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. IMPORTANCE 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.
Sheenu Mittal, Saikat Mandal, Mark A. Farrugia et al.· bioRxiv· 0 citations
Bacterial gene expression depends on the coordinated regulation of RNA synthesis, processing, and decay. The conserved RNA degradosome scaffold, Ribonuclease E (RNase E), assembles into phase-separated bacterial ribonucleoprotein bodies (BR-bodies) through its C-terminal intrinsically disordered region (IDR). This IDR also scaffolds recruitment of degradosome client proteins that carry out post-transcriptional gene regulation. Whether BR-bodies regulate virulence programs to support infection, however, is largely undefined. We show that RNase E of the intracellular pathogen Brucella ovis forms RNA-dependent condensates in vivo and phase-separates with RNA in vitro, with the IDR necessary and sufficient for BR-body assembly. Deleting the IDR (rne(ΔIDR)) did not impair growth but sensitized Brucella to host-relevant oxidative and cell-envelope stressors. Transcriptome-wide profiling that simultaneously resolved mRNA decay and processing revealed that BR-bodies primarily accelerate mRNA turnover while stabilizing a distinct subset of transcripts. Notably, BR-bodies control the processing and levels of virB type IV secretion system (T4SS) mRNA and the turnover of its key activators, linking condensate-based RNA regulation to a core virulence pathway. Consistent with virB dysregulation, the rne(ΔIDR) mutant was severely attenuated in mammalian macrophages. To test whether phase separation is sufficient for BR-body function, we replaced the B. ovis IDR with the highly divergent Caulobacter crescentus IDR. This chimera assembled BR-bodies but rescued fitness incompletely, fully restoring oxidative-stress resistance but not cell-envelope stress resistance or intracellular fitness. BR-bodies therefore link RNA metabolism to Brucella stress resistance and infection, and their full function requires both phase separation and additional native IDR-specific activities such as degradosome interactions. Importance Biomolecular condensates are non-membrane bound organelles that organize biological processes across all domains of life, but their role in bacterial infection is not well characterized. We show that Brucella, an intracellular bacterial pathogen and causative agent of the disease brucellosis, relies on biomolecular condensates called BR-bodies to control RNA stability and regulate gene expression. Without BR-bodies, Brucella is sensitized to host-relevant chemical stresses, cannot properly regulate essential infection machinery, and has severely diminished fitness within mammalian host cells. These results indicate that control of RNA levels by biomolecular condensates is required for Brucella survival within animal hosts, providing evidence that phase separation is a fundamental mechanism underlying Brucella adaptation to the hostile environment encountered during infection.
Kaveendya S. Mallikaarachchi, Rosemary Northcote, Thomas Kim et al.· bioRxiv· 1 citation
