Near-real-time analysis of the phenotypic responses of Escherichia coli to 1-butanol exposure using Raman Spectroscopy

Abstract

Raman spectroscopy was used to study the time course of phenotypic responses of Escherichia coli (DH5α) to 1-butanol exposure (1.2% [vol/vol]). Raman spectroscopy is of interest for bacterial phenotyping because it can be performed (i) in near real time, (ii) with minimal sample preparation (label-free), and (iii) with minimal spectral interference from water. Traditional off-line analytical methodologies were applied to both 1-butanol-treated and control cells to draw correlations with Raman data. Here, distinct sets of Raman bands are presented that characterize phenotypic traits of E. coli with maximized correlation to off-line measurements. In addition, the observed time course phenotypic responses of E. coli to 1.2% (vol/vol) 1-butanol exposure included the following: (i) decreased saturated fatty acids levels, (ii) retention of unsaturated fatty acids and low levels of cyclopropane fatty acids, (iii) increased membrane fluidity following the initial response of increased rigidity, and (iv) no changes in total protein content or protein-derived amino acid composition. For most phenotypic traits, correlation coefficients between Raman spectroscopy and traditional off-line analytical approaches exceeded 0.75, and major trends were captured. The results suggest that near-real-time Raman spectroscopy is suitable for approximating metabolic and physiological phenotyping of bacterial cells subjected to toxic environmental conditions.

FIG 1

Raman spectra of the biological region (600 to 1,800 cm⁻¹) (a) and the CH region (2,800 to 3,100 cm⁻¹) (b) at time zero (before the application of 1-butanol; solid black line), time equal to 180 min for the control cells (gray line), and time equal to 180 min after treatment for the 1-butanol-treated cells (dashed black line).

FIG 2

Culture growth (OD₆₀₀) (a) and Raman (I₁₄₄₉) measure of broader metabolic activity (b) as functions of time for 1-butanol-treated cells (black circles) and control cells (open circles). 1-Butanol (1.2% [vol/vol]) was added to the treated cells at 0 min. Error bars represent 1 standard deviation for data from at least 3 biological replicates.

FIG 3

(a) Saturated fatty acids measured by GC-FID; (b) unsaturated fatty acids measured by GC-FID, (c) cyclopropane fatty acids measured by GC-FID, (d) saturated fatty acids measured by Raman spectroscopy (I₂₈₇₀), (e) unsaturated fatty acids measured by Raman spectroscopy (I₁₂₆₃), and (f) cyclopropane fatty acids measured by Raman spectroscopy (I₁₅₅₄) as functions of time for 1-butanol-treated cells (black circles) and control cells (open circles). 1-Butanol (1.2% [vol/vol]) was added to the treated cells at 0 min. Error bars represent 1 standard deviation for data from at least 3 biological replicates.

FIG 4

(a) Change in fluorescence anisotropy (as a percentage) for 1-butanol-treated cells relative to the control cells, (b) experimentally measured total protein content, (c) membrane fluidity (I₂₈₅₂/I₂₉₂₄) measured by Raman spectroscopy, and (d) total protein content measured by Raman spectroscopy (I₂₉₅₄) as functions of time for 1-butanol-treated cells (black circles) and control cells (open circles) (b and d); diamonds indicate the percent changes between 1-butanol-treated and control cells (a and c). 1-Butanol (1.2% [vol/vol]) was added to the treated cells at 0 min. Error bars represent 1 standard deviation for data from at least 3 biological replicates.