Our results in wild type Salmonella indicate that, when grown in conditions that mimic those inside the small intestine - the flagellar, SPI1, and Type I fimbriae systems are expressed sequentially. Next, we wanted to study if the three systems control the dynamics of expression of each other or not. Many reports have shown the regulatory links between the flagellar, SPI1, and Type I fimbriae networks. Therefore, we wanted to investigate the impact of this regulation on the timing of expression of these systems.
141 We studied gene expression dynamics in mutants where one of the three systems was knocked out. Specifically, we monitored the PflgA, PhilA, and PfimA promoter activities in an
∆flhDC mutant, a ∆SPI1 mutant, and a ∆fimYZ mutant.
Our results show that absence of one of the three systems has an impact on the timing of expression of all three systems. In particular, we note that in a ∆flhDC mutant, the PflgA promoter was inactive, the PhilA promoter dynamics were slower, and the PfimA
promoter activity “off” to “on” transition was faster as compared to the wild type
(Figure 34A). In a ∆SPI1 mutant, the PflgA promoter’s “on” to “off” transition was
delayed, the PhilA promoter was inactive, and the PfimA promoter activation was delayed
(Figure 34B). Finally, in a ∆fimYZ mutant, there was no impact on flagellar gene
expression dynamics, the “on” to “off” step for the PhilA promoter activity was delayed,
and the PfimA promoter activity was about 70-80% lower than the wild type (Figure 34C).
These results clearly demonstrate that apart from being expressed at different times in the growth phase, the timing of expression of the flagellar, SPI1, and Type I fimbriae systems is also controlled by the cross-talk between the three systems. In this report, we focus on the role of FliZ, RtsB, and FimZ in conducting this cross-talk and orchestrating the dynamics of gene expression in the three systems. In addition, we also wanted to test the impact of the three systems on end-point expression of each other.
142
FliZ-, RtsB-, and FimZ-Mediated Cross-Talk Controls Fla, SPI1, and Fim
Levels
FliZ from the flagellar network is a post-translational activator of the flagellar master regulator FlhD4C2 complex. In addition, FliZ is also known to be a positive regulator of the SPI1-encoded genes via an unknown mechanism. To understand the effect of FliZ on flagellar, SPI1, and fimbrial gene expression, we measured end-point expression levels of the PflgA, PhilA, and PfimA promoter activity in wild type, a ΔfliZ mutant
and a ΔfliZ mutant constitutively expressing FliZ from an inducible PLTetO-1 promoter. Our results indicate that deletion of fliZ leads to about a two-fold decrease in PflgA and PhilA
promoter activity as compared to the wild type. On the other hand, deletion of fliZ leads to an approximately thirty percent increase in PfimA promoter activity. Consistent with
these results, over-expression of FliZ from a high-copy plasmid resulted in a roughly two-fold increase in the flagellar and SPI1 network gene expression and about a two- fold decrease in the Type I fimbriae network gene expression (Figure 35A).
Next, we studied the impact of RtsB on gene expression of the three systems. RtsB - encoded in the same operon as the SPI1 regulator, RtsA - binds to the flagellar class 1 promoter, PFlhD4C2 promoter, and represses activation of the flagellar cascade.
The PrtsA promoter, which controls RtsB expression, is activated by SPI1 regulators HilD,
HilC, and RtsA. Therefore, cues that turn on SPI1 gene expression also trigger RtsB expression. Deletion of rtsB resulted in a roughly 40% increase in the PflgA promoter
143 Interestingly, while knocking out rtsB was found to have minimal impact on SPI1 and Type I fimbriae gene expression, over-expression of RtsB resulted in roughly 40% decrease and increase in the SPI1 and Type I fimbriae activity, respectively (Figure 35B).
The Type I fimbriae regulator, FimZ, in conjunction with another regulator in the fimbriae network, FimY, activates transcription from the PfimA promoter. The PfimA
promoter controls expression of six genes which form the export apparatus and the structural components of the Type I fimbriae. In addition, FimZ is also known to activate the PhilE promoter. The resulting HilE, then, binds to HilD and prevents HilD-dependent
activation of SPI1 genes. FimZ is also known to bind to the PFlhD4C2 promoter and repress
expression of the flagellar genes. Consistent with these previous results, deletion and over-expression of FimZ resulted in an increase and decrease of flagellar and SPI1 gene expression, respectively. In addition, consistent with previous reports, deletion of FimZ leads to an approximately 70% decrease in the PfimA promoter activity (Figure 35C).
While the characterization of the molecular interactions of FliZ, RtsB, and FimZ in these three systems has been a subject of a large number of studies, little is known about the role of these cross-talk interactions in controlling the dynamics of gene expression. With this understanding of the infection process, we hypothesized that FliZ-, RtsB-, and FimZ-mediated cross-talk interactions serve to fine-tune the timing of activation and deactivation of the three systems. Therefore, we next systematically studied the dynamics of gene expression in the three systems in different regulatory
144 mutants where the FliZ-, RtsB-, and FimZ-mediated cross-talk elements have been altered.