Studi biofisici su fotorecettori da batteri associati alle piante

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

BLUE LIGHT PHOTORECEPTORS FROM PLANT SYMBIOTIC BACTERIA

In recent years novel and largely unforeseen biological photoreceptors have been discovered in many bacteria, in most cases with poorly understood in vivo functions. Bacterial photoreceptors mainly belong to two superfamilies: blue light (BL) sensing LOV proteins and red/far red (R/FR) light sensing bacteriophytochromes (BphP), binding respectively flavin mononucleotide (FMN) and biliverdin-IXa (BV) as chromophores. LOV proteins and phytochromes are also the main photoreceptors of plants, and it is clear that many bacteria that are plant pathogens or symbionts are able to detect the same colors as their natural host. In this paper we will present the biophysical characterization of novel BL receptors from Methylobacterium radiotolerans, a radiation resistant, nitrogen fixing bacterium, able to promote plant growth and grow facultatively on methanol (2). In addition, M. radiotolerans is an opportunistic human pathogen and has a high potential for being employed in soil bioremediation. As other Methylobacteria, M. radiotolerans bears genes for several BphP and BL receptors, that we started to investigate during the last months by means of steady state and time-resolved spectroscopy. In particular we focus here on a LOV protein that show high structural stability and an extremely long photocycle in its wild type form. Sequence analysis revealed some peculiarities with respect to the majority of LOV domains; point mutations evidenced that this M. radiotolerans LOV photoreceptor is a promising candidate for biophysical applications, chiefly as fluorescent reporter and as genetically encoded photosensitizer (3). The possible roles of photoreceptors in the physiology of M. radiotolerans is also discussed, on the basis of bioinformatics analysis

Biophysical studies on bacterial biliverdin-binding photoreceptors

Bacterial photoreceptors binding open-chain tetrapyrroles (bilins) as chromophores are related to plant phytochromes (phy) as they are photochromic and their primary photochemistry consists of a Z/E isomerisation around the bilin 15=16 double bond. The chromophore is embedded in all cases within a so-called GAF domain with a typical α/β fold. Different to the canonical plant phys which invariably bind phytochromobilin and switch between a red (R) and a far red (FR) absorbing form, the bacterial bilin-binding photoreceptors exhibit a much wider variety of spectroscopic and functional properties, and bind diverse bilin chromophores, e.g., phycocyanobilin (PCB) and biliverdin (BV). In particular, BV-binding photoreceptors present the most red-shifted spectrum, reaching the near infra-red (NIR) range in the photoactive form. This makes these phytochromes very well suited for biomedical applications (1). Here we report steady-state and time-resolved spectroscopic measurements on selected bacterial BV-binding photoreceptors, representatives for four variations of this photoreceptor family: a. a phy and a bathy-phy from Pseudomonas strains with R/FR photochromism; b. a “bacterio” phytochrome from the fungus Aspergillus nidulans, a eukaryotic organism with photochemistry akin to the Pseudomonas syringae protein; c. a novel phy from Methylobacterium radiotolerans with FR/NIR photochromism. In particular, nanosecond time-resolved absorption spectroscopy has revealed kinetics and spectral features of transient species after photoactivation for both the directions of conversion: the conversions of all these BV-phytochromes in the time range 1 μs – 400 ms seem to be more simple than those from plant phytochromes (oat phyA) or from cyanobacteria (Cph1, CphA) (2)(3), in some cases travelling through only one observable intermediate in the R to FR conversion. References (1) Chernov, K.G. et al. (2017) Chem. Rev. 117 6423-6446. (2) Gärtner, W. and Braslavsky, S.E. (2003) In: Photoreceptors and light signalling, Batschauer, A. (ed.). Compr. Series Photochem. Photobiol. Sci., Vol. 3, Batschauer, A. (ed.), Häder, D.-P. and Jori, G. (series eds.), Royal Soc. Chemistry, Cambridge, UK, pp. 136- 180. (3) Remberg, A. et al. (1997) Biochemistry 36 13389-13395

The first molecular characterisation of blue- and red-light photoreceptors from Methylobacterium radiotolerans

Methylobacteria are facultative methylotrophic phytosymbionts of great industrial and agronomical interest, and are considered as opportunistic pathogens a health threat for humans. So far only few reports mention photoreceptors coding sequences in Methylobacteria genomes, but no investigation at the molecular level has been performed yet. We here present a comprehensive in silico research of potential photoreceptors in this bacterial phylum and report photophysical and photochemical characterisation of two representatives of the most wide-spread photoreceptor classes, a blue-light sensing LOV (Light, Oxygen, Voltage) protein and and red/far red light sensing BphP (biliverdin-binding bacterial phytochrome) from M. radiotolerans JCM 2831. Overall, both proteins undergo the expected light-triggered reactions, but peculiar features were also identifed. The LOV protein Mr4511 has an extremely long photocycle and lacks a tryptophan conserved in ca. 75% of LOV domains. Mutation I37V accelerates the photocycle by one order of magnitude, while the Q112W change underscores the ability of tryptophan in this position to perform efficient energy transfer to the flavin chromophore. Time-resolved photoacoustics experiments showed that Mr4511 has a higher triplet quantum yield than other LOV domains and that formation of the photoproduct results in a volume expansion, in sharp contrast to other LOV proteins. Mr4511 was found astonishingly resistant to denaturation by urea, still showing the light-triggered reactions after incubation in urea for more than 20 h. The phytochrome MrBphP1 exhibits the so far most red-shifted absorption maxima for its Pr- and Pfr forms (λmax = 707 nm, 764 nm for the Pr, Pfr-forms). The light-driven conversions in both directions occur with relatively high quantum yields of 0.2. Transient ns absorption spectroscopy (μs-ms time range) identifies the decay of the instantaneously formed lumi-intermediate, followed by only one additional intermediate before formation of the respective final photoproduct for Pr-to-Pfr or Pfr-to-Pr photoconversion, in contrast to other BphPs. The relatively simple photoconversion patterns suggest the absence of shunt pathways reported for other bacteria

Kinetics of conversion between Pr and Pfr states in three prototypical bacteriophytochromes

Bacteriophytochromes (PphPs) show strong structural similarity to canonical phytochromes, however, they employ as chromophore biliverdin IX instead of phytochromobilin (plant phytochromes) or phycocyanobilin (cyanobacterial phytochromes). This change of chromophore shifts the absorption maxima – compared to those of the plant phytochromes – for both Pr and Pfr state further into the red- / far red range of the spectrum (to 700 and 750 nm, respectively), and thereby makes these phytochromes very well suited for biomedical applications (1). Both photochromic states are reported, however, the dynamics of laser flash-induced conversion between both states has not been studied in detail. Here, we present the kinectics of Pr-to-Pfr conversion (and vice versa) for three prototypal bacteriophytochromes, as such phy1 from Pseudomonas (P.) syringae pv. tomato (PstBphP1), phytochrome from P. aeruginosa (PaBphP), and phytochrome from the fungus Aspergillus nidulans (FphA). Whereas the phytochrome from P. syringae and ist fungal ortholog are formed biosynthetically in the Pr state with absorption maxima at 700 nm, the phytochrome from P. aeruginosa is generated in its Pfr state (750 nm), thus being called a bathy-phytochrome. The conversions between both states of all three bacteriophytochromes in the time range of ca. 1 us up to 20 ms are much more simple than comparable kinetics found for canonical phytochromes from plants (oat phyA) or from cyanobacteria (Cph1, CphA, 2,3). Whereas in the latter proteins a sequence of intermediates can be clearly identified, the bacteriophytochromes run through one intermediate, or in some cases a direct formation of the final photoproduct is immediately observed. 1) Chernov, K.G. et al. (2017) Chem. Rev. 117 6423-6446. 2) Gärtner, W. and Braslavsky, S.E. (2003) In: Photoreceptors and light signalling, Batschauer, A. (ed.). Compr. Series Photochem. Photobiol. Sci., Vol. 3, Batschauer, A. (ed.), Häder, D.-P. and Jori, G. (series eds.), Royal Soc. Chemistry, Cambridge, UK, pp. 136-180. 3) Remberg, A. et al. (1997) Biochemistry 36 13389-13395

Single mutation in a novel bacterial LOV protein yields a singlet oxygen generator

Mr4511 from Methylobacterium radiotolerans is a 164 amino acid protein built of a flavin mononucleotide (FMN) binding, blue-light responsive LOV (Light, Oxygen, Voltage) core domain plus flanking regions. In contrast to the majority of LOV domains, Mr4511 lacks a tryptophan residue that was previously identified as a major quencher for the FMN triplet state in photosensitizers for singlet oxygen (SO) engineered from these photoreceptors. Here we show 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 photosensitizer: 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. The introduction of Trp112 in the canonical position for LOV domains dramatically lowers ΦΔ to values comparable to miniSOG, one of the early FMN binding proteins touted as a SO sensitizer. Besides its SO properties, Mr4511 is also exceedingly robust against denaturation with urea and is more photostable than free FMN

Green, red, near-infrared: biophysical investigations on bacterial bilin-binding photoreceptors

Bacterial photoreceptors binding open-chain tetrapyrroles (bilins) as chromophores are related to plant phytochromes (phy) as they are photochromic and the primary photochemistry consists of a Z/E isomerisation around the bilin 15=16 double bond. The chromophore is embedded in all cases within a so-called GAF domain with a typical α/β fold. Different to the canonical plant phys that invariably bind phytochromobilin and switch between a red and a far red absorbing form (R/FR), the bacterial bilin-photoreceptors exhibit a much wider variety of spectroscopic and functional properties, and bind diverse bilin chromophores, e.g. phycocyanobilin (PCB) and biliverdin (BV). In particular, isolated GAF domains of cyanobacteriochromes (CBCRs) take on great importance, because they are photochromic as standalone units. Beyond their intrinsic interest as light-sensing systems in prokaryots, these proteins show features, as a larger fluorescence quantum yield (F) and broader spectral ranges, that render them good candidates for biotechnological applications. Here we report steady-state and time-resolved spectroscopic measurements on selected bacterial bilin-photoreceptors: a. BV-binding phy from Pseudomonas strains with FR/NIR photochromism; b. PCB-binding GAF3 domain of the R/G (Red/Green) switching CBCR 1393 (Slr1393g3) from Synechocystis; c. GAF1 (R/FR) and GAF3 (R/Orange) domains of Anabaena 2699 (All2699). For CBCR GAF domains, both wild-type and mutated proteins, for which the dynamics of light-triggered reactions is altered, were analyzed. Within this pool of bacterial bilin-photoreceptors, a good correlation was found between F and fluorescence lifetimes. Nanosecond time-resolved absorption spectroscopy revealed the kinetics and spectral features of transient species after photoactivation. The most unusual behavior was found for the G-form of Slr1393g3: this form exhibits steady-state fluorescence heterogeneity and an up to now undescribed optical transient with a lifetime of ca. 60 ns at 20°C, upon green-light excitation. This transient was first uncovered by means of a photothermal method, but was now tracked by careful inspections of transient optical kinetic traces. Definitely, further investigations will be necessary to enlighten a possible link between the detected heterogeneity of Slr1393g3-G and this novel transient species

Dynamics and efficiency of photoswitching in biliverdin-binding phytochromes

The light-driven conversions between the dark-adapted and the photoproduct state were recorded for bacteriophytochromes (BphP) carrying biliverdin IXα (BV) as chromophore by time-resolved absorption spectroscopy. BphPs can be photoswitched between a red absorbing (Pr, maximum at ca. 700 nm) and a far-red/near-infrared (Pfr, maximum at ca. 750 nm) absorbing state, thereby showing a considerable red-shift with respect to plant phytochromes. Representatives for BphPs studied here are: PstBphP1 from Pseudomonas syringae pv. tomato, for which Pfr is the photoproduct; the bathy-phytochrome PaBphP from Pseudomonas aeruginosa for which instead Pfr is the thermally stable parental state. The third BphP-like protein was FphA from the fungus Aspergillus nidulans, a eukaryotic protein also carrying BV as chromophore, for which Pr is considered to be the dark-adapted state. All three BphPs show a canonical modular arrangement with a three domain photosensory module (PAS-GAF-PHY) and a histidine-kinase (HK) signalling domain. The quantum yields for Pr-to-Pfr photoconversion are in the range 0.02-0.12, and 0.04-0.08 for the Pfr-to-Pr route. Photoproducts of both bacterial phytochromes thermally recovered in the dark, whereas for the fungal protein (FphA) both Pr and Pfr forms are thermally stable for days and could be interconverted only by selective irradiation. The photoinduced reactions of all three BV-phytochromes are in general kinetically less complex than those of plant phytochromes, with the notable exception of the Pr-to-Pfr route for PstBphP1. By contrast in the Pfr-to-Pr conversion of FphAN753 the final product is already formed during the very early steps of the process, without formation of any further intermediates: to our knowledge it is the first phytochrome showing this behavior. All three proteins investigated are weakly fluorescent in the Pr form, with a maximum fluorescence quantum yield of 0.02 (PaBphP), and have undetectable fluorescence in the Pfr state