Myxococcus xanthus uses short-range C-signaling to coordinate multicellular mound formation with sporulation during fruiting body development.  A csgA mutant deficient in C-signaling can cheat on wild type (WT) in mixtures and form spores disproportionately, but our understanding of cheating behavior is incomplete.  We subjected mixtures of WT and csgA cells at different ratios to co-development and used confocal microscopy and image analysis to quantify the arrangement and morphology of cells.  At a ratio of one WT to four csgA cells (1:4), mounds failed to form. At 1:2, only a few mounds and spores formed.  At 1:1, mounds formed with a similar number and arrangement of WT and csgA rods early in development, but later the number of csgA spores near the bottom of these nascent fruiting bodies (NFBs) exceeded that of WT.  This cheating after mound formation involved csgA forming spores at a greater rate while WT disappeared at a greater rate, either lysing or exiting NFBs.  At 2:1 and 4:1, csgA rods were more abundant than expected throughout the biofilm both before and during mound formation, and cheating continued after mound formation.  We conclude that C-signaling restricts cheating behavior by requiring sufficient WT cells in mixtures.  Excess cheaters may interfere with positive feedback loops that depend on the cellular arrangement to enhance C-signaling during mound building.  Since long-range signaling could not likewise communicate the cellular arrangement, we propose that C-signaling was favored evolutionarily and that other short-range signaling mechanisms provided selective advantages in bacterial biofilm and multicellular 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. Images near the bottom of NFBs were the first optical section above the bottom of the well, in which cells could be clearly visualized (0.25 to 0.5 μm above the bottom of the well).