The eps1 region is organized in multiple transcriptional units. (A) Comparison of the genetic structures of the B. cereus eps1 region and the B. subtilis epsA-O operon. Conserved genes are in black and weave and unique genes are marked in grey. Top, a gene cluster predicted to be involved in the production of an EPS in B. cereus. Bottom, the operon involved in the synthesis of the biofilm EPS in B. subtilis. Automatic annotation of the genes in the eps1 region is shown. (B) qPCR with cDNA obtained by reverse transcribing RNA isolated from a broth culture of B. cereus grown for 24 h at 30 °C.

The eps2 region is organized in a single transcriptional unit. (A) qPCR with cDNA obtained by reverse transcribing RNA isolated from a broth culture of B. cereus grown for 24 h at 30 °C. The arrow indicates the hypothetical promoter region of the operon. (B) The genetic structure determined from the RT-PCR results and the automatic annotation of the genes included in the operon. (C) Upstream putative ORFs found via ORF-Finder analysis and included as an integrant of the operon.

Biofilm phenotypes of the WT and eps-mutant strains. (A) Liquid cultures in TY medium incubated for 24 h at 30 °C with 150 rpm shaking showed that the WT strain yielded the largest amount of biomass attached to the walls of the flask compared to the strain mutated in the eps2 region. (B) Phase contrast micrographs of the spent medium from liquid cultures show the presence of bacterial clumps in the media from the WT and Δeps1 strains. The bottom pictures are closer views of the squared areas in the original pictures (C) Transmission electron micrographs of negatively stained cells decorated with fibers reactive to anti-TasA antibodies. (D) Congo Red biofilm assays in: Top, TY liquid medium supplemented with Congo Red and incubated without shaking at 30 °C for 6 days, and bottom, TY medium solidified with 1.5% agar supplemented with Congo Red and incubated at 30 °C for 72 h. The bars in D = 1 μm.

Eps1 and Eps2 prevent cellular auto-aggregation. The optical densities (ODs) of cultures grown in TY medium incubated at 30 °C for 16 h with shaking at 150 rpm were adjusted to OD = 3 before the experiments were initiated. (A) Top, representative images of the tubes after 5 h of incubation; bottom, OD values of the air-liquid interface in the cell suspensions under static conditions at room temperature. (B) Top, representative images of the cultures after 9 h of incubation; bottom, OD values of the liquid cultures after 9 h of incubation, with sampling at the height of the grade bar. Means compared using t-test (P < 0.05).

EPS1 and EPS2 contribute differently to bacterial cell motility. Cell suspensions were spotted in the centers of the plates, which were then incubated at 28 °C before examining the motility. (A) Images taken from the swimming agar plates (0.3% TY agar plates, 24 h). (B) Images taken from the swarming agar plates (0.7% TY agar plates, 72 h). The graphs represent the values of the colony diameters at the end of each experiment. A Tukey statistical analysis was realized (p-value < 0.01) and the significant differences are indicated with letters. (C,D) Bacterial strains constitutively expressing YFP or CFP were mixed at a 1:1 ratio and then spotted in the centers of the plates. (C) The motility and Congo Red binding phenotypes were analyzed on TY swarming agar plates supplemented with Congo Red. The drawing indicates the zone occupied by each strain in the corresponding complementation experiment and marked with squares in the original picture (bottom). (D) Fluorescence microscopy images showing the presence of each bacterial strain in the same section indicated in panel C, bottom.

EPS2 is more important for the adhesion of B. cereus cells to human epithelial cells and to the zebrafish gut. (A) Adhesion assays in cultures of the human HeLa and MDA epithelial cell lines. Means compared using t-student (P < 0.05). (B) Top left, representative image of a zebrafish larva 6 days post-fertilization (dpf). Bottom left graph, rates (±SD) of 6-dpf zebrafish larvae harboring fluorescent bacteria in the GI tract 24 h after feeding with the WT, Δeps1, Δeps2 or double-mutant B. cereus strains (1 E8 ucf/ml). Means compared using t-student (P < 0.05). Right panel, bacterial localization in the gut of the zebrafish larvae after infection. The bacterial strains were transformed with a plasmid expressing the yfp gene under the control of a constitutive promoter. a-d´: Fluorescent Z-stack confocal microscopy images (the bacteria marked with asterisks in a-d are magnified in a´-d´). Scale bars = 200 μm in a,b,c,d, 100 μm in a-d, and 1 μm in a´-d´.