Microbiome characterization by high-throughput transfer RNA sequencing and modification analysis

題目：通過(guò) tRNA 高通量測(cè)序和修飾分析進(jìn)行微生物組學(xué)表征

作者及單位：

Michael H. Schwartz谈息， Haipeng Wang遍愿， […], Tao Pan&A. Murat Eren

Tao Pan

• Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, 60637, USA
• Committee on Microbiology, University of Chicago, Chicago, IL, 60637, USA

A. Murat Eren

• Committee on Microbiology, University of Chicago, Chicago, IL, 60637, USA
• Department of Medicine, University of Chicago, Chicago, IL, 60637, USA
• Marine Biological Laboratory, Woods Hole, MA, 02543, USA

發(fā)表期刊及時(shí)間：

Nature Communicationsvolume 9, Article number: 5353 (2018) Published: 17 December 2018

摘要：

Advances in high-throughput sequencing have facilitated remarkable insights into the diversity and functioning of naturally occurring microbes; however, current sequencing strategies are insufficient to reveal physiological states of microbial communities associated with ==protein translation dynamics==（此處我認(rèn)為可以理解為蛋白質(zhì)翻譯的動(dòng)態(tài)過(guò)程）. Transfer RNAs (tRNAs) are core components of protein synthesis machinery, present in all living cells, and are ==phylogenetically tractable==（此處我認(rèn)為可以理解為進(jìn)化上保守）, which make them ideal targets to gain physiological insights into environmental microbes. Here we report a direct sequencing approach, tRNA-seq, and a software suite, tRNA-seq-tools, to recover sequences, abundance profiles, and post-transcriptional modifications of microbial tRNA transcripts. Our analysis of cecal samples using tRNA-seq distinguishes high-fat- and low-fat-fed mice in a comparable fashion to 16S ribosomal RNA gene amplicons, and reveals taxon- and diet-dependent variations in tRNA modifications. Our results provide taxon-specific ==in situ==（原位的） insights into the dynamics of tRNA gene expression and post-transcriptional modifications within complex environmental microbiomes.

高通量測(cè)序的發(fā)展促進(jìn)了對(duì)天然微生物的多樣性和功能的深刻洞察; 然 而廷粒，目前的測(cè)序策略不足以揭示與蛋白質(zhì)翻譯動(dòng)態(tài)過(guò)程相關(guān)的微生物群落的生理狀態(tài)力麸。 轉(zhuǎn)運(yùn) RNA（tRNA）是蛋白質(zhì)合成機(jī)器的核心組成部分豹休，存 在于所有活細(xì)胞中尔艇，并且在進(jìn)化上是保守的伤提，因此易于追溯题涨，這使它們成為獲得對(duì)環(huán)境微生物的生理學(xué)洞察的理想目標(biāo)蜈彼。 在這里筑辨，我們報(bào)告了直接測(cè)序方法--tRNA-seq 和軟件套件--tRNA-seq-tools，以復(fù)原微生物 tRNA 轉(zhuǎn)錄物的序列幸逆，豐度譜和轉(zhuǎn)錄后修飾棍辕。 我們使用tRNA?seq分析盲腸樣品區(qū)分高脂肪和低脂肪喂養(yǎng)的小鼠，并將該方法與利用16S核糖體RNA基因擴(kuò)增子的方式相比較还绘，揭示了tRNA修飾中的分類和飲食依賴性變異楚昭。我們的結(jié)果提供 了對(duì)復(fù)雜環(huán)境微生物組內(nèi) tRNA 基因表達(dá)和轉(zhuǎn)錄后修飾的動(dòng)態(tài)的分類群 特異性原位洞察。

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Fig. 1 tRNA modifications of bacterial cultures. Red and black lines show mutation fractions in representative tRNA sequences with (+DM) and without (-DM) demethylase treatment, respectively. a E. coli tRNAPro(CGG) shows m1G37 and s4U8. b E. coli tRNAPhe(GAA) shows acp3U47, ms2i6A37 and s4U8. The additional peak denoted by asterisk (*) may represent an unknown modification. c B. subtilis tRNASer(UGA) shows m1A22, and s4U8. d B. subtilistRNAGlu(UUC) shows m1A22. e S. aureus tRNALeu(UAG) shows m1G37, m1A22 and s4U8. f S. aureus tRNASer(GCU) shows m1A22 and s4U8. g B. viscericola tRNAArg(ACG) shows m1G37 and I34

圖一 細(xì)菌培養(yǎng)物的 tRNA 修飾拍顷。紅色和黑色線分別表示具有（+ DM）和不含（-DM）去甲基化酶處理的代表性 tRNA 序列中的 突變部分抚太。 a. 大腸桿菌脯氨酸 tRNA（CGG） 展示出 m1G37 and s4U8 修飾。 b. 大腸桿菌 苯丙氨酸 tRNA（GAA）顯示 acp3U47, ms2i6A37 and s4U8 修飾。 由星號(hào)（*）表示的附加峰值可表示未 知修飾尿贫。 c. 枯草芽孢桿菌絲氨酸 tRNA（UGA）顯示 m1A22 和 s4U8 修飾电媳。 d.枯草芽孢桿菌谷氨酸 tRNA（UUC）顯示 m1A22 修 飾。 e.金黃色葡萄球菌的亮氨酸 tRNA （UAG） 顯示出 m1G37, m1A22 and s4U8 修飾庆亡。f.金黃色葡萄球菌的絲氨酸 tRNA（GCU） 顯示出 m1A22 and s4U8 修飾匾乓。 g. B.viscericola（Barnesiella viscericola） 精氨酸 tRNA（ACG）顯示 m1G37 和 I34 修飾

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Fig 2. Mutation fractions of two tRNA sites. Heatmaps of mutation fractions for positions 22 and 8 (using standard, canonical tRNA nomenclature) are shown. tRNAs with different anticodons are grouped by their sequences at the respective position of modification (in parenthesis) and in alphabetical orders. Only E. coli and B. subtilis tRNA modifications have been mapped previously by 2D-TLC and LC/MS, but the mapping was not done for every tRNA species12. Every E. coli and B. subtilis tRNA species with mutation fraction at10-times above background is marked with a circle on the right with the following designations: Purples correspond to those known to be present and also identified by sequencing here; blacks correspond to those supposed to be absent but identified by sequencing; greens correspond to those not mapped previously but identified by sequencing; oranges correspond to those known to be present but were not found by sequencing. a m1A22; R corresponds to A or G. b s4U8

圖 2。 兩個(gè) tRNA 位點(diǎn)的突變分?jǐn)?shù)又谋。位置 22 和 8（使用標(biāo)準(zhǔn)拼缝、權(quán)威 tRNA 命名法） 突變分?jǐn)?shù) 的熱力圖在圖中被畫出。不同反密碼子的 tRNA 按照他們各自修飾的位置（在圓括號(hào)中）和 字母順序被分組彰亥。過(guò)去只有 E.coli 和 B. subtilis的 tRNA 修飾被 2D-TLC 和 LC/MS 對(duì)比咧七，但 并沒(méi)有對(duì)所有的 tRNA 種類進(jìn)行比對(duì)。每一個(gè) E.coli 和 B. subtilis中 tRNA 種類任斋， 突變分?jǐn)?shù)超 過(guò)背景 10 倍時(shí)會(huì)在右邊用圓圈標(biāo)記出來(lái)猪叙。圓圈這樣設(shè)計(jì)：已知會(huì)出現(xiàn)并且也被測(cè)序識(shí)別的 用紫色標(biāo)記；已知會(huì)出現(xiàn)但并沒(méi)有被測(cè)序識(shí)別出的用黑色標(biāo)記仁卷；過(guò)去沒(méi)有比對(duì)但被測(cè)序識(shí)別 出的用綠色標(biāo)記；已知會(huì)出現(xiàn)但測(cè)序時(shí)沒(méi)有發(fā)現(xiàn)的用橙色標(biāo)記犬第。a m1A22锦积；R 對(duì)應(yīng)于 A 或 G。 b s4U8歉嗓。

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Fig 3. Microbiome tRNA-seq workflow and taxonomy analysis. a Workflow of tRNA sequencing of gut microbiome samples fed with a high-fat (HF) or low-fat (LF) diet and ==de novo==（從頭分析） tRNA assignment. Conserved tRNA residues that were searched for in this work are shown in red. b Dendrograms compare relationships between HF and LF samples that were inferred based on community profiles of tRNA transcripts, or 16S rRNA gene amplicons. c Class-level taxonomy for averaged HF and LF samples based on tRNA-seq (top) and 16S rRNA gene amplicons (bottom). All bacterial classes at >1% level are shown in distinct colors, all other bacterial classes are grouped together and shown in purple. d tRNAGly taxonomy for anticodons GCC, UCC, and CCC. e tRNAGlu taxonomy for anticodons UUC and CUC. Among the other category for GCC/UCC/CCC and UUC, no class has an abundance of ≥1%; for CUC, other classes with an abundance of ≥1% include Alphaproteobacteria, Gemmatimonadetes, and Ignavibacteria. tRNAs decoding these two amino acids are the most abundant in our tRNA-seq results

圖3. 微生物組tRNA-seq工作流程以及分類學(xué)分析 a 用高脂肪飲食組（HF）或低脂肪飲食組（LF）的腸道微生物組樣品的tRNA測(cè)序以及tRNA全新匹配的工作流程丰介。此工作中尋找到的保守tRNA殘基被標(biāo)注為紅色。 b 系統(tǒng)樹(shù)圖鉴分，比較HF組和LF租兩個(gè)樣品的關(guān)系哮幢，這些關(guān)系基于tRNA轉(zhuǎn)錄物或16S rRNA基因擴(kuò)增子的群組特征推斷得到。 c 基于tRNA?seq（上圖）和16S rRNA基 因擴(kuò)增子（下圖）的平均HF和LF樣品的類級(jí)分類志珍。所有> 1％水平的細(xì)菌類別以不同的顏色顯示橙垢，所有其他細(xì) 菌類別組合在一起并以紫色顯示。 d 甘氨酸t(yī)RNA反密碼子GCC, UCC, 和 CCC 的分類. e 谷氨酸t(yī)RNA反密碼 子UUC和CUC的分類伦糯。在GCC / UCC / CCC和UUC的其他類別中柜某，沒(méi)有一個(gè)類別的豐度≥1％；對(duì)于CUC敛纲，其他豐度≥1％的類別包括甲型變形菌（Alphaproteobacteria）喂击，芽單胞菌（Gemmatimonadetes）和 Ignavibacteria（無(wú)中文名）。在我們的tRNA?seq結(jié)果中這兩種氨基酸的tRNA是最豐富的淤翔。

==de novo==（從頭分析）：不依賴于已有的注釋文件全新分析

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Fig 4. Microbiome tRNA modification analysis. a Workflow for modification assignment using mutation ==signatures==（信號(hào)）. b–e Representative positional plots showing m1A and s4U modifications for transcripts of tRNASer(GCU) (b), tRNASer(UGA) (c), tRNASer(GGA) (d), and tRNASer(CGA) (e), HF-fed mouse sample. The peak numbers correspond to those described in the text with peak 1 called for s4U8 and peak 2 for m1A22. Peak 3 is located around nucleotide 73–79 in the type II tRNASers, but is m1A59 in the standard tRNA nomenclature. Red and black lines show mutation fractions in tRNASer sequences with (+DM) and without (?DM) demethylase treatment, respectively

圖 4 微生物組 tRNA 修飾分析翰绊。 A. 使用變異信號(hào)進(jìn)行修飾分配的工作流程。 B-E. 代表性的位點(diǎn)圖展示了 HF 喂食小鼠樣本 tRNA Ser（GCU）(b), UGA(c),GGA(d),CGA(e)） 轉(zhuǎn)錄本的 m1A 和 s4U 修飾。峰的數(shù)量與文中描述的相當(dāng)监嗜，峰 1 為 s 4 U8谐檀，峰 2 為 m 1 A22 。峰 3 定位在二 型 tRNA Sers 核苷酸 73-79 周圍秤茅，但在標(biāo)準(zhǔn) tRNA 系統(tǒng)命名中卻是 m 1 A59 稚补。紅色和黑色的線 分別展示了 tRNA 測(cè)序中有（+DM） 和沒(méi)有（-DM） 去甲基化酶處理的變異部分

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Fig 5. Taxonomic differences of modification sites. a Examples of aligning tRNA sequencing reads to two seed sequences of tRNASer(UGA) from Lactobacillus, class Bacilli, and Bifidobacterium, class Actinobacteria without and with demethylase treatment. Modification sites identified (s4U and m1A) are highlighted between the white lines. bm1A22, m1A58/59, and s4U8 identified in the abundant bacterial classes from Fig. 3c in the context of their phylogenetic relationship. Large fonts indicate bacterial classes in which the majority of the modifications are found (m1A22 in Clostridia and Bacilli, m1A58/59 in Actinobacteria, and s4U8 in Clostridia and Bacilli). c Proximal location of the m1A22 (red), m158 (blue), and m159 (green) modifications in a tRNA three-dimensional structure

圖5. a. 去甲基化酶處理會(huì)使得 修飾丟失，其中 和 修飾是在多物種間都存在的 b. 圖3c中各類細(xì)菌的進(jìn)化關(guān)系及其所包含的修飾類型 c. 三維結(jié)構(gòu)展示不同修飾位點(diǎn)

==個(gè)人比較認(rèn)可這種方式框喳，用自己的話把圖表描述一下课幕，不用翻譯其中的每一句話，而是解釋圖表的橫縱坐標(biāo)或線條的意思五垮，更容易理解==

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Fig 6. Comparisons of mutation fractions of HF versus LF samples. Bacterial families with the highest numbers of modifications at the respective nucleotides are shown. Each pair shows HF and LF samples with distinct anticodons marked on top. The amino acid whose codons are read by the corresponding tRNA with designated anticodon can be found in Supplementary Table 4. Box-and-whisker plots show median as a line, upper and lower quartiles in the box, and outliers outside of the line. a m1A22 from Lachnospiraceae, class Clostridia. b m1A58 and m1A59 from Bifidobacteriaceae, class Actinobacteria. c s4U8 from Lachnospiraceae, class Clostridia

圖6. HF 和 LF 樣品突變分?jǐn)?shù)的比較乍惊。 顯示了在各自的核苷酸上修飾最多的細(xì)菌科。 每一對(duì)都顯 示了頂部標(biāo)記有不同反密碼子的 HF 和 LF 樣品放仗。 其密碼子由指定反密碼子的相應(yīng) tRNA 讀 取的氨基酸見(jiàn)補(bǔ)充表 4润绎。 箱線圖以直線表示中間值，盒中的上诞挨、下四分位數(shù)莉撇，以及線外的異 常值。 a： 來(lái)自 Lachnospiraceae, class Clostridia 的 m1A22惶傻。 b： m1A58 和 m1A59 來(lái)自 Bifidobacteriaceae, class Actinobacteria.棍郎。 c： 來(lái)自 Lachnospiraceae, class Clostridia 的 s4U8

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Fig. 7. Analysis of differential protein expression and tRNA modification. Proteomics data from HF- and LF-fed mice were from reference36. a Average differential expression of 849 proteins between HF- and LF-fed mouse gut microbiome from day 43 mice that most mimics the experimental condition of our tRNA-seq experiment. b BLASTp protein sequence analysis shows that most of these proteins are from class ==Clostridia（梭菌 ）==. c Quantitative difference between the clostridia proteins from day 43 mice. Lines show the boundaries of the proteins used for downstream analysis that are highly enriched (log >1, 88 proteins) or depleted (log<??1, 105 proteins) in HF over LF samples. The difference in amino acid (d) or codon content (e) determined by subtracting the compositions of HF over-expressed proteins minus the HF under-expressed proteins. The amino acids or codons for which their decoding tRNAs were found to contain m1A modifications are in red: Cys, Glu, Gln, Ser. The difference in amino acid pair (f) and codon pair content (g) determined by subtracting the pair compositions of HF over-expressed proteins minus the HF under-expressed proteins. The first amino acid represents the N-terminal residue and the first codon represents the 5’ codon

圖7. 差異的蛋白表達(dá)和 tRNA 修飾的分析。a 高脂和低脂喂養(yǎng)小鼠 43 天后银室， 其腸微生物組中 849 個(gè)蛋白的平均差異表達(dá)涂佃，第 43天的實(shí)驗(yàn)條件和本文 tRNA-seq實(shí)驗(yàn)的條件最相似。 b 蛋白序列比對(duì)分析顯示測(cè)得的大多數(shù)蛋白來(lái)源于 Clostridia 梭菌類蜈敢。 c 兩列黑線表示用于后續(xù)分析的蛋白挑選分界線： log >1 有 88 個(gè) 蛋白辜荠， 在 HF 中高豐度。 log < -1,105 個(gè)蛋白抓狭， HF 中低豐度伯病。 log 是 基于高脂與低脂喂養(yǎng)小鼠樣本的比值， 通過(guò)減去 HF 過(guò)表達(dá)蛋白的組成辐宾、減去 HF 低表達(dá)蛋白的組成從而 確定氨基酸(d)或密碼子含量(e)的差異狱从。 一些氨基酸或密碼子相應(yīng)的 tRNAs 被發(fā)現(xiàn)含有 m1A 修飾， 這些氨基酸或密碼子標(biāo)為紅色： Cys叠纹、 Glu季研、 Gln、 Ser誉察。 通過(guò)減去 HF 過(guò)表達(dá)蛋白和 HF 低表達(dá)蛋白的配對(duì)組成与涡， 確定了氨 基酸對(duì)(f)和密碼子對(duì)(g)含量的差異。 f 中橫坐標(biāo) first amino acid 代表 N-末端殘基， g 中橫坐標(biāo) first codon 代表 5’ 密碼子驼卖。

==個(gè)人比較認(rèn)可這種方式氨肌，用自己的話把圖表描述一下，不用翻譯其中的每一句話酌畜，而是解釋圖表的橫縱坐標(biāo)或線條的意思怎囚，更容易理解==

翻譯小組：

李碧琪、陳凱星桥胞、葉名琛恳守、鄭易民、王俊豪贩虾、倪豪辰催烘、黃敬潼、黃子亮缎罢、鄧峻瑋伊群、常彥琪、鄭凌伶