Phytochrome photoreceptors sense red and far-red light through photointerconversion between two stable conformations, a process mediated by a linear tetrapyrrole chromophore. Originally, phytochromes were thought to be confined to photosynthetic organisms including cyanobacteria, but they have been recently discovered in heterotrophic bacteria and fungi, where little is known about their functions. It was shown previously in the ascomycetous fungus Aspergillus nidulans that asexual sporulation is stimulated and sexual development repressed by red light. The effect was reminiscent of a phytochrome response, and indeed phytochrome-like proteins were detected in several fungal genomes. All fungal homologs are more similar to bacterial than plant phytochromes and have multifunctional domains where the phytochrome region and histidine kinase domain are combined in a single protein with a C-terminal response-regulator domain. Here, we show that the A. nidulans phytochrome FphA binds a biliverdin chromophore, acts as a red-light sensor, and represses sexual development under red-light conditions. FphA-GFP is cytoplasmic and excluded from the nuclei, suggesting that red-light photoperception occurs in the cytoplasm. This is the first phytochrome experimentally characterized outside the plant and bacterial kingdoms and the second type of fungal protein identified that functions in photoperception.

Phytochromes are bimodal photoswitches composed of a photosensor and an output module. Photoactivation of the sensor is initiated by a double bond isomerization of the tetrapyrrole chromophore and eventually leads to protein conformational changes. Recently determined structural models of phytochromes identify differences between the inactive and the signalling state but do not reveal the mechanism of photosensor activation or deactivation. Here, we report a vibrational spectroscopic study on bathy phytochromes that demonstrates that the formation of the photoactivated state and thus (de)activation of the output module is based on proton translocations in the chromophore pocket coupling chromophore and protein structural changes. These proton transfer steps, involving the tetrapyrrole and a nearby histidine, also enable thermal back-isomerization of the chromophore via keto-enol tautomerization to afford the initial dark state. Thus, the same proton re-arrangements inducing the (de)activation of the output module simultaneously initiate the reversal of this process, corresponding to a negative feedback mechanism.

Although the oceanic cyanobacterium Prochlorococcus harvests light with a chlorophyll antenna [1-3] rather than with the phycobilisomes that are typical of cyanobacteria, some strains express genes that are remnants of the ancestral Synechococcus phycobilisomes [4]. Similarly, some Prochlorococcus cyanophages, which often harbor photosynthesis-related genes [5], also carry homologs of phycobilisome pigment biosynthesis genes [6, 7]. Here, we investigate four such genes in two cyanophages that both infect abundant Prochlorococcus strains [8]: homologs of heme oxygenase (ho1), 15,16-dihydrobiliverdin:ferredoxin oxidoreductase (pebA), ferredoxin (petF) in the myovirus P-SSM2, and a phycocyanobilin:ferredoxin oxidoreductase (pcyA) homolog in the myovirus P-SSM4. We demonstrate that the phage homologs mimic the respective host activities, with the exception of the divergent phage PebA homolog. In this case, the phage PebA single-handedly catalyzes a reaction for which uninfected host cells require two consecutive enzymes, PebA and PebB. We thus renamed the phage enzyme phycoerythrobilin synthase (PebS). This gene, and other pigment biosynthesis genes encoded by P-SSM2 (petF and ho1), are transcribed during infection, suggesting that they can improve phage fitness. Analyses of global ocean metagenomes show that PcyA and Ho1 occur in both cyanobacteria and their phages, whereas the novel PebS-encoding gene is exclusive to phages.

Dispersion is a process used by bacteria to successfully transit from a biofilm to a planktonic growth state and to spawn novel communities in new locales. Alterations in bis-(3=-5=)-cyclic dimeric GMP (c-di-GMP) levels have been shown to be associated with biofilm dispersal in a number of different bacteria. The signaling molecule nitric oxide (NO) is known to induce biofilm dispersion through stimulation of c-di-GMP-degrading phosphodiesterase (PDE) activity. However, no c-di-GMP modulating enzyme directly involved in NO-induced dispersion has yet been described in the opportunistic pathogen Pseudomonas aeruginosa. Here, we characterized MucR (PA1727) and NbdA (PA3311, NO-induced biofilm dispersion locus A), two membranebound proteins with identical domain organization consisting of MHYT-GGDEF-EAL, with respect to their role in NO-induced dispersion. Inactivation of mucR impaired biofilm dispersion in response to NO and glutamate, whereas inactivation of nbdA only impaired biofilm dispersion upon exposure to NO. A specific role of NbdA in NO-induced dispersion was supported by increased PDE activity, resulting in decreased c-di-GMP levels in biofilms expressing nbdA upon exposure to NO, a response that was absent in the ⌬nbdA strain. Moreover, increased PDE activity was mainly due to a transcriptional activation of nbdA upon addition of NO. Biochemical analyses of recombinant protein variants lacking the membrane-anchored MHYT domain support NbdA being an active PDE. In contrast, MucR displayed both diguanylate cyclase and PDE activity in vitro, which seemed regulated in a growth-dependent manner in vivo. This is the first description of a PDE specifically involved in NO-induced biofilm dispersion in P. aeruginosa.

Background:The Pr and Pfr parent states of prototypical and bathy bacteriophytochromes exhibit different thermal stabilities. Results: Unlike bathy phytochromes, the biliverdin cofactor of prototypical phytochromes displays distinct conformational heterogeneity in Pfr. Conclusion: This heterogeneity enables thermal Pfr to Pr conversion in prototypical phytochromes. Significance: Understanding thermal deactivation of the signaling Pfr state is essential for elucidating the molecular function of phytochromes.