Starving Myxococcus xanthus bacteria use short-range C-signaling to coordinate their movements and construct multicellular mounds, which mature into fruiting bodies as rods differentiate into spherical spores. Differentiation requires efficient C-signaling to drive the expression of developmental genes, but how the arrangement of cells within nascent fruiting bodies (NFBs) affects C-signaling is not fully understood. Here, we used confocal microscopy and cell segmentation to visualize and quantify the arrangement, morphology, and gene expression of cells near the bottom of NFBs at much higher resolution than previously achieved. We discovered that “transitioning cells” (TCs), intermediate in morphology between rods and spores, comprised 10 to 15% of the total population. Spores appeared midway between the center and the edge of NFBs early in their development and near the center as maturation progressed. The developmental pattern as well as C-signal-dependent gene expression in TCs and spores were correlated with cell density, the alignment of neighboring rods, and the tangential orientation of rods early in the development of NFBs. These dynamic radial patterns support a model in which the arrangement of cells within the NFBs affects C-signaling efficiency to regulate precisely the expression of developmental genes and cellular differentiation in space and time. Developmental patterns in other bacterial biofilms may likewise rely on short-range signaling to communicate multiple aspects of cellular arrangement, analogous to juxtacrine and paracrine signaling during animal development.

Images of nascent fruiting bodies were acquired with a Nikon A1 Laser Scanning Confocal Microscope, which was configured on a Nikon Ti inverted platform with an XY automated stage and a 100X objective.  Fluorescence from tdTomato were examined using a 560-nm laser for excitation and a 595/50 band pass emission filter.  Fluorescence from mNeonGreen was examined using a 488 nm laser for excitation and a 525/50 band pass emission filter.  Time-lapse confocal images were taken every 15 min.  Images near the bottom of NFBs were the first optical section above the bottom of the well, in which cells could be clearly visualized, so ~0.25 to 0.5 μm above the bottom of the well.