Respiration Response Imaging for Real-Time Detection of Microbial Function at the Single-Cell Level

Konopka, Michael; Strovas, Tim; Ojala, David S.; Chistoserdova, Ludmila; Lidstrom, Mary E.; Kalyuzhnaya, Marina G.

doi:10.1128/aem.01166-10

Cited by 24 publications

(23 citation statements)

References 19 publications

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“…On this background, the Bac Light RedoxSensor Green vitality stain, 3,8-diamino-5-[3-(diethylmethylammonio)propyl]-6-phenyl di-iodide (RSG reagent), was used to diagnose the metabolic state of individual cells. This reagent can be transformed by cell reductases into a highly fluorescent compound, a reliable descriptor of changes in the electron transport chain activity and other vital cellular processes ( 39 , 40 ). As such, the metabolic heftiness of individual cells could be directly related to the intensity of the RSG signal ( 17 ).…”

Section: Resultsmentioning

confidence: 99%

The Glycerol-Dependent Metabolic Persistence of Pseudomonas putida KT2440 Reflects the Regulatory Logic of the GlpR Repressor

Nikel

Romero–Campero

Zeidman

et al. 2015

mBio

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The growth of the soil bacterium Pseudomonas putida KT2440 on glycerol as the sole carbon source is characterized by a prolonged lag phase, not observed with other carbon substrates. We examined the bacterial growth in glycerol cultures while monitoring the metabolic activity of individual cells. Fluorescence microscopy and flow cytometry, as well as the analysis of the temporal start of growth in single-cell cultures, revealed that adoption of a glycerol-metabolizing regime was not the result of a gradual change in the whole population but rather reflected a time-dependent bimodal switch between metabolically inactive (i.e., nongrowing) and fully active (i.e., growing) bacteria. A transcriptional Φ(glpD-gfp) fusion (a proxy of the glycerol-3-phosphate [G3P] dehydrogenase activity) linked the macroscopic phenotype to the expression of the glp genes. Either deleting glpR (encoding the G3P-responsive transcriptional repressor that controls the expression of the glpFKRD gene cluster) or altering G3P formation (by overexpressing glpK, encoding glycerol kinase) abolished the bimodal glpD expression. These manipulations eliminated the stochastic growth start by shortening the otherwise long lag phase. Provision of glpR in trans restored the phenotypes lost in the ΔglpR mutant. The prolonged nongrowth regime of P. putida on glycerol could thus be traced to the regulatory device controlling the transcription of the glp genes. Since the physiological agonist of GlpR is G3P, the arrangement of metabolic and regulatory components at this checkpoint merges a positive feedback loop with a nonlinear transcriptional response, a layout fostering the observed time-dependent shift between two alternative physiological states.

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Section: Resultsmentioning

confidence: 99%

The Glycerol-Dependent Metabolic Persistence of Pseudomonas putida KT2440 Reflects the Regulatory Logic of the GlpR Repressor

Nikel

Romero–Campero

Zeidman

et al. 2015

mBio

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“…While not directly tested in this study, there are a number of promising future applications of BONCAT. For example, combining this technique with fluorescenceactivated cell sorting would enable physical separation of translationally active cells, an approach analogous to the respiration response imaging method using redox sensor green (Kalyuzhnaya et al, 2008;Konopka et al, 2011). Quantification of the relationship between spatial organization and anabolic activity within structured microbial communities such as microbial mats, biofilms or consortia is also possible.…”

Section: Outlook: Combining Boncat With Established Techniquesmentioning

confidence: 99%

“…In addition to visualizing cellular protein and RNA expression, a complementary approach for identifying active cells is based on the incorporation of the indicator dye RedoxSensor green (RSG; Invitrogen), a nontoxic fluorescent indicator of bacterial reductase activity. While the full utility of RSG for microbial ecosystems has not yet been investigated, recent publications have successfully used this assay to detect respiratory activity in cultured aerobic microbial cells and environmental samples in near real time (Kalyuzhnaya et al, 2008;Konopka et al, 2011;Orman and Brynildsen, 2013).…”

Section: Introductionmentioning

confidence: 99%

In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry

Hatzenpichler

Scheller

Tavormina

et al. 2014

Environmental Microbiology

203

233

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Here we describe the application of a new click chemistry method for fluorescent tracking of protein synthesis in individual microorganisms within environmental samples. This technique, termed bioorthogonal non-canonical amino acid tagging (BONCAT), is based on the in vivo incorporation of the non-canonical amino acid L-azidohomoalanine (AHA), a surrogate for l-methionine, followed by fluorescent labelling of AHA-containing cellular proteins by azide-alkyne click chemistry. BONCAT was evaluated with a range of phylogenetically and physiologically diverse archaeal and bacterial pure cultures and enrichments, and used to visualize translationally active cells within complex environmental samples including an oral biofilm, freshwater and anoxic sediment. We also developed combined assays that couple BONCAT with ribosomal RNA (rRNA)-targeted fluorescence in situ hybridization (FISH), enabling a direct link between taxonomic identity and translational activity. Using a methanotrophic enrichment culture incubated under different conditions, we demonstrate the potential of BONCAT-FISH to study microbial physiology in situ. A direct comparison of anabolic activity using BONCAT and stable isotope labelling by nano-scale secondary ion mass spectrometry (15NH3 assimilation) for individual cells within a sediment-sourced enrichment culture showed concordance between AHA-positive cells and 15N enrichment. BONCAT-FISH offers a fast, inexpensive and straightforward fluorescence microscopy method for studying the in situ activity of environmental microbes on a single-cell level.

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“…RSG has been used as a vitality indicator so far, but we find out that its use would be extended to the detection of metabolic activities at a single‐cell level. Indeed, RSG is reduced by the intracellular reductases involved in the aerobic metabolism , leading to the release of a green fluorescent compound that can be easily detected by flow cytometry. RSG could thus be considered as a key indicator of the metabolic state of the microbial cells in different bioprocessing conditions.…”

Section: Introductionmentioning

confidence: 99%

Microbial population heterogeneity versus bioreactor heterogeneity: Evaluation of Redox Sensor Green as an exogenous metabolic biosensor

Baert

Delepierre

Telek

et al. 2016

Engineering in Life Sciences

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Practical applicationIt is known that isogenic microbial population is able to display phenotypic diversification upon environmental changes. In this study, we developed a high throughput methodology based on the use of flow cytometry and the preliminary staining of the cells with Redox Sensor Green (RSG). We demonstrated that RSG is related to the activity of the electron transport chain and www.els-journal.com Page 2 Engineering in Life SciencesThis article is protected by copyright. All rights reserved.2 can be used as an efficient tracker of metabolic activity at the single cell level in different bioreactor configurations. We showed that RSG can be efficiently used to detect carbon limitation and dissolved oxygen limitation in bioreactor. More specifically, our methodology was used in order to assess a potential relationship between bioreactor heterogeneity and microbial population heterogeneity. Unexpectedly, no such correlation was found based on scale-down analysis. AbstractMicrobial heterogeneity in metabolic performances has attracted a lot of attention, considering its potential impact on industrial bioprocesses. However, little is known about the impact of extracellular perturbations (i.e. bioreactor heterogeneity) on cell-to-cell variability in metabolic performances (i.e. microbial population heterogeneity). In this work, we have evaluated the relevance of Redox Sensor Green (RSG) as an exogenous biosensor of metabolic activity at the single cell level. RSG signal is proportional to the activity of the electron transport chain and its signal is strongly affected by metabolic burden, availability of electron final acceptor and side metabolisms (i.e. overflow and mixed acid fermentation). RSG can also be used for the estimation of the impact of scale-down conditions on microbial metabolic robustness. The relationship linking averaged RSG activity and its cell-to-cell variability (noise) has been highlighted but seems unaffected by environmental perturbations.

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Respiration Response Imaging for Real-Time Detection of Microbial Function at the Single-Cell Level

Cited by 24 publications

References 19 publications

The Glycerol-Dependent Metabolic Persistence of Pseudomonas putida KT2440 Reflects the Regulatory Logic of the GlpR Repressor

The Glycerol-Dependent Metabolic Persistence of Pseudomonas putida KT2440 Reflects the Regulatory Logic of the GlpR Repressor

In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry

Microbial population heterogeneity versus bioreactor heterogeneity: Evaluation of Redox Sensor Green as an exogenous metabolic biosensor

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