Biophysical studies on photoreceptors from plant-associated bacteria

The project here presented aims at understanding the photophysical and photochemical properties of: a. biliverding-binding phytochromes, i.e. photoreceptors for red light, from the plant-associated bacterial species Pseudomonas syringae pv tomato (Pst), Pseudomonas aeruginosa (Pa), Xanthomonas campestris pv. campestris (Xcc) and the fungus Aspergillus nidulans: b. photoreceptors for red and blue light from Methylobacterium radiotolerans (Mr), belonging to the plant microbiota. The former are important and well-studied pathogens of agronomic plants, while Mr is a methylotrophic phytosymbiont of great industrial and agronomical interest. This work starts with biliverdin-bindig photoreceptors, 1 as recent works have shown that the photoreceptors from Pst and Xcc play a role in controlling infectivity, virulence and invasiveness towards the model plant Arabidopsis thaliana and citrus plants. In the case of Mr, instead, the functional role of these photoactive proteins still awaits investigation, despite the finding that a genomic survey indicates that these organisms may possess quite a large number of potential and diverse photoreceptors for visible light. The proteins were studied at the molecular level with an array of biophysical techniques: primarily steady-state and time-resolved optical spectroscopy, then time-resolved photoacoustics. Beside the molecular and spectroscopic characterisation of these photoreceptors and their interest as photosensors in prokaryots, the blue-light photoreceptor Mr4511 showed a particular feature: in contrast to the majority of LOV domains, this protein lacks the, in other LOV photoreceptors highly conserved, tryptophan residue, which was previously identified as the major quencher for the FMN triplet-state in LOV-based singlet oxygen (SO) photosensitisers. This experimental work demonstrates that “for Mr4511 it is sufficient to only mutate the reactive cysteine responsible for the photocycle (Cys71) in the native protein to generate an efficient SO photosensitiser: both C71S and C71G variants exhibit SO quantum yields of formation ΦΔ around 0.2 in air-saturated solutions. Under oxygen saturated conditions, ΦΔ reaches ~ 0.5 in deuterated buffer. Also, this protein showed to be exceedingly robust against denaturation with urea and it is more photostable than free FMN.” 2 As a whole, future continuation on this work could reveal the novel potential of photoreceptors from bacteria that are part of plant microbiota, i.e. for environmental, agronomical and biotechnological applications.