Feedback control of the pleiotropic regulators and H-NS antagonists LeuO and RcsB-BglJ in Escherichia coli
Final Report Abstract
LeuO and BglJ-RcsB are pleiotropic transcription regulators and H-NS antagonists in Escherichia coli. However, the leuO and bglJ genes are silenced by H-NS (and StpA) at standard laboratory growth conditions and signals that activate their expression remain elusive. In this project we addressed several aspects of leuO and bglJ regulation we well as LeuO and BglJ protein function. One focus was on the regulation of leuO and bglJ expression. Intriguingly, BglJ-RcsB activates leuO and vice versa LeuO activates bglJ, while LeuO in addition autoregulates leuO and inhibits activation of leuO by BglJ-RcsB. This control resembles a double-positive feedback loop that is interlocked with a negative feedback. This network motif may be stable in the OFF state, triggered by H-NS (and StpA) mediated repression, or exhibit bi-stability. Our characterization of antagonistic regulation of the leuO promoter by BglJ-RcsB and LeuO revealed that its dependence on the relative amount of BglJ and LeuO is in accordance with a straightforward model of independent regulation by BglJ-RcsB and by LeuO. The leuO promoter activity is not affected by the presence or absence of feedback elements, such as the native leuO and/or bglJ loci, indicating the double-positive feedback regulation is of minor relevance at least at laboratory conditions. In a screen for signals that may de-silence leuO or bglJ, we identified and characterized an additional transcription regulator that activates leuO transcription. Further, we found that terminator Rho and Rhoinhibitor Rof are involved in co-transcriptional control of yjjQ-bglJ expression. In further approaches to characterize the pleiotropic regulators LeuO and BglJ, we focused on the LeuO protein function and on defining the BglJ-RcsB regulon. For LeuO, which is a LysR-type regulator, which are in general controlled by a co-effector, we established a sensitive cas promoter based reporter fusion, and we used this reporter to identify and characterize hyperactive LeuO mutants. In addition, we solved the crystal structure of the putative LeuO effector-binding domain of the wildtype and one hyperactive mutant. Interestingly, all residues mutated in hyper-active LeuO map to the dimerization interface of the effector binding domain and thus may stabilize the dimer. These data suggest that a putative co-effector may act similarly by stabilization the effector-binding domain dimer. For BglJ-RcsB we characterized the mechanism of regulation of several target promoters and we found that BglJ-RcsB acts both as a canonical transcription regulator that presumably recruits RNA polymerase and as an H-NS antagonist. The analysis of the BglJ as auxiliary regulator of RcsB was complemented by the identification and characterization of two additional auxiliary regulators of RcsB, which are MatA (= EcpR) and DctR. The data add a further level of control at the output level to the complex Rcs signaling phosphorelay.
Publications
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(2013). RcsB- BglJ-mediated activation of Cascade operon does not induce the maturation of CRISPR RNAs in E. coli K12. RNA Biology 10, 708–715
Arslan, Z., Stratmann, T., Wurm, R., Wagner, R., Schnetz, K., and Pul, Ü.
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(2014). Transcriptional regulation by BglJ– RcsB, a pleiotropic heteromeric activator in Escherichia coli. Nucleic Acids Res 42, 2999-3008
Salscheider, S.L., Jahn, A., and Schnetz, K.
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(2015). YjjQ represses transcription of flhDC and additional loci in Escherichia coli. J Bacteriol 197, 2713-2720
Wiebe, H., Gürlebeck, D., Groß, J., Dreck, K., Pannen, D., Ewers, C., Wieler, L.H., and Schnetz, K.
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(2016). Interaction of the RcsB response regulator with auxiliary transcription regulators in Escherichia coli. J Biol Chem 291, 2357-2370
Pannen, D., Fabisch, M., Gausling, L., and Schnetz, K.
