҭѱƏɚkκ>8Lkκ<D#8Q8LRÝҚѴ˨Ϝ1κ>;ðĨKQzsµӶԍѣűɕO"zsµӶƁɯӴ϶1Ӧ<DӴ϶ǥΛɚͱk˗>8Lkʡ
ʈ>g`QK+GD#\D"zsµӶƁɯӴ϶"ATPzsµӶ°n¸"°± CoA °n¸QәŘΛԏûOH,J`˼ѹ<D#8QϾ˫"zsµӶƁɯӴ϶Qұ͏ķǭ>g8LRƥԧK+g`QQ"zsµӶƁɯӴ϶0e°± CoA °n¸1Ҋ;<"BQɉ"ATP zsµӶ°n¸1Ҋ;<D8L
1κ:hD#8Q8LR"ӫęΛ TCA Ƣҷ1әŘQĸ˛OҊ;<DQK+hS"BhRʹήQ ATP øǣΛNÚϼ]KRN4"˟βώKκ:hDd-NzsµӶƁɯӴ϶QӒűɕOøǣ<J,DźЭɚ1Ր,
8LkκƔ>g# ¹ӘQѴ˨Ͼ˫R"T. takaii 1"zsµӶƁɯӴ϶kƇ^Ġ4ƃ=Ӵ϶k΀,gO`ԏje?"ƢҷOųfӈ\hgƷҭQψՂLӽOɕ=JƢҼʶƅ1
Ǉő>gźӒΛ TCA Ƣҷk˗>g8Lkκ>#\D"˟ β ώ L ƃ ˆ O Deltaproteobacteria O ȍ > g
Desulfurella 0eѮ,E:hDƃ́QźӒΛ TCA ƢҷKR"˟эLRΌNf"ATP zsµӶ°n¸өæǡ1ǂhDɉO"zsµӶƁɯӴ϶1BQÝ˔̈
̀L<J̈Э>g8LK"ӫęΛ TCA ƢҷQ̈Э1
Ƣɍ<D8L1κ:hD 7՟#8heQβώϾ˫R8
h\KQήѮLƁj@"źӒΛűɕQәўkźЭL>gÚϼ]1ǣƮ>gԕf"TCA Ƣҷ1Ɋ˥ήeh
J,Ddf`ӣ0OˮһK+f"Ă$QӴ϶űɕE
6KN4"ƢҷĠð1źӒΛK+g8Lkκ<J,
g#ͰO"źӒΛ TCA Ƣҷ+g,RӫęΛ TCA Ƣҷ1;=gOR"ԮйΗΛűɕK+g POR a OGORQӫęʶƅQűɕkʡ.g Electron bifurcation Od
GJӫęƲr²xµkĚĴӽ÷Ё>gs±y¸ÝҚLQӘʝ1¿ź̉K+g#; ƎRӕɚ́Ɓ˰Չ;ͯL<JҊ;<Dկ
 ;Ǝ1Ɋȍ˰Չ;ͯL<JҊ;<DQ0"+g,RͲϒ˰Չ;ͯL<JҊ;<DQ0"Ԋ,ҖÏ1Ѕ
,J,g 6)#;ƎQҊ;OR˗̈ͯQ͜Ԥ1ɓѬK+g8L0e"Ɋȍ˰ՉɚͯL<JQҊ;RйͧN
d-Oə.g1"BQƼƁ";Ǝ̷őйðOdg˗̈ͯ̽ҧOdf";Ǝ̷őйð1Ѓ."̤ЅɚOƗ

61

Ծ1+gLʉʞ:hg#\D"ӒO"Ͳϒ˰Չ;Ǝ
L<JQҊ;OR"NAԤϋ<D˗̈ͯk;Ǝ1Ļ
΀<N,Q0կL,-ΏƗ1Ɖ:hJ2DQK+g#
B8K"ØƢʽe0O:hDTCA ƢҷQ˟ҭΛNźӒɚRǌ2NɤƌkʈH#˴Ӷan¦ӶϜQ;ƁɯQұͣLNg TCA Ƣҷ1ŘǥәŘQˆÝ0
eǣƮ<"\D"BQͽǀ1̢ͫͽǀQd-O"ͺǣQ;ƎO/6gs±y¸ÝҚOΣȿ>gŘǥΛ+g,RԪ̡ΛNs±y¸÷Ёaͥ̈͢϶1ĚĴ
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Journal of Japanese Society for Extremophiles (2018) Vol.17

Journal of Japanese Society for Extremophiles (2018) Vol.17, 62-68

MINI REVIEW

Kuruma Y

Study of Origin of Life through construction of an artificial cell,
focusing on the membraneз OͦͣkИ,D×ȚϹЬQ̀ϣL;ƎQұ͏βώ˦ÖȚ˽ǌǥ Ưͻ;ƎβώɳҺ ġ͘
Earth-Life Science Institute, Tokyo Institute of Technology, 2-12-1 Ookayama, Meguroku, Tokyo 152-8550, JapanJST PRESTO, Japan
Corresponding Author: Yutetsu Kuruma, kuruma@elsi.jp
Phone: +81-3-5734-2708, FAX: +81-3-5734-3416
Received: Feb. 16, 2019 / Revised: Feb. 21, 2019 / Accepted: Feb. 21, 2019
Abstract The first terrestrial cellular organism isthought to have emerged in the early earth environment
during the Hadean era 4.0-4.1 Gyr ago. Protocells, which
consist of a simple fatty acid membrane andself-reproducing RNA, might have emerged throughchemical evolution and further evolved into the last
universal common ancestor (LUCA). However, we still
do not understand what cellular functions the protocells
acquired on the way as they evolved into the LUCA. For
this question, artificial cell research is trying to find an
answer through reproducing the process of the birth oflife in the laboratory. Although the first man-made cellwould be a simple, containing only the necessarycomponents and genes, such a simple life form could bealmost identical to the first life form that emerged in past.
Additionally, we will have a chance to define theminimal essential genes and functions necessary forcellular life and to constrain the chemical or physicalenvironmental conditions allowing for the emergence oflife. An artificial cell can be constructed byencapsulating a cell-free protein synthesis system andexpressing genes inside lipid vesicles. Based on thistechnique, we could develop an artificial cell into a realliving cell by introducing a designed artificial genomethat consists of all the necessary genes. One of the big
problems for artificial cell construction is, however,reproduction of the cell membrane. Here, we show asynthetic biological approach imparting the properties ofself-growth and self-division to the artificial cell bysynthesizing new lipids inside the vesicle.
Key words: artificial cell, origin of life, vesicle, cell-freesystem, membrane
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62

Journal of Japanese Society for Extremophiles (2018) Vol.17

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Journal of Japanese Society for Extremophiles (2018) Vol.17

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Journal of Japanese Society for Extremophiles (2018) Vol.17

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Journal of Japanese Society for Extremophiles (2018) Vol.17

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67

Journal of Japanese Society for Extremophiles (2018) Vol.17

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ŬСʯʹ
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Journal of Japanese Society for Extremophiles (2018) Vol.17

MINI REVIEWJournal of Japanese Society for Extremophiles (2018) Vol.17, 69-76

Ishida A.

Geochemical studies on origin and evolution of life:
evidence of life in Archaean era and their frontier researches;ƎQұ͏LәŘQƯͻŘǥխǎŶÝQ;ƎQΓҶk_5gβώLӊȬQβώOH,J˦řǌǥ ͼǥβώσ ƯǥǾʣΰ΁ ϔϳ
Department of Earth Science, Tohoku University, Aoba-ku Aramaki Aza Aoba 6-3, Sendai, Miyagi, 9808578 Japan.
Corresponding author: Akizumi Ishida, ishidaz@tohoku.ac.jp
Phone & FAX: +81-22-795-5789
Received: Dec. 27, 2018 / Revised: Jan. 15, 2019 / Accepted: Jan. 17, 2019

Key words: Microfossil, Archean, Secondary Ion Mass
Spectrometry, Carbon stable isotope composition,
metabolism

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;Ǝ+g,RBQΓҶ0eũǚ;Ǝkʖǭ>gɴ̰;ƎLŘΰQβώ +gŶ,ˆÝO;Ǝ1ǣƮ<D8Lk˕`΢ʕΛOҀʽK2gҀʆQ¹H1ŘΰK+gաnµ©paɛϓQՏՠǉΝՠ˻ͯQһϼЖNMЧΩK`ѲǹźЭNՠ,jbg“Řΰ”RՠŶÝy°nՠn°
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ɵjhJ2Dա1800 ȬÝO Charles Darwin ŢǄaՠ

Abstract
Microfossils, graphite and organic matter inthe Archean sedimentary rocks, which are direct
evidence of life on the early Earth, have been reported byprevious studies. However, as the sedimentary age getsolder, those evidence have been deformed, destroyed andlost, leading “mislead” with abiotic signals. In thisreview paper, research history about evidence of life on
the early Earth are reviewed from the point of geoscience,
including geology, mineralogy, isotope geochemistry,and organic geochemistry. Recent studies of microscalemeasurements, which are suitable for searching meager
evidence in the old rocks, are also reviewed.

69

Journal of Japanese Society for Extremophiles (2018) Vol.17

Charles Lyell ŢǄ՜1 NM¹ӮQйͧσǥТӡRΏƗkʀ2HH`ՠѲǹɴ̰aѤИQԕΈ`+fՠŘΰ1̗
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h 10μm70

Journal of Japanese Society for Extremophiles (2018) Vol.17

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Fischer-Tropsch ƲűɕLƍShg¹ӶŘ͢϶L̢϶
0e͢Ř̢϶kƁɯ>gűɕKƁɯ1źЭK+g8
L1QFOκ:hDա8hOǼ<ՠSchopf ŢǄ`¯¥µĴĜĴ˨a͢϶ƃëð̟Ĵ˨NM;ͯO΂˥>gʬ$QҀʆkʙκ<J,g1ՠͺƮOлg\KҜ
ҖO̧ΪRÜ,J,N,ա8Q˕ŶQŘΰk_5g
ҜҖOԏ<JRǌů(2016)aäѯԸ(2016)K҆<4
Ѵҏ:hJ,gա ӊȬՠ8hk˒ʵ>gϯ 38 ĖȬłQȒΰ0e«¸ͱɎŘΰ1ƻƊ:hJ,g 4)աɀͱLȒΰQͿ
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Journal of Japanese Society for Extremophiles (2018) Vol.17ͱ0eՠ̼ȳ̢ͫƠĳŵӊčO;ɝ<J,Dñe0
QɎ;ͯLʖ͈:hJ,gա<0<8QΗѮ`ӟūQƯҭɡƻLQάΤ1ʉʞ:hJ/fՠʖ͈Qƴk
ĳJ,N,աƄŶȊǌǥQˠҝĊ¹ӭŢǄRu¸¯°nQϯ 34 ĖȬłQ Strelley Pool Ȏ0e²µͱQɎŘΰkΗѮ<ՠɉӍ>gÑ̊puµҭӽĴ˨ѸNMk΀,JϫňΛOĴ˨<J,g 3) 23)աͺƮQ
L8iˠҝŢǄQΗѮkϋ˾ΛOƆǭ>gҀʆRͥ
4ՠØɉQȌԍ1˛ɇ:hgա ɎŘΰLL`O˗ŎNҀʆQ¹HL:hJ,gQ
1͢϶aϑ϶QǨǭƃëð̟(δ13C, δ15N)ćK+g#3ա
;ͯ1͢϶aϑ϶Qƨǭkў-ԡ"ҽ,ƃëðkӨɾΛO͜В>g8L1ήehJ,gա͢϶QƼƁR;ͯψ9LQ͢϶ƨǭϽҷQӢ,1űˁ:hgD_ՠδ13C ćOdg;ͯψQʖǭ1źЭK+g 19) (Ƨ 2)ա
46T#_&%R I[2XKS 7:
S<A^ I[FPCY_@[2S&%* PQ[ 22)`HLDMN_0D"G\NAQAOW]JCR"G\L!(X'6^OE\U+ S.9QZB[PHN>?QS0!(^8
R13DQG\NA[`#3: δ13C R͢϶QǨǭƃëð 12C, 13C Q̟k΀,J̊Qd-Oѡ:hgաδ13C = (Rsample / Rstandard – 1 ) * 1000, R = 13C/ 12CաR҂ʳ/dV̂͐҂ʳQ 13C/ 12C ̟kκ>ա
Rsample, Rstandardՠδ15N `ƃ́Oѡ:hՠδ15N = (Rsample / Rstandard – 1 ) * 1000, R =15
N/ 14N KǭН:hgա δ13C ćQ]Kʡʈ:hg˕`Ŷ,;ƎQΓҶRՠϯ 41 ĖȬQȬÝkκ>±~µÄOՠ{¯mp
L<JƻƊ:hJ,g 1)աѐТeR"ƯѡO+GD
˗̈ͯ1zzOdfƯ¾̀4O͗fӈ]Č
ͧ±~µOųfӈ\hDLѴӹ<J,g1ՠՐ͇
Kɀɯ:hgR?Q±~µOBQd-NΓҶ1̗
gQ0ɭΏΛNɶĹ1Ǌ4ՠ҂ʳóɯQԡQ~µ¦¸­µQźЭɚ1ΏjhJ,g.

Ƨ 3 ϯ 38 ĖȬłQ{°¸µ¯µ·pnƯƴQғ˯ĭΧLȒΰ҂ʳա(a) pnƯƴRȬẠ̈ԨOѭjhJ,Jՠί,ǈQԎOQ]Ȓΰ1ԫĳ<ғ˯1źЭK+gաĭΧQǔO
Ṛ̪1Ѯ.gա(b) ˦řǌǥQβώ{±¸1ʵDOΗѮ<DՙтͬȒQԫԻաϯ 38 ĖȬłQ;ƎQΓҶ1̗gաa,b QĭΧQ×ͯRʒȘ̏ŢǄա(c) ʓų<DȒΰաՙ,ӮĴO͢϶1͜Ԥ<J,gա˦řǌǥЋƁǥџŢͯՊOŲђ·Ȍκ:hJ,gա(d) c QՀɎԉĭΧաΰчaϩƭԂͯOdgȎͱ̀ӗ1Ѯehgաՙ,ӮĴO͢϶1ǣƮ<ՠ;ͯ΂˥Qҽ,͢϶ƃëð̟ć1Ѳ͈:hDա
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Journal of Japanese Society for Extremophiles (2018) Vol.17¹ʶKՠƺῢӗa δ13C ćOŊ."ӓӟƲԪǡՀɎԉ(TEM)Odg͢϶QϾˋ̀ӗѴ˨NMǊʶԯQĴ˨aҁùOУ."˕ŶQ;ƎQΓҶL<J¹пO҉қ:hJ,gQR{°¸µ¯µ"Isua ƯƴOͿ>gϯ 38 ĖȬłQƺϋȒÄQ{¯mpK+g
18).Isua Ưƴ՞ͺƯQѶяK“ƯQ˫J”kɤƌ>g՟KRՠ˕ŶQ;ƎQΓҶkʔgβώ1ΞlOўjh
J2D1ՠ͟ȏɚԂͯаkɀɯ>g FeCO3՞¯p՟QĴѴOdgͥ̈ΛN{¯mpQ̨̛k
ǚ_L<JՠB`B`ƺϋȒQƃǭQƗԾNM`+fՠΗѮ:hg{¯mpQZLlM1ͥ̈ΛNɯƣkʉʞ:hJ,g 13) 20)աB-<DÄՠ1999 ȬOµ¥¸z͹ϒβώɳQ Minik Rosing ŢǄ1ʽηN
ƺῢӗLBQȎÄO{¯mpQϨǡkΗѮ<ՠδ13C ć0e`H,O;ͯұ͏K+gLϾҖI6D 18)աǌůԛǡŢǄe˦řǌǥQβώ{±¸RՠIsua ƯƴKQƯҭғ˯kϫňΛOў,ՠʵDOΗѮ<DƺϋȎÄO;ƎQΓҶkΗѮ<D 17) ՞Ƨ 3՟ա8Qβ
ώKRɊ˥Q δ13C Ĵ˨OLM\e?ՠ¯¥µĴĜĴ˨OdgͫǇɚȵQҁùՠͥ̈\DR;ͯΛN{¯mpɀɯˆQ δ13C ĴĺŎ˫QѮϋϜՠѩƁΛNСǹOdgұ͏Qʖǭ1ҁù:hJ,gա:eOՠ
TEM Odg|¸±Q{¯mpϾˋ̀ӗѴ˨kў,ՠ;ͯұ͏{¯mpRͥ̈ұ͏QB
hL̟Ҿ<J͢϶Ͼˋ̀ӗȎ]ảԘ1]ehg
8LkʵDNҀʆL<Jʙκ<DաIsua ƯƴO/6g;ƎQΓҶQ˕ʵβώOԏ<JRǌů(2016)O҆Ϲ1ϺÙ:hJ,gա :eOӊȬKRȂǳŃŢǄe˦ÖǌǥQβώ{±¸OdfvՠLabrador Ưƴ0eϯ 40 ĖȬQ
ȬÝkκ>ƺϋȎÄO δ13C ćQì,{¯mp
1ƻƊ:hJ,g 25)ա8QβώK`ƺῢӗQȬÝ͈ǭՠĤǣԂͯQƃǭa˗̈ͯQǇɯȵOdg δ13C
ĴĺQѮϋ`fNMOŊ.ՠɉӍQÑ̊puµҭӽĴ˨ѸOdg{¯mpQ δ13C ćĴ˨`ў-NMՠΗѮ:hD{¯mp1;ͯұ͏K+g8LkѩƁΛҀʆ0eʡʈ<J,gաͺƮQL8iՠ˕
Ŷ;ƎQΓҶQ˗ňNąѧK+g1ՠ>KOɶĹΛNɤѮ`ĳJ/fՠØɉQҜҖOУ.gɓѬ1+gա
ϯ 38 ĖȬłQ Isua aϯ 40 ĖȬłQ Labrador KΗѮ:hD{¯mpQ δ13C ć1ՠӫęΛn±
CoA Ͻҷav±µƢҷOdgÝҚĴĺkκ>ϢƦOğg8LRՠȿˆBheÝҚϭk˗>g;Ǝ1ǣƮ<DҀʆLҐ]˔.g8L1K2g(Ƨ 2)ա<0<ՠ
ö.SB-<DˆÝQĜƁɯ1ՠͺÝLƃ́QӶ϶Η;ƲEGDQ0+g,RӶ϶ԮΗ;ƲEGDQ0
NMOԏ<JRʹĳQɡƻE6KRηǭK2?ՠØɉ`ɨӻN˼ѹ1ɓѬK+gա

73Ñ ̊puµҭӽĴ˨ѸOdgɎȂԺƴĴ˨
 2000 ȬÝOğfՠĴ˨ɴ̰QәŘOç,ɎŘΰQ
βώRʵDNȇԯOϐğ<DաÑ̊puµҭӽĴ˨Ѹ(Secondary Ion Mass Spectrometer: SIMS)Rՠ΢Ɇ 10¦z³µχȵOғʭ<D 1 ̊puµ¸§k҂ʳѡ
ԯOͨǿ<ՠΗ;<D 2 ̊puµkҭӽԪы̟OƷ
I,JθƼKĴԦɉՠm¯¸vaԪǡǁăϠϜQ˼ĳƟk΀,͈ǭkў-ѤИK+gա¥pz³¨¸±u¸¸QϏԎĴѴЭՠ0HՐҭӽĴѴЭOdgƃëð̟͈ǭՠppm-ppb u¸¸QՐɦȵ͈ǭNMՠSIMS QͰɚR94ē0O<0̗:hN,ĸ˛;ƎQΓҶQĴ˨LΣɚ1с,ա8hOdfՠ
8h\K¿źЭK+GDՠɎŘΰ 1 ĂšëKQ δ13C
ć͈ǭ1źЭLNGDա8Qɴ̰Rwµ°µɎŘΰkǼҟL<Jĸ_JɎŘΰ҂ʳQĴ˨OӦ΀:hՠBQɀͱOdg δ13C ćQӢ,1+g8L1κ:hD 6)ա8QβώOdfϯ 19 ĖȬłK`;ɧϭOd
gÝҚɀȹQ°s¸­µ1+GDźЭɚ1ʽe
0O:hDա̗ɘN1eՠ˕ĸ˛OўjhD8QĴ
˨RՠѤИOdgƃëðĴĺŎ˫kСɩ<J/e?
ͺƮKRΏjhJ,g1ՠ8hÞԔ́$NƯҭȬÝQɎŘΰkǼҟL<J SIMS OdgĴ˨1ўjhJ,g 5)14)23)25)ա
 ӊȬՠѐТeR8-<DɎӽQ˗̈ͯOǼ<J͢
϶Lϑ϶Qƃëð̟ćk 12 ¥pz³¨¸±QĴ˨¤ 1 ͣKƃˆO̧ǭźЭNɴ̰QԍΗOɯŉ<D 8)(Ƨ 4)ա͈ǭźЭNǌ2:1ԕehJ,gɎŘΰQĴ˨O/,Jՠ1 ͣKѩʬQƃëðɡƻkɋehg¨°Rǌ24ՠ8h\KɎŘΰK͈ǭ:
hJ8N0GDϑ϶ƃëð̟Qɡƻ1Ŋjg8LK;ͯψaͽǀOʵDNĽϯˤákÀ.g8L1K2gաĴ˨¤ɆdfȂ:,ɎŘΰBQ`QkĴ
˨>gD_OR\EʢсQɓѬ1+g1ՠØɉQΗȌ1˛ɇ:hgա\DՠSIMS dfϏԎĴѴЭQՐ
, NanoSIMS k΀,DĴ˨KRՠɎŘΰQɀͱOɕ=Dҽę϶QĴȡ(C, N, S Ϝ)kp¨¸Ř>g8L
1K2ՠɎŘΰk̀ɯ>gę϶ĴȡOԏ>gʵ<,ƻƊ`ʬǊ4N:hJ,g 5)14) (Ƨ 4)ա8-<DĂ$QɎŘΰ² ±KQĴ˨Rՠ\E\EΗȌӔ½K+
g1ՠ8h\Kǌ˭K<0ʎ.ehJ8N0GDǎŶÝQ;ɧϭkdf҆ϹOʽe0OK2gźЭɚk
τ_J,gաö.Sՠ8heQɴ̰OdfͺÝQɎ;ͯk̟ҾĴ˨>g8LKՠĥðΛN;ͯψaÝҚ́ȹOӌg8L`źЭONg0`<hN,աØɉQȌ˚ ɎŘΰa˗̈ͯՠ{¯mpNMՠ;ƎQΓҶkʔgβώO/6gƃëðՠę϶QĴȡՠȝΌNMRՠękӉhS;ƎQÝҚűɕaBhOÜԞ<DϹ
ЬзO/6gę϶Qųfӈ]QϾ˫kѮJ/fՠӴJournal of Japanese Society for Extremophiles (2018) Vol.17

Ƨ 4 Ñ̊puµҭӽĴ˨Ѹ(SIMS)OdgɎȂԺƴĴ˨ö(a)-(b) SIMS k΀,D͢϶ϑ϶ƃëð̟Q 1 ͣƃˆ͈ǭ̰QԍΗ(Ishida e. 2018)ա(a) SIMS Ĵ˨¤ա΢Ɇ 12μmՠ̀:R 3μm χȵա(b) ƺϋȒOƇ\hg˗̈ͯ(ՙт)Qү˯ԪǡՀɎԉĭΧա˗̈ͯQ δ13C ćRZ[¹́K+gQOǼ<ՠδ15N ćR¿ƱҭK+gաĴ˨ҎȝR δ13C K±0.16‰ՠδ15N K± 0.56‰(2SD)K+gա cal:v±pՠdol:³¥pՠqtz:ΰчա(c)-(f) ɎŘΰQՀɎԉĭΧLՐϏԎĴѴЭÑ̊puµҭӽĴ˨Ѹ(NanoSIMS)Odgę϶¥µ{ĕա(c)ӓӟĜOdgɎŘΰQՀɎԉĭΧա(d)-(f) NanoSIMS Odgę϶¥µ{ĕաd:͢϶ՠe:ϑ϶ՠf:ζ՘աϑ϶RšũǡKRǨǭ<JpuµŘ<N,QKĴǡpuµ CN K˼ĳ>gաɎŘΰз̀ӗQę϶ĴȡR¹́KN
4ՠ\DՠзQĨӮ0eRZLlMę϶1˼ĳ:hN,8L1j0gա|¸±¸R 5μmա

϶NMOdg;ŘǥűɕQϾ˫kѲǹ<J,gLС
.g8L1K2gաӊȬՠB-<D;ͯͰ˗QӴ϶űɕLӴ϶OõjhgӾȍę϶O̲Ρ>g8LKՠǎŶÝȒΰQɎӽӾȍę϶Ƈ˗ӽ0eƺϋȿˆQ̼
̵Ӿȍę϶͜ȵkʖǭ<ȿˆ̷ΗK+GDɎ;ͯ;
ɧϭkʖǭ>gβώaՠɎ;ͯ1΀,J,DӴ϶Q
Ӣ,OdgƃëðĴĺŎ˫Qȝ0eǎŶÝ̵̼ͽǀkʖǭ>gβώ1̲Ρ:hHH+g 10)11)24) աSIMS
kǚ_L<DɎȂԺƴՐɦȵĴ˨̰R8-<DɎӽɯĴQĴ˨LΣɚ1с4ՠØɉɎŘΰa˗̈ͯBQ`QkǼҟL<DɎӽӾȍę϶͜ȵ͈ǭkў-8LKՠ½ѻQʖǭkdfĥðŘ<ǎŶÝ;ƎƫՠU,
JR˕ŶQ;ƎQ;ɝƼOʵDNĽϯkÀ.g8L
1K2gEi-ա ƯͻσǥKRƯҭaԂͯQͿͱLϼɯՠ˗̈ͯaɎŘΰQŘǥĴ˨QϾ˫0eĸ;ΛNɡƻkɍę<ՠ
MlNͽǀKMlN;ͯψ1MlNÝҚkўNGJ,D0kʖǭ>g̙ԝ\Kβώ1әlK2Dա<0<ՠǰԡOBQd-NͽǀK;ͯ1;ɝźЭ0ՠB`B`;ͯ1΀,gӴ϶QɎӽӾȍę϶1˗̈ͯaɎŘΰÄO̗gQ0ՠNMՠǰԡQΰkѮJ,gE
6KRn³¸K2N,Сǹ1Ǌ4ǣƮ>gաĴ
74˨ɸџQΗӡOdfՠ҆ϹNǎŶÝ;ͯĕ1Ѯ.J
2D0e8BՠͺÝ;ͯǥLQΣÒѧǩ1ɓѬ¿ź̉NȇԯO+gLϛТRС.gաwµ°µɎŘΰkΗѮ<D Tyler ŢǄRՠȿˆĠ4QǾԋǉK+
GDɎ;ͯŘΰkÅɘNѲǹOdGJѮӑ:?ՠ;
ͯǥQβώТLQĤƃβώkȌԍ<D8LK;Ǝұ͏βώO¯p§k`De<Dա;ƎQұ͏kϲѴ4D_ORՠǼҟkºǺOѲǹ·ѻҿ<H
HՠĴӼQƳ˵kҳ.g8LkŪjN,ћƁσǥΛNǜŔLɴ̰1ɓѬK+gաҚӅ ˾ԕͽǀ;ͯǥå/dVµ¤q§XQŬŊՠ:eO˟ҖʯƵϛQ̈åk4E:GDхÓˍŢǄO
8QƼkӕ<JҚɤkѡ<\>աĤƃβώТQʒȘ̏ŢǄՠǌůԛǡŢǄՠϚ˜ˇȫ̠0eR҂ʳ/dV¸Qʙ÷kŴ6\<Dա˟ҖʯKϺÙ<Dѐ
ТQβώɯ˫Q¹ӮRʺ˟ǥџʌнåхɴβώ
(18K13645)QŋɯkŴ6J,\>ա

Journal of Japanese Society for Extremophiles (2018) Vol.17Ŭ Сʯʹ

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Extremophiles 2018Ηѡp±խEuryarchaea-specific RecJ-like protein may work in stalled fork repair with HefɳȍխÉșǌǥǌǥԖ ;ͯҩ͏ͽǀσǥȴ ;Ǝ̈ЭσǥǾʣ̠Ƅխ̤΁ ՗˹ǡ Extremophiles2018 ͆оŋɯӾƷӾXQʓɾk+f1L-9;,\<D#˾ԕͽǀ;ͯǥåQʶ$OR͆оÄ`ǌǇ/Á҄ONf\<D"ӻPJɌλ΄<½7\>#ρRǥå  ʺΡO½ѻQp±K¤¸Ηѡ
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Journal of Japanese Society for Extremophiles (2018) Vol.17

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Journal of Japanese Society for Extremophiles (2018) Vol.17Ηѡp±խClarification of Cesium tolerant mechanisms of alkaliphilic Microbacterium sp. TS-1ɳȍխ˦̵ǌǥԖ ;Ǝσǥβώσ ;ƎσǥǾʣ äіβώǲ ŢǄҒχł˛ 1 Ȭ̠Ƅխ˅ɜ ҥǪ ØƢŬŊ<DǥåR Extremophile2018 L,-ƩԡΛNǥåK"p°nQpxnȕKўjhD#ƩԡΛNǥåL,-8LK"õ΀ѶҌRчҌK+GD#̼ǉXQΉǥ"ǥåŬŊϜRR=_JQðՍEGDD_"
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Journal of Japanese Society for Extremophiles (2018) Vol.17

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ξɲǌǥ йͧσǥϭěϕћƁβώͽÄȘ ХÖӱǌǥǌǥԖ ӆǥβώσȫ΁ ϔɥǟǌǥǌǥԖ ͼȚǥβώσ̢ŵ ŝϊ˦Öǌǥ ;ͯ;ͿȚǥβώµ¸îі ƚϔÖӱǌǥǌǥԖȚǥβώσӰ΁ π¹ӭ

ԊȖǌǥ ȚǥӮ

Oleg GUSEVƩϒβώԍΗ̰×ͼŘǥβώɳȘ˟ ϳÖӱǌǥŘǥβώɳ

Journal of Japanese Society for Extremophiles (2018) Vol.17ЏԤǛƒåǛƒԊ խΰÓ̬̎՞˦ǌ՟ŅǛƒԊխΰӼсϳ՞Éǌ՟Ǜ  ƒխԓӮʯɗ՞Ԭǥǌ՟"äі Ԝ՞ͼβ՟"ԇɀ̵¹՞ͿЋβ՟"̪ũ˪Ѧ՞ͿЋβ՟"ՐƐήĦ՞˦̵ǌ՟"Äˢ Х՞˦Țǌ՟"Äˢ Հ՞ϝ̱ǌ՟"¼ӂƓÊ՞JAMSTEC՟"͌˟ Ŗ՞ͿЋβ՟ǥåÐŒȇ
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