Andrew T. Ulijasz

Structural basis for the photoconversion of a phytochrome to the activated Pfr form

Phytochromes are a collection of bilin-containing photoreceptors that regulate numerous photoresponses in plants and microorganisms through their ability to photointerconvert between a red-light-absorbing, ground state (Pr) and a far-red-light-absorbing, photoactivated state (Pfr). Although the structures of several phytochromes as Pr have been determined, little is known about the structure of Pfr and how it initiates signalling. Here we describe the three-dimensional solution structure of the bilin-binding domain as Pfr, using the cyanobacterial phytochrome from Synechococcus OSB′. Contrary to predictions, light-induced rotation of the A pyrrole ring but not the D ring is the primary motion of the chromophore during photoconversion. Subsequent rearrangements within the protein then affect intradomain and interdomain contact sites within the phytochrome dimer. On the basis of our models, we propose that phytochromes act by propagating reversible light-driven conformational changes in the bilin to altered contacts between the adjacent output domains, which in most phytochromes direct differential phosphotransfer.

Regulation of Iron Transport in Streptococcus pneumoniae by RitR, an Orphan Response Regulator

RitR (formerly RR489) is an orphan two-component signal transduction response regulator in Streptococcus pneumoniae that has been shown to be required for lung pathogenicity. In the present study, by using the rough strain R800, inactivation of the orphan response regulator gene ritR by allele replacement reduced pathogenicity in a cyclophosphamide-treated mouse lung model but not in a thigh model, suggesting a role for RitR in regulation of tissue-specific virulence factors. Analysis of changes in genome-wide transcript mRNA levels associated with the inactivation of ritR compared to wild-type cells was performed by the use of high-density DNA microarrays. Genes with a change in transcript abundance associated with inactivation of ritR included piuB, encoding an Fe permease subunit, and piuA, encoding an Fe carrier-binding protein. In addition, a dpr ortholog, encoding an H(2)O(2) resistance protein that has been shown to reduce synthesis of reactive oxygen intermediates, was activated in the wild-type (ritR(+)) strain. Microarray experiments suggested that RitR represses Fe uptake in vitro by negatively regulating the Piu hemin-iron transport system. Footprinting experiments confirmed site-specific DNA-binding activity for RitR and identified three binding sites that partly overlap the +1 site for transcription initiation upstream of piuB. Transcripts belonging to other gene categories found to be differentially expressed in our array studies include those associated with (i) H(2)O(2) resistance, (ii) repair of DNA damage, (iii) sugar transport and capsule biosynthesis, and (iv) two-component signal transduction elements. These observations suggest that RitR is an important response regulator whose primary role is to maintain iron homeostasis in S. pneumoniae. The name ritR (repressor of iron transport) for the orphan response regulator gene, rr489, is proposed.

Cyanochromes are blue/green light photoreversible photoreceptors defined by a stable double cysteine linkage to a phycoviolobilin-type chromophore.

Phytochromes are a collection of bilin-containing photoreceptors that regulate a diverse array of processes in microorganisms and plants through photoconversion between two stable states, a red light-absorbing Pr form, and a far red light-absorbing Pfr form. Recently, a novel set of phytochrome-like chromoproteins was discovered in cyanobacteria, designated here as cyanochromes, that instead photoconvert between stable blue and green light-absorbing forms Pb and Pg, respectively. Here, we show that the distinctive absorption properties of cyanochromes are facilitated through the binding of phycocyanobilin via two stable cysteine-based thioether linkages within the cGMP phosphodiesterase/adenyl cyclase/FhlA domain. Absorption, resonance Raman and infrared spectroscopy, and molecular modeling of the Te-PixJ GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA) domain assembled with phycocyanobilin are consistent with attachments to the C31 carbon of the ethylidene side chain and the C4 or C5 carbons in the A–B methine bridge to generate a double thioether-linked phycoviolobilin-type chromophore. These spectroscopic methods combined with NMR data show that the bilin is fully protonated in the Pb and Pg states and that numerous conformation changes occur during Pb → Pg photoconversion. Also identified were a number of photochromically inactive mutants with strong yellow or red fluorescence that may be useful for fluorescence-based cell biological assays. Phylogenetic analyses detected cyanochromes capable of different signaling outputs in a wide range of cyanobacterial species. One unusual case is the Synechocystis cyanochrome Etr1 that also binds ethylene, suggesting that it works as a hybrid receptor to simultaneously integrate light and hormone signals.

Phosphorylation of the RitR DNA-binding domain by a Ser–Thr phosphokinase: implications for global gene regulation in the streptococci

We report selective phosphorylation of the DNA‐binding domain of the Streptococcus pneumoniae transcriptional regulator RitR. RitR is annotated as a two‐component response regulator, but lacks a cognate His kinase as a neighbouring locus in the genome. In addition, Asn replaces Asp at the expected acceptor site. By the use of combinatorial phage display, we identified PhpP, a S. pneumoniae Ser–Thr eukaryotic‐like PP2C phosphatase as an interacting partner of RitR. RitR interacts with the phage‐displayed peptide VADGMGGR which forms a part of the active‐site sequence of PhpP. RitR is phosphorylated in vitro by StkP, the presumed cognate kinase of PhpP, and the site on RitR that is phosphorylated has been localized to the RitR DNA‐binding domain. PhpP together with its cognate kinase StkP appear to be necessary for Piu haem transporter expression. In vitro studies suggest that PhpP and StkP interact competitively with RitR in that RitR–PhpP–piu promoter ternary complexes are disrupted by StkP. Our findings indicate a regulatory link between RitR and Ser–Thr kinase–phosphatase‐based bacterial signal transduction.

Phytochrome structure and photochemistry: recent advances toward a complete molecular picture.

Phytochromes are natures primary photoreceptors dedicated to detecting the red and far-red regions of the visible light spectrum, a region also essential for photosynthesis and thus crucial to the survival of plants and other photosynthetic organisms. Given their roles in measuring competition and diurnal/seasonal light fluctuations, understanding how phytochromes work at the molecular level would greatly aid in engineering crop plants better suited to specific agricultural settings. Recently, scientists have determined the three-dimensional structures of prokaryotic phytochromes, which now provide clues as to how these modular photoreceptors might work at the atomic level. The models point toward a largely unifying mechanism whereby novel knot, hairpin, and dimeric interfaces transduce photoreversible bilin isomerization into protein conformational changes that alter signal output.

Characterization of Two Thermostable Cyanobacterial Phytochromes Reveals Global Movements in the Chromophore-binding Domain during Photoconversion

Photointerconversion between the red light-absorbing (Pr) form and the far-red light-absorbing (Pfr) form is the central feature that allows members of the phytochrome (Phy) superfamily to act as reversible switches in light perception. Whereas the chromophore structure and surrounding binding pocket of Pr have been described, those for Pfr have remained enigmatic for various technical reasons. Here we describe a novel pair of Phys from two thermophilic cyanobacteria, Synechococcus sp. OS-A and OS-B′, that overcome several of these limitations. Like other cyanobacterial Phys, SyA-Cph1 and SyB-Cph1 covalently bind the bilin phycocyanobilin via their cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) domains and then assume the photointerconvertible Pr and Pfr states with absorption maxima at 630 and 704 nm, respectively. However, they are naturally missing the N-terminal Per/Arndt/Sim domain common to others in the Phy superfamily. Importantly, truncations containing only the GAF domain are monomeric, photochromic, and remarkably thermostable. Resonance Raman and NMR spectroscopy show that all four pyrrole ring nitrogens of phycocyanobilin are protonated both as Pr and following red light irradiation, indicating that the GAF domain by itself can complete the Pr to Pfr photocycle. 1H-15N two-dimensional NMR spectra of isotopically labeled preparations of the SyB-Cph1 GAF domain revealed that a number of amino acids change their environment during photoconversion of Pr to Pfr, which can be reversed by subsequent photoconversion back to Pr. Through three-dimensional NMR spectroscopy before and after light photoexcitation, it should now be possible to define the movements of the chromophore and binding pocket during photoconversion. We also generated a series of strongly red fluorescent derivatives of SyB-Cph1, which based on their small size and thermostability may be useful as cell biological reporters.

Dynamic Structural Changes Underpin Photoconversion of a Blue/Green Cyanobacteriochrome between Its Dark and Photoactivated States

Background: Phytochromes are photochromic bili-proteins vital to microbial and plant photoperception. Results: NMR spectroscopy generated three-dimensional structures of the photosensing module from a cyanobacterial variant in the dark and photoactivated states. Conclusion: Photoconversion involves thioether bond rupture, bilin isomerization and sliding, and increased protein disorder. Significance: Combined with crystallographic models, these paired NMR structures provide an unprecedented view into photoconversion of a phytochrome-type photoreceptor. The phytochrome superfamily of photoreceptors exploits reversible light-driven changes in the bilin chromophore to initiate a variety of signaling cascades. The nature of these alterations and how they impact the protein moiety remain poorly resolved and might include several species-specific routes. Here, we provide a detailed picture of photoconversion for the photosensing cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) domain from Thermosynechococcus elongatus (Te) PixJ, a member of the cyanobacteriochrome clade. Solution NMR structures of the blue light-absorbing dark state Pb and green light-absorbing photoactivated state Pg, combined with paired crystallographic models, revealed that the bilin and GAF domain dynamically transition via breakage of the C10/Cys-494 thioether bond, opposite rotations of the A and D pyrrole rings, sliding of the bilin in the GAF pocket, and the appearance of an extended region of disorder that includes Cys-494. Changes in GAF domain backbone dynamics were also observed that are likely important for inter-domain signal propagation. Taken together, photoconversion of T. elongatus PixJ from Pb to Pg involves complex structural changes within the GAF domain pocket that transduce light into a mechanical signal, many aspects of which should be relevant to others within the extended phytochrome superfamily.

Regulation of transcription by eukaryotic-like serine-threonine kinases and phosphatases in Gram-positive bacterial pathogens

Bacterial eukaryotic-like serine threonine kinases (eSTKs) and serine threonine phosphatases (eSTPs) have emerged as important signaling elements that are indispensable for pathogenesis. Differing considerably from their histidine kinase counterparts, few eSTK genes are encoded within the average bacterial genome, and their targets are pleiotropic in nature instead of exclusive. The growing list of important eSTK/P substrates includes proteins involved in translation, cell division, peptidoglycan synthesis, antibiotic tolerance, resistance to innate immunity and control of virulence factors. Recently it has come to light that eSTK/Ps also directly modulate transcriptional machinery in many microbial pathogens. This novel form of regulation is now emerging as an additional means by which bacteria can alter their transcriptomes in response to host-specific environmental stimuli. Here we focus on the ability of eSTKs and eSTPs in Gram-positive bacterial pathogens to directly modulate transcription, the known mechanistic outcomes of these modifications, and their roles as an added layer of complexity in controlling targeted RNA synthesis to enhance virulence potential.

The Aspartate-Less Receiver (ALR) Domains: Distribution, Structure and Function

Two-component signaling systems are ubiquitous in bacteria, Archaea and plants and play important roles in sensing and responding to environmental stimuli. To propagate a signaling response the typical system employs a sensory histidine kinase that phosphorylates a Receiver (REC) domain on a conserved aspartate (Asp) residue. Although it is known that some REC domains are missing this Asp residue, it remains unclear as to how many of these divergent REC domains exist, what their functional roles are and how they are regulated in the absence of the conserved Asp. Here we have compiled all deposited REC domains missing their phosphorylatable Asp residue, renamed here as the Aspartate-Less Receiver (ALR) domains. Our data show that ALRs are surprisingly common and are enriched for when attached to more rare effector outputs. Analysis of our informatics and the available ALR atomic structures, combined with structural, biochemical and genetic data of the ALR archetype RitR from Streptococcus pneumoniae presented here suggest that ALRs have reorganized their active pockets to instead take on a constitutive regulatory role or accommodate input signals other than Asp phosphorylation, while largely retaining the canonical post-phosphorylation mechanisms and dimeric interface. This work defines ALRs as an atypical REC subclass and provides insights into shared mechanisms of activation between ALR and REC domains.

Differential Assay for High-Throughput Screening of Antibacterial Compounds

The previously described Bacillus subtilis reporter strain BAU-102 is capable of detecting cell wall synthesis inhibitors that act at all stages of the cell wall synthesis pathway. In addition, this strain is capable of detecting compounds with hydrophobic/ surfactant activity and alternative mechanisms of cell wall disruption. BAU-102 sequesters preformed β-gal in the periplasm, suggesting leakage of β-gal as the means by which this assay detects compound activities. A model is proposed according to which β-gal release by BAU-102 reflects activation of pathways leading to autolysis. The authors also report a simplified high-throughput assay using BAU-102 combined with the fluorogenic substrate N-methylumbelliferyl-β-D-galactoside as a single reagent. Cell wall inhibitors release β-gal consistently only after 60 min of incubation, whereas compounds with surfactant activity show an almost immediate release. A high-throughput screen of a 480-compound library of known bioactives yielded 8 compounds that cause β-gal release. These results validate the BAU-102 assay as an effective tool in antimicrobial drug discovery. (Journal of Biomolecular Screening 2007:1102-1108)