Supreet Saini

The role of coupled positive feedback in the expression of the SPI1 type three secretion system in Salmonella.

Salmonella enterica serovar Typhimurium is a common food-borne pathogen that induces inflammatory diarrhea and invades intestinal epithelial cells using a type three secretion system (T3SS) encoded within Salmonella pathogenicity island 1 (SPI1). The genes encoding the SPI1 T3SS are tightly regulated by a network of interacting transcriptional regulators involving three coupled positive feedback loops. While the core architecture of the SPI1 gene circuit has been determined, the relative roles of these interacting regulators and associated feedback loops are still unknown. To determine the function of this circuit, we measured gene expression dynamics at both population and single-cell resolution in a number of SPI1 regulatory mutants. Using these data, we constructed a mathematical model of the SPI1 gene circuit. Analysis of the model predicted that the circuit serves two functions. The first is to place a threshold on SPI1 activation, ensuring that the genes encoding the T3SS are expressed only in response to the appropriate combination of environmental and cellular cues. The second is to amplify SPI1 gene expression. To experimentally test these predictions, we rewired the SPI1 genetic circuit by changing its regulatory architecture. This enabled us to directly test our predictions regarding the function of the circuit by varying the strength and dynamics of the activating signal. Collectively, our experimental and computational results enable us to deconstruct this complex circuit and determine the role of its individual components in regulating SPI1 gene expression dynamics.

Role of Cross Talk in Regulating the Dynamic Expression of the Flagellar Salmonella Pathogenicity Island 1 and Type 1 Fimbrial Genes

Salmonella enterica, a common food-borne pathogen, differentially regulates the expression of multiple genes during the infection cycle. These genes encode systems related to motility, adhesion, invasion, and intestinal persistence. Key among them is a type three secretion system (T3SS) encoded within Salmonella pathogenicity island 1 (SPI1). In addition to the SPI1 T3SS, other systems, including flagella and type 1 fimbriae, have been implicated in Salmonella pathogenesis. In this study, we investigated the dynamic expression of the flagellar, SPI1, and type 1 fimbrial genes. We demonstrate that these genes are expressed in a temporal hierarchy, beginning with the flagellar genes, followed by the SPI1 genes, and ending with the type 1 fimbrial genes. This hierarchy could mirror the roles of these three systems during the infection cycle. As multiple studies have shown that extensive regulatory cross talk exists between these three systems, we also tested how removing different regulatory links between them affects gene expression dynamics. These results indicate that cross talk is critical for regulating gene expression during transitional phases in the gene expression hierarchy. In addition, we identified a novel regulatory link between flagellar and type 1 fimbrial gene expression dynamics, where we found that the flagellar regulator, FliZ, represses type 1 fimbrial gene expression through the posttranscriptional regulation of FimZ. The significance of these results is that they provide the first systematic study of the effect of regulatory cross talk on the expression dynamics of flagellar, SPI1, and type 1 fimbrial genes.

FliZ Is a Posttranslational Activator of FlhD4C2-Dependent Flagellar Gene Expression

Flagellar assembly proceeds in a sequential manner, beginning at the base and concluding with the filament. A critical aspect of assembly is that gene expression is coupled to assembly. When cells transition from a nonflagellated to a flagellated state, gene expression is sequential, reflecting the manner in which the flagellum is made. A key mechanism for establishing this temporal hierarchy is the sigma(28)-FlgM checkpoint, which couples the expression of late flagellar (P(class3)) genes to the completion of the hook-basal body. In this work, we investigated the role of FliZ in coupling middle flagellar (P(class2)) gene expression to assembly in Salmonella enterica serovar Typhimurium. We demonstrate that FliZ is an FlhD(4)C(2)-dependent activator of P(class2)/middle gene expression. Our results suggest that FliZ regulates the concentration of FlhD(4)C(2) posttranslationally. We also demonstrate that FliZ functions independently of the flagellum-specific sigma factor sigma(28) and the filament-cap chaperone/FlhD(4)C(2) inhibitor FliT. Furthermore, we show that the previously described ability of sigma(28) to activate P(class2)/middle gene expression is, in fact, due to FliZ, as both are expressed from the same overlapping P(class2) and P(class3) promoters at the fliAZY locus. We conclude by discussing the role of FliZ regulation with respect to flagellar biosynthesis based on our characterization of gene expression and FliZs role in swimming and swarming motility.

Role of FimW, FimY, and FimZ in regulating the expression of type i fimbriae in Salmonella enterica serovar Typhimurium.

Type I fimbriae in Salmonella enterica serovar Typhimurium are surface appendages that facilitate binding to eukaryotic cells. Expression of the fim gene cluster is known to be regulated by three proteins--FimW, FimY, and FimZ--and a tRNA encoded by fimU. In this work, we investigated how these proteins and tRNA coordinately regulate fim gene expression. Our results indicate that FimY and FimZ independently activate the P(fimA) promoter which controls the expression of the fim structural genes. FimY and FimZ were also found to strongly activate each others expression and weakly activate their own expression. FimW was found to negatively regulate fim gene expression by repressing transcription from the P(fimY) promoter, independent of FimY or FimZ. Moreover, FimW and FimY interact within a negative feedback loop, as FimY was found to activate the P(fimW) promoter. In the case of fimU, the expression of this gene was not found to be regulated by FimW, FimY, or FimZ. We also explored the effect of fim gene expression on Salmonella pathogenicity island 1 (SPI1). Our results indicate that FimZ alone is able to enhance the expression of hilE, a known repressor of SPI1 gene expression. Based on our results, we were able to propose an integrated model for the fim gene circuit. As this model involves a combination of positive and negative feedback, we hypothesized that the response of this circuit may be bistable and thus a possible mechanism for phase variation. However, we found that the response was continuous and not bistable.

The interaction dynamics of a negative feedback loop regulates flagellar number in Salmonella enterica serovar Typhimurium.

Each Salmonella enterica serovar Typhimurium cell produces a discrete number of complete flagella. Flagellar assembly responds to changes in growth rates through FlhD4C2 activity. FlhD4C2 activity is negatively regulated by the type 3 secretion chaperone FliT. FliT is known to interact with the flagellar filament cap protein FliD as well as components of the flagellar type 3 secretion apparatus. FliD is proposed to act as an anti‐regulator, in a manner similar to FlgM inhibition of σ28 activity. We have found that efficient growth‐dependent regulation of FlhD4C2 requires FliT regulation. In turn, FliD regulation of FliT modulates the response. We also show that, unlike other flagellar‐specific regulatory circuits, deletion of fliT or fliD did not lead to an all‐or‐nothing response in FlhD4C2 activity. To investigate why, we characterized the biochemical interactions in the FliT : FliD :  FlhD4C2 circuit. When FlhD4C2 was not bound to DNA, FliT disrupted the FlhD4C2 complex. Interestingly, when FlhD4C2 was bound to DNA it was insensitive to FliT regulation. This suggests that the FliT circuit regulates FlhD4C2 activity by preventing the formation of the FlhD4C2:DNA complex. Our data would suggest that this level of endogenous regulation of FlhD4C2 activity allows the flagellar system to efficiently respond to external signals.

The rate of protein secretion dictates the temporal dynamics of flagellar gene expression

Flagellar gene expression is temporally regulated in response to the assembly state of the growing flagellum. The key mechanism for enforcing this temporal hierarchy in Salmonella enterica serovar Typhimurium is the σ28‐FlgM checkpoint, which couples the expression of the late flagellar (Pclass3) genes to the completion of the hook–basal body. This checkpoint is triggered when FlgM is secreted from the cell. In addition to the σ28‐FlgM checkpoint, a number of other regulatory mechanisms respond to the secretion of late proteins. In this work, we examined how middle (Pclass2) and late (Pclass3) gene expression is affected by late protein secretion. Dynamic analysis of flagellar gene expression identified a novel mechanism where induction of Pclass2 activity is delayed either when late protein secretion is abolished or when late protein secretion is increased. Using a number of different approaches, we were able to show that this mechanism did not involve any known flagellar regulator. Furthermore, the changes in Pclass2 activity were not correlated with the associated changes in Pclass3 activity, which was found to be proportional to late protein secretion rates. Our data indicate that both Pclass2 and Pclass3 promoters are continuously regulated in response to assembly and late protein secretion rates. These results suggest that flagellar regulation is more complex than previously thought.

FliZ Induces a Kinetic Switch in Flagellar Gene Expression

FliZ is an activator of class 2 flagellar gene expression in Salmonella enterica. To understand its role in flagellar assembly, we investigated how FliZ affects gene expression dynamics. We demonstrate that FliZ participates in a positive-feedback loop that induces a kinetic switch in class 2 gene expression.

Role of extracellular cues to trigger the metabolic phase shifting from acidogenesis to solventogenesis in Clostridium acetobutylicum.

Clostridium acetobutylicum exhibits a two-step metabolic pathway where substrates are first converted to organic acids accompanied by a decrease in pH. The acids are then assimilated to organic solvents. The transition from the acid-producing (acidogenesis) to the solvent-producing phase (solventogenesis) is controlled by integration of a number of cellular and environmental cues, whose precise mode of action are not well understood. In this study, a series of batch experiments were performed to understand the impact of extracellular cues in regulating the dynamics of acidogenesis and solventogenesis. It is demonstrated that the two phases operate independently of each other and the growth phase of the cell, i.e. the cues controlling a phase are not linked to the status of the other phase or the growth phase of the cell. Kinetic experiments demonstrated that there exist two previously uncharacterized negative feedback loops controlling the amounts of acids produced in the acidogenesis phase.

SprB Is the Molecular Link between Salmonella Pathogenicity Island 1 (SPI1) and SPI4

Salmonella pathogenicity island 1 (SPI1) and SPI4 have previously been shown to be jointly regulated. We report that SPI1 and SPI4 gene expression is linked through a transcriptional activator, SprB, encoded within SPI1 and regulated by HilA. SprB directly activates SPI4 gene expression and weakly represses SPI1 gene expression through HilD.

Continuous control of flagellar gene expression by the σ28-FlgM regulatory circuit in Salmonella enterica.

The flagellar genes in Salmonella enterica are expressed in a temporal hierarchy that mirrors the assembly process itself. The σ28–FlgM regulatory circuit plays a key role in controlling this temporal hierarchy. This circuit ensures that the class 3 genes are expressed only when the hook‐basal body (HBB), a key intermediate in flagellar assembly, is complete. In this work, we investigated the role of the σ28–FlgM regulatory circuit in controlling the timing and magnitude of class 3 gene expression using a combination of mathematical modelling and experimental analysis. Analysis of the model predicted that this circuit continuously controls class 3 gene expression in response to HBB abundance. We experimentally validated these predictions by eliminating different components of the σ28–FlgM regulatory system and also by rewiring the transcriptional hierarchy. Based on these results, we conclude that the σ28–FlgM regulatory circuit continuously senses the HBB assembly process and regulates class 3 gene expression and possibly flagellar numbers in response.