scholarly journals Use of a Fluorescent Analog of Glucose (2-NBDG) To Identify Uncultured Rumen Bacteria That Take Up Glucose

2019 ◽  
Vol 85 (7) ◽  
Author(s):  
Junyi Tao ◽  
Courtney McCourt ◽  
Halima Sultana ◽  
Corwin Nelson ◽  
John Driver ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Stable Isotope Probing ◽  
Fluorescent Label ◽  
Major Advance ◽  
New Paradigm ◽  
Fluorescent Substrate ◽  
Substrate Analogs ◽  
Uncultured Bacteria ◽  
Agar Media ◽  
Natural Communities

ABSTRACTFew characteristics are more important to a bacterium than the substrates it consumes. It is hard to identify what substrates are consumed by bacteria in natural communities, however, because most bacteria have not been cultured. In this study, we developed a method that uses fluorescent substrate analogs, cell sorting, and DNA sequencing to identify substrates taken up by bacteria. We deployed this method using 2[N-(7-nitrobenz-2-oxa-1,2-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG), a fluorescent glucose analog, and bacteria of the bovine rumen. This method revealed over 40 different bacteria (amplicon sequence variants [ASVs]) from the rumen that take up glucose. Nearly half of these ASVs represent previously uncultured bacteria. We attempted to grow these ASVs on agar media, and we confirmed that nearly two-thirds resisted culture. In coculture experiments, the fluorescent label of 2-NBDG was not transferred to nontarget bacteria by cross-feeding. Because it is not affected by cross-feeding, our method has an advantage over stable isotope probing. Though we focus on glucose, many substrates can be labeled with the fluorophore NBD. Our method represents a new paradigm for identifying substrates used by uncultured bacteria. It will help delineate the niche of bacteria in their environment.IMPORTANCEWe introduce a method for identifying what substrates are consumed by bacteria in natural communities. Our method offers significant improvement over existing methods for studying this characteristic. Our method uses a fluorescently labeled substrate which clearly labels target bacteria (glucose consumers in our case). Previous methods use isotope-labeled substrates, which are notorious for off-target labeling (due to cross-feeding of labeled metabolites). Our method can be deployed with a variety of substrates and microbial communities. It represents a major advance in connecting bacteria to the substrates they take up.

Complex environments alter competitive dynamics in fungi

2021 ◽  
Author(s):  
Justin Chan ◽  
Stephen Bonser ◽  
Michael M. Kasumovic ◽  
Jeff Powell ◽  
William Kirkham Cornwell
Keyword(s):  
Interspecific Interactions ◽  
Fungal Species ◽  
Wood Block ◽  
Complex Environments ◽  
Biotic Factor ◽  
Pairwise Interactions ◽  
Agar Media ◽  
Contextual Environment ◽  
Natural Settings ◽  
Natural Communities

Competition is a key biotic factor that often structures natural communities. Many attempts to disentangle how competition shapes natural communities have relied on experiments on simplified systems or through simple mathematical models. But these simplified approaches are limited in their ability to represent the complexity seen in more natural settings. Here, we considered the competitive pairwise dynamics between four saprotrophic fungal species. We tested whether the contextual environment changed these dynamics, repeating competitive experiments in a simple agar media and a more ecologically realistic wood block setting. We found that the competitive outcomes on agar media differed from those within the wood blocks. While superior competitors were identified across all pairwise interactions on agar, within the wood blocks, two of six interactions resulted in deadlock, where neither competitor could breach territory of the other, and one interaction resulted in a reversed competitive outcome. These results suggest that the complexity within natural substrates can alter the strength of interspecific interactions and may contribute to coexistence and the resulting high diversity of fungi often observed within wood.

scholarly journals Advancing Genome-Resolved Metagenomics beyond the Shotgun

mSystems ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Rex R. Malmstrom ◽  
Emiley A. Eloe-Fadrosh
Keyword(s):  
Single Molecule ◽  
Cell Sorting ◽  
Stable Isotope Probing ◽  
Natural Environments ◽  
Natural Ecosystems ◽  
Shotgun Metagenomics ◽  
Short Read ◽  
Future Developments ◽  
Long Read ◽  
Shed Light

ABSTRACT Exploration of environmental microbiomes has shed light on the ecological and evolutionary principles at play in natural ecosystems and has been further accelerated through the reconstruction of population genomes to provide genome-centric context. Yet technical challenges with traditional shotgun metagenomics remain for computationally intense short-read assembly, strain heterogeneity within communities, and depth of coverage required for low-abundance microbes. In this Perspective, we highlight three main avenues for promising future developments, including coupling stable isotope probing and genome-resolved metagenomics, applying fluorescence-activated cell sorting approaches to target mini-metagenomes within a larger community, and utilizing single-molecule long-read and synthetic long-read technology to link mobile elements to host microbial cells. These developments on the horizon will undoubtedly advance genome-resolved metagenomic approaches and enable a better understanding of uncultivated microbes in their natural environments.

scholarly journals Use of Combined Microautoradiography and Fluorescence In Situ Hybridization To Determine Carbon Metabolism in Mixed Natural Communities of Uncultured Bacteria from the GenusAchromatium

2000 ◽  
Vol 66 (10) ◽  
pp. 4518-4522 ◽  
Author(s):  
N. D. Gray ◽  
R. Howarth ◽  
R. W. Pickup ◽  
J. Gwyn Jones ◽  
I. M. Head
Keyword(s):  
In Situ Hybridization ◽  
Organic Carbon ◽  
Fluorescence In Situ Hybridization ◽  
Carbon Metabolism ◽  
Carbon Sources ◽  
Uncultured Bacteria ◽  
Sulfur Bacteria ◽  
Organic Carbon Sources ◽  
Natural Communities

ABSTRACT Combined microautoradiography and fluorescence in situ hybridization (FISH) was used to investigate carbon metabolism in uncultured bacteria from the genus Achromatium. All of theAchromatium species identified in a freshwater sediment from Rydal Water, Cumbria, United Kingdom, which were distinguishable only by FISH, assimilated both [14C]bicarbonate and [14C]acetate. This extends previous findings thatAchromatium spp. present at another location could only utilize organic carbon sources. Achromatium spp., therefore, probably exhibit a range of physiologies, i.e., facultative chemolithoautotrophy, mixotrophy, and chemoorganoheterotrophy, similar to other large sulfur bacteria (e.g., Beggiatoa spp.).

scholarly journals Grazing weakens competitive interactions between active methanotrophs and nitrifiers modulating greenhouse-gas emissions in grassland soils

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Hong Pan ◽  
Haojie Feng ◽  
Yaowei Liu ◽  
Chun-Yu Lai ◽  
Yuping Zhuge ◽  
...  
Keyword(s):  
Methane Oxidation ◽  
Ghg Emissions ◽  
Stable Isotope Probing ◽  
Competitive Interactions ◽  
Major Advance ◽  
Grassland Soils ◽  
Grassland Ecosystems ◽  
Underlying Mechanisms ◽  
Grazed Grassland

AbstractGrassland soils serve as a biological sink and source of the potent greenhouse gases (GHG) methane (CH4) and nitrous oxide (N2O). The underlying mechanisms responsible for those GHG emissions, specifically, the relationships between methane- and ammonia-oxidizing microorganisms in grazed grassland soils are still poorly understood. Here, we characterized the effects of grazing on in situ GHG emissions and elucidated the putative relations between the active microbes involving in methane oxidation and nitrification activity in grassland soils. Grazing significantly decreases CH4 emissions while it increases N2O emissions basing on 14-month in situ measurement. DNA-based stable isotope probing (SIP) incubation experiment shows that grazing decreases both methane oxidation and nitrification processes and decreases the diversity of active methanotrophs and nitrifiers, and subsequently weakens the putative competition between active methanotrophs and nitrifiers in grassland soils. These results constitute a major advance in our understanding of putative relationships between methane- and ammonia-oxidizing microorganisms and subsequent effects on nitrification and methane oxidation, which contribute to a better prediction and modeling of future balance of GHG emissions and active microbial communities in grazed grassland ecosystems.

scholarly journals Novel Fluorescence-Assisted Whole-Cell Assay for Engineering and Characterization of Proteases and Their Substrates

2010 ◽  
Vol 76 (22) ◽  
pp. 7500-7508 ◽  
Author(s):  
George Kostallas ◽  
Patrik Samuelson
Keyword(s):  
Cell Sorting ◽  
Fluorescent Substrate ◽  
Whole Cell ◽  
E Coli ◽  
Tev Protease ◽  
Fold Enrichment ◽  
Substrate Peptide ◽  
Subsequent Identification

ABSTRACT We have developed a sensitive and highly efficient whole-cell methodology for quantitative analysis and screening of protease activity in vivo. The method is based on the ability of a genetically encoded protease to rescue a coexpressed short-lived fluorescent substrate reporter from cytoplasmic degradation and thereby confer increased whole-cell fluorescence in proportion to the protease's apparent activity in the Escherichia coli cytoplasm. We demonstrated that this system can reveal differences in the efficiency with which tobacco etch virus (TEV) protease processes different substrate peptides. In addition, when analyzing E. coli cells expressing TEV protease variants that differed in terms of their in vivo solubility, cells containing the most-soluble protease variant exhibited the highest fluorescence intensity. Furthermore, flow cytometry screening allowed for enrichment and subsequent identification of an optimal substrate peptide and protease variant from a large excess of cells expressing suboptimal variants (1:100,000). Two rounds of cell sorting resulted in a 69,000-fold enrichment and a 22,000-fold enrichment of the superior substrate peptide and protease variant, respectively. Our approach presents a new promising path forward for high-throughput substrate profiling of proteases, engineering of novel protease variants with desired properties (e.g., altered substrate specificity and improved solubility and activity), and identification of protease inhibitors.

scholarly journals Rational design of a microbial consortium of mucosal sugar utilizers reduces Clostridiodes difficile colonization

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Fátima C. Pereira ◽  
Kenneth Wasmund ◽  
Iva Cobankovic ◽  
Nico Jehmlich ◽  
Craig W. Herbold ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Rational Design ◽  
Microbial Consortium ◽  
Stable Isotope Probing ◽  
Therapeutic Interventions ◽  
Clostridioides Difficile ◽  
Poor Understanding ◽  
Intestinal Pathogens ◽  
Pathogen Colonization

Abstract Many intestinal pathogens, including Clostridioides difficile, use mucus-derived sugars as crucial nutrients in the gut. Commensals that compete with pathogens for such nutrients are therefore ecological gatekeepers in healthy guts, and are attractive candidates for therapeutic interventions. Nevertheless, there is a poor understanding of which commensals use mucin-derived sugars in situ as well as their potential to impede pathogen colonization. Here, we identify mouse gut commensals that utilize mucus-derived monosaccharides within complex communities using single-cell stable isotope probing, Raman-activated cell sorting and mini-metagenomics. Sequencing of cell-sorted fractions reveals members of the underexplored family Muribaculaceae as major mucin monosaccharide foragers, followed by members of Lachnospiraceae, Rikenellaceae, and Bacteroidaceae families. Using this information, we assembled a five-member consortium of sialic acid and N-acetylglucosamine utilizers that impedes C. difficile’s access to these mucosal sugars and impairs pathogen colonization in antibiotic-treated mice. Our findings underscore the value of targeted approaches to identify organisms utilizing key nutrients and to rationally design effective probiotic mixtures.

144 Use of the CRISPR/CAS 9 system to produce porcine adipose-derived stem cells expressing enhanced green fluorescent protein

2021 ◽  
Vol 33 (2) ◽  
pp. 180
Author(s):  
Q. Xu ◽  
D. J. Milner ◽  
M. B. Wheeler
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Green Fluorescent Protein ◽  
Cell Sorting ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Fluorescent Label ◽  
Adipose Derived Stem Cells ◽  
Egfp Gene ◽  
Green Fluorescent

The goal of our project is to produce porcine adipose-derived stem cells (ASCs) stably expressing enhanced green fluorescent protein (eGFP) by using the clustered regularly interspaced short palindromic repeats (CRIPSR) technique. Fluorescent stem cells can facilitate the tracing and visualisation of stem cell migration, fusion, and participation in tissue regeneration after stem cell injection therapy, and represent a useful tool for tissue engineering research. The production of stem cells containing eGFP from ASCs using the CRISPR gene editing technique is able to reduce the time and labour requirement necessary for harvesting fluorescent cells from transgenic pigs. To generate fluorescent, edited cells, we utilised the ROSA 26 locus of pigs for insertion of the eGFP gene by homology-directed repair of Cas9-cleaved DNA at the ROSA 26 locus. The critical steps of producing stem cells expressing eGFP are (1) cloning of guide oligos into a Cas9 cutting vector and producing a repair template vector to insert GFP; (2) transfecting porcine stem cells with CRISPR plasmids; (3) cell sorting with flow cytometry to isolate colonies expressing GFP. A Rosa 26 Cas9-gRNA cutting vector was produced by cloning a guide RNA sequence into the vector backbone of plasmid pX458-GFP, and the donor vector was produced by the combination of the eGFP gene flanked with ROSA 26 genomic DNA inserted into plasmid pUC57. To isolate cells edited to contain the eGFP gene inserted into the ROSA-26 locus, we transfected 250,000 cells with a 1:1 mass mixture of Cas9-gRNA and eGFP repair plasmid using Lipofectamine STEM reagent (Invitrogen) in three trials. GFP+ cells were isolated by fluorescence-activated cell sorting, plated in 96-well plates, and monitored for colony growth and GFP expression. These trials produced an average of ∼70 colonies from sorting, and ∼1% GFP+ colonies. As pX458 drives expression of GFP as a marker for transfection, we hypothesised that we would potentially isolate more GFP+ edited colonies if we utilised a Cas9-gRNA cutting vector expressing mCherry and sorted for cells expressing both mCherry and GFP. This would allow enrichment of edited cells expressing GFP early after transfection, without interference of cells expressing GFP from the Cas9-gRNA vector alone. Utilising this method, we again obtained an average of ∼70 colonies from sorting, and 3% GFP+ colonies. Results were subjected to Student’s t-test. The comparisons were colonies/cell sorted and GFP+ colonies/cell sorted. All data were expressed as quadratic means+mean SE. When we compared groups, no differences were found for colonies/cell sorted: P=0.53 (1.11 E-03±9.16E-04 and 5.39 E-04±3.77 E-04, respectively, for green-green or red-green) and for GFP+ colonies/cell sorted: P=0.44 (1.94 E-05±2.15E-05 and 4.59 E-05±2.46 E-05, respectively, for green-green or red-green). In conclusion, our attempts to isolate ASC edited to express GFP have been successful, and our initial results suggest that utilising a dual fluorescent label sorting strategy does not enhance the number of GFP+ ASC colonies isolated. Future studies will verify that our GFP+ ASC retain normal stem cell properties.

Substrate Uptake by Uncultured Bacteria from the Genus Achromatium Determined by Microautoradiography

1999 ◽  
Vol 65 (11) ◽  
pp. 5100-5106 ◽  
Author(s):  
N. D. Gray ◽  
R. Howarth ◽  
R. W. Pickup ◽  
J. Gwyn Jones ◽  
I. M. Head
Keyword(s):  
Protein Hydrolysate ◽  
Sediment Cores ◽  
Sulfide Oxidation ◽  
Sodium Molybdate ◽  
Organic Substrate ◽  
Sulfur Cycle ◽  
Substrate Uptake ◽  
Physiological Diversity ◽  
Uncultured Bacteria ◽  
Natural Communities

ABSTRACT Microautoradiography was used to investigate substrate uptake by natural communities of uncultured bacteria from the genusAchromatium. Studies of the uptake of14C-labelled substrates demonstrated thatAchromatium cells from freshwater sediments were able to assimilate 14C from bicarbonate, acetate, and protein hydrolysate; however, 14C-labelled glucose was not assimilated. The pattern of substrate uptake by Achromatiumspp. was therefore similar to those of a number of other freshwater and marine sulfur-oxidizing bacteria. Different patterns of radiolabelled bicarbonate uptake were noted for Achromatium communities from different geographical locations and indicated that one community (Rydal Water) possessed autotrophic potential, while the other (Hell Kettles) did not. Furthermore, the patterns of organic substrate uptake within a single population suggested that physiological diversity existed in natural communities of Achromatium. These observations are consistent with and may relate to the phylogenetic diversity observed in Achromatium communities. Incubation of Achromatium-bearing sediment cores from Rydal Water with35S-labelled sulfate in the presence and absence of sodium molybdate demonstrated that this bacterial population was capable of oxidizing sulfide to intracellular elemental sulfur. This finding supported the role of Achromatium in the oxidative component of a tightly coupled sulfur cycle in Rydal Water sediment. The oxidation of sulfide to sulfur and ultimately to sulfate byAchromatium cells from Rydal Water sediment is consistent with an ability to conserve energy from sulfide oxidation.

Some Problems in Understanding the Solar Corona

1994 ◽  
Vol 144 ◽  
pp. 1-9
Author(s):  
A. H. Gabriel
Keyword(s):  
Solar Corona ◽  
Solar Atmosphere ◽  
Theoretical Modelling ◽  
Total System ◽  
Major Advance ◽  
X Ray ◽  
Proper Perspective ◽  
Space Observations

The development of the physics of the solar atmosphere during the last 50 years has been greatly influenced by the increasing capability of observations made from space. Access to images and spectra of the hotter plasma in the UV, XUV and X-ray regions provided a major advance over the few coronal forbidden lines seen in the visible and enabled the cooler chromospheric and photospheric plasma to be seen in its proper perspective, as part of a total system. In this way space observations have stimulated new and important advances, not only in space but also in ground-based observations and theoretical modelling, so that today we find a well-balanced harmony between the three techniques.

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