Analyzing the social factors that influence willingness to pay for the management of invasive alien species under two different strategies: eradication and prevention

This an accepted author's manuscript (AAM) version of an article published in Environmental Management, 48 (2011): 418-43

Cryo-EM and the elucidation of new macromolecular structures: Random Conical Tilt revisited

Cryo-Electron Microscopy (cryo-EM) of macromolecular complexes is a fundamental structural biology technique which is expanding at a very fast pace. Key to its success in elucidating the three-dimensional structure of a macromolecular complex, especially of small and non-symmetric ones, is the ability to start from a low resolution map, which is subsequently refined with the actual images collected at the microscope. There are several methods to produce this first structure. Among them, Random Conical Tilt (RCT) plays a prominent role due to its unbiased nature (it can create an initial model based on experimental measurements). In this article, we revise the fundamental mathematical expressions supporting RCT, providing new expressions handling all key geometrical parameters without the need of intermediate operations, leading to improved automation and overall reliability, essential for the success of cryo-EM when analyzing new complexes. We show that the here proposed RCT workflow based on the new formulation performs very well in practical cases, requiring very few image pairs (as low as 13 image pairs in one of our examples) to obtain relevant 3D maps.We thank Dr. Llorca for his support during the acquisition of the C3b images and Dr. Shaikh for his support in the use of Spider for the RCT reconstructions. The authors would like to acknowledge economical support from the Spanish Ministry of Economy and Competitiveness through grants AIC-A-2011-0638 and BIO2013-44647-R, the Comunidad de Madrid through grant CAM (S2010/BMD-2305), as well as a postdoctoral Juan de la Cierva grant with reference JCI-2011-10185 to Javier Vargas. Vahid Abrishami is a holder of La Caixa scholarship and C.O.S. Sorzano is recipient of a Ramon y Cajal fellowship

Spatial variation and biovectoring of metals in gull faeces

We assessed the spatial variation in concentrations of ten metals in faeces of the lesser black-backed gull (LBBG) Larus fuscus wintering at seven localities in South-West Spain. We found high concentrations of metals in gull faeces, with several elements (As, Cu, Mo, Pb, Zn) locally exceeding (by 2 to 11 times) derived Lowest Effect Level (LEL) values. We also found strong spatial variation, related to the main pollution sources associated with the different sites. Faeces from Chipiona Port (Gulf of C?adiz) showed the highest levels of As; Cetina saltpans (Bay of C?adiz) ranked first for Pb, Zn and Mo, which was consistent with historic mining and industrial pollution; Don?ana ricefields showed the highest levels of Mn, a highly available element in flooded areas; while landfills ranked first for Cd, Co, Cr, Cu and Ni, potentially associated with electronic waste. Furthermore, we demonstrate how faecal analysis can be used to quantify biovectoring of metals into specific localities, using LBBG movement ecology and census data. At Fuente de Piedra, a shallow, closed-basin lake important for waterbirds, we show that metal inputs by LBBG have increased in recent years, and long-term deposition (e.g., of Pb) may impact aquatic communities and ecological processes in this Ramsar site

Estudio de la interacción de la proteína p6 con el DNA del bacteriófago [phi] 29 de "Bacillus subtilis"

Tesis Doctoral inédita leida en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura 30-03-2007Protein p6 of Bacillus subtilis bacteriophage Φ29 is essential for phage development. It activates the initiation of DNA replication and promotes the early to late transcriptional switch. These activities require the formation of a nucleoprotein complex in which the DNA forms a right-handed superhelix wrapping around a multimeric protein core. However, there was no evidence of p6 binding to Φ29 DNA in vivo. In this work, protein p6 binding to DNA has been studied by crosslinking, chromatin immunoprecipitation (X-ChIP) and real-time PCR. The results have shown that protein p6 binds to most, if not all, the viral genome in vivo, although with higher affinity for both DNA ends, which contain the replication origins. The p6 binding to different Φ29 DNA regions is modulated by the structural properties of their nucleotide sequences and the higher affinity for DNA ends is possibly related to the presence of sequences in which their bendability properties favour the formation of the p6-DNA complex. Moreover, protein p6 binds to Φ29 DNA with higher affinity than plasmid DNA. This specificity could be due to the p6 binding preference for less negatively supercoiled DNA. Thus, the p6 binding dependence on DNA topology could explain its preferential binding to viral with respect to plasmid or bacterial DNA, whose level of negative supercoiling is presumably higher than that of Φ29 DNA. Protein p6 binding to Φ29 DNA is greatly increased when negative supercoiling is decreased by novobiocin. The fact that gyrase is required for viral DNA replication indicates that, although non-covalently closed, the viral genome is topologically constrained in vivo. Viral proteins p1 and p17 seem to be required to restrain the proper topology of Φ29 DNA. Binding of protein p6 all along the Φ29 genome strongly suggests that its functions in replication and transcription control could be outcomes of a more global role as a histone-like protein. The in vivo interaction with DNA of protein p6 of the Φ29-related bacteriophage GA-1 has been also studied with the finding that it has a much lower dependence on supercoiling than Φ29 p6 and that is able to bind to Φ29 DNA, although it does not functionally replace the Φ29 counterpart. We have taken advantage of the binding properties of protein p6 to monitor the entry of Φ29 DNA into the cell by X-ChIP, measuring p6 binding to both viral genome ends. With this approach, we have studied the Φ29 DNA ejection mechanism, demonstrating that the early viral membrane protein p16.7 is required, together with protein p17, for efficient execution of the second pull step of Φ29 DNA ejection. Finally, the quantitative in vitro characterization of reversible protein p6- DNA association in solution, in terms of stoichiometry, affinity and cooperativity, has been carried out using analytical ultracentrifugation. This analysis has shown that p6-DNA complex formation could be fitted by the empirical Hill function. The effect of macromolecular crowding on the p6-DNA complex formation has been also studied using sedimentation equilibrium analysis.El presente trabajo se ha llevado a cabo en el Centro de Biología Molecular “Severo Ochoa” bajo la dirección del Doctor José Miguel Hermoso Núñez y la tutoría de la Doctora Margarita Salas Falgueras. Para su realización se ha contado con una beca de formación de profesorado universitario del Ministerio de Educación y Ciencia.Peer reviewe

Structural Characterization of the Essential Cell Division Protein FtsE and Its Interaction with FtsX in Streptococcus pneumoniae

FtsEX is a membrane complex widely conserved across diverse bacterial genera and involved in critical processes such as recruitment of division proteins and in spatial and temporal regulation of muralytic activity during cell division or sporulation. FtsEX is a member of the ABC transporter superfamily. The component FtsX is an integral membrane protein, whereas FtsE is an ATPase and is required for the transmission of a conformational signal from the cytosol through the membrane to regulate the activity of cell wall hydrolases in the periplasm. Both proteins are essential in the major human respiratory pathogenic bacterium Streptococcus pneumoniae, and FtsX interacts with the modular peptidoglycan hydrolase PcsB at the septum. Here, we report high-resolution structures of pneumococcal FtsE bound to different nucleotides. Structural analysis revealed that FtsE contains all the conserved structural motifs associated with ATPase activity and afforded interpretation of the in vivo dimeric arrangement in both the ADP and ATP states. Interestingly, three specific FtsE regions with high structural plasticity were identified that shape the cavity in which the cytosolic region of FtsX would be inserted. The residues corresponding to the FtsX coupling helix, responsible for contacting FtsE, were identified and validated by in vivo mutagenesis studies showing that this interaction is essential for cell growth and proper morphology.publishedVersio

In Vivo DNA Binding of Bacteriophage GA-1 Protein p6▿

Bacteriophage GA-1 infects Bacillus sp. strain G1R and has a linear double-stranded DNA genome with a terminal protein covalently linked to its 5′ ends. GA-1 protein p6 is very abundant in infected cells and binds DNA with no sequence specificity. We show here that it binds in vivo to the whole viral genome, as detected by cross-linking, chromatin immunoprecipitation, and real-time PCR analyses, and has the characteristics of a histone-like protein. Binding to DNA of GA-1 protein p6 shows little supercoiling dependency, in contrast to the ortholog protein of the evolutionary related Bacillus subtilis phage φ29. This feature is a property of the protein rather than the DNA or the cellular background, since φ29 protein p6 shows supercoiling-dependent binding to GA-1 DNA in Bacillus sp. strain G1R. GA-1 DNA replication is impaired in the presence of the gyrase inhibitors novobiocin and nalidixic acid, which indicates that, although noncovalently closed, the viral genome is topologically constrained in vivo. GA-1 protein p6 is also able to bind φ29 DNA in B. subtilis cells; however, as expected, the binding is less supercoiling dependent than the one observed with the φ29 protein p6. In addition, the nucleoprotein complex formed is not functional, since it is not able to transcomplement the DNA replication deficiency of a φ29 sus6 mutant. Furthermore, we took advantage of φ29 protein p6 binding to GA-1 DNA to find that the viral DNA ejection mechanism seems to take place, as in the case of φ29, with a right to left polarity in a two-step, push-pull process

Binding of phage Φ29 architectural protein p6 to the viral genome: evidence for topological restriction of the phage linear DNA

Bacillus subtilis phage Φ29 protein p6 is required for DNA replication and promotes the switch from early to late transcription. In vivo it binds all along the viral linear DNA, which suggests a global role as an architectural protein; in contrast, binding to bacterial DNA is negligible. This specificity could be due to the p6 binding preference for less negatively supercoiled DNA, as is presumably the case with viral (with respect to bacterial) DNA. Here we demonstrate that p6 binding to Φ29 DNA is greatly increased when negative supercoiling is decreased by novobiocin; in addition, gyrase is required for DNA replication. This indicates that, although non-covalently closed, the viral genome is topologically constrained in vivo. We also show that the p6 binding to different Φ29 DNA regions is modulated by the structural properties of their nucleotide sequences. The higher affinity for DNA ends is possibly related to the presence of sequences in which their bendability properties favor the formation of the p6–DNA complex, whereas the lower affinity for the transcription control region is most probably due to the presence of a rigid intrinsic DNA curvature

Structural basis for the stabilization of the complement alternative pathway C3 convertase by properdin

33 p.-4 fig.-2 fig. supl.Complement is an essential component of innate immunity. Its activation results in the assembly of unstable protease complexes, denominated C3/C5 convertases, leading to inflammation and lysis. Regulatory proteins inactivate C3/C5 convertases on host surfaces to avoid collateral tissue damage. On pathogen surfaces, properdin stabilizes C3/C5 convertases to efficiently fight infection. How properdin performs this function is, however, unclear. Using electron microscopy we show that the N-and C-terminal ends of adjacent monomers in properdin oligomers conform a curly vertex that holds together the AP convertase, interacting with both the C345C and vWA domains of C3b and Bb, respectively. Properdin also promotes a large displacement of the TED (thioestercontaining domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domains of C3b, which likely impairs C3-convertase inactivation by regulatory proteins. The combined effect of molecular cross-linking and structural reorganization increases stability of the C3 convertase and facilitates recruitment of fluid-phase C3 convertase to the cell surfaces. Our model explains how properdin mediates the assembly of stabilized C3/C5-convertase clusters, which helps to localize complement amplification to pathogen surfaces.This work was funded by the Autonomous Region of Madrid (S2010/BMD-2316 to S.R.d.C. and O.L.), the Ramón Areces Foundation (O.L.), and the Spanish government (SAF2011-22988 to O.L. and SAF2011-26583 to S.R.d.C.). O.L. is additionally supported by Red Temática de Investigación Cooperativa en Cáncer (RD06/0020/1001), and S.R.d.C. is also supported by the Fundación Renal Iñigo Alvarez de Toledo and the Seventh Framework Programme European Union Project EURenOmics (European Consortium for High-Throughput Research in Rare Kidney Diseases-305608).Peer Reviewe