Induction and Relaxation Dynamics of the Regulatory Network Controlling the Type III Secretion System Encoded within Salmonella Pathogenicity Island 1

Abstract

SummaryBacterial 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 promotergreen 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 structure, 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 into non-inducing conditions and measuring the 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 minutes. In contrast, the gfp expressed from a promoter controlling the expression of effectors (sicA) persists for 110 ± 9 minutes. This promoter is controlled by a genetic circuit formed by a transcription factor (InvF), chaperone (SicA) and 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 ΔsicA knockout where sicA is complemented with and without the feedback loop. The delay is eliminated when the feedback loop is deleted. Further, a robustness analysis of the model predicts that the delay time can be tuned by changing the affinity of SicA:InvF multimers to 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 to predict how genetic perturbations lead to changes in the global dynamics of a regulatory network.

Localization of Proteins to the 1,2-Propanediol Utilization Microcompartment by Non-native Signal Sequences Is Mediated by a Common Hydrophobic Motif

“…We measured the response of each reporter to the presence of 1,2-propanediol, to the presence of ethanolamine and vitamin B 12 , and to neither or both conditions by sampling cultures grown in appropriate media hourly and interrogating the samples by flow cytometry (20). The P pdu reporter showed activation in response to 55 mM 1,2-propanediol but not in response to 30 mM ethanolamine and 150 nM vitamin B 12 as expected ( Fig.…”

“…Flow Cytometry-Cultures were grown as described above, and at the indicated time points, aliquots of the samples were diluted to an A 600 of ϳ0.01 in phosphate-buffered saline (PBS) supplemented with 2 g/liter kanamycin (to halt translation) and stored at 4°C (20). Samples were then diluted 1:40 into PBS supplemented with 2 g/liter kanamycin in 96-well plates for flow cytometry.…”

“…To observe Eut MCP formation, we used a fluorescent reporter of encapsulation in which EutC 1-20 is fused to GFP followed by a C-terminal ssrA tag, which mediates degradation of the fluorophore by the ClpXP protease in the cytosol (encapsulation in an MCP thus rescues the reporter from proteolysis). When expression of the encapsulation reporter construct pBADeutC [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] -gfp-ssrA was induced by the addition of 0.02% arabinose, fluorescent puncta were not observed in a ⌬eutR::FRT strain upon the addition of 30 mM ethanolamine and 150 nM vitamin B 12 , but puncta were observed in the ⌬pocR::FRT strain under the same conditions (Figs. 2 and 3).…”

“…3). A Western blot against GFP of purified Pdu MCPs from S. enterica LT2 ⌬eutR::FRT expressing EutC [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] -GFP-ssrA and EutE [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] -GFP-ssrA confirmed that these two Eut signal sequences target GFP to the Pdu MCP (Fig. 4).…”

“…5). The assay using PduP 1-20 -GFP-ssrA was somewhat less sensitive likely due to lower expression of the PduP 1-20 -tagged construct, but significant competition (p Ͻ 0.05) was still observed for EutC [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and for the positive control PduD 1-224 -FLAG. Flow cytometry results were confirmed for EutC 1-20 -PduD 21-224 -FLAG by a Western blot of purified Pdu MCPs and cell lysates against GFP and against the FLAG epitope, which shows the same trends as the flow cytometry data (Fig.…”