Blue-Light Inhibition of Listeria monocytogenes Growth Is Mediated by Reactive Oxygen Species and Is Influenced by σB and the Blue-Light Sensor Lmo0799

Abstract

Listeria monocytogenes senses blue light via the flavin mononucleotide-containing sensory protein Lmo0799, leading to activation of the general stress response sigma factor SigB (σ(B)). In this study, we investigated the physiological response of this foodborne pathogen to blue light. We show that blue light (460 to 470 nm) doses of 1.5 to 2 mW cm(-2) cause inhibition of growth on agar-based and liquid culture media. The inhibitory effects are dependent on cell density, with reduced effects evident when high cell numbers are present. The addition of 20 mM dimethylthiourea, a scavenger of reactive oxygen species, or catalase to the medium reverses the inhibitory effects of blue light, suggesting that growth inhibition is mediated by the formation of reactive oxygen species. A mutant strain lacking σ(B) (ΔsigB) was found to be less inhibited by blue light than the wild type, likely indicating the energetic cost of deploying the general stress response. When a lethal dose of light (8 mW cm(-2)) was applied to cells, the ΔsigB mutant displayed a marked increase in sensitivity to light compared to the wild type. To investigate the role of the blue-light sensor Lmo0799, mutants were constructed that either had a deletion of the gene (Δlmo0799) or alteration in a conserved cysteine residue at position 56, which is predicted to play a pivotal role in the photocycle of the protein (lmo0799 C56A). Both mutants displayed phenotypes similar to the ΔsigB mutant in the presence of blue light, providing genetic evidence that residue 56 is critical for light sensing in L. monocytogenes Taken together, these results demonstrate that L. monocytogenes is inhibited by blue light in a manner that depends on reactive oxygen species, and they demonstrate clear light-dependent phenotypes associated with σ(B) and the blue-light sensor Lmo0799.

Importance: Listeria monocytogenes is a bacterial foodborne pathogen that can cause life-threatening infections in humans. It is known to be able to sense and respond to visible light. In this study, we examine the effects of blue light on the growth and survival of this pathogen. We show that growth can be inhibited at comparatively low doses of blue light, and that at higher doses, L. monocytogenes cells are killed. We present evidence suggesting that blue light inhibits this organism by causing the production of reactive oxygen species, such as hydrogen peroxide. We help clarify the mechanism of light sensing by constructing a "blind" version of the blue-light sensor protein. Finally, we show that activation of the general stress response by light has a negative effect on growth, probably because cellular resources are diverted into protective mechanisms rather than growth.

FIG 1

Growth inhibition of EGD-e by blue light. EGD-e was illuminated with blue light (460 to 470 nm, 1.5 to 2.0 mW cm⁻²) either on BHI agar (A) or in a BHI liquid culture (B). (B) White bars represent growth following continuous illumination, and the black bars represent a dark control. The top graph shows the final OD₆₀₀ after 24 h, and the bottom graph shows the difference in lag times between the two conditions. Overnight cultures were standardized to an OD₆₀₀ of 1.0 and diluted to 10⁻⁵ (approximately 10⁴ cells ml⁻¹). Cells were incubated at 30°C for 24 h. The values represent the means of the results from three independent replicates. The error bars represent the standard deviations between replicates. Student's t test was carried out to determine the statistical difference (P ≤ 0.05, indicated with an asterisk) between cultures grown in light and dark.

FIG 2

Cell density influences the extent of growth inhibition of EGD-e by blue light. Dilutions of EGD-e were illuminated with blue light (460 to 470 nm, 1.5 to 2.0 mW cm⁻²) either on BHI agar (A) or in BHI liquid culture (B). (A) Overnight cultures were standardized to an OD₆₀₀ of 1.0 and diluted to 10⁻⁸. Four microliters of each dilution was spotted in triplicate onto BHI agar and grown at 30°C for 24 h. (B) White bars represent growth in the presence of light, and the black bars represent the dark control. The graphs show final OD₆₀₀ measurements (left) and lag times (right). The number over the lag time indicates the time taken to reach an OD₆₀₀ of 0.1. Starting cells were equalized to an OD₆₀₀ of 0.05 and diluted to 10⁻⁶. Cultures were grown in 96-well plates at 30°C for 24 h. The values represent the means of the results from three individual replicates. The error bars represent the standard deviations between replicates. Student's t test was carried out to determine the statistical difference (P ≤ 0.05, indicated with an asterisk) between cultures grown in light and dark.

FIG 3

ROS scavenger DMTU mitigates the inhibitory effect of blue light. EGD-e cells were illuminated with blue light (460 to 470 nm, 1.5 to 2 mW cm⁻²) either on BHI agar (A) or in liquid culture (B) with or without 20 mM DMTU. (A) Overnight cultures were standardized to an OD₆₀₀ of 1.0 and diluted to 10⁻⁸. Four microliters of each dilution was spotted in triplicate onto BHI agar (−) or BHI agar supplemented with 20 mM DMTU (+) and grown at 30°C for 24 h. (B) Final OD measurements (left) and difference in lag time (right). Starting cells were equalized to an OD₆₀₀ of 0.05 and diluted to 10⁻⁶. Cultures were grown in 96-well plates at 30°C for 24 h in BHI broth with or without 20 mM DMTU. The values represent the means of the results from three individual replicates. The error bars represent the standard deviations between samples. Student's t test was carried out to determine the statistical difference between cultures grown with and without DMTU. (B) Asterisks indicate significant differences (P ≤ 0.05).

FIG 4

Catalase alleviates growth inhibition of L. monocytogenes by blue light. (A) Wild-type overnight cultures were standardized, diluted serially, and spotted on BHI agar spread with catalase or on agar plates without catalase. The plates were incubated in the presence of light for 24 h. (B) Wild-type overnight cultures were diluted to an OD₆₀₀ of 0.05 and serially diluted 10-fold to a 10⁻⁶ dilution. The dilutions were grown for 24 h in the presence or absence of light, with or without catalase at a concentration of 125 U ml⁻¹. The 24-h endpoint and the time taken to reach an OD₆₀₀ of 0.1 were calculated for each strain condition at each dilution. The error bars represent standard deviations between samples. Unpaired t tests were used to determine the significance of differences (P ≤ 0.05, indicated by asterisks) between endpoint and lag-phase values between cultures with and without catalase.

FIG 5

Cells lacking SigB have decreased sensitivity to blue light. Shown is the influence of blue light (470 nm, 1.5 to 2 mW cm⁻²) on the growth of L. monocytogenes ΔsigB compared to the wild-type EGD-e on BHI agar (A) and in BHI liquid culture (B). Overnight cultures were standardized to an OD₆₀₀ of 1.0 and diluted to 10⁻⁸. Four microliters of each dilution was spotted in triplicate onto BHI agar and grown at 30°C for 24 h. (B) Final OD measurements (left) and difference in lag time (right). The number over the lag time indicates the time taken to reach 0.1. Starting cells were equalized to an OD₆₀₀ of 0.05 and diluted to 10⁻⁶. Cultures were grown in 96-well plates at 30°C for 24 h. The values represent the means of the results from three individual replicates. The error bars represent the standard deviations between samples. Student's t test was carried out to determine the statistical difference between EGD-e and ΔsigB. The asterisks in panel B indicate a P value of ≤0.05.

FIG 6

L. monocytogenes ΔsigB mutant displays a survival defect in higher-intensity blue light. Overnight cultures were washed and resuspended in PBS and exposed to 8 mW cm⁻² blue light. Viable cell counts were performed at the 0-, 6-, and 12-h time points. WT, wild type. The values represent the means of the results from six individual replicates. The error bars represent the standard deviations between samples.

FIG 7

The lmo0799 C56A blind mutant displays derepressed motility in light. Strains were inoculated on 0.3% agar, and colony diameter was measured 60 h after exposure to ambient white light/incubation in dark at 30°C. The values represent the means of the results from four biological replicates. The error bars represent the standard deviations between samples. The asterisk indicates a P value of ≤0.05.

FIG 8

Light-dark ring formation is abolished for the blind lmo0799 C56A mutant. Overnight cultures were standardized and spotted onto 0.3% BHI agar. The plates were incubated in the dark for 60 h or exposed to five consecutive 12-h periods of ambient light and dark.

FIG 9

Removal of SigB or Lmo0799 decreases the inhibitory effect of light on cell growth. (A) Cultures were standardized to an OD₆₀₀ of 1 and diluted, first 1:10, followed by 1:5 dilutions in PBS. The dilutions were spotted on BHI agar and incubated in the presence or absence of light. (B) Overnight cultures were diluted to an OD₆₀₀ of 0.05 and serially diluted 10-fold to a 10⁻⁶ dilution. The dilutions were grown for 24 h in the presence or absence of light. The 24-h endpoint and the time taken to reach an OD₆₀₀ of 0.1 were calculated for each strain at each dilution. Student's t tests were used to identify endpoint and lag-phase values that differed significantly from those of the wild-type strain. The values represent the means of the results from three individual replicates. The error bars represent the standard deviations between samples. The asterisks in panel B indicate a P value of ≤0.0167, adjusted for Bonferroni correction.