An integrated analysis of structural organization, design principles, and evolution across multiple genomes of a model developmental network
Richard Losick and Dennis Vitkup
The molecular network controlling spore formation by bacteria provides a unique model for understanding the systems biology of developmental organization, transcriptional regulation, and evolution. The functional organization of the sporulation network is also of the major public health and bio-defense importance due to its central role in the virulence of B. anthracis, a close relative of Bacillus subtilis. We have studied the developmental network underlying sporulation in B. subtilis, which includes more than 500 genes and 20 known regulators. We used flow cytometry to track individual cells to trace the origins of varying sporulation response across the population. In another project, we examined how evolution has shaped various parts of the sporulation network. We found that the evolution of various genes depends strongly on their role in the network.
Spore formation in B. subtilis is an ideal model system for investigating the role of regulatory networks in shaping cell development. The master regulator of sporulation is Spo0A, which is activated by phosphorylation via a multicomponent phosphorelay comprising three positive feedback loops. SpoOA is also essential for controlling bacterial cannibalism and biofilm formation. Yet, the ultimate decision to sporulate is stochastic, in that only a portion of the population sporulates, even under optimal conditions and even though the bacteria are genetically identical.
We used fluorescence-activated cell sorting (FACS) to build a computational model for the Spo0A regulatory network and the Gillespie algorithm to model the system using stochastic equations. Activation of Spo0A, and thus sporulation, was thought to be controlled by a bi-stable switch, mediated by one or more of the feedback loops. Based on experimental and computational modeling results, we concluded that genes under the control of Spo0A do not exhibit a bimodal pattern of expression, as expected for a bi-stable switch. Rather, we observed a highly heterogeneous pattern of Spo0A activation, increasing non-linearly with time. In contrast to a classical bistable switch, the rate-limiting factor in the Spo0A network is not the transcription-factor concentration but the phosphate flux. This ensures that en route to committing to sporulation, B. subtilis constantly monitors environmental conditions through the persistence of this flux. The cells enter sporulation only if adverse environmental conditions persist (M. Fujita, in preparation).
We also used the extensively characterized network regulating sporulation to study the evolution of biological networks in a range of endospore-forming bacteria which diverged as long as a billion years ago. Our analysis shows that evolution of gene regulation in the sporulation network proceeds more slowly than evolution of gene presence. This observation contrasts with higher organisms, in which genes tend to be conserved between species, and differences in regulation are believed to be primarily responsible for phenotypic differences. Importantly, the evolutionary pattern of gene and regulatory conservation follow the structural hierarchy of the sporulation system well-established in B. subtilis . Specifically, the modular organization and related sequential activation of the modules is conserved in all considered species. The feed-forward loops and corresponding regulatory interactions are less conserved as these motifs are likely to fine-tune the waves of gene expression.
The network-specific essentiality of a gene proved to be a particularly strong predictor of its conservation. We found that both gene presence and gene regulation were more strongly conserved for essential genes than for non-essential genes. The regulatory interactions tend to be particularly conserved for genes whose presence is highly conserved across organisms. However, genes encoding spore coat proteins are an exception, and vary widely between organisms.