Starving Myxococcus xanthus bacteria use short-range C-signaling to coordinate building of multicellular mounds with differentiation from rods into spores 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 report that cheating requires excess WT cells in the initial mixture and occurs during the mound-building phase of development. 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 near the bottom of nascent fruiting bodies (NFBs). At a ratio of one WT to four csgA cells (1:4), NFBs failed to form. At 1:2, broad mounds formed with half the normal cell density and very few spores. At 1:1, NFBs formed normally with a similar number of WT and csgA rods early in development and a similar number of spores later, so C-signaling by WT rescued csgA development efficiently, but the mutant did not cheat. In contrast, at 2:1 and 4:1 excess WT starting ratios, csgA rods were more abundant than expected in early NFBs, indicative of cheating during mound formation. As NFBs matured, csgA and WT eventually formed spores with similar efficiency, although csgA began sporulation earlier and closer to the radial center. Our results reveal restrictions on cheating behavior, which may have selected C-signaling evolutionarily, and may explain the prevalence of short-range signaling 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, so ~0.25 to 0.5 μm above the bottom of the well.