Development and optimization of an EGFP-based reporter for measuring the general stress response in Listeria monocytogenes

Abstract

A characteristic of the food-borne pathogen Listeria monocytogenes is its tolerance to the harsh conditions found both in minimally processed foods and the human gastrointestinal tract. This trait is partly under the control of the alternative sigma factor sigma B (σ(B)). To study the mechanisms that trigger the activation of σ(B) , and hence the development of stress tolerance, we have developed a fluorescent reporter fusion that allows the real-time activity of σ(B) to be monitored. The reporter, designated Plmo2230::egfp, fuses the strong σ(B)-dependent promoter from the lmo2230 gene (which encodes a putative arsenate reductase) to a gene encoding enhanced green fluorescence protein (EGFP). The reporter was integrated into the genomes of the wild-type strain L. monocytogenes EGD-e as well as two mutant derivatives lacking either sigB or rsbV. The resulting strains were used to study σ(B) activation in response to growth phase and hyperosmotic stress. The wild-type was strongly fluorescent in stationary phase or in cultures with added NaCl and this fluorescence was abolished in both the sigB and rsbV backgrounds, consistent with the σ(B)-dependency of the lmo2230 promoter. During sudden osmotic upshock (addition of 0.5 M NaCl during growth) a real-time increase in fluorescence was observed microscopically, reaching maximal activation after 30 min. Flow cytometry was used to study the activation of σ(B) at a population level by hyperosmotic stress during exponential growth. A strong and proportional increase in fluorescence was observed as the salt concentration increased from 0 to 0.9 M NaCl. Interestingly, there was considerable heterogeneity within the population and a significant proportion of cells failed to induce a high level of fluorescence, suggesting that σ(B) activation occurs stochastically in response to hyperosmotic stress. Thus the Plmo2230::egfp is a powerful tool that will allow the stress response to be better studied in this important human pathogen.

Figure 1. Design of the reporter plasmid pKSV7-P_lmo2230::egfp containing a fusion of the σ^B–dependent promoter region of lmo2230 with egfp (A). Site-directed integration of the reporter fusion vector into the chromosome of L. monocytogenes EGD-e wild type and derivative integrants (B) occurring at homologous site of lmo2230 promoter region (443 bp) after incubation at 42°C (non-permissive for autonomous plasmid maintenance in L. monocytogenes) in the presence of chloramphenicol selection.

Figure 2. Fluorescence of egfp-containing cells of L. monocytogenes EGD-e wild type, ΔsigB and ΔrsbV mutants after transformation with pKSV7-P_lmo2230::egfp. Phase-contrast and fluorescence microscopy of corresponding fields were performed after 1 ml of stationary phase culture was centrifuged, resuspended in 50 μl of sterile PBS and 5 μl was smeared on the slides for visualization. Images are representative of a minimum of ten randomly selected fields captured for three biological replicates for each strain.

Figure 3. EGFP is expressed of L. monocytogenes EGD-e wild type but not in ΔsigB and ΔrsbV backgrounds. Western blot analyses were performed using protein extracts from stationary phase cultures (normalized to 5 mg ml⁻¹ total protein concentrations and 10 µl of these samples separated by SDS-PAGE) with chicken polyclonal anti-GFP antibody (Abcam) HRP-conjugated goat anti-chicken antibody (Promega) and chemiluminescent detection.

Figure 4. σ^B -dependent expression of EGFP is increased in L. monocytogenes EGD-e wild-type integrant in stationary phase and by osmotic stress. Cells of integrants bearing P_lmo2230::egfp fusion were grown from starting OD₆₀₀ = 0.05 up to either exponential (OD₆₀₀ = 0.6) or stationary phase (16 h) in BHI or BHI supplemented with 0.5 M NaCl. A similar number of cells (equivalent to 1 ml of OD₆₀₀ = 0.6 and diluted 1:2 by the fixing procedure) was diluted 1:10 in PBS and concentrated on 10 mm diameter, 0.2 μm pore polycarbonate membrane (Millipore) by filtration. For each experimental condition three biological replicates were analyzed and a minimum ten randomly selected fields of each membrane were captured with a CCD camera attached to Nikon Eclipse E600 with B-2A and ND8 filters used at a fixed exposure time of 2 sec with representative fields shown (A). Fluorescence levels of cells were quantified with automated image processing of microscopic fields by counting numbers of detectable particles (B) and their relative fluorescence intensities (C) with ImageJ 1.44 software with appropriate manipulations as described by others.^,

Figure 5. σ^B -dependent EGFP expression is induced after osmotic upshock in L. monocytogenes EGD-e wild-type P_lmo2230::egfp integrant. Cells bearing P_lmo2230::egfp fusion grown in BHI from OD₆₀₀ = 0.05 up to OD₆₀₀ = 0.6 when 0.5 M NaCl was added. Similar number of cells (equivalent to 1 ml of OD₆₀₀ = 0.6 and diluted 1:2 by the fixing procedure) was taken at appropriate intervals from untreated and osmotically shocked cultures. Fixed cells were diluted 1:10 in PBS and concentrated on 10 mm diameter, 0.2 μm pore polycarbonate membrane (Millipore) by filtration. For each experimental condition three biological replicates were analyzed and a minimum of ten randomly selected fields were captured with a CCD camera attached to Nikon Eclipse E600 with B-2A and ND8 filters were used at a fixed exposure time of 2 s with representative fields shown (A). Fluorescence levels of cells were quantified with automated image processing of captured microscopic fields by counting relative fluorescence intensities (B) with ImageJ 1.44 software with appropriate manipulations as described by others.^,

Figure 6. Heterogeneity of fluorescence within EGFP-expressing population and σ^B activation proportional to the extent of osmotic stress was revealed with FCM. Cells of parent wild-type strain (WT), wild-type-P_lmo2230::egfp (WT-egfp) and ΔsigB-P_lmo2230::egfp (Δσ^B-egfp) derivative strains were grown from a starting OD₆₀₀ = 0.05 up to OD₆₀₀ = 0.6 in BHI (no salt) or BHI supplemented with 0.3 M, 0.6 M or 0.9 M NaCl. FCM was performed using BD Accuri C6 flow cytometer on fixed cells (concentration equivalent to 1 ml of OD₆₀₀ = 0.6, fixed and resuspended in sterile PBS) for three technical replicates of two biological replicates for each strain and each experimental condition. Levels of intrinsic fluorescence (autofluorescence) were determined for wild-type cells without a reporter fusion (WT) and were used to define the gate range of EGFP expressing wild-type-egfp strain population. Mean fluorescence intensities (MFI) for egfp gate and particles located outside the gate (autofluorescence) were calculated by BD CFlow^® software.