scholarly journals Targeted Access to the Genomes of Low-Abundance Organisms in Complex Microbial Communities

2007 ◽  
Vol 73 (10) ◽  
pp. 3205-3214 ◽  
Author(s):  
Mircea Podar ◽  
Carl B. Abulencia ◽  
Marion Walcher ◽  
Don Hutchison ◽  
Karsten Zengler ◽  
...  
Keyword(s):  
Cell Separation ◽  
Genomic Dna ◽  
Microbial Consortia ◽  
Bacterial Cells ◽  
Metabolic Potential ◽  
Low Frequencies ◽  
Powerful Approach ◽  
Laboratory Cultures ◽  
First Time

ABSTRACT Current metagenomic approaches to the study of complex microbial consortia provide a glimpse into the community metabolism and occasionally allow genomic assemblies for the most abundant organisms. However, little information is gained for the members of the community present at low frequencies, especially those representing yet-uncultured taxa, which include the bulk of the diversity present in most environments. Here we used phylogenetically directed cell separation by fluorescence in situ hybridization and flow cytometry, followed by amplification and sequencing of a fraction of the genomic DNA of several bacterial cells that belong to the TM7 phylum. Partial genomic assembly allowed, for the first time, a look into the evolution and potential metabolism of a soil representative from this group of organisms for which there are no species in stable laboratory cultures. Genomic reconstruction from targeted cells of uncultured organisms isolated directly from the environment represents a powerful approach to access any specific members of a community and an alternative way to assess the community's metabolic potential.

scholarly journals Eukaryotic Cell Capture by Amplified Magnetic in situ Hybridization Using Yeast as a Model

2021 ◽  
Vol 12 ◽  
Author(s):  
Fabiola Bastian ◽  
Delphine Melayah ◽  
Mylène Hugoni ◽  
Nora M. Dempsey ◽  
Pascal Simonet ◽  
...  
Keyword(s):  
Eukaryotic Cell ◽  
Morphological Characterization ◽  
Superparamagnetic Nanoparticles ◽  
Eukaryotic Cells ◽  
Hybridization Chain Reaction ◽  
Bacterial Cells ◽  
Cell Capture ◽  
Near Future ◽  
First Time

A non-destructive approach based on magnetic in situ hybridization (MISH) and hybridization chain reaction (HCR) for the specific capture of eukaryotic cells has been developed. As a prerequisite, a HCR-MISH procedure initially used for tracking bacterial cells was here adapted for the first time to target eukaryotic cells using a universal eukaryotic probe, Euk-516R. Following labeling with superparamagnetic nanoparticles, cells from the model eukaryotic microorganism Saccharomyces cerevisiae were hybridized and isolated on a micro-magnet array. In addition, the eukaryotic cells were successfully targeted in an artificial mixture comprising bacterial cells, thus providing evidence that HCR-MISH is a promising technology to use for specific microeukaryote capture in complex microbial communities allowing their further morphological characterization. This new study opens great opportunities in ecological sciences, thus allowing the detection of specific cells in more complex cellular mixtures in the near future.

scholarly journals MetFish: A Metabolomics Platform for Studying Microbial Communities in Chemically Extreme Environments

2019 ◽  
Author(s):  
Chengdong Xu ◽  
Sneha P. Couvillion ◽  
Ryan L. Sontag ◽  
Nancy G. Isern ◽  
Yukari Maezato ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Mineral Content ◽  
Extreme Environments ◽  
Cell Communication ◽  
Microbial Consortia ◽  
Native Prairie ◽  
Prairie Soil ◽  
Fractured Well ◽  
First Time

Metabolites have essential roles in microbial communities, including as mediators of nutrient and energy exchange, cell-to-cell communication, and antibiosis. However, detecting and quantifying metabolites and other chemicals in samples having extremes in salt or mineral content using liquid chromatography-mass spectrometry (LC-MS)-based methods remains a significant challenge. Here we report a facile method based on in situ chemical derivatization followed by extraction for analysis of metabolites and other chemicals in hypersaline samples, enabling for the first time direct LC-MS-based exo-metabolomics analysis in sample matrices containing up to 2 molar total dissolved salts. The method, MetFish, is applicable to molecules containing amine, carboxylic acid, carbonyl, or hydroxyl functional groups, and can be integrated into either targeted or untargeted analysis pipelines. In targeted analyses, MetFish provided limits of quantification as low as 1 nM, broad linear dynamic ranges (up to 5-6 orders of magnitude) with excellent linearity, and low median inter-day reproducibility (e.g. 2.6%). MetFish was successfully applied in targeted and untargeted exo-metabolomics analyses of microbial consortia, quantifying amino acid dynamics in the exo-metabolome during community succession; in situ in a native prairie soil, whose exo-metabolome was isolated using a hypersaline extraction; and in input and produced fluids from a hydraulically fractured well, identifying dramatic changes in the exo-metabolome over time in the well.

scholarly journals Characterization of the first cultured free-living representative of Candidatus Izimaplasma uncovers its unique biology

2020 ◽  
Author(s):  
Rikuan Zheng ◽  
Rui Liu ◽  
Yeqi Shan ◽  
Ruining Cai ◽  
Ge Liu ◽  
...  
Keyword(s):  
Deep Sea ◽  
Deep Ocean ◽  
Extracellular Dna ◽  
Phenotypic Traits ◽  
The Novel ◽  
Metabolic Potential ◽  
Free Living ◽  
Living Wall ◽  
First Time

AbstractCandidatus Izimaplasma, an intermediate in the reductive evolution from Firmicutes to Mollicutes, was proposed to represent a novel class of free-living wall-less bacteria within the phylum Tenericutes found in deep-sea methane seeps. Unfortunately, the paucity of marine isolates currently available has limited further insights into their physiological and metabolic features as well as ecological roles. Here, we present a detailed description of the phenotypic traits, genomic data and central metabolisms tested in both laboratorial and deep-sea environments of the novel strain zrk13, which allows for the first time the reconstruction of the metabolic potential and lifestyle of a member of the tentatively defined Candidatus Izimaplasma. On the basis of the description of strain zrk13, the novel species and genus Xianfuyuplasma coldseepsis is proposed. Notably, DNA degradation driven by X. coldseepsis zrk13 was detected in both laboratorial and in situ conditions, strongly indicating it is indeed a key DNA degrader. Moreover, the putative genes determining degradation broadly distribute in the genomes of other Izimaplasma members. Given extracellular DNA is a particularly crucial phosphorus as well as nitrogen and carbon source for microorganisms in the seafloor, Izimaplasma bacteria are thought to be important contributors to the biogeochemical cycling in the deep ocean.

Probing the Dynamic Self-Assembly Behaviour of Photoswitchable Wormlike Micelles in Real Time

2018 ◽  
Author(s):  
Elaine A. Kelly ◽  
Judith E. Houston ◽  
Rachel Evans
Keyword(s):  
Real Time ◽  
Absorption Spectroscopy ◽  
Self Assembly ◽  
Uv Light ◽  
Wormlike Micelles ◽  
Tetraethylene Glycol ◽  
Light Illumination ◽  
First Time ◽  
Dependent Processes

Understanding the dynamic self-assembly behaviour of azobenzene photosurfactants (AzoPS) is crucial to advance their use in controlled release applications such as<i></i>drug delivery and micellar catalysis. Currently, their behaviour in the equilibrium <i>cis-</i>and <i>trans</i>-photostationary states is more widely understood than during the photoisomerisation process itself. Here, we investigate the time-dependent self-assembly of the different photoisomers of a model neutral AzoPS, <a>tetraethylene glycol mono(4′,4-octyloxy,octyl-azobenzene) </a>(C<sub>8</sub>AzoOC<sub>8</sub>E<sub>4</sub>) using small-angle neutron scattering (SANS). We show that the incorporation of <i>in-situ</i>UV-Vis absorption spectroscopy with SANS allows the scattering profile, and hence micelle shape, to be correlated with the extent of photoisomerisation in real-time. It was observed that C<sub>8</sub>AzoOC<sub>8</sub>E<sub>4</sub>could switch between wormlike micelles (<i>trans</i>native state) and fractal aggregates (under UV light), with changes in the self-assembled structure arising concurrently with changes in the absorption spectrum. Wormlike micelles could be recovered within 60 seconds of blue light illumination. To the best of our knowledge, this is the first time the degree of AzoPS photoisomerisation has been tracked <i>in</i><i>-situ</i>through combined UV-Vis absorption spectroscopy-SANS measurements. This technique could be widely used to gain mechanistic and kinetic insights into light-dependent processes that are reliant on self-assembly.

The Total Synthesis of Rhabdastrellic Acid A

2018 ◽  
Author(s):  
Yaroslav Boyko ◽  
Christopher Huck ◽  
David Sarlah
Keyword(s):  
Total Synthesis ◽  
Late Stage ◽  
Cross Coupling ◽  
Side Chains ◽  
General Strategy ◽  
Modular Approach ◽  
Linear Sequence ◽  
Generating Sequence ◽  
First Time

<div>The first total synthesis of rhabdastrellic acid A, a highly cytotoxic isomalabaricane triterpenoid, has been accomplished in a linear sequence of 14 steps from commercial geranylacetone. The prominently strained <i>trans-syn-trans</i>-perhydrobenz[<i>e</i>]indene core characteristic of the isomalabaricanes is efficiently accessed in a selective manner for the first time through a rapid, complexity-generating sequence incorporating a reductive radical polyene cyclization, an unprecedented oxidative Rautenstrauch cycloisomerization, and umpolung 𝛼-substitution of a <i>p</i>-toluenesulfonylhydrazone with in situ reductive transposition. A late-stage cross-coupling in concert with a modular approach to polyunsaturated side chains renders this a general strategy for the synthesis of numerous family members of these synthetically challenging and hitherto inaccessible marine triterpenoids.</div>

Природные материалы для сорбционной очистки хром- и кадмийсодержащих инфильтратов полигонов ТБО

2019 ◽  
pp. 13-19
Author(s):  
A. Safonov ◽  
N. Andriushchenko ◽  
N. Popova ◽  
K. Boldyrev
Keyword(s):  
Sorption Capacity ◽  
Electron Acceptors ◽  
Bacterial Cells ◽  
Maximum Sorption Capacity ◽  
Sorption Material ◽  
Maximum Sorption ◽  
Properties Of Materials ◽  
Solid Waste Landfills ◽  
Sorption Characteristics

Проведен анализ сорбционных характеристик природных материалов (вермикулит, керамзит, перлит, цеолит Трейд ) при очистке кадмий- и хромсодержащих сточных вод с высокой нагрузкой по ХПК. Установлено, что цеолит обладает максимальными сорбционными характеристиками для Cd и Cr и наименьшим биологическим обрастанием. При использовании вермикулита и керамзита или смесей на их основе можно ожидать увеличения сорбционной емкости для Cd и Сr при микробном обрастании, неизбежно происходящем в условиях контакта с водами, загрязненными органическими соединениями и биогенами. При этом биообрастание может повысить иммобилизационную способность материалов для редоксзависимых металлов за счет ферментативных ресурсов бактериальных клеток, использующих их в качестве акцепторов электронов. Эффект микробного обрастания разнонаправленно изменял параметры материалов: для Cr в большинстве случаев уменьшение и для Cd значительное увеличение. При этом дополнительным эффектом иммобилизации Cr является его биологическое восстановление биопленками. Варьируя состав сорбционного материала, можно подбирать смеси, оптимально подходящие для очистки вод инфильтратов с полигонов твердых бытовых отходов с высокой нагрузкой по ХПК и биогенным элементам как при использовании in situ, так и в системах на поверхности.The analysis of the sorption characteristics of natural materials (vermiculite, expanded clay, perlite, Trade zeolite) during the purification of cadmium and chromium-containing leachate with a high COD load was carried out. It was determined that zeolite had the maximum sorption capacity for Cd and Cr and the lowest biological fouling. When using vermiculite and expanded clay or mixtures on their basis, one can expect an increase in the sorption capacity for Cd and Cr during microbial fouling that inevitably occurs during contacting with water polluted with organic compounds and nutrients. In this case biofouling can increase the immobilization properties of materials for redox-dependent metals due to the enzymatic resources of bacterial cells that use them as electron acceptors. The effect of microbial fouling changed the parameters of materials in different directions: for Cr, in most cases, downward, and for Cd, significantly upward. Moreover, chromium biological recovery by biofilms is an additional effect of immobilization. Varying the composition of the sorption material provides for selecting mixtures that are optimally suitable for the purification of leachates from solid waste landfills with high COD and nutrients load, both when used in situ and in surface systems.

scholarly journals Metagenome-assembled genomes infer potential microbial metabolism in alkaline sulphidic tailings

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Wenjun Li ◽  
Xiaofang Li
Keyword(s):  
Community Structure ◽  
Mine Tailings ◽  
Fluorescent In Situ Hybridization ◽  
High Throughput Sequencing ◽  
Microbial Consortia ◽  
Metabolic Reconstruction ◽  
Metal Release ◽  
Community Members ◽  
Oxidative Stresses

Abstract Background Mine tailings are hostile environment. It has been well documented that several microbes can inhabit such environment, and metagenomic reconstruction has successfully pinpointed their activities and community structure in acidic tailings environments. We still know little about the microbial metabolic capacities of alkaline sulphidic environment where microbial processes are critically important for the revegetation. Microbial communities therein may not only provide soil functions, but also ameliorate the environment stresses for plants’ survival. Results In this study, we detected a considerable amount of viable bacterial and archaeal cells using fluorescent in situ hybridization in alkaline sulphidic tailings from Mt Isa, Queensland. By taking advantage of high-throughput sequencing and up-to-date metagenomic binning technology, we reconstructed the microbial community structure and potential coupled iron and nitrogen metabolism pathways in the tailings. Assembly of 10 metagenome-assembled genomes (MAGs), with 5 nearly complete, was achieved. From this, detailed insights into the community metabolic capabilities was derived. Dominant microbial species were seen to possess powerful resistance systems for osmotic, metal and oxidative stresses. Additionally, these community members had metabolic capabilities for sulphide oxidation, for causing increased salinity and metal release, and for leading to N depletion. Conclusions Here our results show that a considerable amount of microbial cells inhabit the mine tailings, who possess a variety of genes for stress response. Metabolic reconstruction infers that the microbial consortia may actively accelerate the sulphide weathering and N depletion therein.

scholarly journals Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 54
Author(s):  
Christine Landlinger ◽  
Lenka Tisakova ◽  
Vera Oberbauer ◽  
Timo Schwebs ◽  
Abbas Muhammad ◽  
...  
Keyword(s):  
Bacterial Vaginosis ◽  
Bactericidal Activity ◽  
High Selectivity ◽  
Ex Vivo ◽  
Vaginal Microbiome ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Promising Alternative ◽  
First Time

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

C-banding and fluorescence in situ hybridization studies of the wheat–alien hybrid 'Agrotana'

Genome ◽  
1994 ◽  
Vol 37 (3) ◽  
pp. 477-481 ◽  
Author(s):  
Jie Xu ◽  
R. L. Conner ◽  
A. Laroche
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Genomic Dna ◽  
Chromosome Engineering ◽  
C Banding ◽  
Chinese Spring Wheat ◽  
Mother Cells ◽  
Alien Chromatin ◽  
Chromosome Segments

'Agrotana', a wheat-alien hybrid (2n = 56), is a potential source of resistance to common root rot, stem rust, wheat streak mosaic virus, and the wheat curl mite. However, the origin of 'Agrotana', reported to be durum wheat × Agropyron trichophorum (pubescent wheatgrass), is uncertain. The objective of this investigation was to determine the chromosome constitution of 'Agrotana' using C-banding and fluorescence in situ hybridization techniques. The F1 hybrid of 'Agrotana' × 'Chinese Spring' wheat showed 7 I + 21 II in 14.9% of the pollen mother cells, evidence of the presence of the A, B, and D genomes in 'Agrotana'. The hybrid had 16 heavily C-banded chromosomes, namely 4A, and 1-7B of wheat, and a translocation that probably involved wheat chromosomes 2A and 2D. In situ hybridization using biotinylated genomic DNA of Ag. trichophorum cv. Greenleaf blocked with CS DNA failed to identify the alien chromosomes in 'Agrotana', indicating that the alien chromosomes were not likely derived from pubescent wheatgrass. In situ hybridization using labelled wheat genomic DNA blocked with 'Agrotana' DNA revealed that 'Agrotana' had 40 wheat, 14 alien, and 2 (a pair) wheat–alien translocated chromosomes. There was no homology between wheat and the alien chromosomes or chromosome segments involved in the wheat–alien recombinant. Two of the seven pairs of alien chromosomes were homoeologous to each other. The ability to identify alien chromatin in wheat using labelled wheat DNA instead of labelled alien DNA will be particularly useful in chromosome engineering of wheat germplasms having alien chromatin of unknown origin.Key words: wheat–alien hybrid, C-banding, fluorescence in situ hybridization, labelled wheat DNA as probe.

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