Induction and relaxation dynamics of the regulatory network controlling the type III secretion system encoded within...

Bacterial pathogenesis requires the precise spatial and temporal control of gene expression, the dynamics of which are controlled by regulatory networks. A network encoded within Salmonella Pathogenicity Island 1 controls the expression of a type III protein secretion system involved in the invasion of host cells. The dynamics of this network are measured in single cells using promoter-green fluorescent protein (gfp) reporters and flow cytometry. During induction, there is a temporal order of gene expression, with transcriptional inputs turning on first, followed by structural and effector genes. The promoters show varying stochastic properties, where graded inputs are converted into all-or-none and hybrid responses. The relaxation dynamics are measured by shifting cells from inducing to noninducing conditions and by measuring fluorescence decay. The gfp expressed from promoters controlling the transcriptional inputs (hilC and hilD) and structural genes (prgH) decay exponentially, with a characteristic time of 50-55 min. In contrast, the gfp expressed from a promoter controlling the expression of effectors (sicA) persists for 110+/-9 min. This promoter is controlled by a genetic circuit, formed by a transcription factor (InvF), a chaperone (SicA), and a secreted protein (SipC), that regulates effector expression in response to the secretion capacity of the cell. A mathematical model of this circuit demonstrates that the delay is due to a split positive feedback loop. This model is tested in a DeltasicA knockout strain, where sicA is complemented with and without the feedback loop. The delay is eliminated when the feedback loop is deleted. Furthermore, a robustness analysis of the model predicts that the delay time can be tuned by changing the affinity of SicA:InvF multimers for an operator in the sicA promoter. This prediction is used to construct a targeted library, which contains mutants with both longer and shorter delays. This combination of theory and experiments provides a platform for predicting how genetic perturbations lead to changes in the global dynamics of a regulatory network.