The soil bacterium Myxococcus xanthus lives in densely packed groups that form dynamic three-dimensional patterns in response to environmental changes, such as droplet-like fruiting bodies during starvation1. The development of these multicellular structures begins with the sequential formation of cell layers in a process that is poorly understood2. Here, using confocal three-dimensional imaging, we find that motile, rod-shaped M. xanthus cells are densely packed and aligned in each layer, forming an active nematic liquid crystal. Cell alignment is nearly perfect throughout the population except at point defects that carry half-integer topological charge. We observe that new cell layers preferentially form at the position of +1/2 defects, whereas holes preferentially open at −1/2 defects. To explain these findings, we model the bacterial colony as an extensile active nematic fluid with anisotropic friction. In agreement with our experimental measurements, this model predicts an influx of cells towards the +1/2 defects and an outflux of cells from the −1/2 defects. Our results suggest that cell motility and mechanical cell–cell interactions are sufficient to promote the formation of cell layers at topological defects, thereby seeding fruiting bodies in colonies of M. xanthus.