放射線抵抗性細菌の生存戦略

誌名誌名 Gamma field symposia

ISSNISSN 04351096

巻/号巻/号 48

掲載ページ掲載ページ p. 69-76

発行年月発行年月 2011年3月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

Gamma Field Symposia, No. 48, 2009 Institure of Radiation Breeding 叫 S,Japan

69

Survival Strategy of a Radioresistant Bacterium: a Review

Issay NARUM!

Quantum Beam Science Directorate, Japan Ato血cEnergy Agency,

1233 Watanuki, Tak:asaki, Gunma 370-1292, Japan

Introduction

Deinococcus radiodurans is a small, red-pigment-

ed, non-spore-forming Eubacterium (Fig. 1). Members

of this species inhabit a wide range of terrestrial and

aquatic environments and are characterized by an ex -

ceptional capacity to survive the normally lethal DNA

damage induced by agents such as ionizing radiation,

UV radiation and desiccation. Deinococcus radiodu-

rans was first isolated in 1956 from canned meat that

had received 1.8 kGy of y radiation regarded as typi-

cally lethal to bacteria (Aゆ ERSONet al. 1956). Cur-

rently, exposure to up to 10 kGy of ionizing radiation

is used to sterilize foods. As in other organisms, the D.

radiodurans genome sustains over 100 DNA double-

strand breaks (DSBs) after exposure to 10 kGy of y

radiation. DSBs are the most lethal form of DNA dam-

age. Although all living organisms possess DNA repair

mechanisms, only a few of the DSBs can be repaired

in most species. Deinococcus radiodurans is capable

Fig. 1 Electron microscope image of Deinococcus radio-

durans (Courtesy of H. WATANABE).

of repairing the fragmented genome during post-irra-

diation incubation (Cox and BATTISTA 2005). Genome

sequence analysis of D. radiodurans has revealed that

the genome encodes almost all the major prokaryotic

proteins involved in DNA repair (WHITE et al. 1999).

However, the molecular mechanisms underlying the ra-

diation resistance of this bacterium remain unclear.

Proteome and transcriptome analyses have re-

vealed that D. radiodurans efficiently coordinates its

recovery from exposure to ionizing radiation through

a complex array of DNA repair and metabolic path-

way switching (LIPTON et al. 2002; LIU et al. 2003).

However, the discovery of numerous additional irra-

diation-response genes has provided new targets for the

identification of genes critical to radiation resistance.

The extensive investigations conducted thus far provide

useful insights into the mechanisms underlying radia-

tion resistance, but a more detailed empirical explana-

tion of why D. radiodurans is so radiation resistant is

still needed. Further research based on alternative ge-

netic and biochemical approaches should help to give

a better understanding of the mechanisms involved in

DNA repair (NARUM! 2003).

Discovery of a Novel DNA Repair-related Protein

To elucidate the efficient DNA repair mecha-

nisms of D. radiodurans, I and my colleagues at Japan

Atomic Energy Agency have, over a 15-year period,

analyzed the mutations in the genes of DNA-repair-de-

ficient strains. The strains analyzed so far are listed in

Table 1.

Analysis of the radiosensitive strain KH311 of D.

70 Issay NARUM!

Table 1. The DNA repair—deficient strains of Deinococcus radiodurans that were analyzed.

Strain Gene Mutation type (genotype) Reference

KH311 pprA Base substitution(p'PrA446) NARUM! et al. (2004)

rec30 recA Base substitution (recA670) NARUMI et al. (1999)

KI696 recA Base substitution (recA424) SATOH et al. (2002)

KH840 ppr/ IS insertion(pprl307::IS830l) HUA et al. (2003)

KH586 recN 1-bp insertion F'UNAYAMA et al. (1999)

UVS9 uvde Base substitution (uvde335) KITAYAMA et al. (2003)

262 uvrA IS insertion (uvr2230: :1S2621) NARUM! et al. (1997)

302 uvrA 144-bp deletion

radiodurans identified the absence of a novel DNA-

repair-promoting protein, PprA (pleiotropic protein

promoting DNA repair), which produced the loss of

radiation resistance. Investigation in vitro showed that

PprA protein became preferentially bound to double-

stranded DNA carrying strand breaks, inhibited E.

coli exonuclease III activity, and stimulated the DNA

end-joining reaction catalyzed by ATP-dependent and

NAO-dependent DNA ligases. These results suggest

that D. radiodurans has a non-homologous end-join-

ing (NHEJ) repair mechanism in which PprA plays a

critical role (NARUM! et al. 2004). This type of path-

way may be error-prone, because DNA ends produced

by irradiation probably undergo clustered damage, the

removal of which can create mutations. Therefore, the

NHEJ pathway must be accompanied by a mechanism

that prevents mutations to achieve accurate DSB repair

in D. radiodurans (NARUM! 2003). This mechanism

requires clarification to better understand the mecha-

nisms involved in DNA repair.

DNA ligase is one of the most frequently used

reagents in genetic engineering. Discovery of PprA

revealed the potential for a new biotech reagent from

a combination of DNA ligase and PprA. As a result

of technology-transfer by the Japan Atomic Energy

Agency to the private sector, the TA-Blunt Ligation

Kit was released in Japan as a commercial product in

November 2005. The inclusion of the PprA technology

in the ligation kit provides 10-fold increase in ligation

efficiency compared with that of conventional products

[http://www.jaea.go.jp/english/news/p06020901/index.

shtml].

NARUM! et al. (1997)

Radiation Response Mechanism

The highly efficient DSB repair process in D. ra-

diodurans is radiation-inducible and is dependent on de

novo protein synthesis following irradiation (KITAYAMA

and MATSUYAMA 1968). It has been shown that both

PprA and another D. radiodurans protein, RecA, are

radiation-inducible (LIU et al. 2003). It appears that D.

radiodurans possesses a novel DNA-damage response-

mechanism. In Escherichia coli, RecA and LexA play

important roles in the DNA-damage response repair-

mechanism (the SOS system) (WALKER, 1984). In E.

coli, RecA is activated by DNA damage to mediate

proteolytic cleavage of the E. coli LexA repressor, re-

sulting in derepression of the SOS regulon. SOS-like

processes have been conserved in a wide variety of eu-

bacterial species (MILLER and KOKJOHN 1990). Neither

LexAl nor LexA2 of D. radiodurans was found to be

involved in the DNA-damage response repair-mecha—

nism, although RecA was the sole protein required for

cleavage of the LexAl and LexA2 proteins in D. radio-

durans (NARUM! et al. 2001; SATOH et al. 2006).

Analysis of the radiosensitive strain KH840 of D.

radiodurans identified the absence of a novel regula-

tory protein, Pprl (inducer of PprA), which is involved

in the induction of PprA. Inactivation of Pprl resulted

in a loss of PprA and RecA induction (HUA et al. 2003).

Pprl therefore appeared to play a critical role in trig-

gering the DNA damage response and cellular survival

network following irradiation in D. radiodurans (HUA

et al. 2003).

In research in which the author was involved,

OHBA et al. (2005) identified the radiation-responsive

SURVIVAL OF RADIORESISTANT BACTERIUM 71

minimal promoter region of the pprA gene and demon-

strated that up-regulation of pprA expression by Pprl

is triggered at the promoter level. However, we were

unable to find evidence to support direct interaction of

Pprl with this promoter region. This result suggested

the existence of hitherto unknown components in the

Pprl-dependent response to radiation stress in D. ra-

diodurans. In an effort to explore this possibility, the

two-dimensional protein profiles of wild-type and ppr!

disruptant strains were compared (OHBA et al. 2009). In

the course of this investigation, a 10-kDa protein spot

was identified during isoelectric focusing analysis, the

isoelectric point of which differed between wild-type

and ppr/ disruptant strains (Fig. 2). The protein spot in

the wild-type strain indicated higher basicity than that

of the ppr/ disruptant strain, suggesting that the protein

may undergo post-translational modification via PprL

We designated this protein PprM (modulator of the

Pprl-dependent DNA damage response, for the reason

that follows). To determine whether PprM is respon-

sible for the radiation resistance of D. radiodurans, a

pprM disruptant strain was generated by direct inser-

tional mutagenesis using double-crossover recombina-

tion. The pprM disruptant strain exhibited markedly

higher sensitivity to y -rays than the wild-type (Fig.

3), suggesting that PprM plays an important role in the

pprl-dependent radiation response in D. radiodurans

(OHBA et al. 2009).

Basic Acidic Basic

Wild-type

Acidic kDa

20

pprl disruptant

15

10

Fig. 2 Two-dimensional PAGE analysis of Deinococcus

radiodurans wild and ppr/ disruptant strains. Based

on the publication of OHBA et al. (2009). Arrows

indicate a 10-kDa protein spot, the isoelectric point

of which differed between wild and ppr/ disruptant

strains.

To determine whether PprM is involved in the

induction of PprA and RecA, changes in the intracel-

lular levels of PprA, RecA and Pprl following irradia-

tion were investigated. Constitutive production of Ppr A

at an elevated level was observed in the mock-irradi-

ated pprM disruptant strain, while the level of PprA

was comparable to that observed in irradiated cells of

wild and pprM disruptant strains. On the other hand,

induction of RecA was not affected by pprM disrup-

tion. These results suggest that PprM is involved in re-

pressing the production of PprA, but not that of RecA.

We proposed that only the basic form of PprM can be

involved in reversing the repression of PprA produc-

tion following irradiation in D. radiodurans (OHBA et

al. 2009).

Loss of PprA renders D. radiodurans sensitive to

radiation (HUA et al. 2003; NARUM! et al. 2004). On the

other hand, the pprM disruptant strain, which produced

high amounts of PprA in the absence of irradiation,

also exhibited high sensitivity to radiation. Two pos-

sible explanations could account for the radiosensitivity

of the pprM disruptant strain: (i) a defect in the pre-

cisely timed induction of PprA following irradiation,

or (ii) a defect in the regulation of hitherto unknown

ー

3

2

。

0

0

1

1

1

UO!

―ue』

:!

BU!>n>』

ns

10-4 0 2 4 6 8

-y ray dose (kGy)

Fig. 3 Survival curves of wild and gene-disruptant strains

of Deinococcus radiodurans in response to dosage

of ionizing radiation. Based on the publication of

OHBA et al. (2009). Open circles, wild-type strain;

filled squares, pprM disruptant strain; filled tri-

angles, pprA disruptant strain; filled circles, pprA-

pprM double-disruptant strain.

72 Issay NARUMI

protein(s) that are necessary for radioresistance in ad-

dition to PprA. The first explanation is supported by

previous experiments demonstrating that the le.xA2 dis-

ruptant strain, in which enhancement of ppr A promoter

activation was observed following irradiation, exhib-

ited much higher resistance to radiation than the wild

strain (SATOH et al. 2006). In order to confirm the latter

possibility, a pprA—pprM double-disruptant strain was

constructed and the survival rate was examined. The

pprA—pprM double-disruptant strain exhibited much

higher sensitivity to radiation than either the pprA or

the pprM single disruptant strain (Fig. 3). These studies

strongly suggest that PprM is involved in the unique

radiation response mediated by PprI and plays a crucial

role in the induction of PprA (OHBA et al. 2009). At

the same time, it was also revealed that PprM regulates

other hitherto unknown proteins important for radio-

resistance, besides PprA. It appears that there is still

much to learn about D. radiodurans.

Future Prospects

More than 40 species of the genus Deinococcus

have been discovered. The genome sequences of three of

these (D. radiodurans, D. geothermalis and D. deserti)

have been determined (WHITE et al. 1999; MAKAROVA

et al. 2007; de GROOT et al. 2009). Further comparative

genomics and molecular biological analysis involving

targeted mutagenesis and plasmid complementation

will provide new insights into our understanding of the

DNA repair mechanisms and radioresistance in Deino-

coccus species.

Acknowledgements

This work was partly supported by a Grant-in-Aid

for Scientific Research (B) 19380054 from the Japan

Society for the Promotion of Science.

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放射線抵抗性細菌の生存戦略

鳴海一成

独立行政法人日本原子力研究開発機構 量子ビーム応用研究部門

バイオ応用技術研究ユニット

〒370-1292 群馬県高崎市綿貫町 1233

生物の中でも極めて放射線に強い微生物群を総

称して，放射線抵抗性細菌と呼んでいる。 1956年，

ガンマ線滅菌したはずの缶詰の中で増殖している

細菌が見つかり，これが放射線抵抗性細菌の最初

の発見となった。 Deinococcusの放射線耐性機構

の研究は，そのほとんどが，最初に分離された D.

radioduransを用いて行われている。電離放射線

による生物効果の中で最も重篤な損傷は DNAニ重鎖切断であり，放射線を照射すると， D.radio-

duransは，大腸菌の様な一般的な細菌と同程度

に，ゲノム DNA内に二重鎖切断を受ける。一般

的な細菌では，細胞内に生じた数個の DNA二重

鎖切断による損傷が致死的効果を与えるのに対し

て， D.radioduransは，細胞内に生じた 100箇所

以上の二重鎖切断損傷を短時間で修復することが

できる。すなわち，D.radioduransの放射線耐性は，

この菌のもつ優れた DNA修復能に大きく依存し

ている。我々の研究グループがとった研究戦略

は， D.radioduransから分離された放射線感受性

変異株の原因遺伝子を同定することであった。そ

の結果， D.radioduransの放射線耐性に重要な新

規遺伝子を同定することに成功した。同定した遺

伝子は，他の生物で解析済みのどの遺伝子とも全

く似ておらず，機能未知遺伝子に分類されていた

もののひとつであった。この遺伝子から作られる

タンパク質 PprAの性質を解析した結果，放射線

照射後の細胞内で生合成が活発になり，放射線に

よって DNA鎖が切れた部分を認識して結合する

ことにより， DNA鎖切断の修復を高効率で促進

する作用をもつことが分かった。 PprAは放射線

誘導性タンパク質であるが， D.radioduransの放

射線応答機構にもユニークなタンパク質群が関与

していた。我々の研究から， DNA修復タンパク

質の放射線誘導制御に係わる新規因子 Pprl及び

PprMが同定されたが，これらの因子によって制

御を受けるまだ未知の重要な DNA修復関連タン

パク質の存在が示唆される。現在， Deinococcus

属細菌の 3菌種についてゲノム配列が解読されて

いるが，放射線抵抗性細菌の放射線耐性の共通原

理を解明するためには， さらなる比較ゲノム解析

と分子生物学的解析が必要である。

SURVIVAL OF RADIORESISTANT BACTERIUM 75

質疑応答

司会：では質疑応答に移りたいと思います。ご質

問ございますか。どうぞ。

風間：理化学研究所の風間と申します。最初のス

ライドの方で 2kGy照射しても 4時間後にはゲ

ノムが元通りに戻ってしまうがすごく印象的で

した。 exonucleaseを阻害して， DNAligaseの

活性を促進するという PprAタンパク質の特性

から考えると，高等生物の endjoiningみたい

なのが，すごく促進されるのかなと。そうする

と元通りになる機構っていうのは， endjoining

の活性が強いからだと思いますが，その辺はど

ういう機構があると考えられているのでしょう

か。

鳴海： DNAligaseの活性を促進するというのは，

真核生物の Kuタンパク質と DNA-PKcsの系，

それから Mrell/Rad50/Xrs2タンパク質複合

体の系の働きにそっくりなのです。ですから，

non-homologous end joining経路のバクテリア版

を，この PprAが 1つのタンパク質でやってい

るのかもしれないと思います。真核生物の Ku

タンパク質のホモログが，枯草菌とかマイコバ

クテリアには存在しますが，これらはアミノ酸

配列が保存されている本当のKuホモログです。

一方， PprAタンパク質は， Kuホモログともア

ミノ酸配列が全然似てないので，全く新しい

ものだと思いますが， endjoiningのDNA修復

機構には係わっているだろうと考えています。

homologous recombinationとの連携については，

RecAタンパク質など homologous recombination

に関係するようなタンパク質と PprAタンパク

質を掛け合わせてどうなるかという実験を行

いましたが ものす..,,.‘くZ:'< recombination活性が

上がったという結果は得られていませんので，

PprAタンパク質と homologous recombinationと

の関係については，まだちょっと分かりませ

ん。

風間：ありがとうございます。

司会：ほかにございませんか。じゃあ中川さん。

中川：生物資源研究所の中川です。今の質問と関

係しますが，ゲノムが何ヶ所も切れて，それが

元通りに戻るというところが，非常に不思議に

思います。先ほどちょっと話があったネムリュ

スリカも乾燥していくと，染色体がばらばらに

なりますが，それを吸水させると，ちゃんと元

どおりに修復されると聞いています。この場合，

ばらばらになった染色体がいったいどのように

修復されたら，逆位や転座などの染色体異常が

起こらないだろうかと不思議に思っています。

染色体異常が起こらない様に何か制御している

メカニズムがあるのでしょうか。

鳴海：バクテリアのゲノムっていうのは，核膜が

ありませんが，核様体という DNAとDNA結

合タンパク質の複合体構造があって，細胞内に

あまり散在しないような状態にあります。そう

すると DNAのdoublestrand breakができても，

切れた DNA断片が散在せずに， DNA末端の

切れ口同士が近くに存在していることになるの

で，このことが染色体異常の起こらないことと

関係するという説もあります。ただ，放射線抵

抗性細菌では， DNA結合能を持つ核タンパク

質をコードするある種の遺伝子を破壊すると，

細胞の中に DNAが散在しますが，その様な遺

伝子破壊株の放射線耐性は野生株と同様だった

という実験結果もありますので，染色体構造の

特異性と染色体異常抑制機構の関係については

まだ分からないことが多いと思います。

谷坂：京大の谷坂です。 DNA型のトランスポゾ

ンの切り出しは，恐らく放射線と同じような切

断を受けて修復すると思いますが，その修復に

も同じような機構が働くと考えていいのでしょ

うか。

鳴海：放射線による DNAの切断点は，制限酵

素などの酵素反応で切ったようにきれいな切

り口ばかりではありません。 5'末端にリン酸

がついていたりいなかったり，様々な，いわ

ゆる汚い切れ方があると思います。このよう

な切断点を修復する際に，単純に DNAligase

で繋げてしまうと， mutationが起こってしま

います。この様なことから，真核生物の non-

homologous end joiningは誤りがちな修復機構と

言われています。一方，放射線抵抗性細菌には，

誤りがちではない endjoining機構があるのか

も知れません。また，それだけでは不十分で，

recombinationが最終的には重要になってくるの

ではないかと思っています。

76 Issay NARUM!

谷坂：ありがとうございました。

司会：ほかにございませんか。では，時間もきて

おるようですし，立体構造までお決めになって

いると，とても面白い，興味深いなと思って聞

いておりました。ますますの研究のご発展をお

祈りしております。嗚海先生ありがとうござい

ました。

（拍手）