c-di-AMP reports DNA integrity during sporulation in Bacillus subtilis

Abstract

The bacterium Bacillus subtilis produces the DNA integrity scanning protein (DisA), a checkpoint protein that delays sporulation in response to DNA damage. DisA scans the chromosome and pauses at sites of DNA lesions. Structural analysis showed that DisA synthesizes the small molecule cyclic diadenosine monophosphate (c-di-AMP). Here, we demonstrate that the intracellular concentration of c-di-AMP rises markedly at the onset of sporulation in a DisA-dependent manner. Furthermore, exposing sporulating cells to DNA-damaging agents leads to a global decrease in the level of this molecule. This drop was associated with stalled DisA complexes that halt c-di-AMP production and with increased levels of the c-di-AMP-degrading enzyme YybT. Reduced c-di-AMP levels cause a delay in sporulation that can be reversed by external supplementation of the molecule. Thus, c-di-AMP acts as a secondary messenger, coupling DNA integrity with progression of sporulation.

Figure 1

Externally added c-di-AMP promotes progression of sporulation. (A) The chemical structure of c-di-AMP. (B) Fluorescence microscopy images of PY79 (wild-type) cells at 2 h of sporulation in the absence (–) and presence (+) of c-di-AMP (1.4 μM) added at 0 h. Cells were treated with the membrane stain FM1-43. Arrows indicate, positions of polar septa. Scale bar, 1 μm. (C) The percentage of sporulating PY79 (wild-type) and YA5 (ΔdisA) cells at 2 h of sporulation in the absence and presence of c-di-AMP or c-di-GMP (1.4 μM) added at 0 h. (D) The percentage of sporulating PY79 (wild-type) cells at 2.5 h of sporulation in the absence and presence of c-di-AMP (1.4 μM) and various concentrations of nalidixic acid as indicated. c-di-AMP and nalidixic acid were added at 0 h. (C,D) Cells were visualized using fluorescence microscopy, with the appearance of polar septa serving as the marker of sporulation. One representative experiment out of three independent experiments is shown. Standard deviation was calculated from the means of several fields of cells. At least 600 cells were counted for each treatment. c-di-AMP, cyclic diadenosine monophosphate; c-di-GMP, bis-(3’–5’)-cyclic dimeric guanosine monophosphate.

Figure 2

Endogenous c-di-AMP levels correspond to DNA damage. (A) HPLC analysis of extracts from PY79 (wild type) and YA5 (ΔdisA) cells and commercially available c-di-AMP (see Methods section). (B,C) Intracellular c-di-AMP concentrations, as determined by HPLC (see Methods section). Each column represents the mean c-di-AMP concentration calculated from three independent biological samples, with bars representing standard deviation. (B) PY79 (wild-type) cells were collected during vegetative (Veg) growth and at 2 h of sporulation. Sporulation was carried out in the presence of 350 μg/ml NAL or 50 ng/ml MMC, as indicated. YA5 (ΔdisA) cells were collected at 2 h of sporulation. (C) PY79 (wild type), YA5 (ΔdisA), YA188 (ΔyybT), MB21 (P_{hyper−spank}-disA; disA o/ex) and YA214 (P_{hyper−spank}-yybT; yybT o/ex) were collected at 2 h of sporulation. c-di-AMP, cyclic diadenosine monophosphate; HPLC, high-performance liquid chromatography; MMC, Mitomycin C; NAL, nalidixic acid.

Figure 3

Diadenylate cyclase activity is required for DisA foci assembly. (A,B) MB3 (disA-gfp) and YA127 (disA_D77N-gfp) cells were induced to sporulate and subjected to fluorescence microscopy. FM4-64 (red) and signal from GFP (green) are shown. (A) MB3 (upper panels) and YA127 (lower panels) cells at 1.5 h of sporulation. (B) YA127 cells at 2.5 h of sporulation in the absence (upper panels) and presence of 350 μg/ml NAL (lower panels), added at 0 h of sporulation. (C) Percentage of sporulating MB3 (disA-gfp), YA5 (ΔdisA) and Y127 (disA_D77N-gfp) cells at 3.5 h of sporulation in the absence (–) or presence (+) of 350 μg/ml NAL, added at 0 h of sporulation. Cells were visualized using fluorescence microscopy, with the appearance of polar septa serving as the marker of sporulation. One representative experiment out of three independent experiments is shown. Standard deviation was calculated from the means of several fields of cells. At least 600 cells were counted for each treatment. GFP, green fluorescent protein; NAL, nalidixic acid.

Figure 4

YybT is expressed on entry into sporulation. (A) YA180 (yybT–gfp) cells were induced to sporulate and then subjected to fluorescence microscopy. Shown are FM4-64 (red), DAPI (blue) and signal from YybT–GFP (green) at 0 h (upper panels), 1.5 h (middle panels) and 3.5 h (lower panels) of sporulation. Images were scaled to the same intensity range. (B) Enlarged fluorescence images of YA180 (yybT–gfp) cells at 1.5 h of sporulation. Shown are FM4-64 (red) and signal from YybT–GFP (green). Scale bars, 1 μm. DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescent protein.

Figure 5

Components of the c-di-AMP pathway. (A) YA180 (yybT–gfp; upper panels) and YA185 (ΔdisA, yybT–gfp; lower panels) cells were induced to sporulate and then subjected to fluorescence microscopy. Shown are FM4-64 (red), DAPI (blue) and signal from YybT–GFP (green) at 3.5 h of sporulation. (B) YA180 (yybT–gfp) cells were induced to sporulate in the absence (upper panels) and presence (lower panels) of 50 ng/ml MMC. Shown are FM4-64 (red), DAPI (blue) and signal from YybT–GFP (green) at 3.5 h of sporulation. Images were scaled to the same intensity range. Scale bars, 1 μm. (C) A schematic illustration of the activity of c-di-AMP as a secondary molecule signalling DNA damage. Upper left cell: At the onset of sporulation, DisA moves within the cell, scanning the chromosome for lesions, and produces c-di-AMP. YybT localizes to the cell periphery and degrades c-di-AMP. The total concentration of c-di-AMP increases signalling sporulation to proceed (lower cell). Upper right cell: In the presence of broken DNA, DisA stalls at the site of lesion and halts c-di-AMP production. Consequently, YybT expression increases, further reducing the cellular c-di-AMP pool. The drop in c-di-AMP level inhibits sporulation (lower cell). c-di-AMP, cyclic diadenosine monophosphate; DAPI; 4,6-diamidino-2-phenylindole; DisA, DNA integrity scanning protein; GFP, green fluorescent protein; MMC, Mitomycin C.