Christina Huber

• The ability to sense and respond to different environmental conditions allows living organisms to adapt quickly to their surroundings. In order to use light as a source of information, plants, fungi, and bacteria employ phytochromes. With their ability to detect far-red and red light, phytochromes constitute a major photoreceptor family. Bacterial phytochromes (BphPs) are composed of an apo-phytochrome and an open-chain tetrapyrrole, the chromophore biliverdin IXα, which mediates the photosensory properties. Depending on the photoexcitation and the quality of the incident light, phytochromes interconvert between two photoconvertible parental states: the red light-absorbing Pr-form and the far-red light-absorbing Pfr-form. In contrast to prototypical phytochromes, with a thermal stable Pr ground state, there is a group of bacterial phytochromes that exhibit dark reversion from the Pr- to the Pfr-form. These special proteins are classified as bathy phytochromes and range across different classes of bacteria. Moreover, the majority of BphPs act as sensor histidine kinases in two-component regulatory systems. The light-triggered conformational change results in the autophosphorylation of the histidine kinase domain and the transphosphorylation of an associated response regulator, inducing a cellular response. Spectroscopic analysis utilizing homologously produced protein identified PaBphP, the histidine kinase of the human opportunistic pathogen Pseudomonas aeruginosa, as a bathy phytochrome. Intensive research on PaBphP revealed evidence that the interconversion between its physiological active and inactive states is influenced by light and darkness rather than far-red and red light. In order to conduct a comprehensive systematic analysis, further bacterial phytochromes were investigated regarding their biochemical and spectroscopic behavior, as well as their autokinase activity. In addition to PaBphP, this work employs the bathy phytochromes AtBphP2, AvBphP2, XccBphP from the non-photosynthetic plant pathogens Agrobacterium tumefaciens, Allorhizobium vitis, Xanthomonas campestris, as well as RtBphP2 from the soil bacterium Ramlibacter tataouinensis. All investigated BphPs displayed a bathy-typical behavior by developing a distinct Pr-form under far-red light conditions and undergoing dark reversion to their Pfr-form. Different Pr/Pfr-fractions can be identified among the BphP populations in varying natural light conditions, including red or blue light. The Pr-form is considered as the active form due to autophosphorylation activity in the heterologously produced phytochromes when exposed to light. In the absence of light, associated with the development of the Pfr-form, the phytochromes exhibited disabled or strongly reduced autokinase activity. Additionally, light-triggered phosphorylation was observed for the response regulator PaAlgB, which is linked to the phytochrome of P. aeruginosa. This study presents the first comparative investigation of numerous bathy phytochromes under identical conditions. The work addressed a gap in the literature by providing quantitative correlation between kinase activity and calculated Pr/Pfr-fractions obtained from spectroscopic measurements. The biological role of PaBphP was partially elucidated through phenotypic characterization employing P. aeruginosa mutant and overexpression strains. The generation of a functional model was possible by considering the postulated functions of the other phytochromes found in the literature. In summary, bathy BphPs are hypothesized to modulate bacterial virulence according to the circadian day/night rhythm of their hosts. The pathogens are believed to reduce their virulence during daylight hours to evade immune and defense reactions, while increasing their virulence during the evening and night, enabling more effective infections.