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ABSTRACT Myxococcus xanthususes short-range C-signaling to coordinate multicellular mound formation with sporulation during fruiting body development. AcsgAmutant 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 andcsgAcells 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 fourcsgAcells (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 andcsgArods early in development, but later the number ofcsgAspores near the bottom of these nascent fruiting bodies (NFBs) exceeded that of WT. This cheating after mound formation involvedcsgAforming spores at a greater rate, while WT disappeared at a greater rate, either lysing or exiting NFBs. At 2:1 and 4:1,csgArods 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. IMPORTANCEBacteria communicate using both long- and short-range signals. Signaling affects community composition, structure, and function. Adherent communities called biofilms impact medicine, agriculture, industry, and the environment. To facilitate the manipulation of biofilms for societal benefits, a better understanding of short-range signaling is necessary. We investigated the susceptibility of short-range C-signaling to cheating duringMyxococcus xanthusbiofilm development. A mutant deficient in C-signaling fails to form mounds containing spores (i.e., fruiting bodies) but cheats on C-signaling by wild type in starved cell mixtures and forms spores disproportionately. We found that cheating requires sufficient wild-type cells in the initial mix and can occur both before mound formation and later during the sporulation stage of development. By restricting cheating behavior, short-range C-signaling may have been favored evolutionarily rather than long-range diffusible signaling. Cheating restrictions imposed by short-range signaling may have likewise driven the evolution of multicellularity broadly. 

Abstract Upon starvation, rod‐shapedMyxococcus xanthusbacteria form mounds and then differentiate into round, stress‐resistant spores. Little is known about the regulation of late‐acting operons important for spore formation. C‐signaling has been proposed to activate FruA, which binds DNA cooperatively with MrpC to stimulate transcription of developmental genes. We report that this model can explain regulation of thefadIJoperon involved in spore metabolism, but not that of the spore coat biogenesis operonsexoA‐I,exoL‐P, andnfsA‐H. Rather, a mutation infruAincreased the transcript levels from these operons early in development, suggesting negative regulation by FruA, and a mutation inmrpCaffected transcript levels from each operon differently. FruA bound to all four promoter regions in vitro, but strikingly each promoter region was unique in terms of whether or not MrpC and/or the DNA‐binding domain of Nla6 bound, and in terms of cooperative binding. Furthermore, the DevI component of a CRISPR‐Cas system is a negative regulator of all four operons, based on transcript measurements. Our results demonstrate complex regulation of sporulation genes by three transcription factors and a CRISPR‐Cas component, which we propose produces spores suited to withstand starvation and environmental insults. 

StarvingMyxococcus xanthusbacteria 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.