Cycles of light and dark co-ordinate reversible colony differentiation in Listeria monocytogenes

Abstract

Recently, several light receptors have been identified in non-phototrophic bacteria, but their physiological roles still remain rather elusive. Here we show that colonies of the saprophytic bacterium Listeria monocytogenes undergo synchronized multicellular behaviour on agar plates, in response to oscillating light/dark conditions, giving rise to alternating ring formation (opaque and translucent rings). On agar plates, bacteria from opaque rings survive increased levels of reactive oxygen species (ROS), as well as repeated cycles of light and dark, better than bacteria from translucent rings. The ring formation is strictly dependent on a blue-light receptor, Lmo0799, acting through the stress-sigma factor, σ(B) . A transposon screening identified 48 mutants unable to form rings at alternating light conditions, with several of them showing a decreased σ(B) activity/level. However, some of the tested mutants displayed a varied σ(B) activity depending on which of the two stress conditions tested (light or H(2) O(2) exposure). Intriguingly, the transcriptional regulator PrfA and the virulence factor ActA were shown to be required for ring formation by a mechanism involving activation of σ(B) . All in all, this suggests a distinct pathway for Lmo0799 that converge into a common signalling pathway for σ(B) activation. Our results show that night and day cycles co-ordinate a reversible differentiation of a L. monocytogenes colony at room temperature, by a process synchronized by a blue-light receptor and σ(B) .

Figure 1

A blue-light receptor and oscillating cycles of light and dark control differentiation of a Listeria monocytogenes colony. A. Ring forming abilities on an agar plate on bench. Wild-type L. monocytogenes was inoculated on low-agar plates and incubated on the bench at room temperature for 96 h. B. Bacterial phenotypes on agar plates at different light and dark conditions. Wild-type L. monocytogenes was inoculated on low-agar plates and exposed to five cycles of 12 h light/12 h darkness (a total of 120 h, left panel), either to 120 h of constant light (middle panel) or to 120 h of constant darkness (right panel). An open bar indicates light conditions whereas a black bar indicates dark conditions. C. Bacterial phenotypes on agar plates with repetitive cycles of light and dark. Wild-type (WT) and cz− strains were inoculated on low-agar plates and exposed to four cycles of 12 h light/12 h darkness. D. Bacterial phenotypes on an agar plate with repetitive cycles of light and dark. Indicated strains were inoculated on a low-agar plate and exposed to eight cycles of 12 h light/12 h darkness. E. Ring-forming abilities of indicated strains on a low-agar plate exposed to four cycles of 12 h light/12 h darkness.

Figure 2

Light decrease Listeria motility in a mechanism requiring Lmo0799 and an antisense RNA. A. Wild-type (WT) and Δlmo0799 strains were inoculated on low-agar plates and incubated for indicated days on the bench (B); under blue-light-enhanced aquarium light (L1); under laboratory light (L2) or at darkness (D). Bacterial motility was scored daily and the difference between the Δlmo0799 mutant and the WT was plotted. The significant differences are denoted by stars: ‘***’ denotes Bonferroni corrected P-values of < 0.001. B. Northern blot analysis of asRNA expression at light and darkness. Indicated strains were grown at darkness (WT) or at light (WT; Δlmo0799 and ΔsigB) before RNA extraction and Northern blot. The membrane was hybridized with an asRNA RNA probe and a tmRNA (control) DNA probe respectively. Multiple bands of the antisense RNA were detected and the most prominent bands are highlighted by arrows.

Figure 3

Opaque rings produce extracellular polymeric substances required for stress and long-term survival. A. A wild-type (WT) strain was inoculated on a low-agar plate containing 25 μg ml⁻¹ Congo red and exposed to four cycles of 12 h light/12 h darkness. Red regions correspond to opaque rings, white regions to translucent rings respectively. B. Wild-type L. monocytogenes was inoculated on a low-agar plate and exposed to eight cycles of 12 h light/12 h darkness before addition of 1 M of H₂O₂ on a cross-section of the plate. The frequency of oxygen sphere formation at opaque and translucent rings was counted for 1 min and plotted as a fraction of 100%. n = 17 [P < 0.001 ***, Student's T-test (two-tailed)]. C. A wild-type strain was inoculated on a low-agar plate and exposed to five cycles of 12 h light/12 h darkness before bacteria were excised from 48-h-old opaque and translucent rings. Bacteria were resuspended in 1 ml of PBS and exposed to 60 mM of H₂O₂ for 90 min before plating. n = 3 [P < 0.05 *, Student's T-test (two-tailed)]. D. A wild-type strain was inoculated on a low-agar plate and exposed to 13 cycles of 12 h light/12 h darkness before bacteria from 2- or 11-day-old opaque and translucent rings were excised and plated. n = 3 [P < 0.05 *, Student's T-test (two-tailed)].

Figure 4

Transposon mutants unable to form rings are deficient for light and ROS induced σ^B activation as well as biofilm formation. A. Northern blot analysis of lmo2230 expression. Indicated strains were grown at light or dark conditions before RNA extraction and Northern blot. The membrane was hybridized with lmo2230 and tmRNA (control) specific DNA probes. B. Northern blot analysis of lmo2230 expression. Indicated strains were grown in darkness in presence (+) or absence (−) of 60 mM H₂O₂ before RNA extraction and Northern blot. The membrane was hybridized with lmo2230 and tmRNA (control) specific DNA probes. C. Western blot analysis of σ^B expression. Indicated strains were grown in darkness in presence (+) or absence (−) of 60 mM H₂O₂ before protein extraction and Western blot. The membrane was hybridized with an α-σ^B specific antibody. D. Indicated strains were inoculated in microtitre plates at light condition for 48 h before staining with crystal violet and A₅₉₅ measurement. The normalized signals of each strain were plotted and compared with the wild-type (WT) strain and the significant differences (Bonferroni corrected P-values < 0.001) were denoted with ‘***’.

Figure 5

ActA is required for ring formation and controls σ^B activity. A. Indicated strains were inoculated on a low-agar plate and exposed to four cycles of 12 h light/12 h darkness. B. Northern blot analysis of lmo2230 expression. Indicated strains were grown in darkness in presence (+) or absence (−) of 60 mM H₂O₂ before RNA extraction and Northern blot. The membrane was hybridized with lmo2230 and tmRNA (control) specific DNA probes. C. Western blot analysis of σ^B expression. Indicated strains were grown in darkness in presence (+) or absence (−) of 60 mM H₂O₂ before protein extraction and Western blot. The membrane was hybridized with an α-σ^B specific antibody. D. Northern blot analysis of lmo2230 expression. Indicated strains were grown at light conditions before RNA extraction and Northern blot. The membrane was hybridized with lmo2230 and tmRNA (control) specific DNA probes.

Figure 6

Bacteria in inner rings enter a resting state due to carbon deficiency. A. Wild-type (WT) L. monocytogenes strains without (lower colony) or with (upper colony) a chromosomal fusion (pPl2luxP_help) were inoculated on low-agar plates and exposed to five cycles of 12 h light/12 h darkness. Luciferase expression was measured at 12- and 60-h-old rings from the luciferase expressing strain and plotted as relative light expression [P < 0.001 ***, Student's T-test (two-tailed)]. B. The strain harbouring the pPl2luxP_help fusion was inoculated on low-agar plates and exposed to cycles of 12 h light/12 h darkness. One micromole of different carbon sources (glucose; glycerol; N-acetylglucosamine: succinate or acetate) were spotted onto the agar plate (black circles) and luciferase expression was measured after 30 min.

Figure 7

Schematic model. At light conditions (sun), Lmo0799 becomes activated, resulting in liberated σ^B. The presence of reactive oxygen species (ROS – in blue) also leads to freed σ^B. Free σ^B activate, among others, genes important for extracellular polymeric substances (EPS), an antisense RNA inhibiting bacterial motility, prfA and lmo0596 expression respectively. PrfA in turn activates actA expression. lmo0798 expression is blocked by l-lysine. ActA, Lmo0596 and Lmo0798 could function independently and later converge into a common σ^B activating pathway. Proteins being important for light-dependent σ^B activation are shown in yellow, proteins being important for ROS-dependent σ^B activation are shown in blue. Dashed lines indicates non-defined pathways.