scholarly journals Single cell stable isotope probing in microbiology using Raman microspectroscopy

2016 ◽  
Vol 41 ◽  
pp. 34-42 ◽  
Author(s):  
Yun Wang ◽  
Wei E Huang ◽  
Li Cui ◽  
Michael Wagner
Keyword(s):  
Stable Isotope ◽  
Single Cell ◽  
Raman Microspectroscopy ◽  
Stable Isotope Probing

Using Stable Isotope Probing and Raman Microspectroscopy to measure growth rates of heterotrophic bacteria

2021 ◽  
Author(s):  
Felix Weber ◽  
Tatiana Zaliznyak ◽  
Virginia P. Edgcomb ◽  
Gordon T. Taylor
Keyword(s):  
Stable Isotope ◽  
Single Cell ◽  
Bacterial Growth ◽  
Growth Rates ◽  
Heterotrophic Bacteria ◽  
Raman Microspectroscopy ◽  
Stable Isotope Probing ◽  
Single Cell Level ◽  
Cell Level ◽  
E Coli

The suitability of stable isotope probing (SIP) and Raman microspectroscopy to measure growth rates of heterotrophic bacteria at the single-cell level was evaluated. Label assimilation into E. coli biomass during growth on a complex 13 C-labeled carbon source was monitored in time course experiments. 13 C-incorporation into various biomolecules was measured by spectral “red shifts” of Raman-scattered emissions. The 13 C- and 12 C-isotopologues of the amino acid phenylalanine (Phe) proved to be a quantitatively accurate reporter molecules of cellular isotopic fractional abundances ( f cell ). Values of f cell determined by Raman microspectroscopy and independently by isotope-ratio mass spectrometry (IRMS) over a range of isotopic enrichments were statistically indistinguishable. Progressive labeling of Phe in E. coli cells among a range of 13 C/ 12 C organic substrate admixtures occurred predictably through time. Relative isotopologue abundances of Phe determined by Raman spectral analysis enabled accurate calculation of bacterial growth rates as confirmed independently by optical density (OD) measurements. Results demonstrate that combining stable isotope probing (SIP) and Raman microspectroscopy can be a powerful tool for studying bacterial growth at the single-cell level when grown on defined or complex organic 13 C-carbon sources even in mixed microbial assemblages. Importance: Population growth dynamics and individual cell growth rates are the ultimate expressions of a microorganism’s fitness to its environmental conditions, whether natural or engineered. Natural habitats and many industrial settings harbor complex microbial assemblages. Their heterogeneity in growth responses to existing and changing conditions is often difficult to grasp by standard methodologies. In this proof of concept study, we tested whether Raman microspectroscopy can reliably quantify assimilation of isotopically-labeled nutrients into E. coli cells and enable determination of individual growth rates among heterotrophic bacteria. Raman-derived growth rate estimates were statistically indistinguishable from those derived by standard optical density measurements of the same cultures. Raman microspectroscopy also can be combined with methods for phylogenetic identification. We report development of Raman-based techniques that enable researchers to directly link genetic identity to functional traits and rate measurements of single cells within mixed microbial assemblages, currently a major technical challenge in microbiological research.

scholarly journals Host-compound foraging by intestinal microbiota revealed by single-cell stable isotope probing

2013 ◽  
Vol 110 (12) ◽  
pp. 4720-4725 ◽  
Author(s):  
D. Berry ◽  
B. Stecher ◽  
A. Schintlmeister ◽  
J. Reichert ◽  
S. Brugiroux ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Single Cell ◽  
Intestinal Microbiota ◽  
Stable Isotope Probing ◽  
Host Compound

scholarly journals Tracking heavy water (D2O) incorporation for identifying and sorting active microbial cells

2014 ◽  
Vol 112 (2) ◽  
pp. E194-E203 ◽  
Author(s):  
David Berry ◽  
Esther Mader ◽  
Tae Kwon Lee ◽  
Dagmar Woebken ◽  
Yun Wang ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Single Cell ◽  
Optical Tweezers ◽  
Heavy Water ◽  
Heterotrophic Bacteria ◽  
Multiple Displacement Amplification ◽  
Raman Microspectroscopy ◽  
Stable Isotope Probing ◽  
Response Patterns ◽  
Microbial Cells

Microbial communities are essential to the function of virtually all ecosystems and eukaryotes, including humans. However, it is still a major challenge to identify microbial cells active under natural conditions in complex systems. In this study, we developed a new method to identify and sort active microbes on the single-cell level in complex samples using stable isotope probing with heavy water (D2O) combined with Raman microspectroscopy. Incorporation of D2O-derived D into the biomass of autotrophic and heterotrophic bacteria and archaea could be unambiguously detected via C-D signature peaks in single-cell Raman spectra, and the obtained labeling pattern was confirmed by nanoscale-resolution secondary ion MS. In fast-growingEscherichia colicells, label detection was already possible after 20 min. For functional analyses of microbial communities, the detection of D incorporation from D2O in individual microbial cells via Raman microspectroscopy can be directly combined with FISH for the identification of active microbes. Applying this approach to mouse cecal microbiota revealed that the host-compound foragersAkkermansia muciniphilaandBacteroides acidifaciensexhibited distinctive response patterns to amendments of mucin and sugars. By Raman-based cell sorting of active (deuterated) cells with optical tweezers and subsequent multiple displacement amplification and DNA sequencing, novel cecal microbes stimulated by mucin and/or glucosamine were identified, demonstrating the potential of the nondestructive D2O-Raman approach for targeted sorting of microbial cells with defined functional properties for single-cell genomics.

scholarly journals Reverse and Multiple Stable Isotope Probing to Study Bacterial Metabolism and Interactions at the Single Cell Level

2016 ◽  
Vol 88 (19) ◽  
pp. 9443-9450 ◽  
Author(s):  
Yun Wang ◽  
Yizhi Song ◽  
Yifan Tao ◽  
Howbeer Muhamadali ◽  
Royston Goodacre ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Single Cell ◽  
Stable Isotope Probing ◽  
Bacterial Metabolism ◽  
Single Cell Level ◽  
Cell Level

scholarly journals Resolving Genetic Functions within Microbial Populations: In Situ Analyses Using rRNA and mRNA Stable Isotope Probing Coupled with Single-Cell Raman-Fluorescence In Situ Hybridization

2008 ◽  
Vol 75 (1) ◽  
pp. 234-241 ◽  
Author(s):  
Wei E. Huang ◽  
Andrew Ferguson ◽  
Andrew C. Singer ◽  
Kathryn Lawson ◽  
Ian P. Thompson ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Stable Isotope ◽  
Fluorescence In Situ Hybridization ◽  
Single Cell ◽  
Natural World ◽  
Stable Isotope Probing ◽  
Culture Independent ◽  
And Function ◽  
Naphthalene Biodegradation

ABSTRACT Prokaryotes represent one-half of the living biomass on Earth, with the vast majority remaining elusive to culture and study within the laboratory. As a result, we lack a basic understanding of the functions that many species perform in the natural world. To address this issue, we developed complementary population and single-cell stable isotope (13C)-linked analyses to determine microbial identity and function in situ. We demonstrated that the use of rRNA/mRNA stable isotope probing (SIP) recovered the key phylogenetic and functional RNAs. This was followed by single-cell physiological analyses of these populations to determine and quantify in situ functions within an aerobic naphthalene-degrading groundwater microbial community. Using these culture-independent approaches, we identified three prokaryote species capable of naphthalene biodegradation within the groundwater system: two taxa were isolated in the laboratory (Pseudomonas fluorescens and Pseudomonas putida), whereas the third eluded culture (an Acidovorax sp.). Using parallel population and single-cell stable isotope technologies, we were able to identify an unculturable Acidovorax sp. which played the key role in naphthalene biodegradation in situ, rather than the culturable naphthalene-biodegrading Pseudomonas sp. isolated from the same groundwater. The Pseudomonas isolates actively degraded naphthalene only at naphthalene concentrations higher than 30 μM. This study demonstrated that unculturable microorganisms could play important roles in biodegradation in the ecosystem. It also showed that the combined RNA SIP-Raman-fluorescence in situ hybridization approach may be a significant tool in resolving ecology, functionality, and niche specialization within the unculturable fraction of organisms residing in the natural environment.

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