[論文紹介] 微生物パンゲノムにおける抗ファージ防御システムの系統的発見 (Systematic discovery of antiphage defense system in the microbial pangenome)

RESEARCH ARTICLE
◥
MICROBIOLOGY
Systematic discovery of
antiphage defense systems
in the microbial pangenome
Shany Doron,* Sarah Melamed,* Gal Ofir, Azita Leavitt, Anna Lopatina,
Mai Keren, Gil Amitai, Rotem Sorek†
The arms race between bacteria and phages led to the development of sophisticated
antiphage defense systems, including CRISPR-Cas and restriction-modification systems.
Evidence suggests that known and unknown defense systems are located in “defense islands”
in microbial genomes. Here, we comprehensively characterized the bacterial defensive arsenal
by examining gene families that are clustered next to known defense genes in prokaryotic
genomes. Candidate defense systems were systematically engineered and validated in model
bacteria for their antiphage activities. We report nine previously unknown antiphage
systems and one antiplasmid system that are widespread in microbes and strongly protect
against foreign invaders. These include systems that adopted components of the bacterial
flagella and condensin complexes. Our data also suggest a common, ancient ancestry
of innate immunity components shared between animals, plants, and bacteria.
B
acteria and archaea are frequently at-
tacked by viruses (phages) and as a result
have developed multiple, sophisticated lines
of active defense (1–3) that can collectively
be referred to as the prokaryotic “immune
system.” Antiphage defense strategies include
tion residing within such defense islands may
also participate in antiphage defense (15, 16).
Indeed, recent studies that focused on individ-
ual genes enriched next to known defense genes
resulted in the discovery of new systems that
protect bacteria against phages (7, 9, 17).
to be associated with known defe
(15), as well as 23 pfams that wer
in the same study as putatively d
did not pass our thresholds, altoget
a list of 335 candidate gene familie
From defense genes to defens
Antiphage defense systems are usua
of multiple genes that work in conce
defense—for example, cas1, cas2, c
cascade genes in type I CRISPR-Cas
and the R, M and S genes in type I
modification systems (3). Genes
within the same defense system ar
encoded on the same operon, and th
within the operon is highly conse
distantly related organisms sharing t
tem (3, 7, 9, 16, 19, 20). To investig
the defense-associated pfams belo
gene systems, we used each such pfa
chor around which we searched fo
associated genes (Fig. 1A). For this,
all the neighboring genes (10 gene
side) from all the genomes in which
the anchor pfam occurred and clu
genes based on sequence homolog
ods). We then searched for cassettes
ters that, together with the anchor
conserved order across multiple d
nomes, marking such cassettes a
multigene systems (see Methods)
The gene annotations in the resu
date systems were manually inspec
out likely false predictions. We found
the cases (129 of 335) represented
mobile genetic elements, such as
微生物パンゲノムにおける抗ファージ防御システムの系統的発見
2018.6.19
PMID: 29371424
M1 永田祥平
© Jeﬀ Dahlsource: goddesses-and-gods.blogspot.com/2010/01/wadjet.html

2
要旨
ファージに対抗するため，バクテリアはCRISPR-Casおよび制限修飾系を含む洗練された抗
ファージ防御システムを開発してきた。これまでの研究で，既知あるいは未知の防御システ
ムは微生物ゲノムの「防衛島」( defense island )に位置していることが示されている。
本研究では，原核生物のゲノムにおいて既知の防御遺伝子の隣にクラスター化されている遺
伝子ファミリーを調べることによって，細菌の防御システム群を包括的に予測した。候補防
御システムは，モデル細菌を用いた実験により抗ファージ作用が検証された。
微生物の多系統に広まっており，外敵から強く保護する作用を持つ，新規の9つの抗ファー
ジ防御システムと1つの抗プラスミド防御システムを発見した。
これらには，細菌性毛およびコンデンシン複合体の構成成分を採用したシステムが含まれ
る。我々のデータはまた，動物，植物および細菌の間で共有される自然免疫機構に共通の古
代の祖先が存在することを示唆している。
背景
結果

3
背景：原核生物の免疫機構
原核生物 (バクテリア・アーキア)は頻繁にウイルス (ファージ)による攻撃を受けており，
それに対抗するために様々な防御システム (免疫システム)を開発してきた。
自然免疫
獲得免疫
・CRISPR-Cas
・制限修飾系 (RM; Restriction-modiﬁcation)
制限酵素が外来性DNAを切断する。
侵入した外来性DNA (ファージ由来等)の一部をCRISPR領域に
保存し，再侵入時に使用する。
・不稔感染系 (Abi; Abortive infection)
ファージ感染時に細胞死・代謝停止を誘導し，
他の細菌に感染が広がらないようにする。
・Toxin-Antitoxin系
Abi同様にファージの侵入によって活性化し細胞死を誘導する。
最近発見された防御機構：
・BREX (Bacteriophage exclusion)
・Prokaryotic Argonautes
・DISARM (Defence island system associated with restriction‒modiﬁcation)
代表例：
VI01CH16-Fineran ARI 21 August 2014 10:42
Adsorption
inhibition
B A C T E R I U M
Injection
blocking
Attachment
DNA injection
CRISPRs
Transcription
and translation
Abortive infection and
toxin-antitoxin systems
Assembly
Lysis
Restriction
modification
Replication
Figure 1
The phage lytic life cycle and the bacterial antiphage systems. Bacteria possess a range of defense strategies that target various phase
the phage life cycle.
MICROBIAL ANTIPHAGE SYSTEMS
Adsorption and DNA Injection Inhibition
There are two antiphage systems known to target the start of the phage infection life cycle. In
Annu.Rev.Virol.2014.1:307-331.Downloadedfromwww.annualreviews.org
AccessprovidedbyKeioUniversityon06/17/18.Forpersonaluseonly.
↑ファージのライフサイクルとバクテリアの防御機構
原核生物中には未発見の防御機構がまだまだ多数存在していると考えられる。
バクテリアの約40%に存在
原核生物の約75%に存在
Dy et al., Annu. Rev. Virol, 2014

4
本研究のコンセプト
ed
ms
ria
re
ed
le,
n-
er
hat
de-
hat
in
in
their fight against phages.
RESULTS: We searched for gene cassettes of
unknown function that are enriched near
known defense systems in more than 45,000
available bacterial and archaeal genome se-
quences. Such gene cassettes were defined as
candidate defense systems and were system-
atically engineered into model bacteria, which
were then infected by an array of phages to
test for antiphage activities. This yielded the
discovery of nine new families of antiphage
defense systems and one additional family of
antiplasmid systems that are widespread in
microbes and shown to strongly protect against
development of important
biotechnological tools, as
exemplified by the use of restriction enzymes
and CRISPR-Cas for biotechnological and bio-
medical applications. One may envision that
some of the systems discovered in the current
study, once their mechanism is deciphered, will
also be adapted into useful molecular tools in
the future.
▪
The list of author affiliations is available in the full article online.
*These authors contributed equally to this work.
†Corresponding author. Email: rotem.sorek@weizmann.ac.il
Cite this article as S. Doron et al., Science 359, eaar4120
(2018). DOI: 10.1126/science.aar4120
science.aar4120
..................................................
onJune6,2018http://science.sciencemag.org/adedfrom
微生物ゲノムにおいて抗ファージ防御システム関連遺伝子は
密集していることが知られている。
→仮説：既知の抗ファージ防御システムの近傍に存在している遺伝子群には，
実は抗ファージ防御を行っているものが他にもあるのではないか？
オレンジ色：既知抗ファージ防御システム
図: 本論文のResearch Article Summaryより
微生物ゲノム1
微生物ゲノム2
微生物ゲノム3
※本論文において微生物 = 原核生物 = バクテリア + アーキア
Defense island と呼ぶ
source: wsj.com

5
本研究のパイプライン
in microbial genomes such that, for example,
genes encoding restriction enzymes common-
ly reside in the vicinity of genes encoding other
phage resistance systems. The observation that
defense systems are clustered in genomic “de-
fense islands” has led to the hypothesis that
genes of unknown function residing within
such defense islands may also participate in
the future.
▪
Doron et al., Science 359, 1008 (2018) 2 March 2018 1 of 1
(2018). DOI: 10.1126/science.aar4120
A pipeline for systematic discovery of defense systems. Microbial genomes (more than 45,000 in the current study) are mined for genetic systems
that are physically enriched next to known defense systems such as restriction-modification and CRISPR-Cas. Candidate predicted systems are
cloned into model bacteria, and these bacteria are then infected by an array of phages from various families to determine whether they provide defense.
①情報学的予測
②実験的検証
that many currently unknown defense systems
reside in the genomes of nonmodel bacteria
and archaea and await discovery.
RATIONALE: Antiphage defense systems are
known to be frequently physically clustered
in microbial genomes such that, for example,
genes encoding restriction enzymes common-
ly reside in the vicinity of genes encoding other
phage resistance systems. The observation that
defense systems are clustered in genomic “de-
fense islands” has led to the hypothesis that
genes of unknown function residing within
such defense islands may also participate in
RESULTS: We searched for gene cassettes of
unknown function that are enriched near
known defense systems in more than 45,000
available bacterial and archaeal genome se-
quences. Such gene cassettes were defined as
biotechnological tools, as
exemplified by the use of restriction enzymes
and CRISPR-Cas for biotechnological and bio-
medical applications. One may envision that
some of the systems discovered in the current
study, once their mechanism is deciphered, will
the future.
▪
(2018). DOI: 10.1126/science.aar4120
A pipeline for systematic discovery of defense systems. Microbial genomes (more than 45,000 in the current study) are mined for genetic systems図: 本論文のResearch Article Summaryより
45,000以上の微生物ゲノムを取得し，既知防御システムの関連遺伝子をマッピングして，その近傍の未知遺伝子を
新規防御システム関連遺伝子群の候補とした。
新規防御システムの候補となる遺伝子群のDNAを合成し，大腸菌 (E. coli)か枯草菌 (B. subtilis)にクローニングして
様々なファージへの抵抗性を検証した。

6
Fig. 1. Defense island 内の新規抗ファージ防御システムの発見
system, and an abortive infection gene of the
AbiH family) for which source organisms were
similarly selected and cloning was performed
into B. subtilis, as well as a sixth control com-
posed of the recently discovered DISARM de-
fense system (9) (table S4).
the 10 B. subtilis phages tested (see Discussion).
We named the nine verified new systems after
protective deities from various world mytholo-
gies. These defense systems comprise between
1 and 5 genes and span between 2 and 12 kb of
genomic DNA (Table 1 and table S5). Where
Below, we focus on further functional analyses
for a selected set of systems.
The Zorya defense system
The Zorya system (named after a deity from
Slavic mythology) was identified based on the
Doron et al., Science 359, eaar4120 (2018) 2 March 2018 2 of 11
Fig. 1. Discovery of new antiphage defense systems in defense islands.
(A) Illustration of the computational analysis employed for each pfam found
to be enriched in defense islands. Pfams that are enriched in the vicinity of
known defense genes are identified, and their neighboring genes are clustered
based on sequence homology to identify conserved cassettes that represent
putative defense systems. (B) Tendency of protein families to occur
next to defense genes. The genomic neighborhood for each member gene
in each pfam is examined, and the fraction of member genes occurring
in the vicinity (10 genes on each side) of one or more known defense genes
is recorded. Pink, a set of 123 pfams known to participate in antiphage
defense (“positive set”); blue, the remaining 13,960 pfams analyzed in this
study. (C) Neighborhood variability score for the analyzed pfams. Score
represents the fraction of pfam members occurring in different defense
neighborhoods out of total occurrences of pfam members (see Methods).
Pink, the 123 positive pfams; blue, a set of 576 pfams that passed the 65%
threshold for fraction of members occurring with defense genes in proximity.
onJune6,2018http://science.sciencemag.org/dedfrom
candidate system we selected source organisms
from which the system was taken and heterolo-
gously cloned into one of the model organisms.
To increase the probability that the cloned system
would be compatible and functionally expressed
within the receiving bacterium, we selected sys-
tems from mesophilic organisms as close phylo-
genetically as possible to E. coli or to B. subtilis
and included the upstream and downstream in-
tergenic regions so that promoters, terminators,
or other regulatory sequences would be preserved.
Where possible, we took at least two instances
of each system (from two different source ge-
nomes), to account for the possibility that some
systems may not be active in their source orga-
nism (21, 22). The DNA of each system, spanning
the predicted genes and the intergenic spaces,
was synthesized or amplified from the source
genome and cloned into the phylogenetically
closest model organism—either to E. coli (on a
plasmid) or to B. subtilis (genomically integrated).
As a control, we repeated the procedure with five
known defense systems (instances of types I, II,
and III R-M systems, a type III toxin/antitoxin
system, and an abortive infection gene of the
AbiH family) for which source organisms were
similarly selected and cloning was performed
into B. subtilis, as well as a sixth control com-
posed of the recently discovered DISARM de-
fense system (9) (table S4).
Altogether, we attempted to heterologously
clone 61 representative instances of the 28 can-
didate new systems, and successful cloning was
verified by whole-genome sequencing (table S4).
For 27 of these 28 systems, there was at least
one candidate locus for which cloning was suc-
cessful, and RNA sequencing (RNA-seq) of the
transformants showed that, for 26 of the sys-
tems, at least one of the candidate loci was ex-
pressed in the receiving E. coli or B. subtilis strain.
The engineered bacteria were then challenged
by an array of phages consisting of 10 B. subtilis
and six E. coli phages, spanning the three major
families of tailed double-stranded DNA (dsDNA)
phages (myo-, sipho-, and podophages), as well as
one single-stranded DNA (ssDNA) phage infecting
E. coli (Fig. 2, B and C). Measuring phage effi-
ciency of plating (EOP) on system-containing bac-
teria versus control cells, we found that 9 of the
26 tested systems (35%) showed protection from
infection by at least one phage (Fig. 2, B and C
and figs. S2 and S3). In comparison, three of the
six positive control systems showed defense, with
the remaining three showing no protection against
the 10 B. subtilis phages tested (see Discussion).
We named the nine verified new systems after
protective deities from various world mytholo-
gies. These defense systems comprise between
1 and 5 genes and span between 2 and 12 kb of
genomic DNA (Table 1 and table S5). Where
possible, we verified system consistency by test-
ing for phage resistance in systems where indi-
vidual genes were deleted (Figs. 3 to 5 and figs.
S4 and S5). We found between several hundred
and several thousand representations of each
of the defense systems in sequenced microbial
genomes, usually with broad phylogenetic dis-
tribution (fig. S6 and tables S6 to S15). Most
systems were detected in >10 taxonomic phyla,
and 7 of them appear in archaea (fig. S6). Some
of the systems seem to target a specific family
of phages (e.g., the Thoeris system appears to
specifically protect from myophages), whereas
others, such as the Hachiman system, provide
broader defense (Fig. 2B). The genes comprising
the new systems encode many protein domains
that are commonly present in antiviral systems
such as CRISPR-Cas and RNA interference (RNAi),
including helicases, nucleases, and nucleic acid
binding domains, in addition to many domains
of unknown function and also atypical domains
as described below. Three of the systems contain
membrane-associated proteins, as predicted by
the presence of multiple transmembrane helices.
Below, we focus on further functional analyses
for a selected set of systems.
The Zorya defense system
The Zorya system (named after a deity from
Slavic mythology) was identified based on the
Fig. 1. Discovery of new antiphage defense systems in defense islands.
(A) Illustration of the computational analysis employed for each pfam found
to be enriched in defense islands. Pfams that are enriched in the vicinity of
in the vicinity (10 genes on each side) of one or more known defense genes
is recorded. Pink, a set of 123 pfams known to participate in antiphage
defense (“positive set”); blue, the remaining 13,960 pfams analyzed in this
onJune6,2018http://science.sciencemag.org/Downloadedfrom
Fig. 1A: 情報学的解析の概要
Fig. 1B: 各タンパク質ファミリーの防御遺伝子近隣に存在する傾向
14,083種類のタンパク質ファミリー (Pfam)を対象に，各ファミリーごとに，構成遺伝子 (メンバー)が既知防御遺伝子の近隣 (10遺伝子
以内)に出現する割合を計算。総メンバーのうち，防衛関連メンバーの割合としてスコア化 ( defense score )
→ スコアが65%以上であった576 Pfamsへと絞り込んだ。
※防御遺伝子 = 防御システム関連遺伝子とする。
Fig. 1C: タンパク質ファミリーの近傍変動性スコア
Pfamメンバーの出現総数のうち，異なる防御システム領域に出現するPfamメンバーの割合をスコア化 ( defense context variability score )
例えば，pfamXがデータセット中の20個の遺伝子内に見出され，そのうち15個が近くにCas9を有し，5個が近くにType I RMを有した場合，スコアは2/20 = 0.1となる。
→ スコアが0.1以上であった277 Pfamsへと絞り込んだ。
2種類のスコアを算出して候補タンパク質ファミリーを絞り込む..
※要は特定の防御系付近にしか出てこない遺伝子を取り除きたい。

7
情報学的解析パイプラインの流れ
14,083 Pfams
Pfam総メンバーのうち，防衛関連メンバー (既知防御遺伝子の近隣に出現)の割合が65%以上のPfam
576 Pfams
Pfamメンバーの出現総数のうち，異なる防御システム領域に出現するPfamメンバーの割合が10%以上のPfam
277 Pfams
先行研究によって防御システムに関連していると推定されている遺伝子ファミリーを追加
335 gene families
※Pfamとして登録されていないもの含む
(タンパク質ファミリーデータベースに登録されているほぼ総数)
Figure S1: Distribution of predicted functions for pfams genomically associated with
known defense genes. (A) Pfams retrieved from prediction cycle #1. (B) Pfams retrieved
from prediction cycle #2.
Figure S1A.
335遺伝子ファミリーの内訳
複数遺伝子より構成されるシステムを予測するため，
各ファミリー (335個)を軸に周辺の遺伝子 (前後10遺伝子)を取得し，
並び順が保存されている遺伝子群を1セットにて防御システム候補としている。
335遺伝子ファミリーの内 Known defense systems と Candidate systems を
正解セット (既知防御遺伝子)にし，予測サイクルをもう1回実行。
著者らが作成した正解セット
41 候補免疫システム
特定系統内にのみ存在する系を除去
28 候補免疫システム

8
Fig. 2. 防御システムの実験的検証（手法）
ciated with tellurium- and stress-resistance genes
(23). The reconstructed system is composed of the
four genes zorABCD, overall encompassing ~9 kb
of DNA, with pfam15611 being the third gene in
the system (zorC) (Fig. 3C and Table 1). A repre-
sentative Zorya operon from E. coli E24377A was
cloned into E. coli MG1655 and provided 10- to
10,000-fold protection against infection by T7,
comprised of the three genes zorABE. A type II
Zorya was cloned from E. coli ATCC8739 into
E. coli MG1655 and provided defense against T7
and the ssDNA phage SECphi17 (Figs. 2C and 3, B
and C, and fig. S3).
The first two genes of the Zorya system, zorA
and zorB, contain protein domains sharing dis-
tant, but clear, homology with domains in motA
of the flagellar motor (the static part wi
which the flagellar rotor swivels) (24). The Mo
complex also forms the proton channel
provides the energy for flagellar rotation,
pling transport of protons into the cell with
rotation (Fig. 3D) (25, 26). Whereas zorB sh
the same size and domain organization w
motB (including the pfam13677 and pfam00
Fig. 2A: 実験的検証の流れ
対象：Escherichia coli str. MG1655, Bacillus subtilis str. BEST7003
1. 候補防御システム遺伝子と周辺遺伝子間領域のDNAを合成し，E. coliかB. subtilisに導入した。
2. 導入に成功し，発現が確認された26候補システム含有バクテリアについて，10種のB. subtilisファージと6種のE. coliファージを感染
させ抵抗性があるか検証した。

9
Fig. 2. 防御システムの実験的検証（結果）
Fig. 2. Experimentally verified defense systems. (A) Flowchart of the
experimental verification strategy. (B) Active defense systems cloned into
B. subtilis. (C) Active defense systems cloned into E. coli. For (B) and (C),
fold protection was measured using serial dilution plaque assays,
comparing the system-containing strain to a control strain that lacks the
system and has an empty vector instead. Data represent average of three
replicates; see figs. S2 and S3. Numbers below phage names represent
phage genome size. On the right, gene organization of the defense
systems, with identified domains indicated (DUF, domain of unknown
function). Gene sizes are drawn to scale; scale bar, 400 amino acids.
Fig. 2B: B. Subtilisでファージ抵抗性が示された防御システム
Fig. 2C: E. coliでファージ抵抗性が示された防御システム
Fig. 2. Experimentally verified defense systems. (A) Flowchart of the
experimental verification strategy. (B) Active defense systems cloned into
B. subtilis. (C) Active defense systems cloned into E. coli. For (B) and (C),
fold protection was measured using serial dilution plaque assays,
comparing the system-containing strain to a control strain that lacks the
system and has an empty vector instead. Data represent average of three
replicates; see figs. S2 and S3. Numbers below phage names represent
phage genome size. On the right, gene organization of the defense
systems, with identified domains indicated (DUF, domain of unknown
function). Gene sizes are drawn to scale; scale bar, 400 amino acids.
ファージゲノムサイズ→
ヒートマップ：防御システムを導入していないコントロールと比較した
際の抵抗性。n=3の平均。
Myoviridae特異的な抵抗性→
広く抵抗→
• いくつかの防御システムは特定のファージ系統を標的にしている。
• 新規防御システムを構成する遺伝子には，一般的な抗ウイルスシステムと共通したドメインを
持つものも多い。
↑防御システムの遺伝子構成
Helicase, nuclease, 核酸結合ドメイン等
DUF: 機能未知ドメイン

10
Table 1. 本研究で発見された防御システムの一覧
The Thoeris defense system
Thoeris (Egyptian protective deity of childbirth
and fertility) is a system that was detected based
on the enrichment of pfam08937 [Toll-interleukin
B. subtilis BEST7003 and were found to confer
defense against myophages (Fig. 2B, Fig. 4, B
and C, and fig. S2). Because the three myophages
that we tested are very different from each other
and share few homologous genes, it is possible
volved in direct recognition of pathogens (31, 32).
Our finding marks a common involvement of TIR
domains in innate immunity across the three
domains of life and implies that the ancestry of
this important component of eukaryotic innate
Table 1. Composition of defense systems reported in this study.
System Operon Associated domains* Domain annotations
No. of
instances
detected within
microbes
No. (%) of
genomes in
which system
is found
Comments
Thoeris ThsAB pfam13289, pfam14519,
pfam08937, pfam13676
SIR2, Macro domain,
TIR domain
2099 2070 (4.0%) Membrane associated
(sometimes)............................................................................................................................................................................................................................................................................................................................................
Hachiman HamAB pfam08878, COG1204,
Helicase 1781 1742 (3.4%)
............................................................................................................................................................................................................................................................................................................................................
Shedu SduA pfam14082 Nuclease 1246 1191 (2.3%)............................................................................................................................................................................................................................................................................................................................................
Gabija GajAB pfam13175, COG3593,
COG0210, pfam13245
ATPase, nuclease, helicase 4598 4360 (8.5%)
............................................................................................................................................................................................................................................................................................................................................
Septu PtuAB pfam13304, COG3950,
ATPase, HNH nuclease 2506 2117 (4.1%)
............................................................................................................................................................................................................................................................................................................................................
Lamassu LmuAB pfam14130, pfam02463 SMC ATPase N-terminal
domain
697 682 (1.3%)
............................................................................................................................................................................................................................................................................................................................................
Zorya
(type I)
ZorABCD pfam01618, pfam13677,
pfam00691, COG1360,
pfam15611, pfam00176,
pfam00271, COG0553,
pfam04471
MotA/ExbB, MotB, helicase,
Mrr-like nuclease
............................................................................................................................................................................................................................................................................................................................................
Zorya
(type II)
ZorABE pfam01618, pfam13677,
pfam00691, COG1360,
COG3183, pfam01844
MotA/ExbB, MotB, HNH
nuclease
............................................................................................................................................................................................................................................................................................................................................
Kiwa KwaAB pfam16162 No annotated domain 934 924 (1.8%) Membrane associated............................................................................................................................................................................................................................................................................................................................................
Druantia DruABCDE (type I)
DruMFGE (type II)
DruHE (III)
pfam14236, pfam00270,
pfam00271, pfam09369,
COG1205, pfam00145,
COG0270
Helicase, methylase 1342 1321 (2.6%)
............................................................................................................................................................................................................................................................................................................................................
Wadjet JetABCD pfam11855, pfam09660,
pfam13835, pfam09661,
pfam13555, pfam13558,
COG4913, COG1196,
pfam11795, pfam09983,
pfam11796, pfam09664,
COG4924
MukBEF condensin,
topoisomerase VI
3173 2880 (5.6%)
............................................................................................................................................................................................................................................................................................................................................
*Pfam and COG domains were assigned according to the information in the IMG database (48) and supplemented using HHpred (52).
Fig. 4
Fig. 3
Fig. 5
本研究で発見された防御システムの中で特徴的だった3つのシステムを解説

11
No. of
instances
detected within
microbes
No. (%) of
genomes in
which system
is found
Comments
SIR2, Macro domain,
TIR domain
(sometimes)............................................................................................................................................................................................................................................................................................................................................
Helicase 1781 1742 (3.4%)
............................................................................................................................................................................................................................................................................................................................................
COG0210, pfam13245
............................................................................................................................................................................................................................................................................................................................................
............................................................................................................................................................................................................................................................................................................................................
domain
697 682 (1.3%)
............................................................................................................................................................................................................................................................................................................................................
Zorya
(type I)
pfam00691, COG1360,
pfam15611, pfam00176,
pfam00271, COG0553,
pfam04471
Mrr-like nuclease
............................................................................................................................................................................................................................................................................................................................................
Zorya
(type II)
pfam00691, COG1360,
COG3183, pfam01844
nuclease
............................................................................................................................................................................................................................................................................................................................................
DruMFGE (type II)
DruHE (III)
pfam14236, pfam00270,
pfam00271, pfam09369,
COG1205, pfam00145,
COG0270
............................................................................................................................................................................................................................................................................................................................................
pfam13835, pfam09661,
pfam13555, pfam13558,
COG4913, COG1196,
pfam11795, pfam09983,
pfam11796, pfam09664,
COG4924
MukBEF condensin,
topoisomerase VI
3173 2880 (5.6%)
............................................................................................................................................................................................................................................................................................................................................
Fig. 3

12
Fig. 3. Zorya system (1/2)
with a helicase domain that in some cases also en-
codes a C-terminal Mrr-like nuclease domain.
Type II Zorya lacks zorC and zorD and instead
contains zorE, a smaller gene encoding an HNH-
endonuclease domain.
The gene composition of the Zorya system may
point to several hypotheses as to its mechanism
of action. It is possible that the system has adopted
esis, Zorya may be a conditional abortive infec-
tion system. Indeed, although Zorya-containing
cells that were infected by phage T7 did not yield
phage progeny in >80% of infection events, in-
fection of Zorya-containing cells in liquid cultures
has led to an eventual culture collapse, suggesting
that Zorya-mediated defense involves death or
metabolic arrest of the infected cells (fig. S7).
ity, because point mutations in residues predicted
to be critical for proton translocation through the
channel (either ZorA:T147A/S184A or ZorB:D26N)
yielded a nonfunctional system (Fig. 3E). Sim-
ilarly, point mutations inactivating the Walker B
motif of the ZorD helicase domain, predicted to
prevent adenosine triphosphate (ATP) hydrolysis,
resulted in loss of protection from phage infection.
RM: Restriction-modiﬁcation system
TA: Toxin-Antitoxin system
Abi: Abortive infection system
DISARM: 最近発見された免疫機構
Wadjet, Druantia: 本研究で発見された免疫機構
→Zoryaシステムはどのようにしてファージに対抗しているのか？ (続く)
Type Iに関わる遺伝子としてZorABCD, Type IIに関わる遺伝子としてZorABEが同定。
Fig. 3A, B: Type I Zoryaシステム (A), Type II Zoryaシステム (B)とその周辺の遺伝子配置を表した模式図。
Type I
Type II

13
Fig. 3. Zorya system (2/2)
←C: Zoryaのドメイン構成
Type IとIIに共通しているのはzorA, B。
zorA, BはmotA, Bと相同性のあるドメインを有している。
Fig. 3. The Zorya system. (A) Representative instances of the type I Zorya
system and their defense island context. Genes known to be involved in defense
are orange. Mobilome genes are in dark gray. RM, restriction-modification;
TA, toxin-antitoxin; Abi, abortive infection; Wadjet and Druantia are systems
identified as defensive in this study (see below). (B) Representative instances of
the type II Zorya system. (C) Domain organization of the two types of Zorya.
(D) Model of the flagellum base. The position of the MotAB complex is
indicated. (E) EOP of phage SECphi27 infecting wild-type (WT) type I Zorya,
deletion strains, and strains with point mutations. Data represent plaque-
forming units per ml; average of three replicates. Error bars, mean ± SD. ZorA:
T147A/S184A and ZorB:D26N are predicted to abolish proton flux; ZorC:
E400A/H443A are mutations in two conserved residues in pfam15611 (EH
domain) whose function is unknown (23); ZorD:D730A/E731A are mutations in
the Walker B motif, predicted to abolish ATP hydrolysis.
June6,2018
arch 2018 4 of 11
e instances of the type I Zorya
known to be involved in defense
RM, restriction-modification;
et and Druantia are systems
. (B) Representative instances of
tion of the two types of Zorya.
of the MotAB complex is
indicated. (E) EOP of phage SECphi27 infecting wild-type (WT) type I Zorya,
deletion strains, and strains with point mutations. Data represent plaque-
forming units per ml; average of three replicates. Error bars, mean ± SD. ZorA:
T147A/S184A and ZorB:D26N are predicted to abolish proton flux; ZorC:
E400A/H443A are mutations in two conserved residues in pfam15611 (EH
domain) whose function is unknown (23); ZorD:D730A/E731A are mutations in
the Walker B motif, predicted to abolish ATP hydrolysis.
←D: 毛基部のモデル図
バクテリアの毛モーターの一部である内膜タンパク質。
MotAB複合体となってプロトンチャネルを形成し，細胞内へのプロ
トン流入を通して毛を回転させる。
MotA, B:
zorC: 機能未知遺伝子
zorD: Helicaseドメインを持つ
Helicase: DNAの二重螺旋やRNA二次構造を解く
zorE: HNH endonucleaseドメインを持つ
Endonucleaseの一種
tures
esting
th or
S7).
ilarly, point mutations inactivating the Walker B
motif of the ZorD helicase domain, predicted to
prevent adenosine triphosphate (ATP) hydrolysis,
resulted in loss of protection from phage infection.
of phage SECphi27 infecting wild-type (WT) type I Zorya,
nd strains with point mutations. Data represent plaque-
←E: Zoryaを構成する遺伝子を欠損/変異導入した際のファージ抵抗性の検証
zorABCDは全てZoryaシステムが機能するのに必須。
予測された各ドメインの活性にとって重大な箇所に変異を導入して検証もしている。
PFU: プラーク形成単位。値が大きいほど細胞が多く溶解している。
ZorC, D, EはファージDNAの感知および不活性化に関与している可能性があり，
ファージの不活性化が失敗すると，ZorABプロトンチャネルが開き，膜脱分極および
細胞死を引き起こすと考えられる。
毛の根本

14
No. of
instances
detected within
microbes
No. (%) of
genomes in
which system
is found
Comments
SIR2, Macro domain,
TIR domain
(sometimes)............................................................................................................................................................................................................................................................................................................................................
Helicase 1781 1742 (3.4%)
............................................................................................................................................................................................................................................................................................................................................
COG0210, pfam13245
............................................................................................................................................................................................................................................................................................................................................
............................................................................................................................................................................................................................................................................................................................................
domain
697 682 (1.3%)
............................................................................................................................................................................................................................................................................................................................................
Zorya
(type I)
pfam00691, COG1360,
pfam15611, pfam00176,
pfam00271, COG0553,
pfam04471
Mrr-like nuclease
............................................................................................................................................................................................................................................................................................................................................
Zorya
(type II)
pfam00691, COG1360,
COG3183, pfam01844
nuclease
............................................................................................................................................................................................................................................................................................................................................
DruMFGE (type II)
DruHE (III)
pfam14236, pfam00270,
pfam00271, pfam09369,
COG1205, pfam00145,
COG0270
............................................................................................................................................................................................................................................................................................................................................
pfam13835, pfam09661,
pfam13555, pfam13558,
COG4913, COG1196,
pfam11795, pfam09983,
pfam11796, pfam09664,
COG4924
MukBEF condensin,
topoisomerase VI
3173 2880 (5.6%)
............................................................................................................................................................................................................................................................................................................................................
Fig. 4

15
Fig. 4. Thoeris system
main gene is responsible for identification of
specific phage patterns, with multiple TIR do-
main genes serving for recognition of differ-
ent phage components. Under this hypothesis,
it is tempting to suggest that Thoeris is the pro-
karyotic ancestral form of pathogen-associated
molecular pattern (PAMP) receptors.
A recent study showed that TIR domains can
have enzymatic NAD+
(oxidized form of NAD)
hydrolase activities (36), which is in line with pre-
ish NAD+
binding, also resulted in system inactiv-
ation (ThsA:N112A and ThsA:D100A/N115A for
the B. cereus and B. amyloliquefaciens systems,
respectively). These results suggest that NAD+
binding and hydrolysis are essential for the
antiphage activity of the Thoeris system.
The Druantia system
Another system worth discussing briefly is the
Druantia system (named after a deity from the
are also associated with a cytosine methylase
(fig. S5, A and B). A type I system cloned from
E. coli UMEA 4076-1 into E. coli MG1655 rendered
the engineered strain resistant against four of
the six phages tested, and by serially deleting
four of the genes in this system, we verified that
all four are essential for its activity (fig. S5C).
Notably, DUF1998-containing genes are among
the components of the recently reported DISARM
(9) and Dpd (40) defense systems, where their
Fig. 4. The Thoeris system. (A) Representative instances of the Thoeris
system and their defense island context. Thoeris genes thsA (containing
NAD+
binding domain) and thsB (TIR domain) are marked dark and
light green, respectively. Genes known to be involved in defense are
orange. Mobilome genes are in dark gray. RM, restriction-modification;
TA, toxin-antitoxin; Abi, abortive infection. (B) The two Thoeris systems
shown in this study to protect against myophages. Locus tag accessions
are indicated for the individual genes. (C) EOP of phage SBSphiJ
infection with WT and mutated versions of the B. amyloliquefaciens Y2
Thoeris (top set) or B. cereus MSX-D12 Thoeris (bottom set) cloned
into B. subtilis BEST7003. Average of three replicates; error bars,
mean ± SD.
it is tempting to suggest that Thoeris is the pro-
karyotic ancestral form of pathogen-associated
molecular pattern (PAMP) receptors.
A recent study showed that TIR domains can
have enzymatic NAD+
(oxidized form of NAD)
hydrolase activities (36), which is in line with pre-
binding and hydrolysis are essential for the
antiphage activity of the Thoeris system.
The Druantia system
Another system worth discussing briefly is the
Druantia system (named after a deity from the
the six phages tested, and by serially deleting
four of the genes in this system, we verified that
all four are essential for its activity (fig. S5C).
Notably, DUF1998-containing genes are among
the components of the recently reported DISARM
(9) and Dpd (40) defense systems, where their
Fig. 4. The Thoeris system. (A) Representative instances of the Thoeris
system and their defense island context. Thoeris genes thsA (containing
NAD+
binding domain) and thsB (TIR domain) are marked dark and
light green, respectively. Genes known to be involved in defense are
orange. Mobilome genes are in dark gray. RM, restriction-modification;
TA, toxin-antitoxin; Abi, abortive infection. (B) The two Thoeris systems
shown in this study to protect against myophages. Locus tag accessions
are indicated for the individual genes. (C) EOP of phage SBSphiJ
infection with WT and mutated versions of the B. amyloliquefaciens Y2
Thoeris (top set) or B. cereus MSX-D12 Thoeris (bottom set) cloned
into B. subtilis BEST7003. Average of three replicates; error bars,
mean ± SD.
Fig. 4A: Thoerisシステムとその周辺の遺伝子配置を表した模式図。
thrA, Bを同定。
特にthrBは複数コピー存在している。
Fig. 4B: Thoerisのドメイン構成
thrAはNAD+結合ドメインを持つ
thrBはTIR-likeドメインを持つ
Fig. 4C: thrA, BはThoerisシステムの機能にとって必須である。
特にNAD+結合・加水分解能が必須である。
TIR: Toll-interleukin receptor
ThsA: NAD+結合サイトへの変異導入
ThsB: TIRドメイン内のNAD切断活性サイトへの変異導入
TIRドメインは動物のToll様受容体，植物のR遺伝子に存在し，病原体の直接的な認識に関与していることが知られている。
R遺伝子: 植物で病原体に対する抵抗性を生み出す。
→ ・TIRドメインが生命全体で自然免疫機構に関わっている。
・このような原核生物の抗ファージ防衛システムが真核生物の自然免疫の祖先になった可能性を示唆。
このシステムの発見によって..
→それぞれ特異的なファージのパターンを認識しているのでは。

16
No. of
instances
detected within
microbes
No. (%) of
genomes in
which system
is found
Comments
SIR2, Macro domain,
TIR domain
(sometimes)............................................................................................................................................................................................................................................................................................................................................
Helicase 1781 1742 (3.4%)
............................................................................................................................................................................................................................................................................................................................................
COG0210, pfam13245
............................................................................................................................................................................................................................................................................................................................................
............................................................................................................................................................................................................................................................................................................................................
domain
697 682 (1.3%)
............................................................................................................................................................................................................................................................................................................................................
Zorya
(type I)
pfam00691, COG1360,
pfam15611, pfam00176,
pfam00271, COG0553,
pfam04471
Mrr-like nuclease
............................................................................................................................................................................................................................................................................................................................................
Zorya
(type II)
pfam00691, COG1360,
COG3183, pfam01844
nuclease
............................................................................................................................................................................................................................................................................................................................................
DruMFGE (type II)
DruHE (III)
pfam14236, pfam00270,
pfam00271, pfam09369,
COG1205, pfam00145,
COG0270
............................................................................................................................................................................................................................................................................................................................................
pfam13835, pfam09661,
pfam13555, pfam13558,
COG4913, COG1196,
pfam11795, pfam09983,
pfam11796, pfam09664,
COG4924
MukBEF condensin,
topoisomerase VI
3173 2880 (5.6%)
............................................................................................................................................................................................................................................................................................................................................
Fig. 5

17
Fig. 5. 対プラスミドWadjet system
Defense against plasmid transformation
Some of the putative defense systems that we
experimentally tested did not show any anti-
phage activity despite being strongly associated
with known defense genes. We reasoned that
some of these systems may defend against other
forms of foreign DNA. To test this hypothesis,
we selected one such system, which we denote
Wadjet (god protector of ancient Egypt), for fur-
ther experimentation. Wadjet is a four-gene sys-
our array, all three consistently and significantly
reduced transformation efficiency of the episomal
plasmid pHCMC05 (Fig. 5C). These results sug-
gest that Wadjet may be a defense system spe-
cifically targeting foreign plasmids.
We identified three different domain compo-
sitions, each encoding a different set of pfams,
but all with common sequence signatures mark-
ing them as three types of Wadjet (Fig. 5B).
Whereas the pfam domains of Wadjet genes are
during replication (42), and mutations in the
housekeeping condensins lead to severe defects
in chromosome segregation and viability (43).
Several versions of housekeeping condensins
appear in bacterial genomes: SMC, MukBEF, and
MksBEF (44); the Wadjet system was previously
noted as a distant homolog of the MksBEF sys-
tem described in P. aeruginosa (45).
Although the domain organization of the
jetABC genes may lead to the hypothesis that
Fig. 5. The Wadjet system provides protection against plasmid trans-
formation in B. subtilis. (A) Representative instances of the Wadjet system
and their defense island context. Genes known to be involved in defense are
orange. RM, restriction-modification; TA, toxin-antitoxin; Abi, abortive infection.
(B) Domain organization of the three types of Wadjet. Pfam and COG
domains were assigned according to the information in the IMG database (48).
(C) Wadjet reduces plasmid transformation efficiency in B. subtilis. Instances
of Wadjet systems were taken from B. cereus Q1 (type I), B. vireti LMG 21834
(type II), and B. thuringiensis serovar finitimus YBT-020 (type III) (table S4) and
cloned into B. subtilis BEST7003. Gene deletions and point mutations are of
the B. cereus Q1 type I Wadjet. Transformation efficiency of plasmid pHCMC05
into Wadjet-containing strains is presented as a percentage of the transfor-
mation efficiency to B. subtilis BEST7003 carrying an empty vector instead of
the Wadjet system. Average of three replicates; error bars, mean ± SD.
Fig. 5A: Wadjetシステムとその周辺の遺伝子配置を表した模式図。
大部分が未知の機能領域 (DUF; Domains of unknown function)
→タンパク質の立体構造をもとに比較したところ，JetA, B, Cは
コンデンシン系のMukF, MukE, MukBと構造的相同性がある。
absence of recognizable domains in its genes
suggests a new mode of defense not shared by
prokaryotic defense systems whose mechanism
is currently understood.
Defense against plasmid transformation
Some of the putative defense systems that we
Three instances representing three different types
of Wadjet (see below) were cloned from three
separate Bacillus species into B. subtilis BEST7003.
Although none of these systems provided pro-
tection against any of the 10 Bacillus phages in
our array, all three consistently and significantly
reduced transformation efficiency of the episomal
and JetC and genes belonging to the housekeep-
ing condensin system MukF, MukE, and MukB,
respectively. Bacterial condensins are chromosome-
organizing complexes that are responsible for
DNA condensation and accurate segregation
during replication (42), and mutations in the
housekeeping condensins lead to severe defects
Wadjetシステムを構成する4つの遺伝子
jetABCDを同定。
コンデンシン系: 染色体の構築と分離に関わる
Fig. 5B: Wadjetのドメイン構成..
jetABCDは全てWadjetシステムの機能に必須。
JetB: MukE-MukF様タンパク質間相互作用を妨害する変異導入
JetC: ATP加水分解活性に関わるサイト (Walker Bモチーフ)に変異導入
JetD: 立体構造予測に基づいてトポイソメラーゼIVドメインがあると推定し，
トポイソメラーゼIVドメインのDNA結合を妨害する変異導入
トポイソメラーゼ: 2本鎖DNAの一方または両方を切断し再結合する酵素
Fig. 5C: Wadjetは枯草菌における
プラスミド形質転換効率を低下させ
る。
←
・Wadjetシステムのドメイン構成は，MukBEFコンデンシン系と類似している。
→MukBEFコンデンシン系を祖先に適応し対プラスミド防御システムとなったのではないか。
・過剰な自然形質転換を防ぐ役割がある，あるいはssDNAファージを対象としている可能性もある。

18
Fig. 3, 4, 5まとめ
Fig. 3. Zoryaシステム：抗ファージ防御システム。
細菌性毛の構成成分を持つシステム。脱分極を通して細胞死を誘導す
る，不稔感染系の一種。
Fig. 4. Thoerisシステム：抗ファージ防御システム。
TIRドメインを持ち，動物・植物および細菌が持つ自然免疫機構に共通の古代の
祖先が存在することを示唆している。
Fig. 5. Wadjetシステム：対プラスミド防御システム。
コンデンシン系の構成成分を持つシステム。過剰な自然形質転換を防ぐ。
本研究で発見された防御システムの中で特徴的だった3つのシステムを解説

19
総括
まとめ/掲載理由
本論文では，多数の原核生物ゲノムを対象に既知防御システムの近隣遺伝子を網羅的に探索
することで新規防御システム候補を予測し，実験的検証を経て新規に9つの抗ファージ防御
システムと1つの抗プラスミド防御システムを発見した。
これらは動物・植物における自然免疫の起源について示唆を与えると共に，制限修飾系や
CRISPR-Casのような革新的な分子ツールとして応用できる可能性がある。
感想
Zorya
source: ancient-origins.net
• バクテリアの毛運動や染色体維持に関わっているような要素が外敵に対する防衛システムにもなっていて興味深
い。
• そもそもなぜ原核生物の免疫システムがゲノム上でクラスターとなっているのか興味深い。
• 今回予測はされたが実験で検証できなかったもの含め，まだまだ未発見の防御システムは多数存在するはず。(今回
使用した2種のモデル生物上では機能しないものだった可能性等)
• 今回発見された防御システム達は微生物ゲノム中の1割未満にしか存在していないが (cf. Table 1)，一体どのようなウ
イルスに対抗するためにこのような機構を獲得したのか。
• たびたびCRISPR-Cas (獲得免疫)の話を持ち出してきているが，今回の実験系はそ
もそも自然免疫系しか対象としていない。一度感染させたファージを再度感染させ
る実験等を行えば，新たな獲得免疫系が発見できる可能性があると思った。

20
防御システムの原核生物内における系統的分布
Figure S6

21
Zorya system 補足
Figure S7. Phage infection of Zorya-containing cells in solid and liquid cultures. (A)
Zorya-containing cells were infected with T7 phage at MOI 0.05 and were incubated for
10 min at 37 °C to allow phage adsorption. Cells with adsorbed phages were then
centrifuged and washed to eliminate unabsorbed phages. These T7-infected cells were
mixed together with E. coli MG1655 cells that lack Zorya, and the mixture was plated
using an agar overlay method. Phage bursts from successful infections are visualized as a
single infection center on a lawn from the T7-sensitive E. coli strain, enabling an
evaluation of the number of phages that have adsorbed and completed a successful
infection cycle. Three replicates of cells containing type I Zorya, type II Zorya or no
Figure S7
MOI: 細胞1個に対するウイルスの数
B, C: Zorya持ち細胞群の培養曲線がNo Zoryaに比べて遅れて下落していることから，
感染したZorya持ち細胞群の多くは不稔感染を被ったのではないか。
A: 感染サイクルを完了させることができたファージ数の評価。
Type I持ちのうち平均して17%，Type IIでは6%が子孫ファージを産生した。

[論文紹介] 微生物パンゲノムにおける抗ファージ防御システムの系統的発見 (Systematic discovery of antiphage defense system in the microbial pangenome)

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[論文紹介] 微生物パンゲノムにおける抗ファージ防御システムの系統的発見 (Systematic discovery of antiphage defense system in the microbial pangenome)