Blue Light Signaling Regulates Escherichia coli W1688 Biofilm Formation and l-Threonine Production

Abstract

Escherichia coli biofilm may form naturally on biotic and abiotic surfaces; this represents a promising approach for efficient biochemical production in industrial fermentation. Recently, industrial exploitation of the advantages of optogenetics, such as simple operation, high spatiotemporal control, and programmability, for regulation of biofilm formation has garnered considerable attention. In this study, we used the blue light signaling-induced optogenetic system Magnet in an E. coli biofilm-based immobilized fermentation system to produce l-threonine in sufficient quantity. Blue light signaling significantly affected the phenotype of E. coli W1688. A series of biofilm-related experiments confirmed the inhibitory effect of blue light signaling on E. coli W1688 biofilm. Subsequently, a strain lacking a blue light-sensing protein (YcgF) was constructed via genetic engineering, which substantially reduced the inhibitory effect of blue light signaling on biofilm. A high-efficiency biofilm-forming system, Magnet, was constructed, which enhanced bacterial aggregation and biofilm formation. Furthermore, l-threonine production was increased from 10.12 to 16.57 g/L during immobilized fermentation, and the fermentation period was shortened by 6 h. IMPORTANCE We confirmed the mechanism underlying the inhibitory effects of blue light signaling on E. coli biofilm formation and constructed a strain lacking a blue light-sensing protein; this mitigated the aforementioned effects of blue light signaling and ensured normal fermentation performance. Furthermore, this study elucidated that the blue light signaling-induced optogenetic system Magnet effectively regulates E. coli biofilm formation and contributes to l-threonine production. This study not only enriches the mechanism of blue light signaling to regulate E. coli biofilm formation but also provides a theoretical basis and feasibility reference for the application of optogenetics technology in biofilm-based immobilized fermentation systems.

Keywords: Escherichia coli; biofilm; blue light signaling; l-threonine; optogenetics.

FIG 1

Effects of blue light on biofilm formation in E. coli. (A) E. coli W1688 and ΔycgF strains were inoculated into a 96-well plate, incubated at 37°C under dark or blue light conditions with different light intensities for 24 h, and then imaged after crystal violet (CV) staining. (B) Corresponding OD₅₇₀ value in the 96-well plate. (C) E. coli W1688 and ΔycgF strains were incubated in 24-well plates with round coverslips under light or dark conditions. The biofilm cells on the coverslips were stained with DAPI, and the state of biofilm formation was observed under a fluorescence microscope. Scale bars, 50 μm. (D) Biofilm cells on the coverslips were subjected to ethanol gradient dehydration and observed via SEM. Scale bars, 50 μm. The values represent the means and standard deviations from three independent experiments. ***, P > 0.001; **, P > 0.01; and *, P > 0.05, by two-way ANOVA.

FIG 2

Decreased expression of biofilm-related genes under blue light conditions. Shown is expression of the genes involved in curli and type I fimbria formation, motility, and c-di-GMP level. The standard deviation of all gene expressions was P < 0.001. The values represent the means and standard deviations of values from three independent experiments. RQ, relative quantity. ***, P > 0.001; **, P > 0.01; and *, P > 0.05, by two-way ANOVA.

FIG 3

E. coli motility assay and curli fiber formation under dark or blue light conditions. (A) Chemotactic rings of E. coli W1688 and ΔycgF strains observed on Eiken agar plates (diameter, 7 cm); (B) staining of E. coli curli fibers with Congo red (CR). (C) CR supernatant was used for full-wavelength analysis. (D) TEM images of E. coli W1688 and ΔycgF strains. Scale bars, 1 μm.

FIG 4

Free-cell fermentation and growth curves of E. coli W1688 and mutants. (A) l-Threonine production and glucose consumption in free-cell fermentation using E. coli W1688 and ΔycgF strains. (B) Growth curves of E. coli W1688 and all mutants. The cell densities were determined by measuring OD₆₀₀ at 2, 4, 6, 8, 10, 12, and 14 h.

FIG 5

Adsorption and adhesion of the optogenetic system Magnet. (A) Biofilm formation in the E. coli W1688, ΔycgF, ΔycgF nMagHigh, and ΔycgF pMagHigh strains; (B) blue light-dependent aggregation of bacteria expressing nMagHigh and pMagHigh. E. coli cells exhibiting nMagHigh (labeled with EGFP) or pMagHigh (labeled with mCherry) were mixed in a 1:1 ratio (OD₆₀₀ = 0.15) and incubated for 4 h under blue light or dark conditions. Scale bars, 20 μm. (C) SEM analysis of sample obtained from immobilized continuous fermentation using the ΔycgF strain and the coculture of ΔycgF nMagHigh and ΔycgF pMagHigh strains.

FIG 6

l-Threonine production and glucose consumption in immobilized continuous fermentation using ΔycgF strain and the coculture of ΔycgF nMagHigh and ΔycgF pMagHigh strains. (A and B) Outcomes of immobilized continuous fermentation using (A) the ΔycgF strain and (B) the coculture of ΔycgF nMagHigh and ΔycgF pMagHigh strains; (C) schematic representation of a blue light oscillation incubator in operation.

FIG 7

E. coli senses blue light via the BluF-EAL protein BluF (YcgF), which controls various functions of biofilm. Solid arrows show activation or inhibition, and dotted arrows show an indirect effect.