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.

Optics-Free Visualization of Proteins in Single Cells by Time of Flight-Secondary Ion Mass Spectrometry Coupled with Genetically Encoded Chemical Tags

2020 ◽  
Author(s):  
Feifei Jia ◽  
Jie Wang ◽  
Yanyan Zhang ◽  
Qun Luo ◽  
Luyu Qi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Secondary Ion Mass Spectrometry ◽  
Single Cells ◽  
Time Of Flight ◽  
E Coli ◽  
Tof Sims ◽  
Ion Mass Spectrometry ◽  
Chemical Tags ◽  
Secondary Ion

<p></p><p><i>In situ</i> visualization of proteins of interest at single cell level is attractive in cell biology, molecular biology and biomedicine, which usually involves photon, electron or X-ray based imaging methods. Herein, we report an optics-free strategy that images a specific protein in single cells by time of flight-secondary ion mass spectrometry (ToF-SIMS) following genetic incorporation of fluorine-containing unnatural amino acids as a chemical tag into the protein via genetic code expansion technique. The method was developed and validated by imaging GFP in E. coli and human HeLa cancer cells, and then utilized to visualize the distribution of chemotaxis protein CheA in E. coli cells and the interaction between high mobility group box 1 protein and cisplatin damaged DNA in HeLa cells. The present work highlights the power of ToF-SIMS imaging combined with genetically encoded chemical tags for <i>in situ </i>visualization of proteins of interest as well as the interactions between proteins and drugs or drug damaged DNA in single cells.</p><p></p>

scholarly journals In Situ Visualizing the Recognition Between Proteins and Platinum-Damaged DNA in Single-Cells by Correlated Optical and Secondary Ion Mass Spectrometric Imaging

2020 ◽  
Author(s):  
Yu Lin ◽  
Kui Wu ◽  
Feifei Jia ◽  
Ling Chen ◽  
Zhaoying Wang ◽  
...  
Keyword(s):  
Single Cells ◽  
Mass Spectrometric ◽  
Dependent Manner ◽  
Mass Spectrometric Imaging ◽  
Novel Approach ◽  
Molecular Mechanism Of Action ◽  
Secondary Ion ◽  
Damaged Dna

<p><b><i>In situ</i> visualization of the recognition and interaction between proteins and drug damaged DNA at single cell level is highly important for understanding the molecular mechanism of action of DNA targeting drugs, yet a great challenge. We herein report a novel approach, termed as correlated optical and secondary ion mass spectrometric imaging (COSIMSi), for exploring the recognition between proteins and cisplatin-damaged DNA in single cells. Genetically encoded EYFP-fused HMGB1, an <i>in vitro</i> well-known specific binder of cisplatin-damaged DNA, and dye-stained DNA, and cisplatin were mapped by LSCM and ToF-SIMS imaging, respectively. The LSCM and SIMS images were aligned with aiding of an addressable silicon wafer to generate fused images, in which the co-localization of the fluorescence and MS signals indicated the formation of HMGB1-Pt-DNA ternary complexes in a dose- and time-dependent manner. In contrast, COSIMSi showed that little HMGB1(F37A)-Pt-DNA complex was produced under the same conditions. Moreover, we demonstrated for the first time that cisplatin lesion on DNA prevented a DNA-binding protein Smad3 from interacting with DNA. These results verify that the COSIMSi is an effective and straightforward tool for <i>in situ</i> visualization of recognition and interaction between proteins and specific damaged DNA in single cells. </b><br></p>

In Situ Visualizing the Recognition Between Proteins and Platinum-Damaged DNA in Single-Cells by Correlated Optical and Secondary Ion Mass Spectrometric Imaging

2020 ◽  
Author(s):  
Yu Lin ◽  
Kui Wu ◽  
Feifei Jia ◽  
Ling Chen ◽  
Zhaoying Wang ◽  
...  
Keyword(s):  
Single Cells ◽  
Mass Spectrometric ◽  
Dependent Manner ◽  
Mass Spectrometric Imaging ◽  
Novel Approach ◽  
Molecular Mechanism Of Action ◽  
Secondary Ion ◽  
Damaged Dna

<p><b><i>In situ</i></b><b> visualization of the recognition and interactions between proteins and drug damaged DNA at single cell level is highly important for understanding the molecular mechanism of action of DNA targeting drugs, yet a great challenge. We report herein a novel approach, termed as correlated optical and secondary ion mass spectrometric imaging (COSIMSi), to explore the recognition between proteins and cisplatin-damaged DNA in single cells. Genetically encoded EYFP-fused HMGB1, an <i>in vitro</i> well-known specific binder of cisplatin-damaged DNA, dye-stained DNA, and platinum were mapped by LSCM and ToF-SIMS imaging, respectively. The LSCM and SIMS images were aligned with aiding of an addressable silicon wafer to generate fused images, in which the co-localization of the fluorescent and MS signals indicated the formation of HMGB1-Pt-DNA ternary complexes in a dose- and time-dependent manner. In contrast, COSIMSi results showed that little HMGB1<a></a><a>(F37A</a>)-Pt-DNA complex was produced under the same conditions. Moreover, we demonstrated for the first time that cisplatin lesions on DNA prevented DNA-binding proteins Smad3 and Smad7 from interacting with DNA. These results verify that the COSIMSi is an effective and straightforward tool for <i>in situ</i> visualization of recognition and interactions between proteins and specific damaged DNA in single cells. </b></p>

scholarly journals In Situ Visualizing the Recognition Between Proteins and Platinum-Damaged DNA in Single-Cells by Correlated Optical and Secondary Ion Mass Spectrometric Imaging

2020 ◽  
Author(s):  
Yu Lin ◽  
Kui Wu ◽  
Feifei Jia ◽  
Ling Chen ◽  
Zhaoying Wang ◽  
...  
Keyword(s):  
Single Cells ◽  
Mass Spectrometric ◽  
Dependent Manner ◽  
Mass Spectrometric Imaging ◽  
Novel Approach ◽  
Molecular Mechanism Of Action ◽  
Secondary Ion ◽  
Damaged Dna

<p><b><i>In situ</i> visualization of the recognition and interaction between proteins and drug damaged DNA at single cell level is highly important for understanding the molecular mechanism of action of DNA targeting drugs, yet a great challenge. We herein report a novel approach, termed as correlated optical and secondary ion mass spectrometric imaging (COSIMSi), for exploring the recognition between proteins and cisplatin-damaged DNA in single cells. Genetically encoded EYFP-fused HMGB1, an <i>in vitro</i> well-known specific binder of cisplatin-damaged DNA, and dye-stained DNA, and cisplatin were mapped by LSCM and ToF-SIMS imaging, respectively. The LSCM and SIMS images were aligned with aiding of an addressable silicon wafer to generate fused images, in which the co-localization of the fluorescence and MS signals indicated the formation of HMGB1-Pt-DNA ternary complexes in a dose- and time-dependent manner. In contrast, COSIMSi showed that little HMGB1(F37A)-Pt-DNA complex was produced under the same conditions. Moreover, we demonstrated for the first time that cisplatin lesion on DNA prevented a DNA-binding protein Smad3 from interacting with DNA. These results verify that the COSIMSi is an effective and straightforward tool for <i>in situ</i> visualization of recognition and interaction between proteins and specific damaged DNA in single cells. </b><br></p>

Optics-Free Visualization of Proteins in Single Cells by Time of Flight-Secondary Ion Mass Spectrometry Coupled with Genetically Encoded Chemical Tags

2020 ◽  
Author(s):  
Feifei Jia ◽  
Yu Lin ◽  
Jie Wang ◽  
Yanyan Zhang ◽  
Qun Luo ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Secondary Ion Mass Spectrometry ◽  
Single Cells ◽  
Time Of Flight ◽  
E Coli ◽  
Tof Sims ◽  
Ion Mass Spectrometry ◽  
Chemical Tags ◽  
Secondary Ion

<p><i>In situ</i> visualization of proteins of interest at single cell level is attractive in cell biology, molecular biology and biomedicine, which usually involves <a></a><a>photon, electron or X-ray</a> based imaging methods. Herein, we report an optics-free strategy that images a specific protein in single cells by time of flight-secondary ion mass spectrometry (ToF-SIMS) following genetic incorporation of fluorine-containing unnatural amino acids as a <a>chemical</a> tag into the protein <i>via</i> genetic code expansion technique. The method was developed and validated by imaging GFP in <i>E. coli</i> and human HeLa cancer cells, and then utilized to visualize the distribution of chemotaxis protein CheA in <i>E. Coli</i> cells and the interaction between high mobility group box 1 protein and cisplatin damaged DNA in HeLa cells. The present work highlights the power of ToF-SIMS imaging combined with genetically encoded chemical tags for <i>in situ</i> visualization of proteins of interest as well as the interactions between proteins and drugs or drug damaged DNA in single cells.<br></p>

scholarly journals Optics-Free Visualization of Proteins in Single Cells by Time of Flight-Secondary Ion Mass Spectrometry Coupled with Genetically Encoded Chemical Tags

2020 ◽  
Author(s):  
Feifei Jia ◽  
Jie Wang ◽  
Yanyan Zhang ◽  
Qun Luo ◽  
Luyu Qi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Secondary Ion Mass Spectrometry ◽  
Single Cells ◽  
Time Of Flight ◽  
E Coli ◽  
Tof Sims ◽  
Ion Mass Spectrometry ◽  
Chemical Tags ◽  
Secondary Ion

<p></p><p><i>In situ</i> visualization of proteins of interest at single cell level is attractive in cell biology, molecular biology and biomedicine, which usually involves photon, electron or X-ray based imaging methods. Herein, we report an optics-free strategy that images a specific protein in single cells by time of flight-secondary ion mass spectrometry (ToF-SIMS) following genetic incorporation of fluorine-containing unnatural amino acids as a chemical tag into the protein via genetic code expansion technique. The method was developed and validated by imaging GFP in E. coli and human HeLa cancer cells, and then utilized to visualize the distribution of chemotaxis protein CheA in E. coli cells and the interaction between high mobility group box 1 protein and cisplatin damaged DNA in HeLa cells. The present work highlights the power of ToF-SIMS imaging combined with genetically encoded chemical tags for <i>in situ </i>visualization of proteins of interest as well as the interactions between proteins and drugs or drug damaged DNA in single cells.</p><p></p>

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 In Situ Visualizing the Recognition Between Proteins and Platinum-Damaged DNA in Single-Cells by Correlated Optical and Secondary Ion Mass Spectrometric Imaging

2020 ◽  
Author(s):  
Yu Lin ◽  
Kui Wu ◽  
Feifei Jia ◽  
Ling Chen ◽  
Zhaoying Wang ◽  
...  
Keyword(s):  
Single Cells ◽  
Mass Spectrometric ◽  
Dependent Manner ◽  
Mass Spectrometric Imaging ◽  
Novel Approach ◽  
Molecular Mechanism Of Action ◽  
Secondary Ion ◽  
Damaged Dna

<p><b><i>In situ</i></b><b> visualization of the recognition and interactions between proteins and drug damaged DNA at single cell level is highly important for understanding the molecular mechanism of action of DNA targeting drugs, yet a great challenge. We report herein a novel approach, termed as correlated optical and secondary ion mass spectrometric imaging (COSIMSi), to explore the recognition between proteins and cisplatin-damaged DNA in single cells. Genetically encoded EYFP-fused HMGB1, an <i>in vitro</i> well-known specific binder of cisplatin-damaged DNA, dye-stained DNA, and platinum were mapped by LSCM and ToF-SIMS imaging, respectively. The LSCM and SIMS images were aligned with aiding of an addressable silicon wafer to generate fused images, in which the co-localization of the fluorescent and MS signals indicated the formation of HMGB1-Pt-DNA ternary complexes in a dose- and time-dependent manner. In contrast, COSIMSi results showed that little HMGB1<a></a><a>(F37A</a>)-Pt-DNA complex was produced under the same conditions. Moreover, we demonstrated for the first time that cisplatin lesions on DNA prevented DNA-binding proteins Smad3 and Smad7 from interacting with DNA. These results verify that the COSIMSi is an effective and straightforward tool for <i>in situ</i> visualization of recognition and interactions between proteins and specific damaged DNA in single cells. </b></p>

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.

Sign in / Sign up

Export Citation Format

Share Document