Using Stable Isotope Probing and Raman Microspectroscopy To Measure Growth Rates of Heterotrophic Bacteria

Abstract

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.

One Cell at a Time: Advances in Single-Cell Methods and Instrumentation for Discovery in Aquatic Microbiology

“…For example, Li et al (2012) demonstrated that resonance Raman spectra of carotenoids can quantify dissolved inorganic carbon assimilation by individual cyanobacteria and coastal phytoplankton cells incubated with 13 C-bicarbonate. Building on this advance, Taylor et al (2017) and Weber et al (2021) demonstrated that SIP-Raman accurately measures growth rates in individual cells of photoautotrophs and heterotrophs, respectively. Growth rates have also been reported in nanoSIMS studies based on enrichment ratios ( Foster et al, 2011 , 2021 ).…”

“…Unique to CRM, a researcher can identify specific microbial taxa within a microscopic field using FISH and the instrument’s epifluorescence optics and then immediately switch optical paths to obtain Raman spectra from each target in a matter of seconds to minutes (Raman-FISH) ( Huang et al, 2007 ). The automated microscope stage can visit each target using stored x-y coordinates or create fixed-point gridded maps ( Taylor, 2019 ; Weber et al, 2021 ), similar to grid systems in MSI. Sampling by Raman spectroscopy is usually non-destructive, so samples can be subjected to other downstream analyses.…”

“…The ability to extract detailed chemical information from individual live or fully hydrated microbial cells under standard laboratory conditions is one of the most outstanding strengths of CRM. This attribute enables mapping distributions of molecular pools within undistorted and unaltered cells in two and three dimensions as well as to track movement of stable isotope tracers through time (e.g., Wakisaka et al, 2016 ; Taylor, 2019 ; Weber et al, 2021 ). Raman scattering, however, is an inherently weak phenomenon (1 in 10 6 to 10 7 photons scattered) meaning that many analytes will be below the method’s detection limits.…”

Anaerobic ciliates as a model group for studying symbioses in oxygen‐depleted environments

“…New diagnostic techniques have recovered complex microbiomes in microbial eukaryotes (Lanzoni et al, 2019; Rossi et al, 2019). Determining the fraction of these that are true symbionts vs. transient associations or ingested prey will require advanced microscopy methods such as Raman spectroscopy (Weber et al, 2021) to track the exchanges of metabolic products between partners.…”

Monitoring and modelling the dynamics of the cellular glycolysis pathway: A review and future perspectives

“…Weber et al. explored the utility of Raman microspectroscopy coupled with stable isotope probing for monitoring the growth rates of heterologous bacteria at single-cell resolution, based on the 13 C- and 12 C-isotopologues of the amino acid phenylalanine [ 267 ], identifiable by the strong phenylalanine ring breathing modes 966 and 1,002 cm −1 ). Using a robust curve fitting process for precise deconvolution of partially overlapping peaks, the identified peaks provided an accurate reporter probe for quantification of cellular isotope fractional abundances (f cell ) for complex 13 C-labelled carbon source assimilation in E. coli in time-course experiments.…”