Sabine Hansen:
A study of bacterial gene regulatory mechanisms

Date: 04-02-2013    Supervisor: Sine Lo Svenningsen

Bacterial cells are capable of rapidly changing their protein expression in response to ever-changing environments and physiological conditions. The cells are able to switch on the expression of proteins that due to changing environmental conditions have become vital to sustain life and likewise switch off the expression of unfavorable proteins. This dynamic regulation requires a coordinated effort by a network of regulatory factors. The regulatory mechanisms employed by bacterial cell to regulate their protein expression have been extensively studied. However, little is known about how the different regulatory mechanisms affect system dynamics. We have designed a synthetic gene regulatory network (GRN) in bacterial cells that enables us to study the dynamics of GRNs. The results presented in this PhD thesis show that model equations based on the established mechanisms of action of each regulator studied accurately reproduced the experimental data. Simulations of system dynamics reveals that even two step regulatory cascades significantly increase response times compared to direct allosteric regulation of a transcription factor. It is observed that while many system behaviors, such as steady-state target levels, can be obtained from different types of regulatory mechanisms by tweaking key parameters such as regulator production rate and target affinity, some system properties, such as ultrasensitive responses and ultrafast turn-on and turn-off dynamics require the employment of a particular type of regulatory mechanism. The synthetic system presented in this thesis is, to our knowledge, the first of its kind to allow a direct comparison of the dynamic behaviors of gene regulatory networks that employ different mechanisms of regulation.

In addition to studying the dynamic behavior of GRNs this thesis also provided the first evidence of the sensor histidine kinase VC1831 being an additional player in the Vibrio cholerae quorum sensing (QS) GRN. Bacteria use a process of cell-cell communication called QS which enable the bacterial cells to collectively control their gene expression using small signaling molecules called autoinducers, thereby coordinating group behavior. At the heart of the V. cholerae QS response lie four small RNA (sRNA) molecules called the quorum regulatory RNAs (Qrr). This PhD thesis provides evidence that the sensor histidine kinase VC1831 is regulated by the Qrr sRNAs. It is further shown that VC1831 feeds back to activate the expression the Qrrs, presumably via phosphorylation of LuxU. Thus, VC1831, which responds to an unknown ligand, is a new player in the V. cholerae QS response. Prior to this report, the two autoinducer sensors CqsS and LuxQ were the only histidine kinases known to have the ability to activate Qrr expression.