Transposon insertion site sequencing (TIS) is a powerful tool that has significantly advanced our knowledge of functional genomics. For example, TIS has been used to identify essential genes of Saccharomyces cerevisiae, screen for antibiotic resistance genes in Klebsiella pneumoniae and determine the set of genes required for virulence of Mycobacterium tuberculosis. While providing valuable insights, these applications of TIS focus on (conditional) gene essentiality and neglect possibly interesting but subtle differences in the importance of genes for fitness. Notably, it has been demonstrated that data obtained from TIS experiments can be used for fitness quantification and the construction of genetic interaction maps, but this potential is only sporadically exploited. Here, we present a method to quantify the fitness of gene disruption mutants using data obtained from a TIS screen developed for the yeast Saccharomyces cerevisiae called SATAY. We show that the mean read count per transposon insertion site provides a metric for fitness that is robust across biological and technical replicate experiments. Importantly, the ability to resolve differences between gene disruption mutants with low fitness depends crucially on the inclusion of insertion sites that are not observed in the sequencing data to estimate the mean. While our method provides reproducible results between replicate SATAY datasets, the obtained fitness distribution differs substantially from those obtained using other techniques. It is currently unclear whether these inconsistencies are due to biological or technical differences between the methods. We end with suggestions for modifications of the SATAY procedure that could improve the resolution of the fitness estimates. Our analysis indicates that increasing the sequencing depth does very little to reduce the uncertainty in the estimates, while replacing the PCR amplification with methods that avoid or reduce the number of amplification cycles will likely be most effective in reducing noise.

How can a self-organized cellular function evolve, adapt to perturbations, and acquire new sub-functions? To make progress in answering these basic questions of evolutionary cell biology, we analyze, as a concrete example, the cell polarity machinery of Saccharomyces cerevisiae. This cellular module exhibits an intriguing resilience: it remains operational under genetic perturbations and recovers quickly and reproducibly from the deletion of one of its key components. Using a combination of modeling, conceptual theory, and experiments, we propose that multiple, redundant self-organization mechanisms coexist within the protein network underlying cell polarization and are responsible for the module’s resilience and adaptability. Based on our mechanistic understanding of polarity establishment, we hypothesize that scaffold proteins, by introducing new connections in the existing network, can increase the redundancy of mechanisms and thus increase the evolvability of other network components. Moreover, our work gives a perspective on how a complex, redundant cellular module might have evolved from a more rudimental ancestral form.

Polarity establishment underlies proper cell cycle completion across virtually all organisms. Much progress has been made in generating an understanding of the structural and functional components of this process, especially in model species. Here we focus on the evolutionary dynamics of the fungal polarization protein network in order to determine general components and mechanistic principles, species- or lineage-specific adaptations and the evolvability of the network. The currently available genomic and proteomic screens in a variety of fungal species have shown three main characteristics: (1) certain proteins, processes and functions are conserved throughout the fungal clade; (2) orthologous functions can never be assumed, as various cases have been observed of homologous loci with dissimilar functions; (3) species have, typically, various species- or lineage-specific proteins incorporated in their polarization network. Further large-scale comparative and experimental studies, including those on non-model species representing the great fungal diversity, are needed to gain a better understanding of the evolutionary dynamics and generalities of the polarization network in fungi.