Temporal, genome-scale analysis of Myxococcus xanthus developmental fate in a mixed population

Myxococcus xanthus bacteria form aggregates when starved on solid surfaces and some cells differentiate into spores. Studies of mutants in monoculture have advanced knowledge of this multi-cellular developmental process, but our understanding of the genetic determinants is incomplete. To assess gene function genomewide, we generated a pool of barcoded transposon insertion mutants, subjected it to starvation, and separated developmental samples into non-aggregated cells, aggregated cells, and spores. We also subjected our pool to chemically-induced unicellular sporulation. Evaluation of changes in the abundance of mutants in samples allowed identification of 200 genes in which insertions reproducibly caused distinct patterns of depletion and/or accumulation over time. Many of these genes have well-established roles in development, validating our approach, while many others have not previously been associated with development. Genes involved in type IV pili (T4P)-dependent motility were more important than gliding motility genes for aggregation and sporulation in the mixed population. Although exopolysaccharide (EPS) synthesis genes are required for aggregation in monoculture, most were dispensable for aggregation in our pool, consistent with EPS sharing between cells, yet these genes were required cell-autonomously for efficient sporulation. Genes for positive regulators of EPS synthesis were important for aggregation as well as sporulation, suggesting functions beyond EPS production. Insertions in several novel genes impaired both starvation- and chemically-induced sporulation. Many genes increased the efficiency of starvation-induced sporulation. Some of these mutants, which we call "developmental winners", are novel cheaters. Our results demonstrate the power of using the newly-created mutant library to elucidate M. xanthus biology.

How cells coordinate their activities to build multicellular structures with differentiated cell types is a fundamental question in developmental biology. Starvation triggers thousands of M. xanthus cells to move coordinately and build mounds in which some cells differentiate into spores, while other cells lyse or persist as rods. We tracked a barcoded transposon mutant library through development with separation of sub-populations based on aggregation and sporulation fates. We discovered gene sets with distinct abundance profiles over time and across sub-populations. Sets contained both known and uncharacterized genes. For known genes, comparison of our results in a developmentally-competent mixture of mutants with published results for mutants in monoculture distinguished social from cell-autonomous functions. The novel genes provide numerous avenues toward deeper understanding of cellular interactions and differentiation. The mutant library offers a platform for further studies aimed at dissecting M. xanthus behaviors functionally, ecologically, and evolutionarily.