Generation of variants in Listeria monocytogenes continuous-flow biofilms is dependent on radical-induced DNA damage and RecA-mediated repair

Abstract

The food-borne pathogen Listeria monocytogenes is a gram-positive microaerophilic facultative anaerobic rod and the causative agent of the devastating disease listeriosis. L. monocytogenes is able to form biofilms in the food processing environment. Since biofilms are generally hard to eradicate, they can function as a source for food contamination. In several occasions biofilms have been identified as a source for genetic variability, which potentially can result in adaptation of strains to food processing or clinical conditions. However, nothing is known about mutagenesis in L. monocytogenes biofilms and the possible mechanisms involved. In this study, we showed that the generation of genetic variants was specifically induced in continuous-flow biofilms of L. monocytogenes, but not in static biofilms. Using specific dyes and radical inhibitors, we showed that the formation of superoxide and hydroxyl radicals was induced in continuous-flow biofilms, which was accompanied with in an increase in DNA damage. Promoter reporter studies showed that recA, which is an important component in DNA repair and the activator of the SOS response, is activated in continuous-flow biofilms and that activation was dependent on radical-induced DNA damage. Furthermore, continuous-flow biofilm experiments using an in-frame recA deletion mutant verified that RecA is required for induced generation of genetic variants. Therefore, we can conclude that generation of genetic variants in L. monocytogenes continuous-flow biofilms results from radical-induced DNA damage and RecA-mediated mutagenic repair of the damaged DNA.

Figure 1. Induced generation of variants in continuous-flow biofilms.

The graph presents the average and standard deviation of the rifampicin resistant fraction (0.05 µg/ml) of continuous-flow biofilms, static biofilms, and static planktonic cultures using three biological independent experiments. *Data points significantly different from the continuous-flow biofilms (p<0.05, t-test).

Figure 2. Induced radical formation during continuous-flow biofilm formation.

The micrographs show fluorescence (1, 3, and 5) and phase contrast (2, 4, and 6) pictures of MitoSOX (A) and HPF (B) stained cells obtained from continuous-flow biofilms (1 and 2), static biofilms (3 and 4), and static planktonic cultures (5 and 6).

Figure 3. Radical inhibitors bipyridyl and thiourea inhibit radical formation during continuous-flow biofilm formation.

Fluorescence (1 and 3) and phase contrast (2 and 4) pictures of MitoSOX (1 and 2) and HPF (3 and 4) stained cells obtained from continuous-flow biofilms grown in BHI with 0.05 mM bipyridyl and 50 mM thiourea.

Figure 4. Radical formation during continuous-flow biofilm formation results in DNA damage.

A) Ethidium bromide-stained agarose gel containing 0.5 µg genomic DNA isolated from cells obtained from continuous-flow biofilms, static biofilms, and planktonic cultures grown in BHI without (-) and with 0.05 mM bipyridyl and 50 mM thiourea (+). B) Relative intensity of each lane of the agarose gel plotted against the migration distance. DNA isolated from cells obtained from continuous-flow biofilms grown in BHI without (blue) and with bipyridyl and thiourea (red), static biofilms grown in BHI without (yellow) and with bipyridyl and thiourea (green), and static planktonic cultures grown in BHI without (purple) and with bipyridyl and thiourea (brown).

Figure 5. Activation of recA during continuous-flow biofilm formation is dependent on radical-induced DNA damage.

Micrographs show fluorescence (1 and 3) and phase contrast (2 and 4) pictures of cells expressing EGFP from the recA promoter. Cells are obtained from continuous-flow biofilms grown in BHI without (1 and 2) and with (3 and 4) 0.05 mM bipyridyl and 50 mM thiourea.

Figure 6. Continuous-flow biofilm formation is dependent on radical- and RecA-induced activation of yneA.

Activation of SOS response member yneA is required to obtain fully grown continuous-flow biofilms that are composed of micro-colonies surrounded by knitted chains composed of elongated cells. A) The graph presents the average and standard deviation of the continuous-flow biofilm produced by the wild-type strain, the ΔrecA mutant, and the ΔyneA mutant grown in BHI, and the wild-type strain grown in BHI with 0.05 mM bipyridyl and 50 mM thiourea using three biological independent experiments. *Data points significantly different from the wild-type strain grown in BHI (p<0.05, t-test). B) The micrographs present phase contrast pictures of continuous-flow biofilms of the wild-type strain grown in BHI without (1) and with 0.05 mM bipyridyl and 50 mM thiourea (2), and of the ΔrecA mutant (3), and the ΔyneA mutant (4) grown in BHI.

Figure 7. Generation of variants in continuous-flow biofilms is dependent on radical-induced DNA damage and RecA-mediated mutagenic DNA repair.

The graph presents the average and standard deviation of the rifampicin resistant fraction (0.05 µg/ml) of continuous-flow biofilms of the wild-type strain and the ΔrecA mutant grown in BHI, and the wild-type strain grown in BHI with 0.05 mM bipyridyl and 50 mM thiourea using three independent biological experiments. *Data points significantly different from the wild-type strain grown in BHI (p<0.05, t-test).

Figure 8. Proposed model for the generation of variants in L. monocytogenes continuous-flow biofilms.

Continuous influx of oxygen saturated BHI in the flow-cells will result in relatively high oxygen concentrations in the attached cells. Intracellular oxygen is converted into superoxide and subsequently hydroxyl radicals. These ROS cause DNA damage and as result recA is activated. RecA subsequently mediates mutagenic repair of the damaged DNA, which results in the generation of genetic variants. Furthermore, activation of recA results in the activation of the SOS response and its regulon member yneA. YneA subsequently accumulates at the midcell to prevent septum formation, which results in cell elongation. Cell elongation is required to reach fully grown continuous-flow biofilms that consist of ball-shaped microcolonies surrounded by a network of knitted-chains composed of elongated cells.