scholarly journals Universal activity-based labeling method for ammonia- and alkane-oxidizing bacteria

2021 ◽  
Author(s):  
Dimitra Sakoula ◽  
Garrett J. Smith ◽  
Jeroen Frank ◽  
Rob J. Mesman ◽  
Linnea F. M. Kop ◽  
...  
Keyword(s):  
Cycloaddition Reaction ◽  
Fluorescent Labeling ◽  
Protein Profiling ◽  
Microbial Biodiversity ◽  
Complex Samples ◽  
Carbon Cycles ◽  
Phylogenetic Identification ◽  
In Situ Detection ◽  
Labeling Method

AbstractThe advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia-, methane-, and other short-chain alkane-oxidizing microorganisms, indicating that our understanding of the microbial biodiversity within the biogeochemical nitrogen and carbon cycles still is incomplete. The in situ detection and phylogenetic identification of novel ammonia- and alkane-oxidizing bacteria remain challenging due to their naturally low abundances and difficulties in obtaining new isolates from complex samples. Here, we describe an activity-based protein profiling protocol allowing cultivation-independent unveiling of ammonia- and alkane-oxidizing bacteria. In this protocol, 1,7-octadiyne is used as a bifunctional enzyme probe that, in combination with a highly specific alkyne-azide cycloaddition reaction, enables the fluorescent or biotin labeling of cells harboring active ammonia and alkane monooxygenases. Biotinylation of these enzymes in combination with immunogold labeling revealed the subcellular localization of the tagged proteins, which corroborated expected enzyme targets in model strains. In addition, fluorescent labeling of cells harboring active ammonia or alkane monooxygenases provided a direct link of these functional lifestyles to phylogenetic identification when combined with fluorescence in situ hybridization. Furthermore, we show that this activity-based labeling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and alkane-oxidizing bacteria from complex environmental samples, enabling the recovery of high-quality metagenome-assembled genomes. In conclusion, this study demonstrates a novel, functional tagging technique for the reliable detection, identification, and enrichment of ammonia- and alkane-oxidizing bacteria present in complex microbial communities.

scholarly journals An activity-based labelling method for the detection of ammonia and methane-oxidizing bacteria

2021 ◽  
Author(s):  
Dimitra Sakoula ◽  
Garrett J. Smith ◽  
Jeroen Frank ◽  
Rob J. Mesman ◽  
Linnea F.M. Kop ◽  
...  
Keyword(s):  
Cycloaddition Reaction ◽  
Immunogold Labelling ◽  
Protein Profiling ◽  
Microbial Biodiversity ◽  
Carbon Cycles ◽  
Methane Oxidizing Bacteria ◽  
Phylogenetic Identification ◽  
Biotin Labelling ◽  
Labelling Method

AbstractThe advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia- and methane-oxidizing microorganisms, indicating that our understanding of the microbial biodiversity within the biogeochemical nitrogen and carbon cycles still is incomplete. However, the in situ detection and phylogenetic identification of novel ammonia- and methane-oxidizing bacteria remains a challenge. Here, we describe an activity-based protein profiling protocol allowing cultivation-independent unveiling of ammonia- and methane-oxidizing bacteria. In this protocol, 1,7-octadiyne is used as a bifunctional enzyme probe that, in combination with a highly specific alkyne-azide cycloaddition reaction, enables the fluorescent or biotin labelling of cells harboring active ammonia and methane monooxygenases. The biotinylation of these enzymes in combination with immunogold labelling reveals the subcellular localization of the tagged proteins, while the fluorescent labelling of cells harboring active ammonia or methane monooxygenases provides a direct link of these functional lifestyles to phylogenetic identification when combined with fluorescence in situ hybridization. Furthermore, we show that this activity-based labelling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and methanotrophs from complex environmental samples, facilitating the retrieval of their high quality metagenome-assembled genomes. In conclusion, this study demonstrates a novel, functional tagging technique for the reliable detection, identification, and enrichment of ammonia- and methane-oxidizing bacteria present in complex microbial communities.

scholarly journals A single-cell view on the ecophysiology of anaerobic phototrophic bacteria

2008 ◽  
Vol 105 (46) ◽  
pp. 17861-17866 ◽  
Author(s):  
Niculina Musat ◽  
Hannah Halm ◽  
Bärbel Winterholler ◽  
Peter Hoppe ◽  
Sandro Peduzzi ◽  
...  
Keyword(s):  
Single Cells ◽  
Abundant Species ◽  
Quantitative Information ◽  
Cell Number ◽  
Carbon Cycles ◽  
Total Uptake ◽  
Phylogenetic Identification ◽  
Metabolic Activities ◽  
Secondary Ion

Quantitative information on the ecophysiology of individual microorganisms is generally limited because it is difficult to assign specific metabolic activities to identified single cells. Here, we develop and apply a method, Halogen In Situ Hybridization-Secondary Ion Mass Spectroscopy (HISH-SIMS), and show that it allows simultaneous phylogenetic identification and quantitation of metabolic activities of single microbial cells in the environment. Using HISH-SIMS, individual cells of the anaerobic, phototropic bacteria Chromatium okenii, Lamprocystis purpurea, and Chlorobium clathratiforme inhabiting the oligotrophic, meromictic Lake Cadagno were analyzed with respect to H13CO3− and 15NH4+ assimilation. Metabolic rates were found to vary greatly between individual cells of the same species, showing that microbial populations in the environment are heterogeneous, being comprised of physiologically distinct individuals. Furthermore, C. okenii, the least abundant species representing ≈0.3% of the total cell number, contributed more than 40% of the total uptake of ammonium and 70% of the total uptake of carbon in the system, thereby emphasizing that numerically inconspicuous microbes can play a significant role in the nitrogen and carbon cycles in the environment. By introducing this quantification method for the ecophysiological roles of individual cells, our study opens a variety of possibilities of research in environmental microbiology, especially by increasing the ability to examine the ecophysiological roles of individual cells, including those of less abundant and less active microbes, and by the capacity to track not only nitrogen and carbon but also phosphorus, sulfur, and other biological element flows within microbial communities.

Rapid quantification and in situ detection of nitrifying bacteria in biofilms by monoclonal antibody method

2000 ◽  
Vol 41 (4-5) ◽  
pp. 301-308 ◽  
Author(s):  
N. Noda ◽  
H. Ikuta ◽  
Y. Ebie ◽  
A. Hirata ◽  
S. Tsuneda ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Fluorescent Antibody ◽  
Nitrifying Bacteria ◽  
Fluorescent Antibody Technique ◽  
Antibody Technique ◽  
Antibody Method ◽  
In Situ Detection ◽  
Rapid Quantification

Fluorescent antibody technique by the monoclonal antibody method is very useful and helpful for the rapid quantification and in situ detection of the specific bacteria like nitrifiers in a mixed baxterial habitat such as a biofilm. In this study, twelve monoclonal antibodies against Nitrosomonas europaea (IFO14298) and sixteen against Nitrobacter winogradskyi (IFO14297) were raised from splenocytes of mice (BALB/c). It was found that these antibodies exhibited little cross reactivity against various kinds of heterotrophic bacteria. The direct cell count method using monoclonal antibodies could exactly detect and rapidly quantify N. europaea and N. winogradskyi. Moreover, the distribution of N. europaea and N. winogradskyi in a biofilm could be examined by in situ fluorescent antibody technique. It was shown that most of N. winogradskyi existed near the surface part and most of N. europaea existed at the inner part of the polyethylene glycol (PEG) gel pellet, which had entrapped activated sludge and used in a landfill leachate treatment reactor. It was suggested that this monoclonal antibody method was utilized for estimating and controlling the population of nitrifying bacteria as a quick and favorable tool.

scholarly journals Single Step In Situ Detection of Surface Protein and MicroRNA in Clustered Extracellular Vesicles Using Flow Cytometry

2021 ◽  
Vol 10 (2) ◽  
pp. 319
Author(s):  
Hee Cheol Yang ◽  
Won Jong Rhee
Keyword(s):  
Extracellular Vesicles ◽  
Liquid Biopsy ◽  
Molecular Beacon ◽  
Disease Diagnosis ◽  
Single Step ◽  
Flow Cytometer ◽  
Surface Proteins ◽  
Biomarker Detection ◽  
In Situ Detection

Because cancers are heterogeneous, it is evident that multiplexed detection is required to achieve disease diagnosis with high accuracy and specificity. Extracellular vesicles (EVs) have been a subject of great interest as sources of novel biomarkers for cancer liquid biopsy. However, EVs are nano-sized particles that are difficult to handle; thus, it is necessary to develop a method that enables efficient and straightforward EV biomarker detection. In the present study, we developed a method for single step in situ detection of EV surface proteins and inner miRNAs simultaneously using a flow cytometer. CD63 antibody and molecular beacon-21 were investigated for multiplexed biomarker detection in normal and cancer EVs. A phospholipid-polymer-phospholipid conjugate was introduced to induce clustering of the EVs analyzed using nanoparticle tracking analysis, which enhanced the detection signals. As a result, the method could detect and distinguish cancer cell-derived EVs using a flow cytometer. Thus, single step in situ detection of multiple EV biomarkers using a flow cytometer can be applied as a simple, labor- and time-saving, non-invasive liquid biopsy for the diagnosis of various diseases, including cancer.

scholarly journals Diversity of Biodeteriorative Bacterial and Fungal Consortia in Winter and Summer on Historical Sandstone of the Northern Pergola, Museum of King John III’s Palace at Wilanow, Poland

2021 ◽  
Vol 11 (2) ◽  
pp. 620
Author(s):  
Magdalena Dyda ◽  
Agnieszka Laudy ◽  
Przemyslaw Decewicz ◽  
Krzysztof Romaniuk ◽  
Martyna Ciezkowska ◽  
...  
Keyword(s):  
Seasonal Changes ◽  
Microbial Community Composition ◽  
Fungal Communities ◽  
Bacterial Strains ◽  
Microbial Biodiversity ◽  
Acidophilic Bacteria ◽  
Almost All ◽  
Generation Sequencing ◽  
King John

The aim of the presented investigation was to describe seasonal changes of microbial community composition in situ in different biocenoses on historical sandstone of the Northern Pergola in the Museum of King John III’s Palace at Wilanow (Poland). The microbial biodiversity was analyzed by the application of Illumina-based next-generation sequencing methods. The metabarcoding analysis allowed for detecting lichenized fungi taxa with the clear domination of two genera: Lecania and Rhinocladiella. It was also observed that, during winter, the richness of fungal communities increased in the biocenoses dominated by lichens and mosses. The metabarcoding analysis showed 34 bacterial genera, with a clear domination of Sphingomonas spp. across almost all biocenoses. Acidophilic bacteria from Acidobacteriaceae and Acetobacteraceae families were also identified, and the results showed that a significant number of bacterial strains isolated during the summer displayed the ability to acidification in contrast to strains isolated in winter, when a large number of isolates displayed alkalizing activity. Other bacteria capable of nitrogen fixation and hydrocarbon utilization (including aromatic hydrocarbons) as well as halophilic microorganisms were also found. The diversity of organisms in the biofilm ensures its stability throughout the year despite the differences recorded between winter and summer.

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