Combining fluorescent transcriptional reporter fusions and 4D-Confocal Laser Scanning Microscopy (CLSM) to decipher spatio-temporal patterns of gene expression within Bacillus subtilis submerged biofilms

The 4D spatio-temporal patterns of expression of 16 genes in Bacillus subtilis NDmed submerged biofilms were aquired by CLSM with single or dual fluorescent reporter fusions. Matrix genes are represented by epsA, tapA, bslA, srfAA, ypqP and capE, motility by hag, exoprotease by aprE, carbon metabolism by ackA, cggR, gapB, competence by comGA, cannibalism by skfA, respiration by ctaA and narG, and sporulation by spoIIGA and spoVC. Bacillus subtilis NDmed submerged biofilms were observed using a Leica SP8 AOBS inverted laser scanning microscope (CLSM, LEICA Microsystems, Wetzlar, Germany) at the INRAE MIMA2 platform (https://doi.org/10.15454/1.5572348210007727E12). Gfp excitation was performed at 488 nm with an argon laser, and the emitted fluorescence was recorded within the ranges 500-550nm. mCherry excitation was performed at 561 nm with an argon laser, and the emitted fluorescence was recorded within the range 600–750 nm. For propidium iodiide (PI) the fluorophore was exited at 488 nm and the emitted fluorescence collected in the range 600-750nm. For 4D (xyzt) acquisitions an image was taken every 1 hour for 48 hours or 1 and half hours for 72 h (512 × 512 pixels, pixel size 0.361 µm, 1 image every z = 1 µm with a scan speed of 600 Hz).