Characterizing metabolic stress-induced phenotypes ofSynechocystisPCC6803 with Raman spectroscopy

Abstract

Background During their long evolution, Synechocystis sp. PCC6803 developed a remarkable capacity to acclimate to diverse environmental conditions. In this study, Raman spectroscopy and Raman chemometrics tools (RametrixTM) were employed to investigate the phenotypic changes in response to external stressors and correlate specific Raman bands with their corresponding biomolecules determined with widely used analytical methods. Methods Synechocystis cells were grown in the presence of (i) acetate (7.5–30 mM), (ii) NaCl (50–150 mM) and (iii) limiting levels of MgSO4 (0–62.5 mM) in BG-11 media. Principal component analysis (PCA) and discriminant analysis of PCs (DAPC) were performed with the RametrixTM LITE Toolbox for MATLABⓇ. Next, validation of these models was realized via RametrixTM PRO Toolbox where prediction of accuracy, sensitivity, and specificity for an unknown Raman spectrum was calculated. These analyses were coupled with statistical tests (ANOVA and pairwise comparison) to determine statistically significant changes in the phenotypic responses. Finally, amino acid and fatty acid levels were measured with well-established analytical methods. The obtained data were correlated with previously established Raman bands assigned to these biomolecules. Results Distinguishable clusters representative of phenotypic responses were observed based on the external stimuli (i.e., acetate, NaCl, MgSO4, and controls grown on BG-11 medium) or its concentration when analyzing separately. For all these cases, RametrixTM PRO was able to predict efficiently the corresponding concentration in the culture media for an unknown Raman spectra with accuracy, sensitivity and specificity exceeding random chance. Finally, correlations (R > 0.7) were observed for all amino acids and fatty acids between well-established analytical methods and Raman bands.

Raman spectroscopy in cell biology and microbiology

“…Raman spectroscopy has been used in near real‐time phenotyping as an alternative to genomics. [ 17 ] Raman studies have been published for bacteria exposed to alcohol [ 18 ] or antibiotics. [ 19 ] Single‐cell phenotyping studies of lipids [ 20 ] and characterization of phenotypic differences between species with and without tolerance [ 21 ] have clearly shown the effectiveness of this method in probing phenotypic changes in near real time under exposure to environmental stimuli.…”

Isolation of bacteria from artificial bronchoalveolar lavage fluid using density gradient centrifugation and their accessibility by Raman spectroscopy

“…Culture conditions such as the concentration of CO 2 during growth can also influence the metabolism and consequently the spectra [11]. Therefore, it is possible to observe the change from aerobic to microaerophilic metabolism in Escherichia coli by Raman spectra [12], and low salt concentrations in the medium are depicted by changes in amino acids and fatty acids [13].…”

Raman spectroscopy-based measurements of single-cell phenotypic diversity in microbial communities

“…Once the spectra have been pre-processed, they can be used to investigate the phenotypic 380 heterogeneity among or within cells. Although Raman spectroscopy has been previously used to 381 detect stress-driven phenotypes (Tanniche et al, 2020), we argue that there is a need for single-cell 382 quantitative measurements for phenotypic diversity and propose the use of Hill numbers. We chose 383…”

“…This technique has 69 been used to study stress-induced phenotypic differences of the cyanobacterium Synechocystis sp. 70 (Tanniche et al, 2020): the fingerprints of cells treated with different concentrations of acetate or 71…”