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1.
Nature ; 620(7976): 1018-1024, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37612503

RESUMO

Coral reefs are highly diverse ecosystems that thrive in nutrient-poor waters, a phenomenon frequently referred to as the Darwin paradox1. The energy demand of coral animal hosts can often be fully met by the excess production of carbon-rich photosynthates by their algal symbionts2,3. However, the understanding of mechanisms that enable corals to acquire the vital nutrients nitrogen and phosphorus from their symbionts is incomplete4-9. Here we show, through a series of long-term experiments, that the uptake of dissolved inorganic nitrogen and phosphorus by the symbionts alone is sufficient to sustain rapid coral growth. Next, considering the nitrogen and phosphorus budgets of host and symbionts, we identify that these nutrients are gathered through symbiont 'farming' and are translocated to the host by digestion of excess symbiont cells. Finally, we use a large-scale natural experiment in which seabirds fertilize some reefs but not others, to show that the efficient utilization of dissolved inorganic nutrients by symbiotic corals established in our laboratory experiments has the potential to enhance coral growth in the wild at the ecosystem level. Feeding on symbionts enables coral animals to tap into an important nutrient pool and helps to explain the evolutionary and ecological success of symbiotic corals in nutrient-limited waters.


Assuntos
Antozoários , Ecossistema , Nitrogênio , Fósforo , Fotossíntese , Simbiose , Animais , Antozoários/crescimento & desenvolvimento , Antozoários/metabolismo , Antozoários/fisiologia , Nitrogênio/metabolismo , Fósforo/metabolismo , Simbiose/fisiologia , Aves/fisiologia
2.
Nat Chem Biol ; 19(7): 865-877, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37277468

RESUMO

Bacterial capsules have critical roles in host-pathogen interactions. They provide a protective envelope against host recognition, leading to immune evasion and bacterial survival. Here we define the capsule biosynthesis pathway of Haemophilus influenzae serotype b (Hib), a Gram-negative bacterium that causes severe infections in infants and children. Reconstitution of this pathway enabled the fermentation-free production of Hib vaccine antigens starting from widely available precursors and detailed characterization of the enzymatic machinery. The X-ray crystal structure of the capsule polymerase Bcs3 reveals a multi-enzyme machine adopting a basket-like shape that creates a protected environment for the synthesis of the complex Hib polymer. This architecture is commonly exploited for surface glycan synthesis by both Gram-negative and Gram-positive pathogens. Supported by biochemical studies and comprehensive 2D nuclear magnetic resonance, our data explain how the ribofuranosyltransferase CriT, the phosphatase CrpP, the ribitol-phosphate transferase CroT and a polymer-binding domain function as a unique multi-enzyme assembly.


Assuntos
Infecções por Haemophilus , Vacinas Anti-Haemophilus , Haemophilus influenzae tipo b , Lactente , Criança , Humanos , Infecções por Haemophilus/microbiologia , Infecções por Haemophilus/prevenção & controle , Vacinas Anti-Haemophilus/metabolismo , Cápsulas Bacterianas/metabolismo , Bactérias Gram-Negativas
4.
Biochemistry ; 59(32): 2934-2945, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32786405

RESUMO

The phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential peripheral membrane glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. PimA undergoes functionally important conformational changes, including (i) α-helix-to-ß-strand and ß-strand-to-α-helix transitions and (ii) an "open-to-closed" motion between the two Rossmann-fold domains, a conformational change that is necessary to generate a catalytically competent active site. In previous work, we established that GDP-Man and GDP stabilize the enzyme and facilitate the switch to a more compact active state. To determine the structural contribution of the mannose ring in such an activation mechanism, we analyzed a series of chemical derivatives, including mannose phosphate (Man-P) and mannose pyrophosphate-ribose (Man-PP-RIB), and additional GDP derivatives, such as pyrophosphate ribose (PP-RIB) and GMP, by the combined use of X-ray crystallography, limited proteolysis, circular dichroism, isothermal titration calorimetry, and small angle X-ray scattering methods. Although the ß-phosphate is present, we found that the mannose ring, covalently attached to neither phosphate (Man-P) nor PP-RIB (Man-PP-RIB), does promote the switch to the active compact form of the enzyme. Therefore, the nucleotide moiety of GDP-Man, and not the sugar ring, facilitates the "open-to-closed" motion, with the ß-phosphate group providing the high-affinity binding to PimA. Altogether, the experimental data contribute to a better understanding of the structural determinants involved in the "open-to-closed" motion not only observed in PimA but also visualized and/or predicted in other glycosyltransfeases. In addition, the experimental data might prove to be useful for the discovery and/or development of PimA and/or glycosyltransferase inhibitors.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Manosiltransferases/química , Manosiltransferases/metabolismo , Movimento , Manose/metabolismo , Modelos Moleculares , Conformação Proteica
5.
Biochem J ; 476(14): 2059-2092, 2019 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-31366571

RESUMO

The evolution of metabolic pathways is a major force behind natural selection. In the spotlight of such process lies the structural evolution of the enzymatic machinery responsible for the central energy metabolism. Specifically, glycogen metabolism has emerged to allow organisms to save available environmental surplus of carbon and energy, using dedicated glucose polymers as a storage compartment that can be mobilized at future demand. The origins of such adaptive advantage rely on the acquisition of an enzymatic system for the biosynthesis and degradation of glycogen, along with mechanisms to balance the assembly and disassembly rate of this polysaccharide, in order to store and recover glucose according to cell energy needs. The first step in the classical bacterial glycogen biosynthetic pathway is carried out by the adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase. This allosteric enzyme synthesizes ADP-glucose and acts as a point of regulation. The second step is carried out by the glycogen synthase, an enzyme that generates linear α-(1→4)-linked glucose chains, whereas the third step catalyzed by the branching enzyme produces α-(1→6)-linked glucan branches in the polymer. Two enzymes facilitate glycogen degradation: glycogen phosphorylase, which functions as an α-(1→4)-depolymerizing enzyme, and the debranching enzyme that catalyzes the removal of α-(1→6)-linked ramifications. In this work, we rationalize the structural basis of glycogen metabolism in bacteria to the light of the current knowledge. We describe and discuss the remarkable progress made in the understanding of the molecular mechanisms of substrate recognition and product release, allosteric regulation and catalysis of all those enzymes.


Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Glucose-1-Fosfato Adenililtransferase/metabolismo , Glicogênio Sintase/metabolismo , Glicogênio/biossíntese , Regulação Alostérica
6.
Proc Natl Acad Sci U S A ; 113(16): 4416-21, 2016 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-27044109

RESUMO

Coral communities in the Persian/Arabian Gulf (PAG) withstand unusually high salinity levels and regular summer temperature maxima of up to ∼35 °C that kill conspecifics elsewhere. Due to the recent formation of the PAG and its subsequent shift to a hot climate, these corals have had only <6,000 y to adapt to these extreme conditions and can therefore inform on how coral reefs may respond to global warming. One key to coral survival in the world's warmest reefs are symbioses with a newly discovered alga,Symbiodinium thermophilum Currently, it is unknown whether this symbiont originated elsewhere or emerged from unexpectedly fast evolution catalyzed by the extreme environment. Analyzing genetic diversity of symbiotic algae across >5,000 km of the PAG, the Gulf of Oman, and the Red Sea coastline, we show thatS. thermophilumis a member of a highly diverse, ancient group of symbionts cryptically distributed outside the PAG. We argue that the adjustment to temperature extremes by PAG corals was facilitated by the positive selection of preadapted symbionts. Our findings suggest that maintaining the largest possible pool of potentially stress-tolerant genotypes by protecting existing biodiversity is crucial to promote rapid adaptation to present-day climate change, not only for coral reefs, but for ecosystems in general.


Assuntos
Antozoários , Dinoflagellida/genética , Ecossistema , Variação Genética , Salinidade , Estresse Fisiológico , Simbiose , Animais , Oceanos e Mares
7.
J Phycol ; 54(5): 762-764, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29981276

RESUMO

The binary designation Symbiodinium thermophilum was invalid due to the absence of an illustration as required by Article 44.2 of the ICN. Herein, it is validated. This species is the most common symbiont in reef corals in the southern Persian/Arabian Gulf, the world's hottest body of water sustaining reef coral growth.


Assuntos
Dinoflagellida/classificação , Terminologia como Assunto , Recifes de Corais , Oceano Índico , Simbiose
8.
Proc Biol Sci ; 284(1858)2017 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-28679724

RESUMO

The depth distribution of reef-building corals exposes their photosynthetic symbionts of the genus Symbiodinium to extreme gradients in the intensity and spectral quality of the ambient light environment. Characterizing the mechanisms used by the coral holobiont to respond to the low intensity and reduced spectral composition of the light environment in deeper reefs (greater than 20 m) is fundamental to our understanding of the functioning and structure of reefs across depth gradients. Here, we demonstrate that host pigments, specifically photoconvertible red fluorescent proteins (pcRFPs), can promote coral adaptation/acclimatization to deeper-water light environments by transforming the prevalent blue light into orange-red light, which can penetrate deeper within zooxanthellae-containing tissues; this facilitates a more homogeneous distribution of photons across symbiont communities. The ecological importance of pcRFPs in deeper reefs is supported by the increasing proportion of red fluorescent corals with depth (measured down to 45 m) and increased survival of colour morphs with strong expression of pcRFPs in long-term light manipulation experiments. In addition to screening by host pigments from high light intensities in shallow water, the spectral transformation observed in deeper-water corals highlights the importance of GFP-like protein expression as an ecological mechanism to support the functioning of the coral-Symbiodinium association across steep environmental gradients.


Assuntos
Aclimatação , Antozoários/efeitos da radiação , Dinoflagellida/efeitos da radiação , Luz , Pigmentação , Animais , Antozoários/fisiologia , Recifes de Corais , Dinoflagellida/fisiologia , Fluorescência , Simbiose
9.
Int J Mol Sci ; 18(7)2017 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-28677653

RESUMO

Photoconvertible fluorescent proteins (pcRFPs) are a group of fluorophores that undergo an irreversible green-to-red shift in emission colour upon irradiation with near-ultraviolet (near-UV) light. Despite their wide application in biotechnology, the high-level expression of pcRFPs in mesophotic and depth-generalist coral species currently lacks a biological explanation. Additionally, reduced penetration of near-UV wavelengths in water poses the question whether light-driven photoconversion is relevant in the mesophotic zone, or whether a different mechanism is involved in the post-translational pigment modification in vivo. Here, we show in a long-term mesocosm experiment that photoconversion in vivo is entirely dependent on near-UV wavelengths. However, a near-UV intensity equivalent to the mesophotic underwater light field at 80 m depth is sufficient to drive the process in vitro, suggesting that photoconversion can occur near the lower distribution limits of these corals. Furthermore, live coral colonies showed evidence of efficient Förster Resonance Energy Transfer (FRET). Our simulated mesophotic light field maintained the pcRFP pool in a partially photoconverted state in vivo, maximising intra-tetrameric FRET and creating a long-range wavelength conversion system with higher quantum yield than other native RFPs. We hypothesise that efficient conversion of blue wavelengths, abundant at depth, into orange-red light could constitute an adaptation of corals to life in light-limited environments.


Assuntos
Antozoários/genética , Antozoários/metabolismo , Transferência Ressonante de Energia de Fluorescência , Luz , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Animais , Expressão Gênica , Genes Reporter , Microscopia de Fluorescência , Simbiose
10.
Mol Ecol ; 24(2): 453-65, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25496144

RESUMO

The genomic framework that enables corals to adjust to unfavourable conditions is crucial for coral reef survival in a rapidly changing climate. We have explored the striking intraspecific variability in the expression of coral pigments from the green fluorescent protein (GFP) family to elucidate the genomic basis for the plasticity of stress responses among reef corals. We show that multicopy genes can greatly increase the dynamic range over which corals can modulate transcript levels in response to the light environment. Using the red fluorescent protein amilFP597 in the coral Acropora millepora as a model, we demonstrate that its expression increases with light intensity, but both the minimal and maximal gene transcript levels vary markedly among colour morphs. The pigment concentration in the tissue of different morphs is strongly correlated with the number of gene copies with a particular promoter type. These findings indicate that colour polymorphism in reef corals can be caused by the environmentally regulated expression of multicopy genes. High-level expression of amilFP597 is correlated with reduced photodamage of zooxanthellae under acute light stress, supporting a photoprotective function of this pigment. The cluster of light-regulated pigment genes can enable corals to invest either in expensive high-level pigmentation, offering benefits under light stress, or to rely on low tissue pigment concentrations and use the conserved resources for other purposes, which is preferable in less light-exposed environments. The genomic framework described here allows corals to pursue different strategies to succeed in habitats with highly variable light stress levels. In summary, our results suggest that the intraspecific plasticity of reef corals' stress responses is larger than previously thought.


Assuntos
Aclimatação/genética , Antozoários/genética , Luz , Proteínas Luminescentes/genética , Pigmentação/genética , Animais , Antozoários/fisiologia , Cor , Dosagem de Genes , Dados de Sequência Molecular , Família Multigênica , Filogenia , Regiões Promotoras Genéticas , Proteína Vermelha Fluorescente
11.
Nat Commun ; 14(1): 2511, 2023 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-37188683

RESUMO

Coral broadcast spawning events - in which gametes are released on certain nights predictably in relation to lunar cycles - are critical to the maintenance and recovery of coral reefs following mass mortality. Artificial light at night (ALAN) from coastal and offshore developments threatens coral reef health by masking natural light:dark cycles that synchronize broadcast spawning. Using a recently published atlas of underwater light pollution, we analyze a global dataset of 2135 spawning observations from the 21st century. For the majority of genera, corals exposed to light pollution are spawning between one and three days closer to the full moon compared to those on unlit reefs. ALAN possibly advances the trigger for spawning by creating a perceived period of minimum illuminance between sunset and moonrise on nights following the full moon. Advancing the timing of mass spawning could decrease the probability of gamete fertilization and survival, with clear implications for ecological processes involved in the resilience of reef systems.


Assuntos
Antozoários , Animais , Poluição Luminosa , Recifes de Corais , Fotoperíodo , Lua , Luz
12.
mBio ; 14(4): e0041423, 2023 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-37409813

RESUMO

Invasive aspergillosis is one of the most serious clinical invasive fungal infections, resulting in a high case fatality rate among immunocompromised patients. The disease is caused by saprophytic molds in the genus Aspergillus, including Aspergillus fumigatus, the most significant pathogenic species. The fungal cell wall, an essential structure mainly composed of glucan, chitin, galactomannan, and galactosaminogalactan, represents an important target for the development of antifungal drugs. UDP (uridine diphosphate)-glucose pyrophosphorylase (UGP) is a central enzyme in the metabolism of carbohydrates that catalyzes the biosynthesis of UDP-glucose, a key precursor of fungal cell wall polysaccharides. Here, we demonstrate that the function of UGP is vital for Aspergillus nidulans (AnUGP). To understand the molecular basis of AnUGP function, we describe a cryoEM structure (global resolution of 3.5 Å for the locally refined subunit and 4 Å for the octameric complex) of a native AnUGP. The structure reveals an octameric architecture with each subunit comprising an N-terminal α-helical domain, a central catalytic glycosyltransferase A-like (GT-A-like) domain, and a C-terminal (CT) left-handed ß-helix oligomerization domain. AnUGP displays unprecedented conformational variability between the CT oligomerization domain and the central GT-A-like catalytic domain. In combination with activity measurements and bioinformatics analysis, we unveil the molecular mechanism of substrate recognition and specificity for AnUGP. Altogether, our study not only contributes to understanding the molecular mechanism of catalysis/regulation of an important class of enzymes but also provides the genetic, biochemical, and structural groundwork for the future exploitation of UGP as a potential antifungal target. IMPORTANCE Fungi cause diverse diseases in humans, ranging from allergic syndromes to life-threatening invasive diseases, together affecting more than a billion people worldwide. Increasing drug resistance in Aspergillus species represents an emerging global health threat, making the design of antifungals with novel mechanisms of action a worldwide priority. The cryoEM structure of UDP (uridine diphosphate)-glucose pyrophosphorylase (UGP) from the filamentous fungus Aspergillus nidulans reveals an octameric architecture displaying unprecedented conformational variability between the C-terminal oligomerization domain and the central glycosyltransferase A-like catalytic domain in the individual protomers. While the active site and oligomerization interfaces are more highly conserved, these dynamic interfaces include motifs restricted to specific clades of filamentous fungi. Functional study of these motifs could lead to the definition of new targets for antifungals inhibiting UGP activity and, thus, the architecture of the cell wall of filamentous fungal pathogens.

13.
Sci Adv ; 9(49): eadj0390, 2023 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-38055814

RESUMO

Global climate change threatens tropical coral reefs, yet local management can influence resilience. While increasing anthropogenic nutrients reduce coral resistance and recovery, it is unknown how the loss, or restoration, of natural nutrient flows affects reef recovery. Here, we test how natural seabird-derived nutrient subsidies, which are threatened by invasive rats, influence the mechanisms and patterns of reef recovery following an extreme marine heatwave using multiyear field experiments, repeated surveys, and Bayesian modeling. Corals transplanted from rat to seabird islands quickly assimilated seabird-derived nutrients, fully acclimating to new nutrient conditions within 3 years. Increased seabird-derived nutrients, in turn, caused a doubling of coral growth rates both within individuals and across entire reefs. Seabirds were also associated with faster recovery time of Acropora coral cover (<4 years) and more dynamic recovery trajectories of entire benthic communities. We conclude that restoring seabird populations and associated nutrient pathways may foster greater coral reef resilience through enhanced growth and recovery rates of corals.


Assuntos
Antozoários , Resiliência Psicológica , Animais , Ratos , Recifes de Corais , Teorema de Bayes , Aves , Ecossistema
14.
Elife ; 112022 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-35801683

RESUMO

Pigments homologous to the green fluorescent protein (GFP) have been proposed to fine-tune the internal light microclimate of corals, facilitating photoacclimation of photosynthetic coral symbionts (Symbiodiniaceae) to life in different reef habitats and environmental conditions. However, direct measurements of the in vivo light conditions inside the coral tissue supporting this conclusion are lacking. Here, we quantified the intra-tissue spectral light environment of corals expressing GFP-like proteins from widely different light regimes. We focus on: (1) photoconvertible red fluorescent proteins (pcRFPs), thought to enhance photosynthesis in mesophotic habitats via wavelength conversion, and (2) chromoproteins (CPs), which provide photoprotection to the symbionts in shallow water via light absorption. Optical microsensor measurements indicated that both pigment groups strongly alter the coral intra-tissue light environment. Estimates derived from light spectra measured in pcRFP-containing corals showed that fluorescence emission can contribute to >50% of orange-red light available to the photosynthetic symbionts at mesophotic depths. We further show that upregulation of pink CPs in shallow-water corals during bleaching leads to a reduction of orange light by 10-20% compared to low-CP tissue. Thus, screening by CPs has an important role in mitigating the light-enhancing effect of coral tissue scattering and skeletal reflection during bleaching. Our results provide the first experimental quantification of the importance of GFP-like proteins in fine-tuning the light microclimate of corals during photoacclimation.


Assuntos
Antozoários , Dinoflagellida , Animais , Antozoários/metabolismo , Dinoflagellida/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Luz , Simbiose , Água/metabolismo
15.
Curr Biol ; 30(13): 2433-2445.e3, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32442463

RESUMO

Coral bleaching, caused by the loss of brownish-colored dinoflagellate photosymbionts from the host tissue of reef-building corals, is a major threat to reef survival. Occasionally, bleached corals become exceptionally colorful rather than white. These colors derive from photoprotective green fluorescent protein (GFP)-like pigments produced by the coral host. There is currently no consensus regarding what causes colorful bleaching events and what the consequences for the corals are. Here, we document that colorful bleaching events are a recurring phenomenon in reef regions around the globe. Our analysis of temperature conditions associated with colorful bleaching events suggests that corals develop extreme coloration within 2 to 3 weeks after exposure to mild or temporary heat stress. We demonstrate that the increase of light fluxes in symbiont-depleted tissue promoted by reflection of the incident light from the coral skeleton induces strong expression of the photoprotective coral host pigments. We describe an optical feedback loop involving both partners of the association, discussing that the mitigation of light stress offered by host pigments could facilitate recolonization of bleached tissue by symbionts. Our data indicate that colorful bleaching has the potential to identify local environmental factors, such as nutrient stress, that can exacerbate the impact of elevated temperatures on corals, to indicate the severity of heat stress experienced by corals and to gauge their post-stress recovery potential. VIDEO ABSTRACT.


Assuntos
Antozoários/fisiologia , Dinoflagellida/fisiologia , Temperatura Alta/efeitos adversos , Pigmentação , Simbiose , Animais , Cor , Recifes de Corais , Retroalimentação
16.
Curr Res Struct Biol ; 2: 89-103, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-34235472

RESUMO

Glycogen and starch are the major carbon and energy reserve polysaccharides in nature, providing living organisms with a survival advantage. The evolution of the enzymatic machinery responsible for the biosynthesis and degradation of such polysaccharides, led the development of mechanisms to control the assembly and disassembly rate, to store and recover glucose according to cell energy demands. The tetrameric enzyme ADP-glucose pyrophosphorylase (AGPase) catalyzes and regulates the initial step in the biosynthesis of both α-polyglucans. AGPase displays cooperativity and allosteric regulation by sensing metabolites from the cell energy flux. The understanding of the allosteric signal transduction mechanisms in AGPase arises as a long-standing challenge. In this work, we disclose the cryoEM structures of the paradigmatic homotetrameric AGPase from Escherichia coli (EcAGPase), in complex with either positive or negative physiological allosteric regulators, fructose-1,6-bisphosphate (FBP) and AMP respectively, both at 3.0 Å resolution. Strikingly, the structures reveal that FBP binds deeply into the allosteric cleft and overlaps the AMP site. As a consequence, FBP promotes a concerted conformational switch of a regulatory loop, RL2, from a "locked" to a "free" state, modulating ATP binding and activating the enzyme. This notion is strongly supported by our complementary biophysical and bioinformatics evidence, and a careful analysis of vast enzyme kinetics data on single-point mutants of EcAGPase. The cryoEM structures uncover the residue interaction networks (RIN) between the allosteric and the catalytic components of the enzyme, providing unique details on how the signaling information is transmitted across the tetramer, from which cooperativity emerges. Altogether, the conformational states visualized by cryoEM reveal the regulatory mechanism of EcAGPase, laying the foundations to understand the allosteric control of bacterial glycogen biosynthesis at the molecular level of detail.

17.
Curr Biol ; 28(21): R1263-R1265, 2018 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-30399353

RESUMO

High levels of phytoplankton visible in satellite imagery are correlated with an increased uptake of carbon compounds by corals. This suggests that corals rely less on carbon production by photosynthetic symbionts when other resources are plentiful, and that the changes in the acquisition mode of carbon can be inferred by remote-sensing techniques.


Assuntos
Antozoários , Animais , Carbono , Dieta , Fotossíntese , Simbiose
18.
FEBS J ; 285(20): 3815-3834, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30106221

RESUMO

Bis-(3'-5')-cyclic diguanylic acid (c-di-GMP) belongs to the class of cyclic dinucleotides, key carriers of cellular information in prokaryotic and eukaryotic signal transduction pathways. In bacteria, the intracellular levels of c-di-GMP and their complex physiological outputs are dynamically regulated by environmental and internal stimuli, which control the antagonistic activities of diguanylate cyclases (DGCs) and c-di-GMP specific phosphodiesterases (PDEs). Allostery is one of the major modulators of the c-di-GMP-dependent response. Both the c-di-GMP molecule and the proteins interacting with this second messenger are characterized by an extraordinary structural plasticity, which has to be taken into account when defining and possibly predicting c-di-GMP-related processes. Here, we report a structure-function relationship study on the catalytic portion of the PA0575 protein from Pseudomonas aeruginosa, bearing both putative DGC and PDE domains. The kinetic and structural studies indicate that the GGDEF-EAL portion is a GTP-dependent PDE. Moreover, the crystal structure confirms the high degree of conformational flexibility of this module. We combined structural analysis and protein engineering studies to propose the possible molecular mechanism guiding the nucleotide-dependent allosteric control of catalysis; we propose that the role exerted by GTP via the GGDEF domain is to allow the two EAL domains to form a dimer, the species competent to enter PDE catalysis.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , GMP Cíclico/análogos & derivados , Guanosina Trifosfato/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Pseudomonas aeruginosa/metabolismo , Regulação Alostérica , Cristalografia por Raios X , GMP Cíclico/metabolismo , Hidrólise , Diester Fosfórico Hidrolases/química , Conformação Proteica , Multimerização Proteica
19.
FEBS J ; 274(10): 2496-505, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17419724

RESUMO

Pigments homologous to the green fluorescent protein (GFP) contribute up to approximately 14% of the soluble protein content of many anthozoans. Maintenance of such high tissue levels poses a severe energetic penalty to the animals if protein turnover is fast. To address this as yet unexplored issue, we established that the irreversible green-to-red conversion of the GFP-like pigments from the reef corals Montastrea cavernosa (mcavRFP) and Lobophyllia hemprichii (EosFP) is driven by violet-blue radiation in vivo and in situ. In the absence of photoconverting light, we subsequently tracked degradation of the red-converted forms of the two proteins in coral tissue using in vivo spectroscopy and immunochemical detection of the post-translational peptide backbone modification. The pigments displayed surprisingly slow decay rates, characterized by half-lives of approximately 20 days. The slow turnover of GFP-like proteins implies that the associated energetic costs for being colorful are comparatively low. Moreover, high in vivo stability makes GFP-like proteins suitable for functions requiring high pigment concentrations, such as photoprotection.


Assuntos
Antozoários/química , Proteínas Luminescentes/metabolismo , Pigmentos Biológicos/metabolismo , Animais , Antozoários/efeitos da radiação , Cor , Escuridão , Cinética , Luz , Proteínas Luminescentes/efeitos da radiação , Pigmentos Biológicos/efeitos da radiação , Espectrometria de Fluorescência
20.
FEBS J ; 274(4): 1102-9, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17244197

RESUMO

For a variety of coral species, we have studied the molecular origin of their coloration to assess the contributions of host and symbiont pigments. For the corals Catalaphyllia jardinei and an orange-emitting color morph of Lobophyllia hemprichii, the pigments belong to a particular class of green fluorescent protein-like proteins that change their color from green to red upon irradiation with approximately 400 nm light. The optical absorption and emission properties of these proteins were characterized in detail. Their spectra were found to be similar to those of phycoerythrin from cyanobacterial symbionts. To unambiguously determine the molecular origin of the coloration, we performed immunochemical studies using double diffusion in gel analysis on tissue extracts, including also a third coral species, Montastrea cavernosa, which allowed us to attribute the red fluorescent coloration to green-to-red photoconvertible fluorescent proteins. The red fluorescent proteins are localized mainly in the ectodermal tissue and contribute up to 7.0% of the total soluble cellular proteins in these species. Distinct spatial distributions of green and cyan fluorescent proteins were observed for the tissues of M. cavernosa. This observation may suggest that differently colored green fluorescent protein-like proteins have different, specific functions. In addition to green fluorescent protein-like proteins, the pigments of zooxanthellae have a strong effect on the visual appearance of the latter species.


Assuntos
Antozoários/metabolismo , Pigmentação/fisiologia , Pigmentos Biológicos/química , Simbiose , Animais , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Imunoquímica , Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Ficoeritrina/química , Especificidade da Espécie , Espectrometria de Fluorescência , Proteína Vermelha Fluorescente
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