Demonstration of Carbon Catabolite Repression in Naphthalene Degrading Soil Bacteria via Raman Spectroscopy Based Stable Isotope Probing

Abstract

Carbon catabolite repression (CCR) is a regulatory phenomenon occurring in both lower organisms like bacteria and higher organisms like yeast, which allows them to preferentially utilize a specific carbon source to achieve highest metabolic activity and cell growth. CCR has been intensely studied in the model organisms Escherichia coli and Bacillus subtilis by following diauxic growth curves, assays to estimate the utilization or depletion of carbon sources, enzyme assays, Western blotting and mass spectrometric analysis to monitor and quantify the involvement of specific enzymes and proteins involved in CCR. In this study, we have visualized this process in three species of naphthalene degrading soil bacteria at a single cell level via Raman spectroscopy based stable isotope probing (Raman-SIP) using a single and double labeling approach. This is achieved using a combination of (2)H and (13)C isotope labeled carbon sources like glucose, galactose, fructose, and naphthalene. Time dependent metabolic flux of (13)C and (2)H isotopes has been followed via semi quantification and 2D Raman correlation analysis. For this, the relative intensities of Raman marker bands corresponding to (2)H and (13)C incorporation in newly synthesized macromolecules like proteins and lipids have been utilized. The 2D correlation analysis of time dependent Raman spectra readily identified small sequential changes resulting from isotope incorporation. Overall, we show that Raman-SIP has the potential to be used to obtain information about regulatory processes like CCR in bacteria at a single cell level within a time span of 3 h in fast growing bacteria. We also demonstrate the potential of this approach in identifying the most efficient naphthalene degraders asserting its importance for use in bioremediation.

Transparent soil microcosms for live-cell imaging and non-destructive stable isotope probing of soil microorganisms

“…Raman spectroscopy is a powerful tool to detect the uptake of 13 C-labeled compounds into cellular biomass by measuring the characteristic ‘red-shift’ of the phenylalanine peak from 1003 cm −1 to 966 cm −1 upon incorporation of 13 C into the cell ( Huang et al, 2004 ; Huang et al, 2009 ; Li et al, 2012 ; Li et al, 2013 ; Kumar B N et al, 2016 ). To test whether we could detect differences in the phenylalanine peak between 12 C- and 13 C-treated cells within TS microcosms, we grew B. subtilis cells in growth medium containing either 12 C- or 13 C-glucose and inoculated them into Nafion and cryolite TS microcosms.…”

“…In this study, we critically evaluate the capabilities of both Nafion and cryolite as TS substrates for advancing experimental research in soil microbial ecology by generating three-dimensional matrices containing pore spaces analogous to those bacteria inhabit in terrestrial soils ( Dal Ferro and Morari, 2015 ; Deng et al, 2015 ; Baveye et al, 2018 ). We show that TS microcosms made of both Nafion and cryolite are amenable to high-resolution, three-dimensional imaging by fluorescence and confocal microscopy, and additionally are compatible with Raman microspectroscopy – a powerful non-destructive method to obtain physiological information about cell states and microbial metabolic activity and nutrient uptake ( Huang et al, 2004 ; Huang et al, 2009 ; Li et al, 2012 ; Li et al, 2013 ; Berry et al, 2015 ; Kumar B N et al, 2016 ). Both TS substrates enable the measurement of deuterium uptake as a marker of microbial activity, while cryolite-based TS microcosms further enable the measurement of microbial uptake of isotopically labeled ( 13 C) carbon.…”

Label-free, simultaneous quantification of starch, protein and triacylglycerol in single microalgal cells

“…For example, as many valuable compounds carry characteristic Raman signal, this method should be able to rapidly provide a landscape-like view of biosynthetic capability of cells at single-cell resolution. Moreover, SCRS can be interpreted for a much greater range of phenotypes beyond metabolite profile: for example, SCRS has recently been used to characterize substrate metabolism [ 37 , 38 ], metabolic activity [ 39 ], stress response [ 18 , 40 ] and interspecies interactions [ 37 , 41 ]. Therefore to gauge and fulfill the potential of the SCRS approach, one research direction is to probe whether, and to what degree, the plethora of phenotypes can be simultaneously measured or modeled via SCRS.…”

Raman Stable Isotope Probing of Bacteria in Visible and Deep UV-Ranges

“…Understanding the dynamics of bacterial metabolism is the key to elucidating biological processes [1][2][3][4]. The identification of metabolically active bacterial cells is of high relevance in clinical diagnosis [5][6][7][8], industrial biotechnology, and microbial ecology [1,[9][10][11][12]. The standard method for identifying metabolically active cells in biomedical samples involves the use of a gas sensor to monitor the increase in CO 2 concentration in bacterial cultures [13,14].…”