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Diversity and composition of the microbiome associated with eggs of the Southern green stinkbug, Nezara viridula (Hemiptera: Pentatomidae) 南方绿臭虫(Nezara viridula)虫卵微生物群的多样性和组成(半翅目:蝽科)
IF 3.4 3区 生物学 Q1 Immunology and Microbiology Pub Date : 2022-12-07 DOI: 10.1002/mbo3.1337
Margot W. J. Geerinck, Sara Van Hee, Gabriele Gloder, Sam Crauwels, Stefano Colazza, Hans Jacquemyn, Antonino Cusumano, Bart Lievens

Although microbial communities of insects from larval to adult stage have been increasingly investigated in recent years, little is still known about the diversity and composition of egg-associated microbiomes. In this study, we used high-throughput amplicon sequencing and quantitative PCR to get a better understanding of the microbiome of insect eggs and how they are established using the Southern green stinkbug Nezara viridula (L.) (Hemiptera: Pentatomidae) as a study object. First, to determine the bacterial community composition, egg masses from two natural populations in Belgium and Italy were examined. Subsequently, microbial community establishment was assessed by studying stinkbug eggs of different ages obtained from laboratory strains (unlaid eggs collected from the ovaries, eggs less than 24 h old, and eggs collected 4 days after oviposition). Both the external and internal egg-associated microbiomes were analyzed by investigating egg washes and surface-sterilized washed eggs, respectively. Eggs from the ovaries were completely devoid of bacteria, indicating that egg-associated bacteria were deposited on the eggs during or after oviposition. The bacterial diversity of deposited eggs was very low, with on average 6.1 zero-radius operational taxonomic units (zOTUs) in the external microbiome and 1.2 zOTUs in internal samples of egg masses collected from the field. Bacterial community composition and density did not change significantly over time, suggesting limited bacterial growth. A Pantoea-like symbiont previously found in the midgut of N. viridula was found in every sample and generally occurred at high relative and absolute densities, especially in the internal egg samples. Additionally, some eggs harbored a Sodalis symbiont, which has previously been found in the abdomen of several insects, but so far not in N. viridula populations. We conclude that the egg-associated bacterial microbiome of N. viridula is species-poor and dominated by a few symbionts, particularly the species-specific obligate Pantoea-like symbiont.

近年来,人们对昆虫从幼虫到成虫阶段的微生物群落进行了越来越多的研究,但对与卵相关的微生物群落的多样性和组成知之甚少。本研究以南方绿臭虫Nezara viridula (L.)(半翅目:Pentatomidae)为研究对象,采用高通量扩增子测序和定量PCR技术,深入了解虫卵微生物组及其建立机制。首先,为了确定细菌群落组成,研究人员检查了比利时和意大利两个自然种群的鸡蛋质量。随后,通过研究实验室菌株不同年龄的臭虫卵(从卵巢采集的未产卵卵、小于24 h的卵和产卵后4 d采集的卵)来评估微生物群落的建立。通过对洗蛋液和表面消毒的洗蛋液,分别分析了外部和内部鸡蛋相关微生物组。来自卵巢的卵完全没有细菌,表明与卵相关的细菌在产卵期间或产卵后沉积在卵上。产卵卵的细菌多样性很低,野外采集的卵团体外微生物群平均为6.1个零半径操作分类单位(zero-radius operational taxonomic units, zOTUs),体内微生物群平均为1.2个。细菌群落组成和密度随时间变化不明显,表明细菌生长有限。在每个样本中都发现了一种在病毒乳杆菌中肠中发现的泛菌状共生体,并且通常以较高的相对密度和绝对密度出现,特别是在内部卵样本中。此外,一些卵还含有一种共生体,这种共生体以前在几种昆虫的腹部被发现,但到目前为止还没有在N. viridula种群中发现。我们得出结论,病毒乳杆菌的卵相关细菌微生物组是物种贫乏的,由少数共生体主导,特别是种特异性的专性pantoea样共生体。
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引用次数: 4
Characterizing viral samples using machine learning for Raman and absorption spectroscopy 利用拉曼和吸收光谱的机器学习表征病毒样本
IF 3.4 3区 生物学 Q1 Immunology and Microbiology Pub Date : 2022-12-05 DOI: 10.1002/mbo3.1336
Miad Boodaghidizaji, Shreya Milind Athalye, Sukirt Thakur, Ehsan Esmaili, Mohit S. Verma, Arezoo M. Ardekani

Machine learning methods can be used as robust techniques to provide invaluable information for analyzing biological samples in pharmaceutical industries, such as predicting the concentration of viral particles of interest in biological samples. Here, we utilized both convolutional neural networks (CNNs) and random forests (RFs) to predict the concentration of the samples containing measles, mumps, rubella, and varicella-zoster viruses (ProQuad®) based on Raman and absorption spectroscopy. We prepared Raman and absorption spectra data sets with known concentration values, then used the Raman and absorption signals individually and together to train RFs and CNNs. We demonstrated that both RFs and CNNs can make predictions with R2 values as high as 95%. We proposed two different networks to jointly use the Raman and absorption spectra, where our results demonstrated that concatenating the Raman and absorption data increases the prediction accuracy compared to using either Raman or absorption spectrum alone. Additionally, we further verified the advantage of using joint Raman-absorption with principal component analysis. Furthermore, our method can be extended to characterize properties other than concentration, such as the type of viral particles.

机器学习方法可以作为强大的技术,为制药行业的生物样品分析提供宝贵的信息,例如预测生物样品中感兴趣的病毒颗粒的浓度。在这里,我们利用卷积神经网络(cnn)和随机森林(rf)基于拉曼和吸收光谱预测含有麻疹、腮腺炎、风疹和水痘-带状疱疹病毒(ProQuad®)的样品浓度。我们制备了已知浓度值的拉曼和吸收光谱数据集,然后分别使用拉曼和吸收信号,并将它们一起用于训练rf和cnn。我们证明了RFs和cnn都可以做出R2值高达95%的预测。我们提出了两种不同的网络来联合使用拉曼光谱和吸收光谱,我们的结果表明,与单独使用拉曼光谱或吸收光谱相比,连接拉曼和吸收数据提高了预测精度。此外,我们进一步验证了联合拉曼吸收与主成分分析的优势。此外,我们的方法可以扩展到表征浓度以外的特性,例如病毒颗粒的类型。
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引用次数: 1
A yeast-based tool for screening mammalian diacylglycerol acyltransferase inhibitors 筛选哺乳动物二酰基甘油酰基转移酶抑制剂的酵母为基础的工具
IF 3.4 3区 生物学 Q1 Immunology and Microbiology Pub Date : 2022-12-01 DOI: 10.1002/mbo3.1334
Peter Gajdoš, Rodrigo Ledesma-Amaro, Jean-Marc Nicaud, Tristan Rossignol

Dysregulation of lipid metabolism is associated with obesity and metabolic diseases but there is also increasing evidence of a relationship between lipid body excess and cancer. Lipid body synthesis requires diacylglycerol acyltransferases (DGATs) which catalyze the last step of triacylglycerol synthesis from diacylglycerol and acyl-coenzyme A. The DGATs and in particular DGAT2, are therefore considered potential therapeutic targets for the control of these pathologies. Here, the murine and the human DGAT2 were overexpressed in the oleaginous yeast Yarrowia lipolytica deleted for all DGAT activities, to evaluate the functionality of the enzymes in this heterologous host and DGAT activity inhibitors. This work provides evidence that mammalian DGATs expressed in Y. lipolytica are a useful tool for screening chemical libraries to identify potential inhibitors or activators of these enzymes of therapeutic interest.

脂质代谢失调与肥胖和代谢性疾病有关,但也有越来越多的证据表明脂质体过量与癌症之间存在关系。脂体合成需要二酰基甘油酰基转移酶(dgat),它催化二酰基甘油和酰基辅酶a合成三酰基甘油的最后一步。因此,dgat,特别是DGAT2,被认为是控制这些病理的潜在治疗靶点。在本研究中,小鼠和人类DGAT2在剔除所有DGAT活性的脂质耶氏酵母中过表达,以评估该酶在异种宿主和DGAT活性抑制剂中的功能。这项工作提供了证据,表明哺乳动物在脂肪瘤中表达的dgat是筛选化学文库的有用工具,可以识别这些酶的潜在抑制剂或激活剂。
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引用次数: 0
Observations on the interaction between plant growth-promoting bacteria and the root-knot nematode Meloidogyne javanica 植物促生菌与爪哇根结线虫相互作用的观察
IF 3.4 3区 生物学 Q1 Immunology and Microbiology Pub Date : 2022-11-26 DOI: 10.1002/mbo3.1319
Aoife Egan, Thomais Kakouli-Duarte

Pseudomonas fluorescens, strains L124, L228, L321, and the positive control strain F113 used in this study, produce compounds associated with plant growth promotion, biocontrol, antimicrobial and antiviral activity, and adaptation to stresses. These bacterial strains were tested in vitro and in vivo in tomato plants, to determine their potential role in Meloidogyne javanica suppression. In laboratory experiments, only 2% of M. javanica eggs hatched when exposed to the metabolites of each bacterial strain. Additionally, 100% M. javanica J2 mortality was recorded when nematodes were exposed to the metabolites of F113 and L228. In greenhouse experiments, M. javanica infected tomato plants, which were also inoculated with the bacterial strains F113 and L124, displayed the highest biomass (height, number of leaves, fresh and dry weight) of all bacterial treatments tested. Results from the development and induced systemic resistance experiments indicated that the bacterial strains F113 and L321 had the most effective biocontrol capacity over nematode infection, delayed nematode development (J3/J4, adults and galls), and reduced nematode fecundity. In addition, these results indicated that the bacterial strain L124 is an effective plant growth promoter of tomato plants. Furthermore, it was determined that the bacterial strain L321 was capable of M. javanica biocontrol. P. fluorescens F113 was effective at both increasing tomato plant biomass and M. javanica biocontrol. In an agricultural context, applying successional drenches with these beneficial plant growth promoting rhizobacteria would ensure bacteria viability in the rhizosphere of the plants, encourage positive plant bacterial interactions and increase biocontrol against M. javanica.

本研究中使用的荧光假单胞菌菌株L124、L228、L321和阳性对照菌株F113产生与植物生长促进、生物防治、抗菌和抗病毒活性以及逆境适应相关的化合物。研究了这些菌株在番茄植株体内和体外的抑菌作用。在实验室实验中,当暴露于每种菌株的代谢物时,只有2%的爪哇支原体卵孵化。此外,当线虫暴露于F113和L228的代谢物时,记录到100%的javanica J2死亡率。在温室试验中,同样接种菌株F113和L124的黑僵菌侵染番茄植株的生物量(株高、叶数、鲜重和干重)在所有细菌处理中最高。发育和诱导抗性实验结果表明,菌株F113和L321对线虫感染、延缓线虫发育(J3/J4、成虫和瘿虫)和降低线虫繁殖力具有最有效的生物防治能力。此外,这些结果表明菌株L124是一种有效的番茄植株生长促进剂。结果表明,菌株L321具有一定的防菌活性。荧光假单胞菌F113对提高番茄植株生物量和防治爪哇菌均有显著效果。在农业环境中,连续施用这些有益植物生长的促进根细菌的雨水将确保植物根际细菌的活力,促进植物细菌的积极相互作用,并增加对爪哇栗霉的生物防治。
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引用次数: 1
The emergence of the tetrathionate reductase operon in the Escherichia coli/Shigella pan-genome 大肠杆菌/志贺氏菌泛基因组中四硫酸还原酶操纵子的出现
IF 3.4 3区 生物学 Q1 Immunology and Microbiology Pub Date : 2022-11-06 DOI: 10.1002/mbo3.1333
Floyd G. Adsit Jr, Thomas A. Randall, Jacqueline Locklear, David M. Kurtz

Escherichia coli pathogenic variants (pathovars) are generally characterized by defined virulence traits and are susceptible to the evolution of hybridized identities due to the considerable plasticity of the E. coli genome. We have isolated a strain from a purified diet intended for research animals that further demonstrates the ability of E. coli to acquire novel genetic elements leading potentially to emergent new pathovars. Utilizing next generation sequencing to obtain a whole genome profile, we report an atypical strain of E. coli, EcoFA807-17, possessing a tetrathionate reductase (ttr) operon, which enables the utilization of tetrathionate as an electron acceptor, thus facilitating respiration in anaerobic environments such as the mammalian gut. The ttr operon is a potent virulence factor for several enteric pathogens, most prominently Salmonella enterica. However, the presence of chromosomally integrated tetrathionate reductase genes does not appear to have been previously reported in wild-type E. coli or Shigella. Accordingly, it is possible that the appearance of this virulence factor may signal the evolution of new mechanisms of pathogenicity in E. coli and Shigella and may potentially alter the effectiveness of existing assays using tetrathionate reductase as a unique marker for the detection of Salmonella enterica.

大肠杆菌致病性变异(病原菌)通常具有明确的毒力特征,并且由于大肠杆菌基因组的相当大的可塑性,易受杂交身份进化的影响。我们已经从用于研究动物的纯化饮食中分离出一株菌株,这进一步证明了大肠杆菌获得新遗传元件的能力,可能导致新病原体的出现。利用下一代测序获得全基因组图谱,我们报道了一株非典型大肠杆菌EcoFA807-17,该菌株具有四硫酸盐还原酶(ttr)操纵子,该操纵子能够利用四硫酸盐作为电子受体,从而促进哺乳动物肠道等厌氧环境中的呼吸。ttr操纵子是几种肠道病原体的有效毒力因子,最突出的是肠沙门氏菌。然而,在野生型大肠杆菌或志贺氏菌中,染色体整合四硫酸还原酶基因的存在似乎并没有先前的报道。因此,这种毒力因子的出现可能标志着大肠杆菌和志贺氏菌致病性新机制的进化,并可能潜在地改变现有使用四硫酸还原酶作为检测肠沙门氏菌的独特标记物的检测方法的有效性。
{"title":"The emergence of the tetrathionate reductase operon in the Escherichia coli/Shigella pan-genome","authors":"Floyd G. Adsit Jr,&nbsp;Thomas A. Randall,&nbsp;Jacqueline Locklear,&nbsp;David M. Kurtz","doi":"10.1002/mbo3.1333","DOIUrl":"10.1002/mbo3.1333","url":null,"abstract":"<p><i>Escherichia coli</i> pathogenic variants (pathovars) are generally characterized by defined virulence traits and are susceptible to the evolution of hybridized identities due to the considerable plasticity of the <i>E. coli</i> genome. We have isolated a strain from a purified diet intended for research animals that further demonstrates the ability of <i>E. coli</i> to acquire novel genetic elements leading potentially to emergent new pathovars. Utilizing next generation sequencing to obtain a whole genome profile, we report an atypical strain of <i>E. coli</i>, EcoFA807-17, possessing a tetrathionate reductase (<i>ttr</i>) operon, which enables the utilization of tetrathionate as an electron acceptor, thus facilitating respiration in anaerobic environments such as the mammalian gut. The <i>ttr</i> operon is a potent virulence factor for several enteric pathogens, most prominently <i>Salmonella enterica</i>. However, the presence of chromosomally integrated tetrathionate reductase genes does not appear to have been previously reported in wild-type <i>E. coli</i> or <i>Shigella</i>. Accordingly, it is possible that the appearance of this virulence factor may signal the evolution of new mechanisms of pathogenicity in <i>E. coli</i> and <i>Shigella</i> and may potentially alter the effectiveness of existing assays using tetrathionate reductase as a unique marker for the detection of <i>Salmonella enterica</i>.</p>","PeriodicalId":18573,"journal":{"name":"MicrobiologyOpen","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2022-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9638481/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10568689","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Comparison of one-step with two-step production of Bacillus atrophaeus spores for use as bioindicators 一步法与两步法生产用作生物指示剂的萎缩芽孢杆菌孢子的比较
IF 3.4 3区 生物学 Q1 Immunology and Microbiology Pub Date : 2022-11-03 DOI: 10.1002/mbo3.1332
Philipp Stier, Ulrich Kulozik

The production method of spores significantly influences the resistance of spores used as bioindicators (BI) in the validation of sterilization of packaging material surfaces in aseptic food manufacturing. Therefore, the standardization of the spore production method represents an important and desirable goal in industrial BI production to ensure reliable validation test results. Previously, we recommended a two-step production approach for submerged spore production, in which the cultivation phase to obtain high cell mass was separate from the sporulation phase. In this work, a one-step manufacturing process was investigated to reduce production complexity and facilitate standardization of spore production. It was found that one-step BI production is technically possible but at the expense of spore yield. The two-step manufacturing process can realize almost 10-fold higher spore yields.

在无菌食品生产中,作为生物指示剂(BI)的孢子在包装材料表面的灭菌验证中,孢子的生产方法对其抗性有显著影响。因此,孢子生产方法的标准化是BI工业生产中重要和理想的目标,以确保可靠的验证试验结果。以前,我们推荐了一种两步生产方法来生产淹没孢子,其中获得高细胞质量的培养阶段与孢子形成阶段分开。在本工作中,研究了一步制造工艺,以降低生产复杂性,促进孢子生产的标准化。研究发现,一步制BI在技术上是可行的,但以牺牲孢子产量为代价。两步制造过程可以使孢子产量提高近10倍。
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引用次数: 0
Brucella ceti and Brucella pinnipedialis genome characterization unveils genetic features that highlight their zoonotic potential 鲸头布鲁氏菌和鳍足布鲁氏菌基因组特征揭示了突出其人畜共患潜力的遗传特征
IF 3.4 3区 生物学 Q1 Immunology and Microbiology Pub Date : 2022-10-26 DOI: 10.1002/mbo3.1329
Massimiliano Orsini, Andrea Ianni, Luca Zinzula

The Gram-negative bacteria Brucella ceti and Brucella pinnipedialis circulate in marine environments primarily infecting marine mammals, where they cause an often-fatal disease named brucellosis. The increase of brucellosis among several species of cetaceans and pinnipeds, together with the report of sporadic human infections, raises concerns about the zoonotic potential of these pathogens on a large scale and may pose a threat to coastal communities worldwide. Therefore, the characterization of the B. ceti and B. pinnipedialis genetic features is a priority to better understand the pathological factors that may impact global health. Moreover, an in-depth functional analysis of the B. ceti and B. pinnipedialis genome in the context of virulence and pathogenesis was not undertaken so far. Within this picture, here we present the comparative whole-genome characterization of all B. ceti and B. pinnipedialis genomes available in public resources, uncovering a collection of genetic tools possessed by these aquatic bacterial species compared to their zoonotic terrestrial relatives. We show that B. ceti and B. pinnipedialis genomes display a wide host-range infection capability and a polyphyletic phylogeny within the genus, showing a genomic structure that fits the canonical definition of closeness. Functional genome annotation led to identifying genes related to several pathways involved in mechanisms of infection, others conferring pan-susceptibility to antimicrobials and a set of virulence genes that highlight the similarity of B. ceti and B. pinnipedialis genotypes to those of Brucella spp. displaying human-infecting phenotypes.

革兰氏阴性菌鲸氏布鲁氏菌和鳍足布鲁氏菌在海洋环境中传播,主要感染海洋哺乳动物,在海洋中引起一种通常致命的疾病,称为布鲁氏菌病。几种鲸类和鳍足类动物中布鲁氏菌病的增加,以及零星人类感染的报告,引起了人们对这些病原体大规模人畜共患的可能性的关注,并可能对世界各地的沿海社区构成威胁。因此,为了更好地了解可能影响全球健康的病理因素,研究鲸头绦虫和鳍足绦虫的遗传特征是一个优先事项。此外,到目前为止,还没有对鲸头绦虫和鳍足绦虫基因组在毒力和发病机制方面进行深入的功能分析。在这幅图中,我们展示了公共资源中所有鲸鲸b.s ceti和鳍足b.s pinnipedialis基因组的比较全基因组特征,揭示了这些水生细菌物种与其人畜共患病的陆地亲戚相比所拥有的遗传工具集合。我们发现鲸头鲸和尖足鲸基因组显示出广泛的宿主感染能力和属内的多种系统发育,显示出符合近亲关系规范定义的基因组结构。功能基因组注释确定了与感染机制相关的几种途径相关的基因,其他基因赋予抗微生物药物的泛易感性,以及一组毒力基因,这些基因突出了鲸头贝氏杆菌和鳍足贝氏杆菌基因型与显示人类感染表型的布鲁氏菌基因型的相似性。
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引用次数: 0
Whole-genome sequencing and genome-scale metabolic modeling of Chromohalobacter canadensis 85B to explore its salt tolerance and biotechnological use 加拿大嗜盐杆菌85B的全基因组测序和基因组尺度代谢建模,探索其耐盐性及其生物技术应用
IF 3.4 3区 生物学 Q1 Immunology and Microbiology Pub Date : 2022-10-26 DOI: 10.1002/mbo3.1328
Blaise Manga Enuh, Belma Nural Yaman, Chaimaa Tarzi, Pınar Aytar Çelik, Mehmet Burçin Mutlu, Claudio Angione

Salt tolerant organisms are increasingly being used for the industrial production of high-value biomolecules due to their better adaptability compared to mesophiles. Chromohalobacter canadensis is one of the early halophiles to show promising biotechnology potential, which has not been explored to date. Advanced high throughput technologies such as whole-genome sequencing allow in-depth insight into the potential of organisms while at the frontiers of systems biology. At the same time, genome-scale metabolic models (GEMs) enable phenotype predictions through a mechanistic representation of metabolism. Here, we sequence and analyze the genome of C. canadensis 85B, and we use it to reconstruct a GEM. We then analyze the GEM using flux balance analysis and validate it against literature data on C. canadensis. We show that C. canadensis 85B is a metabolically versatile organism with many features for stress and osmotic adaptation. Pathways to produce ectoine and polyhydroxybutyrates were also predicted. The GEM reveals the ability to grow on several carbon sources in a minimal medium and reproduce osmoadaptation phenotypes. Overall, this study reveals insights from the genome of C. canadensis 85B, providing genomic data and a draft GEM that will serve as the first steps towards a better understanding of its metabolism, for novel applications in industrial biotechnology.

耐盐生物由于具有比中温微生物更好的适应性,越来越多地被用于高价值生物分子的工业生产。加拿大嗜盐杆菌是早期显示出生物技术潜力的嗜盐菌之一,迄今尚未被探索。先进的高通量技术,如全基因组测序,可以深入了解生物体的潜力,同时处于系统生物学的前沿。同时,基因组尺度代谢模型(GEMs)通过代谢的机制表征实现表型预测。在此,我们对加拿大C. canadensis 85B的基因组进行了测序和分析,并利用它重建了一个GEM。然后,我们使用通量平衡分析对GEM进行分析,并与加拿大大麻的文献数据进行验证。我们发现加拿大C. canadensis 85B是一种代谢多样的生物,具有许多应激和渗透适应的特征。同时还预测了产外托因和聚羟基丁酸酯的途径。GEM揭示了在最小的培养基中在几种碳源上生长并繁殖渗透适应表型的能力。总的来说,本研究揭示了加拿大C. canadensis 85B基因组的见解,提供了基因组数据和GEM草案,这将是更好地了解其代谢的第一步,用于工业生物技术的新应用。
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引用次数: 4
Comparison of DNA purification methods for high-throughput sequencing of fungal communities from wine fermentation 葡萄酒发酵真菌群落高通量测序的DNA纯化方法比较
IF 3.4 3区 生物学 Q1 Immunology and Microbiology Pub Date : 2022-10-25 DOI: 10.1002/mbo3.1321
Antoine Gobert, Marie Sarah Evers, Christophe Morge, Céline Sparrow, Vincent Delafont

High-throughput sequencing approaches, which target a taxonomically discriminant locus, allow for in-depth insight into microbial communities’ compositions. Although microorganisms are historically investigated by cultivation on artificial culture media, this method presents strong limitations, since only a limited proportion of microorganisms can be grown in vitro. This pitfall appears even more limiting in enological and winemaking processes, during which a wide range of molds, yeasts, and bacteria are observed at the different stages of the fermentation course. Such an understanding of those dynamic communities and how they impact wine quality therefore stands as a major challenge for the future of enology. As of now, although high-throughput sequencing has already allowed for the investigation of fungal communities, there is no available comparative study focusing on the performance of microbial deoxyribonucleic acid (DNA) extraction in enological matrixes. This study aims to provide a comparison of five selected extraction methods, assayed on both must and fermenting must, as well as on finished wine. These procedures were evaluated according to their extraction yields, the purity of their extracted DNA, and the robustness of downstream molecular analyses, including polymerase chain reaction and high-throughput sequencing of fungal communities. Altogether, two out of the five assessed microbial DNA extraction methods (DNeasy PowerSoil Pro Kit and E.Z.N.A.® Food DNA Kit) appeared suitable for robust evaluations of the microbial communities in wine samples. Consequently, this study provides robust tools for facilitated upcoming studies to further investigate microbial communities during winemaking using high-throughput sequencing.

高通量测序方法,其目标是一个分类上的区别位点,允许深入了解微生物群落的组成。虽然微生物历来是通过在人工培养基上培养来研究的,但这种方法存在很强的局限性,因为只有有限比例的微生物可以在体外培养。在酿酒和酿酒过程中,这个陷阱显得更加有限,在发酵过程的不同阶段,可以观察到各种各样的霉菌、酵母和细菌。因此,对这些动态群落的理解,以及它们如何影响葡萄酒质量,是未来酿酒学的一个重大挑战。到目前为止,虽然高通量测序已经可以用于真菌群落的研究,但还没有针对微生物脱氧核糖核酸(DNA)在微生物基质中提取性能的比较研究。本研究的目的是提供五种选择的提取方法的比较,分析了两种葡萄汁和发酵葡萄汁,以及成品葡萄酒。根据提取率、提取DNA的纯度和下游分子分析的稳健性,包括聚合酶链反应和真菌群落的高通量测序,对这些方法进行了评估。总的来说,五种评估的微生物DNA提取方法中的两种(dnasy PowerSoil Pro Kit和E.Z.N.A.®Food DNA Kit)似乎适合于对葡萄酒样品中的微生物群落进行可靠的评估。因此,本研究为未来的研究提供了强大的工具,以促进利用高通量测序进一步研究酿酒过程中的微生物群落。
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引用次数: 0
Localization of the CotY and ExsY proteins to the exosporium basal layer of Bacillus anthracis 炭疽芽孢杆菌外孢子基底层CotY和ExsY蛋白的定位
IF 3.4 3区 生物学 Q1 Immunology and Microbiology Pub Date : 2022-10-14 DOI: 10.1002/mbo3.1327
Jorge Durand-Heredia, George C. Stewart

Spores are an infectious form of the zoonotic bacterial pathogen, Bacillus anthracis. The outermost spore layer is the exosporium, comprised of a basal layer and an external glycoprotein nap layer. The major structural proteins of the inner basal layer are CotY (at the mother cell central pole or bottlecap) and ExsY around the rest of the spore. The basis for the cap or noncap specificity of the CotY and ExsY proteins is currently unknown. We investigated the role of sequence differences between these proteins in localization during exosporium assembly. We found that sequence differences were less important than the timing of expression of the respective genes in the positioning of these inner basal layer structural proteins. Fusion constructs with the fluorescent protein fused at the N-terminus resulted in poor incorporation whereas fusions at the carboxy terminus of CotY or ExsY resulted in good incorporation. However, complementation studies revealed that fusion constructs, although accurate indicators of protein localization, were not fully functional. A model is presented that explains the localization patterns observed. Bacterial two-hybrid studies in Escherichia coli hosts were used to examine protein–protein interactions with full-length and truncated proteins. The N-terminus amino acid sequences of ExsY and CotY appear to be recognized by spore proteins located in the spore interspace, consistent with interactions seen with ExsY and CotY with the interspace proteins CotE and CotO, known to be involved with exosporium attachment.

孢子是人畜共患的细菌病原体,炭疽芽孢杆菌的一种传染性形式。孢子最外层为外孢子层,由基底层和外糖蛋白绒毛层组成。内基底层的主要结构蛋白是CotY(在母细胞中心极或瓶盖处)和ExsY(在孢子的其余部分)。CotY和ExsY蛋白的帽或非帽特异性的基础目前尚不清楚。我们研究了这些蛋白之间的序列差异在外孢子组装过程中的定位作用。我们发现,在这些内层基底层结构蛋白的定位中,序列差异不如各自基因的表达时间重要。与在n端融合的荧光蛋白的融合构建体结合不良,而在CotY或ExsY的羧基端融合则结合良好。然而,互补研究表明,融合构建体虽然是蛋白质定位的准确指标,但功能并不完全。提出了一个模型来解释观测到的定位模式。大肠杆菌宿主的细菌双杂交研究用于检测蛋白质与全长和截短蛋白质的相互作用。ExsY和CotY的n端氨基酸序列似乎可以被位于孢子间隙的孢子蛋白识别,这与ExsY和CotY与间隙蛋白CotE和CotO的相互作用一致,这些蛋白已知与孢子附着有关。
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引用次数: 4
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