Wojnowska, M; (2013) Biochemical and structural characterisation of a two-component signalling system downstream of bacteriophytochrome photoreceptor 1 in Rhizobium NT-26. Doctoral thesis , UCL (University College London).

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Abstract

Prokaryotic signal transduction frequently involves two-component systems (TCSs), typically comprising a sensor histidine kinase (HK) and a response regulator (RR). Via a conserved phosphotransfer reaction, TCSs couple the detection of diverse stimuli with appropriate responses. The initial aim of this project was to characterise the two-component “signalome” of an arsenite oxidiser, Rhizobium NT-26, in the context of the environmental niche, and compare it to “signalomes” of other bacterial species. A light-sensing HK, bacteriophytochrome photoreceptor 1 (BphP1), was thus identified. Previous studies indicated that BphP1 and the members of its gene cluster - two single-domain RRs and a hybrid RR/HK protein, ExsG - may constitute a TCS. Functional characterisation revealed that BphP1 initiates a branched signalling pathway; however, the mediated output could not be identified. ExsG, a HWE-type HK, was shown to possess dual HK/RR activity and act as a novel type of signalling switch: phosphorylation of the N-terminal receiver domain downregulates the autokinase activity of the C-terminal kinase core. Ion mobility-mass spectrometry analysis indicated that while Asp62 phosphorylation stabilises the “closed” form of ExsG, nucleotide binding stabilises the “open” conformation. These observations led to a model in which phosphorylation of the receiver domain precludes ATP binding and thus inhibits autokinase activity. Furthermore, ExsG was demonstrated to hexamerise via the HWE core, which makes it the first non-dimeric HK. Notably, however, the basic unit of the hexameric assembly is a homodimer, and the HWE core shares homology with canonical HK cores. The results presented herein broaden the current knowledge on TCSs and identify previously unreported mechanisms involved in two-component signal transduction. The members of the BphP1 signalling cascade enrich the pool of modular communication units that can be exploited in engineering artificial signalling networks, biosensors and microorganisms with novel functionalities.

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