Impact of low-intensity 463 nm blue light on proliferation and adaptive mutation of Escherichia coli DH5α cells

Abstract

The growth rate of many microorganisms decreases when exposed to blue light (BL) at around 450 nm. This growth retardation is likely caused by the photosensitivity of endogenous photosensitizers, flavins, which absorb BL and generate reactive oxygen species (ROS) that cause inactivation and mutations of microorganisms. In this study, the effect of weak (0-0.12 mW/cm²) 463 nm BL on E. coli (DH5α) cell proliferation was investigated. Surprisingly, BL of only 0.03 mW/cm² reduced the DH5α colony number by 52%, and about 5% of cells (BL-resistant cells) formed colonies under 0.1 mW/cm² BL. Five-generation subculturing suggested that the BL-resistant cells underwent mutation(s) and adapted to grow under a BL environment. Fewer superoxide anion radicals were generated by BL irradiation in BL-resistant cells than in control DH5α cells because of the lower amount of intracellular flavins in BL-resistant cells. Our data suggest that the main photosensitizer of 463 nm BL is flavins, and ROS generated by BL-activated flavins induces inactivation and mutation(s) of DH5α cells. BL-resistant mutant cells with low amounts of intracellular flavins may reproducibly be generated by mutation(s) and preferentially proliferate under BL conditions.

Keywords: E. coli (DH5α); Blue light; Flavins; Reactive oxygen species (ROS); Stress-induced adaptive mutation.

Fig. 1

Irradiation systems and the effects of visible light. (a) The emission spectra of blue (blue solid line) and green (green solid line) LED systems, and the absorption spectra of FMN (orange dashed line) and uroporphyrin (red dashed line). (b) The survival rates (S) under dark (gray bar) and green (green bar) or blue (blue bar) lights (0.1 mW/cm²).

Fig. 2

Survival rates of DH5α cells under BL irradiation. (a) Survival rates of control DH5α cells under BL irradiation of 0–0.12 mW/cm² for 24 h. (b) Survival rates under 0.1 mW/cm² BL irradiation for 0–24 h for control DH5α cells (gray squares), BL-tolerant cells (blue square), and fifth-generation BL-resistant cells (red square).

Fig. 3

BL-induced production of O₂^•–. (a) Relationship between ΔA560 and concentration of FMN under 2 mW/cm² BL for 5 min. (b) ΔA560 before and after incubation under 2 mW/cm² BL for 5 min for insoluble (dotted black bar) and soluble (gray bar) fractions of control DH5α cells and for insoluble (dotted purple bar) and soluble (blue bar) fractions of BL-resistant cells. The asterisk indicates a statistically significant difference (p < 0.05).

Fig. 4

Degradation of O₂^•– by SOD activity. (a) Relationship between ΔA560_FMN and SOD concentration (U/mL) in the presence of 0.4 µM FMN under 2 mW/cm² BL for 5 min. (b) ΔA560_FMN before and after 2 mW/cm² BL for 5 min in the presence of 0.4 µM FMN with no cellular fraction (orange bar), with insoluble (dotted black bar) and soluble (gray bar) fractions of control DH5α cells and insoluble (dotted purple bar) and soluble (blue bar) fractions of BL-resistant cells. The asterisk indicates a statistically significant difference (p < 0.05).

Fig. 5

Analyses of flavins by fluorescent spectra. (a) Representative fluorescence spectra excited at 460 nm (solid lines), and excitation spectra monitored by the fluorescence at 550 nm (dashed lines) of FMN (orange lines), insoluble (black lines) and soluble (gray lines) fractions of control DH5α cells and soluble fractions of BL-resistant cells (blue lines). (b) The maximum fluorescence intensities for soluble fractions of control (gray bar) and BL-resistant (blue bar) cells. The asterisk indicates a statistically significant difference (p < 0.05).

Fig. 6

Analyses of flavins by HPLC. (a) Representative elution profiles of the four flavin standards (orange line), and control (gray lines) and BL-resistant (blue lines) cells monitored at 450 nm. The elution peak volumes of FAD in soluble fractions are shifted to the left, probably because of the presence of large amounts of hydrophilic components. (b) The correlation of flavin amounts estimated from HPLC analyses and the maximum fluorescence intensities of soluble fractions of control (gray circles) and BL-resistant (blue squares) cells. (c) The total amounts of flavins estimated from HPLC analyses. The asterisk indicates a statistically significant difference (p < 0.05, n = 8).