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生物の教科書が書き換わる!?人類史上初めて原核生物なのに核を持つ細菌が見つかる。
http://www.asyura2.com/16/eg3/msg/168.html
投稿者 ピノキ 日時 2021 年 1 月 02 日 03:07:52: /cgEbzQ/iEx0c g3ODbYNM
 

またまた日本で世紀の大発見がありました。
原核生物なのに人間のような細胞のつくりになっている細菌が見つかりました。


この細菌の性質を解明していくことで、天然ガス田の開発や効率利用の促進など日本のエネルギー産業に大きく貢献する可能性があります。
将来は意図的に天然ガスを生み出せるようになるかもしれません。
生物界の常識を覆したうえ、エネルギー生成にもかかわっているとは驚きです。



ひつじさん【明るいニュースch】


【画像の出典】
@産総研:地下で発見!ゲノムが膜で包まれたバクテリア (aist.go.jp)
 https://www.aist.go.jp/aist_j/press_release/pr2020/pr20201214/pr20201214.html


A真核細胞の構造 | バイオハックch (manabu-biology.com)
 http://manabu-biology.com/archives/42005065.html


B水溶性天然ガス田の操業における課題解決への取り組み|JOGMEC石油・天然ガス資源情報ウェブサイト
 https://oilgas-info.jogmec.go.jp/info_reports/1008604/1008819.html


C「生命の起源」ついに明らかに? その想像以上にシンプルなメカニズム | WIRED.jp
 https://wired.jp/2017/05/09/how-life-began/


D「生物基礎教科書解説」原核細胞と真核生物の細胞の構造|高校生物の学び舎 (biology-manabiya.net)
 https://biology-manabiya.net/basicbiology-a/


E三ドメイン説による生物の分類とは【古細菌・真正細菌・真核細胞】 | 透析note (clinical-engineers.com)
 https://clinical-engineers.com/third-domain-theory/


Fメタンハイドレート 世界 - Bing
 http://matome.naver.jp/odai/214176932...(リンク切れ)


G石炭を天然ガスに変えるメタン生成菌を発見 | つくばサイエンスニュース (tsukuba-sci.com)
 http://www.tsukuba-sci.com/?p=992


H石炭を天然ガスに変える、メタン生成菌を新発見 - スマートジャパン (itmedia.co.jp)
 https://www.itmedia.co.jp/smartjapan/articles/1610/17/news042.html


Iメタンハイドレート - Bing
 https://matome.naver.jp/odai/21419484...(リンク切れ)


J職人の勘をデータベースに。海上石油プラントではAIが何でも教えてくれる | ギズモード・ジャパン (gizmodo.jp)
 https://www.gizmodo.jp/2017/07/mugendai-oilrig.html


K真核細胞と原核細胞の違い 【第2回】細胞の多様性 - 偏差値30からの生物基礎まとめ (hatenablog.com)
 https://shimasensei.hatenablog.com/entry/2018/02/25/140233


L【60秒解説】日本周辺に眠る「燃える氷」(METI/経済産業省)
 https://www.meti.go.jp/main/60sec/2016/20160216001.html


M花見川流域を歩く HANAMIGAWA RYUIKI wo ARUKU: 発見 花見川川底から天然ガス湧出 (hanamigawa2011.blogspot.com)
 https://hanamigawa2011.blogspot.com/2013/05/blog-post_5.html


N水溶性天然ガス田の操業における課題解決への取り組み|JOGMEC石油・天然ガス資源情報ウェブサイト
 https://oilgas-info.jogmec.go.jp/info_reports/1008604/1008819.html


Oパネェ速報 : 希少金属使わず、ヨウ素を触媒にクロスカップリング反応 (livedoor.jp)
 http://blog.livedoor.jp/sakuratanakasakura/archives/1249822.html


P国内シェア75%のヨウ素、千葉県で産学連携広がる 医療・太陽電池への活用模索(2/2ページ) - 産経ニュース (sankei.com)
 https://www.sankei.com/region/news/181204/rgn1812040032-n2.html
 

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1. 2021年5月20日 02:29:25 : jXbiWWJBCA : Rm5WWGpiTzAwU2c=[672] 報告

この人は、相変わらずいい加減な記事だな

この研究自体は、科学だけでなく実用性にもつながる非常に優れたもので、少なくとも大学の生物学の教科書は書き換わるだろうが


別にRT761自体は始祖ではないし、生命の起源が明らかになるわけでもない

極限環境下で生存する、極めて特殊な生物(配列)が新たに見つかったということだ

DNAを覆う膜構造があることは、真核生物と原核生物をつなぐ可能性はあるが

全く独立して進化してきた可能性もあるから、今後の研究次第だろう

https://academist-cf.com/journal/?p=15780

https://academist-cf.com/journal/?p=15780
ゲノムが膜で包まれた常識外れのバクテリア – 最先端の顕微鏡で鮮やかにとらえた膜構造
片山 泰樹 2021年4月6日 ゲノムが膜で包まれた常識外れのバクテリア – 最先端の顕微鏡で鮮やかにとらえた膜構造2021-04-02T09:49:51+09:00研究コラム 微生物, 生物学, 細胞

微生物のマジョリティは「培養できない菌」という未知の領域
「原核生物」はバクテリアやアーキアと呼ばれる微生物の仲間です。肉眼で見ることができないため馴染みの薄い存在ですが、その種類と存在量はもう一方の生物群である「真核生物」(動物、植物、菌類、原生生物)よりも多いと推定されています。ところが、性質や実物の写真などを記載してカタログ化すると、上記5つのグループのなかで最も薄い図鑑になってしまうのは原核生物です。善玉・悪玉菌と呼ばれる乳酸菌や大腸菌などのように、性質がよく知られている菌は原核生物全体でみるとごくわずかしかいません。
ある菌種の性質を知るためには、他の菌から完全に分離して人工的に培養し増殖させる必要があります。大まかに見積もって全原核生物の99%は人工的に培養することができない「未培養」の菌のため、性質が未知なのです。
1千万種は下らないといわれている原核生物の大多数が未知のまま——そこにロマンが詰まっている……もとい、社会に有益な知見や材料が得られるはず、というのが今回紹介する研究の原点です。
地下環境の未培養菌を約5年かけて分離培養
私は、地上から数100 m深くの地下環境に棲息する微生物を培養することで、その生態を知ろうとしています。地下に埋まっている天然ガスは我々の生活や社会を支えるエネルギー源です。天然ガスの主成分のメタンの一部は、地下の微生物によってつくられたと推定されています(量にして約20%)。
動植物の死がいに由来する有機物が地下に埋没する過程で微生物によって分解されメタンができ、地層に貯まって天然ガス田となるわけです。どのような菌がどのようにして、何の有機物を利用してメタンをつくっているのかを明らかにすることができれば、天然ガスの有効利用につながります。
そこで私たちは、微生物由来のメタンが埋蔵する千葉県の南関東ガス田に行き、地下水と泥試料を採取し、実験室で試行錯誤を繰り返しながら培養を行いました。約5年の歳月をかけて今回の発見の目玉となるバクテリアRT761株を分離培養し、その性質を明らかにすることができました。RT761株は非常に増殖の遅い菌だったので、長い年月を要しました。
はじめは常識よりも自分を疑った
菌を1種類に分離培養して最初にすることは、どのような菌の形か、他の菌がまだ混ざっていないかを顕微鏡で観察することです。菌を見やすくするためにDNAを染色しRT761株の細胞を観察したとき、思わず首をかしげました。DNAの染色のされ方がおかしい…。普通、というより、どの原核生物も細胞全体がDNAで染まるはずが、RT761株は一部しか染まりません。
RT761株細胞の光学顕微鏡写真。蛍光染色剤(緑)でゲノムDNAを染色。原核生物の代表選手、大腸菌(左上)のように細胞全体が染まっていない様子がはっきりとわかる。RT761株のサイエンスはここからはじまった。
いやいや、染色剤がおかしい? と新たに調製し直したり、たまたま菌がおかしい? と培養をやり直したりと、人為的なミスを疑いました。ところが何度やっても同じ……光学顕微鏡ではらちがあかないので、電子顕微鏡をつかって観察しました。すると、もっと驚きました。細胞の中に「ゲノムDNAを包む膜」があったのです。同時に腑に落ちました。この膜があるから上の図ではDNAが隔離されて染色されていたのです。道理で細胞全体が染まらないはずです。
極薄くスライスしたRT761株細胞の透過性電子顕微鏡写真。白くモヤっと見えるゲノムDNAを包むようにして膜(矢印)が観察される。幾多もの原核生物を長年電顕観察している孟 憲英さん(論文の共著者)に実験を依頼。そんなベテランの彼女が観察中に血相を変えて飛んできた衝撃の写真。
生命の設計図であるゲノムが細胞内の膜(核膜)で包まれているのは真核生物だけです。真核生物の核膜とは似ても似つかぬものでしたが、真核・原核生物を区別する根本的な特徴という、疑いようもなかった常識に当てはまらない菌である可能性が高まりました。
RT761株はグラム陰性菌というタイプなので、細胞を包む膜として外膜と内膜(細胞膜)という2つの生体膜を持ちます。さらに細胞内膜があれば、計3枚の生体膜を持つことになります。しかし、上図ではその点がはっきりしませんでした。そこで、日本電子株式会社の協力を得て、クライオ電子顕微鏡を使って観察することにしました。
私たちは本社にお邪魔して、デモをしてもらいました。3つの膜が観察されるのか、研究のインパクトを左右する瞬間に大きな期待と不安が入り交じりました。固唾をのんでモニターを見つめるなか、期待通りの結果が得られ、大いに沸きました。
世界最高レベルの分解能を持つクライオ電子顕微鏡CRYO ARMTM 300(日本電子株式会社)で観察されたRT761株細胞の一部。@外膜、A内膜、B細胞内膜がくっきりと観察される。普段の光学顕微鏡では小さな黒い点にしか見えない存在が、最先端の技術でここまで鮮やかに可視化できたことに驚嘆と感動をよんだ写真。
RT761株のカタログ化と生態研究
遺伝子を解析することによってRT761株を分類してみると、原核生物の分類階級で最も上位の「門」で新しい菌であることが判明しました。既知の菌が属するどの系統グループにも当てはまらなかったのです。そこで、新しい系統群として記載を行いました。複数の膜を持つ特徴にちなんで学名をAtribacter laminatus(Atribacterota門)と命名提案し、正式に承認されました。命名は最初に発見した研究者の特権です。
新門のRT761株は、既知の菌とは非常に遠縁であり、進化の道をずっとさかのぼらないと共通の祖先がいない、といえます。バクテリア進化の初期の段階で分かれたグループAtribacterota門はなぜ細胞内膜を持つに至ったのか? 今後は、細胞内膜の機能をその進化と関連付けて解き明かしていきたいと考えています。
天然ガスをつくる地下の微生物の生態を知ろうとして未培養菌を培養していたところ、細胞構造がおかしい菌を発見したため、当初の研究ミッションがいささかなおざりになってしまいました。培養をせず環境から直接遺伝子を解析することによって、Atribacterota門は地下環境に優占する系統グループであることがすでに明らかとなっています。遺伝子として最初に発見されたのが1998年なので、RT761株は約20年の時を経て最初の培養株となったわけです。地下環境で何をしているのか? この優占グループの生態研究にも着手したところです。
おわりに
バクテリアを一番大まかに、門という階級で分類すると111門あると考えられています。このなかですでに培養されカタログ化された菌がいる門は33つです。残り約7割もの門は、遺伝子配列だけでしかその存在が知られていない未培養菌で構成されています。世界には既知の菌とは非常に遠縁で未知な系統群がまだまだたくさんいるということです。
新門のRT761株がそうだったように、これら未培養門のなかに私たちの想像を超えた菌がいるであろうことは想像に難くありません。その実態を解き明かすことで原核生物の多様性と普遍性、そしてその根底にある進化を体系的に理解することができるでしょう。環境微生物の世界は、未培養菌という大きなナゾにロマンと可能性が満ちあふれています。
参考文献
・Taiki Katayama, Masaru K. Nobu, Hiroyuki Kusada, Xian-Ying Meng, Naoki Hosogi, Katsuyuki Uematsu, Hideyoshi Yoshioka, Yoichi Kamagata & Hideyuki Tamaki. 2020. Isolation of a member of the candidate phylum ‘Atribacteria’ reveals a unique cell membrane structure. Nature Communications, 11, 6381.
https://www.nature.com/articles/s41467-020-20149-5
本論文が同誌Editors’ Highlightsに選出 https://www.nature.com/collections/jedgcgeija
・産総研プレスリリース「地下で発見!ゲノムが膜で包まれたバクテリア」(2020年12月14日)https://www.aist.go.jp/aist_j/press_release/pr2020/pr20201214/pr20201214.html
この記事を書いた人

片山 泰樹
産業技術総合研究所・地質調査総合センター・地圏微生物研究グループ・主任研究員。
生まれはタイ・バンコク、育ちは名古屋。北海道大学農学部にて博士号取得後、産総研ポスドクを経て、2011年より現職。
三度の飯と同じくらい顕微鏡で菌を観察、時に観賞するのが好き。地下という可視化できない広大な暗黒の空間で人知られず黙々と地球を動かす小さな生物の生き様を探求しています。好きな作曲家はJ. Brahms。

https://www.nature.com/articles/s41467-020-20149-5
Isolation of a member of the candidate phylum ‘Atribacteria’ reveals a unique cell membrane structure
Taiki Katayama, Masaru K. Nobu, Hiroyuki Kusada, Xian-Ying Meng, Naoki Hosogi, Katsuyuki Uematsu, Hideyoshi Yoshioka, Yoichi Kamagata & Hideyuki Tamaki
Nature Communications volume 11, Article number: 6381 (2020) Cite this article

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Abstract
A key feature that differentiates prokaryotic cells from eukaryotes is the absence of an intracellular membrane surrounding the chromosomal DNA. Here, we isolate a member of the ubiquitous, yet-to-be-cultivated phylum ‘Candidatus Atribacteria’ (also known as OP9) that has an intracytoplasmic membrane apparently surrounding the nucleoid. The isolate, RT761, is a subsurface-derived anaerobic bacterium that appears to have three lipid membrane-like layers, as shown by cryo-electron tomography. Our observations are consistent with a classical gram-negative structure with an additional intracytoplasmic membrane. However, further studies are needed to provide conclusive evidence for this unique intracellular structure. The RT761 genome encodes proteins with features that might be related to the complex cellular structure, including: N-terminal extensions in proteins involved in important processes (such as cell-division protein FtsZ); one of the highest percentages of transmembrane proteins among gram-negative bacteria; and predicted Sec-secreted proteins with unique signal peptides. Physiologically, RT761 primarily produces hydrogen for electron disposal during sugar degradation, and co-cultivation with a hydrogen-scavenging methanogen improves growth. We propose RT761 as a new species, Atribacter laminatus gen. nov. sp. nov. and a new phylum, Atribacterota phy. nov.

Introduction
Cultivation of uncultured microorganisms is a critical step in uncovering their phenotypic features, such as cell structure and metabolic function. However, most lineages of the domains Bacteria and Archaea remain uncharacterized1 due to difficulties in cultivation2,3. While omics-based cultivation-independent characterization can circumvent cultivation and provide insight into metabolism and ecology4,5, metabolic reconstruction is generally based on genes characterized in cultured organisms and, thus, prediction of novel phenotypic features of uncultured microorganisms remains challenging6.

In this study, we have isolated an anaerobic bacterium that belongs to the bacterial candidate phylum “Ca. Atribacteria”4 (“Ca. Caldatribacteriota” in Genome Taxonomy Database7). “Ca. Atribacteria” was originally discovered through 16S rRNA gene clones in sediments from the hot spring in Yellowstone National Park and designated as OP9 in 19988, and recently proposed to include the JS1 lineage5. The members of this phylum are globally distributed and, in some cases abundant, in anaerobic environments5. Here, we report structural, genomic and physiological characterization of the cultured representative of “Ca. Atribacteria”.

Results and discussion
Multilayered cell structure
A bacterium, designated RT761, was isolated after 3 years of enrichment from saline formation water and sediments derived from deep aquifers in natural-gas deposits in Japan. Phylogenetic analysis based on 16S rRNA gene and conserved protein-coding markers revealed that strain RT761 was assigned to the clade OP9 of “Ca. Atribacteria” (Supplementary Figs. 1 and 2). RT761 cells are Gram-stained negative, nonspore forming, and tapered rod or ovoid-shaped with pointed ends (Fig. 1a, b). Although typical gram-negative cells only have two lipid bilayers (cytoplasmic membrane [CM] and outer membrane [OM]), cryo-electron tomography (CET) revealed that RT761 possesses three layers with appearances characteristic of lipid bilayers: a 4 nm thickness and two leaflets (Fig. 1c, d and Supplementary Figs. 3, 4 and Supplementary Movies 1 and 2). Between the outer lipid membrane-like layer (LML) and middle LML, we observe a narrow space (7.0 nm [SD 0.3]) with a thin layer of 2.2 nm width (SD 0.1) (Fig. 1c and Supplementary Fig. 4). The outer LML was occasionally observed (15 out of 31 cells) to have small embedded jagged structures (periodicity of ca. 4 nm) comprising between 1.9 and 13.7% of cell outline in two-dimensional images (Supplementary Figs. 3, 4 and Supplementary Movie 3). The inner LML has an amorphous structure (including occasional small invaginations) that does not follow the inside of the other LML, and is likely separated from the other LMLs (Fig. 1d, e and Supplementary Movies 1, 2, 4). We observe ribosomes in both the inner LML-bounded space [19.8 nm diameter (SD 0.7)] and middle LML-bounded space [20.1 nm (SD 0.8)] (Supplementary Fig. 5). More are observed to be present in the former based on CET and fluorescence microscopy (Supplementary Figs. 5 and 6). The inner LML is most distant from the other membranes at the cell poles, creating large polar spaces (Fig. 1d, e). Additional observation through transmission electron microscopy shows that the inner LML appears to envelopes the RT761 nucleoid (Fig. 1f). This was further supported by the fluorescence microscopy-based observation of bulk DNA/RNA in the center of the cell bounded by a lipid membrane structure (stained with FM4-64), leaving open polar spaces (Fig. 2). (In some photomicrographs, the RT761 outer two LML may not be stained well by FM4-64 as, in the presence of multiple membrane layers, the dye can tend to stain outermost layers less9,10,11). During cell division, the three LML are observed to be clearly maintained at the site of binary fission, and the inner LML apparently continues to surround the nucleoid, invaginates following the two other LML (but does not directly attach), and splits into the daughter cells as division completes (Supplementary Figs. 7 and 8). At the site of division, we observe three distinct sets of layers, unlike typical gram-negative bacteria (Supplementary Fig. 7). These observations suggest that the three LMLs are not connected to each other. While the exact identity/composition of the three LMLs remain unclear, the results point to two possibilities. The jagged structures in the outer LML suggest that the outer layer may be proteinaceous, in which case, the layers would represent a surface protein layer (outer LML), OM (middle LML), and CM (inner LML). On the other hand, the lipid bilayer-like characteristics of the observed layers suggest RT761 possesses three lipid membranes, in which case, the observed layers would represent the OM (outer LML with high protein density like Thermotoga12), peptidoglycan layer (2.2 nm thick layer), CM (middle LML), and a intracytoplasmic membrane (inner LML) surrounding the cell’s nucleoid.

Fig. 1: Morphology and membrane structure in RT761 cells showing the presence of three lipid membrane-like layers (LMLs) with the innermost layer surrounding the nucleoid.
figure1
a Phase-contrast microscopy. b Scanning electron microscopy. c–e Cryo-electron tomography (also see Supplementary Movies 1 and 2). c The original slice picture is shown in Supplementary Fig. 4. Black arrowheads indicate the outer (1), middle (2), and inner (3) LMLs. White arrowheads indicate the 2.2 nm thick layer (1) and faint layers (2). d White arrow indicates inner LML invagination. e 3D-rendered reconstruction of the cell in d. (also see Supplementary Movie 4). Color code: outer LML, orange; middle LML, blue; inner LML, yellow; ribosome, green. f Transmission electron micrograph of a thin section of RT761 cells. N nucleoid. Scale bars, 5.0 μm (a), 1.5 μm (b), 0.2 μm (c), 0.1 μm (d, e), and 0.5 μm (f).

Full size image
Fig. 2: Confocal-laser microscopy showing the localization of DNA and RNA within the intracytoplasmic membrane structure. DNA, RNA, and membrane lipids were stained by Hoechst (blue), SYTO RNAselect (green) and FM4-64 (red), respectively.
figure2
Outlines of the cell from a are included in all panels. a Phase contrast image. b–d Confocal-laser images. e–h Image overlays. i Line profiles of fluorescence intensity plotted longitudinally along white arrow in h. Membrane staining maxima are indicated by arrowheads in h. Source data are provided as a Source Data file. Scale bars, 1 μm.

Full size image
Unique genomic features
The potential importance of localization and membranes in RT761 was supported by genomic and transcriptomic analyses. Alignment of all RT761 protein-coding genes with a reference sequence database revealed that 34 genes in the RT761 genome contained unique N-terminal extensions (NTE; 10–73 amino acids in length) compared to the top 250 hits in the NCBI RefSeq database and some of them were conserved among “Ca. Atribacteria” OP9 genomes (Supplementary Datas 1 and 2). The genes with NTE included those involved in critical cellular processes: cell division (FtsZ), Lipid A biosynthesis (UDP-3-O-acyl-N-acetyglucosamine deacetylase—LpxC), DNA replication, DNA repair, transcriptional regulation, tRNA processing, transmembrane signaling, and H2 generation (FeFe hydrogenase subunit alpha—HydA). Notably, several facilitate central functions in their respective processes: FtsZ recruits other cell division proteins to the fission site13, LpxC performs the committing step in Lipid A biosynthesis14, and HydA catalyzes the reduction of protons to H2 in the hydrogenase complex15. NTEs in prokaryotes have so far been only found in enzymes that localize to the lumen of subcellular compartments called bacterial microcompartment (BMC), and are necessary for the encapsulation of enzymes with NTE into BMC shells16,17. Among 34 genes with NTE in RT761, only one enzyme (deoxyribose-phosphate aldolase) may be related to BMC because of its occurrence in the gene cluster encoding homologs of BMC (although BMC-like compounds were not visible in electron microscopy). Interestingly, RT761 and other “Ca. Atribacteria” possessed two FtsZ genes—one conventional FtsZ and another with an NTE that is predicted to form an amphipathic helix (Supplementary Fig. 9). RT761 expressed both FtsZ genes (RT761_00112 and RT761_02154) during exponential growth (Supplementary Data 3). While the NTE-lacking FtsZ gene is adjacent to FtsA, the NTE-possessing FtsZ lacks a corresponding FtsA (i.e., only one copy of ftsA in the genome). FtsA facilitates association between FtsZ and the CM through a C-terminal amphipathic helix18, suggesting that the NTE-possessing FtsZ may be capable of binding to the membrane. Although 9 out of 6751 cultured bacterial type strains possess both a typical and NTE-possessing FtsZ, putative amphipathic helices were not found in any of these sequences. Such unique features may play key roles in binary fission through multi-layers in RT761 (Supplementary Figs. 7 and 8), but additional investigations are necessary for verification.

Further analysis reveals unique genomic features of RT761 related to membrane-mediated physiology. Based on transcriptomic analysis of RT761 under exponential growth phase, membrane-associated proteins comprised 5 out of 10 of the mostly highly expressed genes (Supplementary Data 3). These include a putative transmembrane protein (RT761_00009), lipoprotein (RT761_00907), periplasmic substrate-binding protein (RT761_02219), and two fasciclin domain-containing transmembrane proteins (RT761_00748 and 01694), all of which have unknown functions. These findings point toward the importance of membrane-centric metabolism in RT761 physiology. The RT761 genome also has a high proportion of proteins with transmembrane helices (29.6% of all proteins) greater than 99.7% of all gram-negative type strains with sequenced genomes available (Fig. 3).

Fig. 3: Unique genomic compositions of membrane-related features observed for phyla with unique cell structures.
figure3
The horizontal axis shows the genomic proportion proteins encoding transmembrane helices. The vertical axis shows the ratio of proportions of proteins encoding Sec signal peptides estimated by SignalP-5.0 and SignalP-4.1. RT761 (red), type strains from Thermotogae (green), Dictyoglomi (orange), Caldiserica (pink), and other gram-negative type strains (gray) are plotted (3502 genomes downloadable from the Joint Genome Institute Integrated Microbial Genomes and Microbiomes database). For Thermotogae and other gram-negative type strains, 95% (green) and 99.9% (gray) confidence ellipses are shown respectively. Source data are provided as a Source Data file. (Bottom) Cell structures of select species are shown for “Ca. Atribacteria”, Thermotogae, and Dictyoglomi. The illustrations indicate the outer membrane (black), cytoplasmic membrane (blue), intracytoplasmic membrane (purple), and nucleoid (yellow). *For RT761, the shown schematic requires further investigation to conclude the identity/role of each layer.

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We also found that RT761 may have unique signal peptide sequences for Sec-secreted proteins. While SignalP-4.119 estimated that RT761 has a low proportion of Sec-secreted proteins (3.4% of all proteins) less than 96.7% of all Gram-negative type strains, SignalP-5.020 predicted 2.67 times more (9.0% of all proteins) (Fig. 3). Evaluation of all gram-negative type strain genomes revealed that most cultured phyla (26 out of 29) have consistent predictions between SignalP-4.1 and SignalP-5.0 (1.1 ± 0.2 [S.D.] times more on average) (Supplementary Table 1). Note that the reference databases used by SignalP-4.1 and SignalP-5.0 are similar in phylogenetic composition and diversity (Supplementary Table 1). Through further analysis of genomes from 33 candidate phyla, we discover that only two candidate phyla, “Ca. Calescamantes” (EM3) and “Ca. Microgenomates” (OP11) within “Ca. Patescibacteria”, have RT761-like signatures (Supplementary Fig. 10) (1.8 ± 0.6 [S.D.] times more in SignalP-5.0 on average for other candidate phyla), indicating that the above features of RT761 are not an artifact from lack of Ca. Atribacteria-derived sequences in the SignalP reference databases. Comparison of hydrophobicity, signal peptide length, and number of NT cationic residues interestingly revealed no differences between Sec-secreted proteins predicted by SignalP-5.0 and SignalP-4.1, suggesting that the Sec-secreted proteins of RT761 may have unique signal peptides that could only be predicted through integration of a recurrent neural network implemented in SignalP-5.0. Intriguingly, we only observed RT761-like signatures (high genomic proportion of proteins with transmembrane helices and underestimation of Sec-secreted proteins by SignalP-4.1) in three other cultured phyla with unique cell structures (Fig. 3): Thermotogae members (outer toga12), Dictyoglomi (multicell-spanning outer envelope21), and Caldiserica (electron-lucent outer envelope22). In total, comparison of genomic transmembrane and extracellular protein abundance signatures may serve as a new approach for identification of bacterial lineages with unique cell membrane structure and is, thus, distinct from currently available genotype-based cell morphology prediction approaches (e.g., RodZ for rod-shape and lipid A synthesis genes for gram-negative structure).

Syntrophic physiology
In addition to the unique cell structure and genomic features, we found that strain RT761 can benefit from syntrophic interaction with methanogenic archaea. RT761 degraded glucose, producing H2, acetate, CO2, and ethanol (trace levels) as end products and could not utilize exogenous electron acceptors for anaerobic respiration (i.e., nitrate, ferric iron, and sulfate). Although RT761 growth was inhibited by accumulation of hydrogen during cultivation with glucose, addition of a hydrogen-consuming methanogenic archaeon increased the growth rate and maximum cell density of RT761 (Supplementary Fig. 11). RT761 can theoretically shift to ethanol fermentation as an alternative electron disposal route but only generates a small amount, indicating that RT761 primarily relies on hydrogen formation to maintain cellular redox balance. Thus, in contrast to most hydrogen-producing bacteria, RT761 highly depends on syntrophic association with a hydrogen-scavenging methanogen for ideal growth. Such dependence of sugar degradation on a syntrophic partner is thought to be important in anoxic ecosystems23,24. Thus far, sugar-degrading organisms that benefit from syntrophic interactions have been identified in Firmicutes (Syntrophococcus sucromutans25, Bacillus stamsii26) and the class Anaerolineae of Chloroflexi (Anaerolinea thermolimosa27, A. thermophila23, Bellilinea caldifistulae28, Flexilinea flocculi29, and Longilinea arvoryzae28). Unlike these organisms, RT761 does not produce lactate, lacks NiFe hydrogenases, and rather possesses energy-conserving complexes often associated with nonsugar-degrading syntrophic organisms—an ion-translocating NADH:ferredoxin oxidoreductase Rnf and both a trimeric and tetrameric FeFe hydrogenase5 (Table 1). This suggests that the sugar-degrading strategy of RT761 differs from hitherto isolated sugar degraders that benefit from syntrophic interaction. We speculate that RT761 may avoid ethanol production as continuous exposure to acetaldehyde generated through ethanol fermentation could cumulatively damage chromosomal DNA, especially due to the slow growth rate (doubling time of 5.1 days). Similar metabolisms that are theoretically possible or thermodynamically required for association with methanogens (e.g., sugar degradation producing hydrogen and syntrophic propionate oxidation) have been predicted in cultivation-independent analyses of “Ca. Atribacteria”4,5,30,31. The observed physiology of RT761 justifies the prevalent detection of environmental clones of “Ca. Atribacteria” across Earth’s anoxic ecosystems favoring fermentation and syntrophy5.

Table 1 Fermentation products and genome-predicted metabolic properties of semi-syntrophic anaerobes that benefit from interaction with methanogenic archaea.
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Based on unique phenotypic, genotypic and phylogenetic characteristics, including cell structure/regulation potentially more complex than a typical prokaryote, we propose strain RT761 as a new species, Atribacter laminatus gen. nov., sp. nov. Based on the phylogenetic and phylogenomic analyses, we further propose a new phylum Atribacterota phy. nov.

Description of Atribacter gen. nov
Atribacter (A.tri.bac’ter. L. masc. adj. ater, black; N.L. masc. n. bacter, a rod; N.L. masc. n. Atribacter, a black bacterium, pertaining to the original candidate phylum name “Ca. Atribacteria”4).

Obligately anaerobic, Gram-negative, non-motile, non-spore-forming, rod cells that are tapered with pointed ends. The major end products from glucose degradation is acetate, hydrogen, and carbon dioxide. No anaerobic respiration with nitrate, sulfate, or Fe(III) is observed. The cellular fatty acids are C15:0 (51% of the total), C18:0 (21%), C16:0 (10%), iso-C15:0 (10%), iso-C13:0 3OH (6%), and C18:1 cis9 (2%). DNA G+C content of the type species is 38.69 mol%. The type species is Atribacter laminatus.

Description of Atribacter laminatus sp. nov
Atribacter laminatus (L. fem. n. lamina, layer; N.L. masc. adj. laminatus, layered, pertaining multilayered cell structure).

Shows the following characteristics in addition to those given for the genus. Cells are a rod or ovoid shape tapered with pointed ends with 0.6–0.8 μm wide and 1.3–1.8 μm long. Grows at 20–50 °C (optimally at 45 °C), at pH 6.4–8.2 (optimally at pH 7.3) and in the presence of 0.01–0.6 M NaCl (optimally in 0.1 M NaCl). Growth occurs with glucose, fructose, galactose, rhamnose, xylose, mannose, sucrose, cellobiose, raffinose, pectin, mannitol, and sorbitol. Ribose, arabinose, lactose, maltose, trehalose, melibiose, starch, cellulose, or gelatin are not utilized. In pure culture, the end products of glucose (1 mM) degradation are acetate (1 mM), hydrogen (1 mM), and carbon dioxide. Yeast extract is required for growth. Peptone or casamino acids did not stimulate growth. Growth is enhanced in co-culture with a hydrogen-scavenging methanogen. Sensitive to chloramphenicol, kanamycin, neomycin, rifampicin, and vancomycin, but resistant to ampicillin and neomycin. Colonies are light brown circular and convex disks on the deep agar slant. The type strain, RT761T (=NBRC 112890T=DSM 105538T), was isolated from a slurry of sediments and formation water derived from a deep sedimentary, natural-gas-bearing saline aquifer in Japan.

Description of Atribacteraceae fam. nov
Atribacteraceae (A.tri.bac.te.ra.ce’ae. N.L. masc. n. Atribacter type genus of the family; suff. -aceae, ending to denote a family; N.L. fem. pl. n. Atribacteraceae the family of the genus Atribacter).

The description is the same as for the genus Atribacter. Type genus is Atribacter.

Description of Atribacterales ord. nov
Atribacterales (A.tri.bac.te.ra’les. N.L. masc. n. Atribacter type genus of the order; suff. -ales, ending to denote an order; N.L. fem. pl. n. Atribacterales the order of the genus Atribacter).

The description is the same as for the genus Atribacter. Type genus is Atribacter.

Description of Atribacteria classis nov
Atribacteria (A.tri.bac.te’ri.a. N.L. masc. n. Atribacter type genus of the type order of the class; suff. -ia, ending to denote a class; N.L. neut. pl. n. Atribacteria the class of the order Atribacterales).

The description is the same as for the genus Atribacter. Type order is Atribacterales.

Description of Atribacterota phyl. nov
Atribacterota (A.tri.bac.te.ro’ta. N.L. neut. pl. n. Atribacteria type class of the phylum; N.L. neut. pl. n. Atribacterota the phylum of the class Atribacteria).

The phylum Atribacterota is defined based on phylogenetic and phylogenomic analyses of the sole isolated strain RT761T and uncultured representatives from various environments. Type order is Atribacterales.

Methods
Sample collection
The sediment and formation water samples were collected from a settling pond that was placed downstream of a commercial gas, and water producing well to remove suspended sand particles from the formation water in Mobara, Chiba prefecture, Japan. The samples came from the gas-bearing aquifers in the screened depth range of 490–900 m that consist of repeating sequences of turbidite (alternating beds of sandstone and mudstone) in the Otadai and Kiwada formations. These sediments were deposited in deep marine environments during the Plio-Pleistocene periods32,33. The water temperature was 24.4 °C, the pH was 7.7, and the redox potential was −213 mV. The Cl− concentration was 17,000 mg l−1, and the sulfate concentration was 30% similarity and >70% coverage. Secondary structure of amino acid sequences of N-terminal extension associated with FtsZ was predicted using JPred440. Transmembrane proteins and signal peptides were predicted using TMHMM v2.041 (default options) and SignalP (v4.119 and v5.020 using the gram-negative option and default options for the remaining settings) correspondingly, and the percentages of these proteins out of the total number of ORFs were compared to those in all gram-negative type strain draft genomes available on the Joint Genome Institute Integrated Microbial Genomes and Microbiomes database41,42, and those in draft genomes of uncultured phyla that were confirmed to encode lipid A synthesis genes (lpxB, lpxC, or lpxD) in at least one draft genome. The 16S rRNA gene sequences were aligned against the SILVA v132 alignment using SILVA SINA Aligner v1.2.1143 with default settings. The phylogenetic tree was constructed using RAxML-NG44 using the generalized time reversible (GTR) model, 4 gamma categories, and 100 bootstrap iterations. Prediction/selection of conserved genes and tree construction was performed through PhyloPhlAn45 using default settings.

RNA was extracted from late exponential growth of both pure culture and co-culture with a methanogen M. thermoautotrophicus str. Delta H using the ISOSPIN Plant RNA kit (NIPPON GENE, Japan) according to the manufacturer’s instructions and was sequenced using an Illumina sequencer NovaSeq 600 system (illumina, USA) at Filgen, Inc. (Nagoya, Japan). Total RNA was depleted of ribosomal RNA via Ribo-Zero rRNA removal kit (illumina). The sequenced RNA was trimmed via Trimmomatic v0.3346 and mapped to the assembled genome through BBmap v37.10 (https://sourceforge.net/projects/bbmap/) to calculate the gene expression levels, which were represented by Reads Per Kilobase of transcript per Million mapped reads.

Microscopic analyses
Cell morphology and structure was observed via phase-contrast and fluorescence microscopy (BX51; Olympus, Japan), confocal laser scanning microscopy (LSM800; ZEISS, Germany), scanning electron microscopy (SEM) (S-4500; Hitachi, Japan), transmission electron microscopy (TEM) (H-7600; Hitachi, Japan) and cryo-electron microscopy (CRYO ARM 300; JEOL, Japan). The cells in exponential phase of growth in pure culture condition were used for all microscopic observation.

Cells were washed with phosphate-buffered saline (PBS) before staining. A Gram-staining kit (BD) was used for Gram staining. Membranes of RT761 cells were strained with FM4-64 (ThermoFisher Scientific, USA) at a final concentration of 40 µg ml−1. DNA was strained with Hoechst 33342 (ThermoFisher Scientific) at final concentrations of 2 µg ml−1. RNA was strained with SYTO RNAselect (ThermoFisher Scientific) at a final concentration of 10 µM. The stained sample was incubated for 1 h at 30 °C and observed under confocal laser scanning microscope.

For fluorescence in situ hybridization, the cells were fixed in 1% paraformaldehyde at 4 °C for overnight and stored in 99% ethanol-PBS (1:1) at −20 °C. The fixed cells were incubated in a moisture chamber with a hybridization buffer (0.9 M NaCl, 0.01% sodium dodecyl sulfate, 20 mM Tris-HCl, pH 7.2 containing fluorescently labeled probes (0.5 pmol µl−1). After incubation at 46 °C for 2.5 h, the buffer was replaced with washing solution (0.9 M NaCl, 0.01% sodium dodecyl sulfate, 20 mM Tris-HCl, pH 7.2). The sample was incubated at 48 °C for 30 min and observed under a fluorescence phase-contrast microscope. An oligonucleotide probe targeting the 16S rRNA gene was Cy-3-labeled EUB338 probe (5′- GCTGCCTCCCGTAGGAGT-3′).

For SEM observation, the cells were fixed with 2% glutaraldehyde in 0.1 M sodium phosphate buffer (pH7.2) at 4 °C for 2 h, postfixed with 1% osmium tetroxide at room temperature for 1 h, dehydrated through a graded ethanol series followed by 3-methylbutyl acetate for 20 min, dried with a critical point dryer (JCPD-5; JEOL), and finally coated with gold.

For cryo-electron microscopy and tomography, 2 μl of the cell culture were placed on glow-discharged holey carbon grid (Quantifoil R 1/4 Cu grid, Quantifoil MicroTools GmbH, Germany), and the grid was automatically blotted at 22 °C and 80% humidity and plunged into liquid ethane using a Leica EM GP (Leica Microsystems, Austria). The frozen grid was mounted onto a liquid-nitrogen cryo-specimen holder and loaded into a CRYO ARM 300 equipped with a cold-field emission electron gun operating at 300 kV, a hole-free phase plate47 and an omega-type in-column energy filter with an energy slit width of 30 eV. The images were recorded on K3 direct detection camera (GATAN, USA) at a nominal magnification of 10,000–15,000× (corresponding to an imaging resolution of 3.3–4.9 Å per pixel, with the total dose under 1.5 electrons per Å2 using a low dose system). For observation of two leaflets of cellular layers, the images were recorded at a magnification of 60,000× (corresponding to an imaging resolution of 0.8 Å per pixel, with the total dose under 25 electrons per Å2 using a low dose system). Tilt series images were collected automatically in a range of ±60° at 2° increments using the SerialEM v3.8.0 beta (http://bio3d.colorado.edu/SerialEM)48. The total electron dose on the specimen per tilt series was kept under approximately 90 electrons per Å2 to minimize radiation damage. The tilt series were aligned using gold fiducials, and tomograms were reconstructed using the IMOD v4.9.1249. The 3D segmentation of eight times binned volumes including surface rendering and smoothing to generate the final tomographic model was performed with Amira v6.3.0 (ThermoFisher Scientific) according to the previously described method50. In brief, segmentation of each structure was traced manually using brush tools in Amira. The thickness of layers, distances between each layer and size of ribosomes were determined from 16 tomographic slice pictures of four different cells. The percentage of jagged structure in outline of outermost layer was measured from 15 cells of two-dimensional tomographic projection images.

For TEM observation, the cells were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH7.4) at 4 °C for 3 h and then postfixed with 1% osmium tetroxide at 4 °C for 90 min. The fixed cells were suspended in 1% aqueous uranyl acetate at room temperature for 1 h. The suspended cells were embedded in 1.5% agarose and dehydrated through a graded ethanol series. The dehydrated blocks were embedded in Epon812 resin. Ultrathin sections were cut with an ultramicrotome (Leica EM UC7), mounted on copper grids, and stained with uranyl acetate and lead citrate.

Physiological characterization
All physiological experiments were performed in triplicate. The effects of temperature, pH, and concentrations of NaCl on cell growth, utilization of carbohydrates and sensitivity to antibiotics were determined by hydrogen production in gas phase of cultures. Hydrogen and methane in gas phase of cultures were measured with a gas chromatography equipped with a thermal conductivity detector (GC-8A; Shimadzu, Japan). Glucose, acetate, and ethanol in liquid phase of cultures were measured with a high-performance liquid chromatography (HPLC) (LC20; Shimadzu) with Shim-pack SPR-H column (Shimadzu) or HPLC (LC-2000Plus, Jasco) with Aminex HPX-87H column (BIO-RAD). Methyl esters of cellular fatty acids were identified and quantified via a gas chromatography-mass spectrometry (M7200A GC/3DQMS system; Hitachi).

Quantitative PCR
SYBR green-based real-time PCR was run on a CFX Connect real-time PCR detection system (Bio-Rad Laboratories Inc., USA) using the PowerUp SYBR green master mix (Applied Biosystems, USA) to quantify the population of RT761 cells. The forward and reverse primers, rt1F (5′-GCTAATACCCCATATGCTCCCTG-3′) and rt1R (5′-ACCTCGCCAACCAGCTGATGGGG-3′), were designed from the 16S rRNA gene sequences of strain RT761. The length of amplified products was 62 bp. Total DNA was extracted from pure- or co-cultures using an ISOSPIN Fecal DNA (NIPPON GENE). Standard curves for quantification were determined based on 10-fold serial dilutions of the target PCR products of strain RT761 at known concentrations. All reactions, including the non-template control, were performed in triplicate. The presence of a single PCR product without any nonspecific amplicons was confirmed via agarose gel and melting curve analyses. The PCR product was sequenced by Sanger sequencing to confirm the amplification of 16S rRNA gene from strain RT761. All qPCR runs showed no PCR amplifications from non-template control and culture samples without adding RT761 cells, and had efficiency levels of approximately 95%, with an R2 of >0.99. Cell growth rate was estimated using 16S rRNA gene copy number as a proxy for cell population.

Statistics and reproducibility
For phase-contrast microscopic observation, a representative section of one field of view (Fig. 1a) was selected from ten fields of view using cells from three independent cultures. For fluorescence microscopic observation, a representative section of one field of view (Fig. 2) was selected from eight fields of view from two independent experiments. For FISH observation, a representative section of one field of view (Supplementary Fig. 6a) was selected from four fields of view from two independent experiments. For SEM observation, a representative section of one field of view (Fig. 1b) was selected from 17 fields of view using cells from three independent cultures. For TEM observation, a representative section of one field of view (Fig. 1f) was selected from 29 fields of view using cells from three independent cultures. For cryo-electron microscopy (at high electron dose), a representative section of one field of view (Supplementary Fig. 3) was selected from four fields of view from two independent cultures. For cryo-electron microscopy (at low electron dose), a representative section of one field of view (Supplementary Fig. 7) was selected from 30 fields of view using cells from two independent cultures. For CET, four different cells from two independent cultures were selected for tomographic analysis, and a representative section of one slice image with different binned volumes (Fig. 1c, d and Supplementary Figs. 4, 5, 7) from original 4092 tilt series in each dataset. Among these four cells, one cell was selected for segmentation and 3D reconstruction (Fig. 1e).

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
The draft genome sequences and annotation data of strain RT761 are available in NBCI BioProject under accession number PRJNA528842. The type strain of Atribacter laminatus, RT761T (=NBRC 112890T=DSM 105538T), has been deposited in two culture collections: NBRC (Japan) and DSMZ (Germany). All micrographic data are available at BioStudies under accession code S-BSST519. The reference genome sequences used in this study are available on the Joint Genome Institute Integrated Microbial Genomes and Microbiomes database (https://img.jgi.doe.gov/). All other data supporting the findings of this study are available within the article and its Supplementary Information. Source data are provided with this paper.

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Acknowledgements
We acknowledge the Kanto Natural Gas Development Co., Ltd. for collecting environmental samples at their facilities. We thank Dr. Aharon Oren and Dr. Aiden C. Parte for their help with the nomenclature of the species. We also thank Hiroyuki Imachi for support of cryo-electron tomography; Naoki Morita for quantification of fermentation products; Chiwaka Miyako for assistance in molecular analyses; Fumie Nozawa for assistance in cultivation experiments. This work was supported by JSPS KAKENHI Grant Numbers JP17K15183, JP18H05295, and JP18H02426 and partly supported by JST ERATO grant number JPMJER1502, Japan.

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Author notes
These authors contributed equally: Taiki Katayama, Masaru K. Nobu.

Affiliations
Geomicrobiology Research Group, Research Institute for Geo-Resources and Environment, Geological Survey of Japan, National Institute of Advanced Industrial Science and Technology (AIST), Central 7, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8567, Japan

Taiki Katayama & Hideyoshi Yoshioka

Bioproduction Research Institute, Central 6-10, 1-1-1 Higashi, AIST, Tsukuba, Ibaraki, 305-8566, Japan

Masaru K. Nobu, Hiroyuki Kusada, Xian-Ying Meng, Yoichi Kamagata & Hideyuki Tamaki

EM Application Department, EM Business Unit, JEOL, Ltd., 3-1-2 Musashino, Akishima, Tokyo, 196-8558, Japan

Naoki Hosogi

Department of Marine and Earth Sciences, Marine Work Japan Ltd., 3-54-1 Oppamahigashi, Yokosuka, Kanagawa, 237-0063, Japan

Katsuyuki Uematsu

Contributions
T.K., M.K.N., Y.K., and H.T. designed the study and wrote the manuscript. T.K. and H.Y. performed enrichment cultures, and T.K. isolated the bacterium. M.K.N. performed bioinformatic analyses. T.K. and H.K. performed phase-contrast, fluorescence and confocal-laser microscopy. X.Y.M. performed scanning and transmission electron microscopy. N.H. performed cryo-electron tomography. K.U. performed segmentation and 3D reconstruction of tomograms. All authors reviewed the results and approved the manuscript.

Corresponding authors
Correspondence to Yoichi Kamagata or Hideyuki Tamaki.

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Katayama, T., Nobu, M.K., Kusada, H. et al. Isolation of a member of the candidate phylum ‘Atribacteria’ reveals a unique cell membrane structure. Nat Commun 11, 6381 (2020). https://doi.org/10.1038/s41467-020-20149-5

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Received
27 August 2020

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14 December 2020

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Bacterial physiology
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