26 - Phytochromes
Florida 2 08:00 - 12:00
|Chair(s): Wolfgang Gartner|
08:00 Cyanobacteriochrome Regulation of Second Messenger-Dependent Processes in Cyanobacteria BL Montgomery*, Michigan State University
Abstract: Microorganisms adapt to changes in their external environment by sensing and amplifying external first messenger signals using second messengers to alter their metabolism and behaviors. Levels of the second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) are regulated by a variety of environmental stimuli and play critical roles in controlling cellular processes such as biofilm formation, motility, central metabolism, and cell differentiation in a range of bacteria. C-di-GMP is a ubiquitous second messenger synthesized and degraded by proteins that contain GGDEF, EAL or HD-GYP domains. GGDEF domain proteins exhibit diguanylate cyclase (DGC) activity, resulting in the synthesis of c-di-GMP from two GTP molecules. EAL and HD-GYP domains have phosphodiesterase (PDE) activity, resulting in c-di-GMP degradation. C-di-GMP signaling systems have been largely characterized in pathogenic gamma proteobacteria; however, DGC or PDE proteins that are prominent in cyanobacterial species remain largely underexplored. Many putative c-di-GMP synthesis or degradation output domains are found in genes that also harbor light-responsive signal input domains, suggesting that light is an important signal for altering c-di-GMP homeostasis. Indeed, such output domains are often the second most common output domain in photoreceptors"only outnumbered by a histidine kinase output domain. Cyanobacteria differ from other bacteria as they have a higher percentage of c-di-GMP signaling enzymes associated with photoreceptors and different photoreceptors are utilized. These results suggest that c-di-GMP plays key roles in adaptation of cyanobacteria to the photoenvironment. Our recent studies demonstrate that light regulates c-di-GMP levels in cyanobacteria and this photoregulation is associated with distinct c-di-GMP-dependent physiological responses. The range of identified cyanobacterial responses under control of light-dependent regulation of c-di-GMP will be discussed.
08:30 Phytochromes in Myxobacteria: Implications for Light-Controlled Morphogenesis KD Gallagher, Northeastern Illinois University
; E Mihalas-Sanchez, Northeastern Illinois University; A Mapara, Northeastern Illinois University; P Duong, Northeastern Illinois University; A Nugent, Northeastern Illinois University; D Bizhga, Northeastern Illinois University; H Patel, Northeastern Illinois University; NC Woitowich, Northeastern Illinois University; EA Stojkovic*, Northeastern Illinois University
Abstract: Bacteriophytochromes (BphPs) are red-light photoreceptors that require biliverdin (BV), open-chain tetrapyrrole as a cofactor for photoactivity. BphPs belong to a large phytochrome family of photoreceptors found in various plants and microorganisms but their role in non-photosynthetic organisms remains largely unknown. Here we present the first structural and functional characterization of two BphPs from the non-photosynthetic myxobacterium Stigmatella aurantiaca. Among prokaryotes, myxobacteria are distinguished by a unique multicellular stage in their life-cycle in which fruiting bodies are formed. In contrast to closely related Myxococcus xanthus, which forms fruiting bodies in the dark, S. aurantiaca produces orange-pigmented fruiting bodies only in the presence of light. Besides BphPs, the S. aurantiaca genome annotation indicates the presence of a putative heme oxygenase, which is essential in BV synthesis. However, the genome of M. xanthus completely lacks BphPs and heme oxygenase genes. Our hypothesis is that BphPs may play a role in the fruiting body formation of S. aurantiaca. Like classical BphPs, SaBphP1 and SaBphP2 are composed of a photosensory module covalently linked to a histidine kinase. They share 41% sequence identity, both bind BV and undergo red to far-red light (Pr/Pfr) photoconversion. Unlike classical BphPs, including SaBphP2, wild-type SaBphP1 lacks a highly conserved His that stabilizes BV, a feature also common to other myxobacterial BphPs. Interestingly, SaBphP1 undergoes limited Pr/Pfr photoconversion that can be restored by a single Thr (Thr289) to His mutation in the BV-binding photosensory module. Currently, we are investigating the role of BphPs in fruiting body formation by inactivating/mutating genes coding for BphPs and screening for expected phenotypes. Our goal is to determine what mechanistic changes accompany light-induced morphogenesis in myxobacteria and the novel role of photoreceptors in these non-photosynthetic microorganisms.
09:00 Structural insights into phytochrome fluorescence S Bhattacharya*
; H Lehtivuori; KT Forest
Abstract: Use of fluorescent proteins in studying in-vivo processes in mammalian systems requires development of near-infrared biomarkers due to clear signals unimpeded by absorption or autofluorescence of biomolecules. Bacteriophytochromes (BphPs) that use biliverdin as their chromophore have been engineered to form monomeric near-infrared biomarkers. The original design template for a fluorescent phytochrome was the chromophore binding domain of Deinococcus radiodurans (DrCBD), with a D207H substitution. This variant proved to be the hallmark for the next generation of phytochrome biomarkers including IFP1.4 (D207H +11substitutions), Wi-Phy (D207HY263F) and the iRFP family based on Rhodopseudomonas palustris. We solved the x-ray crystal structures of IFP1.4 and several monomeric DrCBD variants, to explain the origins of fluorescence in derived BphPs. A comparative study revealed two important themes. First, hydrophobic packing around the D-ring increases fluorescence by limiting D-ring motion. Second, while the 207th residue is critical to photochemistry it certainly does not need to be His in order to maximize fluorescence. In fact, the highest fluorescence quantum yield to date in this family belongs to a D207L variant in which waters are excluded from the chromophore vicinity (WiPhy2). Higher quantum yield and longer excited state lifetimes than in the parent suggest the loss of a de-excitation pathway via proton transfer. Continuing our structural analysis, we have turned to the iRFP family of phytofluors and uncovered a surprisingly stable interaction between phytochrome and the heme oxygenase partner that provides biliverdin. We have also engineered a smaller phytochrome variant without the characteristic figure of eight knot topology. Our engineered knotless chromophore binding domain folds and binds bilin. Combining structural insights with protein engineering will help us understand how holo-phytochrome is assembled and design better near-infrared fluorophores.
09:30 Structural Basis of Color Perception and Signal Integration in Cyanobacteriochromes H Shin, University of Illinois at Chicago
; X Zeng, University of Illinois at Chicago; Z Ren, University of Illinois at Chicago; X Yang*, University of Illinois at Chicago
Abstract: Phytochromes are bilin-binding photoreceptors that were first characterized as red-light photoreceptors in plants. Thanks to large-scale genome sequencing efforts and recent discovery of cyanobacteriochromes, the phytochrome superfamily has been significantly expanded. Bilin-binding photoreceptors collectively exhibit extraordinary versatility in action spectra, domain architecture and signaling properties. We will present our ongoing crystallographic and biochemical studies that aim to understand how color perception, signal integration and long-range signaling are achieved in multi-sensor bilin-based photoreceptors at the molecular level.
10:30 The GAF-3 domain from Slr 1393: Photochemistry and Insight into Structural Changes W. Gaertner*, MPI Chemical Energy Conversion
Abstract: Cyanobacteriochromes are photochromic photoreceptors carrying bilins as chromophores. Their light-driven reactions are similar to those of canonical photochromes. They have attracted strong attention of scientists as CBCRs show a much broader absorbance range and, in addition, these proteins collate the entire photochemistry and chromophore binding capability in one single GAF domain. GAF3 of Slr1393 from Synechocystis PCC6803 generates a red light-absorbing parental state and a green light-absorbing photoproduct state (Î»max = 650nm, 535 nm, respectively). Both forms can be fully photo-converted into each other. Time-resolved absorption spectroscopy in the us-to-ms time range identified a single intermediate for each conversion direction. Three dimensional structure of both parental and photoproduct state could be solved (with 1.6 and 1.8 A resolution), and also an intermediate state was trapped with a chromophore isomerized into the red-absorbing state and the protein surrounding still fixed in the green-absorbing state. The observable fluorescence, the small size, and the efficient photochemistry make CBCR-GAF promising tools for optogenetic applications.
11:00 Synthetic Photobiology in Near-infrared M Gomelsky*, University of Wyoming
; MARK GOMELSKY
Abstract: Several branches of medicine, including cancer immunotherapy, neurology and regenerative medicine, are rapidly adapting the use of engineered cells. A major concern in using engineered cells lies in our poor ability to control them. Synthetic photoregulated systems responsive to light in the near-infrared optical window (NIRW; ~670-900 nm) can help overcoming this problem. NIRW light is harmless and penetrates deep into mammalian tissues. Microbial photoreceptors, bacteriophytochromes, naturally respond to NIRW light. We will present several types of engineered bacteriophytochrome-based optogenetic (synthetic photobiology) tools. One toolset involves NIRW light-activated nucleotide cyclases that can be used to control cAMP and cGMP levels with high spatial and temporal precision. These cyclases have been engineered by fusing bacteriophytochrome photosensory modules with homodimeric nucleotide cyclases. We have engineered NIRW light-activated adenylate cyclases with various levels of enzymatic activities and different lit form stabilities. Another set of engineered bacteriophytochrome-based tools involves NIRW light-dependent systems to control gene expression in mammalian cells. Two prototype systems will be discussed. One system is based on the NIRW light-activated cyclic dinucleotide-mediated expression, while another one is based on the NIRW light-dependent bacterial antirepressor-repressor system. Performance of the newly developed bacteriophytochrome tools will be discussed.