Water level changes, subsidence, and sea level rise in the Ganges-Brahmaputra-Meghna delta.

Being one of the most vulnerable regions in the world, the Ganges-Brahmaputra-Meghna delta presents a major challenge for climate change adaptation of nearly 200 million inhabitants. It is often considered as a delta mostly exposed to sea-level rise and exacerbated by land subsidence, even if the local vertical land movement rates remain uncertain. Here, we reconstruct the water-level (WL) changes over 1968 to 2012, using an unprecedented set of 101 water-level gauges across the delta. Over the last 45 y, WL in the delta increased slightly faster (∼3 mm/y), than global mean sea level (∼2 mm/y). However, from 2005 onward, we observe an acceleration in the WL rise in the west of the delta. The interannual WL fluctuations are strongly modulated by El Niño Southern Oscillation (ENSO) and Indian Ocean Dipole (IOD) variability, with WL lower than average by 30 to 60 cm during co-occurrent El Niño and positive IOD events and higher-than-average WL, by 16 to 35 cm, during La Niña years. Using satellite altimetry and WL reconstructions, we estimate that the maximum expected rates of delta subsidence during 1993 to 2012 range from 1 to 7 mm/y. By 2100, even under a greenhouse gas emission mitigation scenario (Representative Concentration Pathway [RCP] 4.5), the subsidence could double the projected sea-level rise, making it reach 85 to 140 cm across the delta. This study provides a robust regional estimate of contemporary relative WL changes in the delta induced by continental freshwater dynamics, vertical land motion, and sea-level rise, giving a basis for developing climate mitigation strategies.

Basin-wide sea level coherency in the tropical Indian Ocean driven by Madden-Julian Oscillation.

Changes in sea level may be attributed either to barotropic (involving the entire water column) or baroclinic processes (governed by stratification). It has been widely accepted that barotropic sea level changes in the tropics are insignificant at intraseasonal time scales (periods of 30-80 days). Based on bottom pressure records, we present evidence for significant basin-wide barotropic sea level variability in the tropical Indian Ocean during December-April with standard deviations amounting to ∼30-60% of the standard deviation in total intraseasonal sea level variability. The origin of this variability is linked to a small patch of wind over the Eastern Indian Ocean, associated with boreal winter Madden-Julian Oscillations (MJO). These large fluctuations are likely to play a prominent role in the intraseasonal sea level and mass budgets. Because of their much faster propagation than baroclinic processes, they allow the basin to adjust to climatic perturbations much more rapidly than was previously thought.

Crystallographic studies of the structured core domain of Knr4 from Saccharomyces cerevisiae.

The potentially structured core domain of the intrinsically disordered protein Knr4 from Saccharomyces cerevisiae, comprising residues 80-340, was expressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method. Selenomethionine-containing (SeMet) protein was also purified and crystallized. Crystals of both proteins belonged to space group P6522, with unit-cell parameters a = b = 112.44, c = 265.21 Šfor the native protein and a = b = 112.49, c = 262.21 Šfor the SeMet protein, and diffracted to 3.50 and 3.60 Šresolution, respectively. There are two molecules in the asymmetric unit related by a twofold axis. The anomalous signal of selenium was recorded and yielded an electron-density map of sufficient quality to allow the identification of secondary-structure elements.

Purification and characterization of a new laccase from the filamentous fungus Podospora anserina.

A new laccase from the filamentous fungus Podospora anserina has been isolated and identified. The 73 kDa protein containing 4 coppers, truncated from its first 31 amino acids, was successfully overexpressed in Pichia pastoris and purified in one step with a yield of 48% and a specific activity of 644Umg(-1). The kinetic parameters, k(cat) and K(M), determined at 37 °C and optimal pH are 1372 s(-1) and 307 µM for ABTS and, 1.29 s(-1) and 10.9 µM, for syringaldazine (SGZ). Unlike other laccases, the new protein displays a better thermostability, with a half life>400 min at 37 °C, is less sensitive to chloride and more stable at pH 7. Even though, the new 566 amino-acid enzyme displays a large homology with Bilirubin oxidase (BOD) from Myrothecium verrucaria (58%) and exhibits the four histidine rich domains consensus sequences of BODs, the new enzyme is not able to oxidize neither conjugated nor unconjugated bilirubin.

Heat and drying time modulate the O2 reduction current of modified glassy carbon electrodes with bilirubin oxidases.

Here we show that the magnitude of the O(2) reduction current of cathodes based on Bilirubin oxidases (BOD) immobilized into a redox hydrogel strongly depends on the drying conditions such as the curing time and temperature of drying as well as the thermostability of the BOD. To illustrate this effect, we performed experiments with two different BODs: one labile BOD from Trachyderma tsunodae and one highly thermostable BOD from Bacillus pumilus with different preparation protocols. The balance between the kinetics of formation of the hydrogel and the enzyme stability leads to optimal drying conditions of 2h at 25°C for both types of BODs when the most widespread protocol uses 18 hours at ambient temperature. For drying times longer than two hours, the catalytic current decreases because of the instability of T. tsunodae. Finally the optimal conditions for BOD from T. tsunodae lead to a faster preparation of electrodes than with the protocol currently in use (2h vs. 18h) and catalytic currents for oxygen reduction 100% higher (1040µA/cm(2) vs. 517µA/cm(2)).

Bilirubin oxidase from Bacillus pumilus: a promising enzyme for the elaboration of efficient cathodes in biofuel cells.

A CotA multicopper oxidase (MCO) from Bacillus pumilus, previously identified as a laccase, has been studied and characterized as a new bacterial bilirubin oxidase (BOD). The 59 kDa protein containing four coppers, was successfully over-expressed in Escherichia coli and purified to homogeneity in one step. This 509 amino-acid enzyme, having 67% and 26% sequence identity with CotA from Bacillus subtilis and BOD from Myrothecium verrucaria, respectively, shows higher turnover activity towards bilirubin compared to other bacterial MCOs. The current density for O(2) reduction, when immobilized in a redox hydrogel, is only 12% smaller than the current obtained with Trachyderma tsunodae BOD. Under continuous electrocatalysis, an electrode modified with the new BOD is more stable, and has a higher tolerance towards NaCl, than a T. tsunodae BOD modified electrode. This makes BOD from B. pumilus an attractive new candidate for application in biofuel cells (BFCs) and biosensors.

Spectroscopic and crystallographic characterization of "alternative resting" and "resting oxidized" enzyme forms of bilirubin oxidase: implications for activity and electrochemical behavior of multicopper oxidases.

While there is broad agreement on the catalytic mechanism of multicopper oxidases (MCOs), the geometric and electronic structures of the resting trinuclear Cu cluster have been variable, and their relevance to catalysis has been debated. Here, we present a spectroscopic characterization, complemented by crystallographic data, of two resting forms occurring in the same enzyme and define their interconversion. The resting oxidized form shows similar features to the resting form in Rhus vernicifera and Trametes versicolor laccase, characterized by "normal" type 2 Cu electron paramagnetic resonance (EPR) features, 330 nm absorption shoulder, and a short type 3 (T3) Cu-Cu distance, while the alternative resting form shows unusually small A(||) and high g(||) EPR features, lack of 330 nm absorption intensity, and a long T3 Cu-Cu distance. These different forms are evaluated with respect to activation for catalysis, and it is shown that the alternative resting form can only be activated by low-potential reduction, in contrast to the resting oxidized form which is activated via type 1 Cu at high potential. This difference in activity is correlated to differences in redox states of the two forms and highlights the requirement for efficient sequential reduction of resting MCOs for their involvement in catalysis.

Bilirubin oxidase from Magnaporthe oryzae: an attractive new enzyme for biotechnological applications.

A novel bilirubin oxidase (BOD), from the rice blast fungus Magnaporthe oryzae, has been identified and isolated. The 64-kDa protein containing four coppers was successfully overexpressed in Pichia pastoris and purified to homogeneity in one step. Protein yield is more than 100 mg for 2 L culture, twice that of Myrothecium verrucaria. The k(cat)/K(m) ratio for conjugated bilirubin (1,513 mM⁻¹ s⁻¹) is higher than that obtained for the BOD from M. verrucaria expressed in native fungus (980 mM⁻¹ s⁻¹), with the lowest K(m) measured for any BOD highly desirable for detection of bilirubin in medical samples. In addition, this protein exhibits a half-life for deactivation >300 min at 37 °C, high stability at pH 7, and high tolerance towards urea, making it an ideal candidate for the elaboration of biofuel cells, powering implantable medical devices. Finally, this new BOD is efficient in decolorizing textile dyes such as Remazol brilliant Blue R, making it useful for environmentally friendly industrial applications.

Effect of substrate inhibition and cooperativity on the electrochemical responses of glucose dehydrogenase. Kinetic characterization of wild and mutant types.

Thanks to its insensitivity to dioxygen and to its good catalytic reactivity, and in spite of its poor substrate selectivity, quinoprotein glucose dehydrogenase (PQQ-GDH) plays a prominent role among the redox enzymes that can be used for analytical purposes, such as glucose detection, enzyme-based bioaffinity assays, and the design of biofuel cells. A detailed kinetic analysis of the electrochemical catalytic responses, leading to an unambiguous characterization of each individual steps, seems a priori intractable in view of the interference, on top of the usual ping-pong mechanism, of substrate inhibition and of cooperativity effects between the two identical subunits of the enzyme. Based on simplifications suggested by extended knowledge previously acquired by standard homogeneous kinetics, it is shown that analysis of the catalytic responses obtained by means of electrochemical nondestructive techniques, such as cyclic voltammetry, with ferrocene methanol as a mediator, does allow a full characterization of all individual steps of the catalytic reaction, including substrate inhibition and cooperativity and, thus, allows to decipher the reason that makes the enzyme more efficient when the neighboring subunit is filled with a glucose molecule. As a first practical illustration of this electrochemical approach, comparison of the native enzyme responses with those of a mutant (in which the asparagine amino acid in position 428 has been replaced by a cysteine residue) allowed identification of the elementary steps that makes the mutant type more efficient than the wild type when cooperativity between the two subunits takes place, which is observed at large mediator and substrate concentrations. A route is thus opened to structure-reactivity relationships and therefore to mutagenesis strategies aiming at better performances in terms of catalytic responses and/or substrate selectivity.

Efficient direct electron transfer of PQQ-glucose dehydrogenase on carbon cryogel electrodes at neutral pH.

We present a comprehensive study of the direct electron transfer reaction of soluble PQQ-GDH from Acinetobacter calcoaceticus. Wild-type PQQ-sGDH nonspecifically adsorbed on carbon cryogel electrodes retained its enzymatic activity for glucose and maltose oxidation at pH 7.2 and 37 °C. The cyclic voltammograms in the absence of enzymatic substrate showed 2 redox peaks that suggest a two-step, one-electron oxidation/reduction of PQQ. Calibration curves showed a linear amperometric response for a wide glucose concentration range, including the values normally found in blood. At saturation, the catalytic current reached 0.93 mA cm(-2). Altogether the experimental results suggest that the amperometric output of the electrodes and the shape of the calibration curves represent a combination of the intrinsic enzyme kinetics, the maximum rate of heterogeneous electron transfer and the substrate accessibility to the enzyme's active center caused by the confinement of the enzyme into the mesoporous structure. A new mutant enzyme, N428C, developed in our group that shows almost twice the maximum catalytic activity in homogeneous experiments in solution, also showed a DET signal on carbon cryogel electrodes for glucose electro-oxidation. The higher activity for the mutant enzyme was also verified on the electrode surface.

Designing a highly active soluble PQQ-glucose dehydrogenase for efficient glucose biosensors and biofuel cells.

We report for the first time a soluble PQQ-glucose dehydrogenase that is twice more active than the wild type for glucose oxidation and was obtained by combining site directed mutagenesis, modelling and steady-state kinetics. The observed enhancement is attributed to a better interaction between the cofactor and the enzyme leading to a better electron transfer. Electrochemical experiments also demonstrate the superiority of the new mutant for glucose oxidation and make it a promising enzyme for the development of high-performance glucose biosensors and biofuel cells.

An atomic force microscopy analysis of yeast mutants defective in cell wall architecture.

Yeast cells are surrounded by a thick cell wall, the composition and structure of which have been characterized by biochemical and genetic methods. In this study, we used atomic force microscopy (AFM) to visualize the cell surface topography and to determine cell wall nanomechanical properties of yeast mutants defective in cell wall architecture. While all mutants investigated showed some alteration in cell surface topography, this alteration was particularly salient in mutants defective in beta-glucan elongation (gas1), chitin synthesis (chs3) and cross-linkages between chitin and beta-glucan (crh1crh2). In addition, these alterations in surface topology were accompanied by increased roughness of the cell. From force-indentation curves, the Young's modulus was determined, as it gives a measure of the elasticity of the cell wall. A value of approximately 1.6 MPa was obtained for the cell walls of the wild-type strain in exponential and stationary phases of growth. The same value was measured in a mnn9 mutant defective in protein mannosylation, and was two-fold reduced in a mutant with reduced beta-glucan (fks1Delta and knr4Delta), only in the stationary phase of growth. In contrast, the elasticity was dramatically reduced in mutants defective in chitin synthesis (chs3Delta), beta-glucan elongation (gas1Delta) and, even more remarkably, in a crh1Deltacrh2Delta mutant defective in the enzymes that catalyse cross-linkages of chitin to beta-glucan. Taken together, these results provide direct physical evidence that the nanomechanical properties of the yeast cell wall are mainly dependent on cross-links and cell wall remodelling, rather than on cell wall composition or thickness.

Functional dissection of an intrinsically disordered protein: understanding the roles of different domains of Knr4 protein in protein-protein interactions.

Knr4, recently characterized as an intrinsically disordered Saccharomyces cerevisiae protein, participates in cell wall formation and cell cycle regulation. It is constituted of a functional central globular core flanked by a poorly structured N-terminal and large natively unfolded C-terminal domains. Up to now, about 30 different proteins have been reported to physically interact with Knr4. Here, we used an in vivo two-hybrid system approach and an in vitro surface plasmon resonance (BIAcore) technique to compare the interaction level of different Knr4 deletion variants with given protein partners. We demonstrate the indispensability of the N-terminal domain of Knr4 for the interactions. On the other hand, presence of the unstructured C-terminal domain has a negative effect on the interaction strength. In protein interactions networks, the most highly connected proteins or "hubs" are significantly enriched in unstructured regions, and among them the transient hub proteins contain the largest and most highly flexible regions. The results presented here of our analysis of Knr4 protein suggest that these large disordered regions are not always involved in promoting the protein-protein interactions of hub proteins, but in some cases, might rather inhibit them. We propose that this type of regions could prevent unspecific protein interactions, or ensure the correct timing of occurrence of transient interactions, which may be of crucial importance for different signaling and regulation processes.

Structure-function analysis of Knr4/Smi1, a newly member of intrinsically disordered proteins family, indispensable in the absence of a functional PKC1-SLT2 pathway in Saccharomyces cerevisiae.

The coordination between cell wall synthesis and cell growth in the yeast Saccharomyces cerevisiae implicates the PKC1-dependent MAP kinase pathway. KNR4, encoding a 505 amino acid long protein, participates in this coordination, since it displays synthetic lethality with all the members of the PKC1 pathway and shows physical interaction with Slt2/Mpk1. The recent finding that KNR4 interacts genetically or physically with more than 100 partners implicated in different cellular processes raised the question of how these interactions may occur and their physiological significance. This called for an in-depth structure-function analysis of the Knr4 protein, which is reported in the present paper. Computational analysis supported by biochemical and biophysical data characterize Knr4 as a newly identified member of the growing family of intrinsically disordered proteins. Despite disordered regions that are located at the N- and C-termini and are probably responsible for fine regulatory function; this protein contains a structured central core (amino acid residues 80-340) that is able to restore wild-type phenotypes of knr4Delta mutant in stress conditions. However, this fragment was unable to complement synthetic lethality between knr4 mutations and deletions of genes encoding protein kinases of the PKC1-dependent pathway. For these crucial events to occur, the presence of the N-terminal part of Knr4 protein is indispensable. Moreover, we demonstrate that this protein is essential for cell viability in the absence of a functional Pkc1-Slt2 pathway, since the lethality caused by KNR4 deletion in such a genetic background could not be compensated by overexpression of any gene from yeast genomic libraries.

Assessment of the genetic diversity of Xanthomonas axonopodis pv. phaseoli and Xanthomonas fuscans subsp. fuscans as a basis to identify putative pathogenicity genes and a type III secretion system of the SPI-1 family by multiple suppression subtractive hybridizations.

Fluorescent amplified fragment length polymorphism revealed that strains of Xanthomonas axonopodis pv. phaseoli and Xanthomonas fuscans subsp. fuscans are genetically distinct and can be grouped into four genetic lineages. Four suppression subtractive hybridizations were then performed to isolate DNA fragments present in these bean pathogens and absent from closely related xanthomonads. Virulence gene candidates were identified such as homologs of hemagglutinins, TonB-dependent receptors, zinc-dependent metalloproteases, type III effectors, and type IV secretion system components. Unexpectedly, homologs of the type III secretion apparatus components (SPI-1 family), usually reported in animal pathogens and insect symbionts, were also detected.

The 'interactome' of the Knr4/Smi1, a protein implicated in coordinating cell wall synthesis with bud emergence in Saccharomyces cerevisiae.

The integrity of the Saccharomyces cerevisiae cell wall requires a functional Pkc1-Slt2 MAP kinase pathway that contributes to transient growth arrest, enabling coordination of cell division with cell wall remodelling. How this coordination takes place is still an open question. Recently, we brought evidence that Knr4 protein, whose absence leads to several cell wall defects, may play a role in this function. Here, we show that Knr4 is a monomeric protein that exhibits an aberrant mobility on a SDS-gel electrophoresis and a non-globular structure. Furthermore, Knr4 is an unstable protein that is degraded as cells enter the stationary phase of growth, while its corresponding gene is constitutively expressed. In exponentially growing cells on glucose, Knr4 appeared to be present in a protein complex that migrates with an apparent Mw superior to 250 kDa. Using the TAP-tag methodology, nine potential partners of Knr4 were identified, which could be distributed into three biological processes. A first group consisted of Slt2 and Pil1, two proteins dedicated to cell wall maintenance and biogenesis. The second group comprised four proteins (Bud6, Act1, Cin8 and Jnm1) implicated in the establishment of cell polarity and bud integrity during mitosis. The last group contained four proteins (Asc1, Ubc1, Hsc82 and Gvp36) that probably deal with the stability/degradation of proteins. Deletion analysis revealed that the domain of interaction covered 2/3 of the Knr4 sequence on the N-terminal side. Moreover, the replacement of the two in vivo phosphorylated Ser(200) and Ser(203) by alanines led to a mutated protein with reduced protein interactions and a weaker complementation ability towards knr4 null mutant phenotypes. These results together with previous data from genome scale two-hybrid and synthetic interaction screens support the notion that Knr4 is a regulatory protein that participates in the coordination of cell wall synthesis with bud emergence, and that this function may be modulated by phosphorylation of this protein.

The [URE3] yeast prion results from protein aggregates that differ from amyloid filaments formed in vitro.

The [URE3] yeast prion is a self-propagating inactive form of the Ure2 protein. Ure2p is composed of two domains, residues 1-93, the prion-forming domain, and the remaining C-terminal part of the protein, which forms the functional domain involved in nitrogen catabolite repression. In vitro, Ure2p forms amyloid filaments that have been proposed to be the aggregated prion form found in vivo. Here we showed that the biochemical characteristics of these two species differ. Protease digestions of Ure2p filaments and soluble Ure2p are comparable when analyzed by Coomassie staining as by Western blot. However, this finding does not explain the pattern specifically observed in [URE3] strains. Antibodies raised against the C-terminal part of Ure2p revealed the existence of proteolysis sites efficiently cleaved when [URE3], but not wild-type crude extracts, were submitted to limited proteolysis. The same antibodies lead to an equivalent digestion pattern when recombinant Ure2p (either soluble or amyloid) was analyzed in the same way. These results strongly suggest that aggregated Ure2p in [URE3] yeast cells is different from the amyloid filaments generated in vitro.