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In addition to oxygenic photosynthetic systems, solar radiation is utilized for energy by diverse anoxygenic photosynthetic systems. Aerobic anoxygenic phototrophic bacteria (AAPB) perform photosynthesis without producing oxygen but still live in aerobic conditions. Typically they have been reported from aquatic ecosystems, but they can also be found from polar and desert soil ecosystems and primary succession communities like soil crusts. Recently, AAPB have been discovered in the metagenomic data of several plant phyllospheres. By utilizing citizen science, we screened plant foliar samples from eleven different locations in Finland for AAPB by near infrared fluorescence imaging of culturable phyllosphere and endosphere bacteria. Near infrared fluorescence reports the presence of AAPB which contain Bacteriochlorophyllamolecules, embedded in Light Harvesting 1 - Reaction Center complex. We found that AAPB were ubiquotous in phyllosphere communities of diverse plant species in all sampling locations. They were also consistently present in the endosphere of plant species with perennial leaves. Most of the AAPB isolated represent alphaproteobacterial genera Sphingomonas and Methylobacterium, but several isolates from genus Lichenihabitans as well as putative novel alphaproteobacterial taxa were also identified. Methylobacterial isolates were mostly detected in the phyllosphere with weak host specificity, while Sphingomonas AAPB were detected also in the endosphere of several plant species, with clear host specific taxa. We studied also the fluorescence spectral properties of several AAPBs. All the observed spectra resemble typical fluorescence spectral properties of Light Harvesting complex 1. Still, slight variation among each spectra could be obtained, revealing some physical difference among the complexes. Our results demonstrate for the first time, that AAPB are common in cold climate plant endophytic as well as epiphytic microbiomes and they build up substantial amounts of Bacteriochlorophyllacontaining Light Harvesting complexes. Their putative role in plant adaptation to strong seasonality in light and temperature or tolerance of abiotic stressors remains to be investigated in future studies.

Signal propagation in photosensory proteins is a complex and multidimensional event. Unraveling such mechanisms site-specifically in real time is an eligible but a challenging goal. Here, we elucidate the site-specific events in a red-light sensing phytochrome using the unnatural amino acid azidophenylalanine, vibrationally distinguishable from all other protein signals. In canonical phytochromes, signal transduction starts with isomerization of an excited bilin chromophore, initiating a multitude of processes in the photosensory unit of the protein, which eventually control the biochemical activity of the output domain, nanometers away from the chromophore. By implementing the label in prime protein locations and running two-color step-scan FTIR spectroscopy on the Deinococcus radiodurans bacteriophytochrome, we track the signal propagation at three specific sites in the photosensory unit. We show that a structurally switchable hairpin extension, a so-called tongue region, responds to the photoconversion already in microseconds and finalizes its structural changes concomitant with the chromophore, in milliseconds. In contrast, kinetics from the other two label positions indicate that the site-specific changes deviate from the chromophore actions, even though the labels locate in the chromophore vicinity. Several other sites for labeling resulted in impaired photoswitching, low structural stability, or no changes in the difference spectrum, which provides additional information on the inner dynamics of the photosensory unit. Our work enlightens the multidimensionality of the structural changes of proteins under action. The study also shows that the signaling mechanism of phytochromes is accessible in a time-resolved and site-specific approach by azido probes and demonstrates challenges in using these labels. peerReviewed

Phytochrome proteins regulate many photoresponses of plants and microorganisms. Light absorption causes isomerization of the biliverdin chromophore, which triggers a series of structural changes to activate the signaling domains of the protein. However, the structural changes are elusive, and therefore the molecular mechanism of signal transduction remains poorly understood. Here, we apply two-color step-scan infrared spectroscopy to the bacteriophytochrome from Deinococcus radiodurans. We show by recordings in H2O and D2O that the hydrogen bonds to the biliverdin D-ring carbonyl become disordered in the first intermediate (Lumi-R) forming a dynamic microenvironment, then completely detach in the second intermediate (Meta-R), and finally reform in the signaling state (Pfr). The spectra reveal via isotope labeling that the refolding of the conserved “PHY-tongue” region occurs with the last transition between Meta-R and Pfr. Additional changes in the protein backbone are detected already within microseconds in Lumi-R. Aided by molecular dynamics simulations, we find that a strictly conserved salt bridge between an arginine of the PHY tongue and an aspartate of the chromophore binding domains is broken in Lumi-R and the arginine is recruited to the D-ring C═O. This rationalizes how isomerization of the chromophore is linked to the global structural rearrangement in the sensory receptor. Our findings advance the structural understanding of phytochrome photoactivation. peerReviewed