Determinants of Cofactor Specificity for the Glucose-6-Phosphate Dehydrogenase from Escherichia coli: Simulation,Kinetics and Evolutionary Studies

Glucose 6-Phosphate Dehydrogenases (G6PDHs) from different sources show varying specificities towards NAD⁺ and NADP⁺ as cofactors. However, it is not known to what extent structural determinants of cofactor preference are conserved in the G6PDH family. In this work, molecular simulations, kinetic characterization of site-directed mutants and phylogenetic analyses were used to study the structural basis for the strong preference towards NADP⁺ shown by the G6PDH from Escherichia coli. Molecular Dynamics trajectories of homology models showed a highly favorable binding energy for residues K18 and R50 when interacting with the 2''-phosphate of NADP⁺, but the same residues formed no observable interactions in the case of NAD⁺. Alanine mutants of both residues were kinetically characterized and analyzed with respect to the binding energy of the transition state, according to the k_cat/K_M value determined for each cofactor. Whereas both residues contribute to the binding energy of NADP⁺, only R50 makes a contribution (about -1 kcal/mol) to NAD⁺ binding. In the absence of both positive charges the enzyme was unable to discriminate NADP⁺ from NAD⁺. Although kinetic data is sparse, the observed distribution of cofactor preferences within the phylogenetic tree is sufficient to rule out the possibility that the known NADP⁺-specific G6PDHs form a monophyletic group. While the β1-α1 loop shows no strict conservation of K18, (rather, S and T seem to be more frequent), in the case of the β2-α2 loop, different degrees of conservation are observed for R50. Noteworthy is the fact that a K18T mutant is indistinguishable from K18A in terms of cofactor preference. We conclude that the structural determinants for the strict discrimination against NAD⁺ in the case of the NADP⁺-specific enzymes have evolved independently through different means during the evolution of the G6PDH family. We further suggest that other regions in the cofactor binding pocket, besides the β1-α1 and β2-α2 loops, play a role in determining cofactor preference.     

Integrative taxonomy of Flatbill Flycatchers (Tyrannidae) reveals a new species from the Amazonian lowlands

Integrative taxonomic studies continue to reveal that many current polytypic species of birds are in fact constituted by two or more species and therefore have been central in uncovering ‘hidden’ or ‘cryptic’ biodiversity. The Olivaceous Flatbill (Aves: Tyrannidae: Rhynchocyclus olivaceus) currently has nine recognized subspecies distributed throughout the Neotropics, but so far, no complete phylogenetic hypothesis exists to test the validity and evolutionary relationships among them. To remedy this, we conducted a multi-character integrative taxonomic revision of the genus Rhynchocyclus, focusing on the polytypic R. olivaceus. The combination of a taxonomically dense sampled multilocus phylogeny (including three mitochondrial and two nuclear genes) with phenotypic analyses including morphological and vocal characters pointed to several taxonomic inconsistencies within R. olivaceus. The analyses strongly support that R. olivaceus is paraphyletic, with an exclusively cis-Andean clade (where the topotypic R. olivaceus is found) clustering as sister to Rhynchocyclus fulvipectus, to the exclusion of a clade grouping trans-Andean and western Amazonian populations currently placed in R. olivaceus—one of which is unnamed and fully diagnosable based on vocal and genetic characters. Consistent with the phylogenetic results, our vocal analyses identified at least four morphologically cryptic lineages within R. olivaceus that can be mutually diagnosed from each other by different loudsongs and call parameters. Therefore, we provide evidence for splitting these four groups into separate species, two of which are sympatric but not syntopic in western Amazonia, including an unnamed species described herein—Rhynchocyclus cryptus, sp. nov. urn:lsid:zoobank.org:act:2DC17190-2BDD-49EC-88E6-4CF2FC2562A3.     

Improvements to resampling measures of group support

Several aspects of current resampling methods to assess group support are reviewed. When the characters have different prior weights or some state transformation costs are different, the frequencies under either bootstrapping or jackknifing can be distorted, producing either under‐ or overestimations of the actual group support. This is avoided by symmetric resampling, where the probability p of increasing the weight of a character equals the probability of decreasing it. Problems with interpreting absolute group frequencies as a measure of the support are discussed; group support does not necessarily vary with the frequency itself, since in some cases groups with positive support may have much lower frequencies than groups with no support at all. Three possible solutions for this problem are suggested. The first is measuring the support as the difference in frequency between the group and its most frequent contradictory group. The second is calculating frequencies for values of p below the threshold under which the frequency ranks the groups in the right order of support (this threshold may vary from data set to data set). The third is estimating the support by using the slope of the frequency as a function of different (low) values of p; when p is low, groups with actual support have negative slopes (closer to 0 when the support is higher), and groups with no support have positive slopes (larger when evidence for and against the group is more abundant).     

Analysis of CAPZA3 localization reveals temporally discrete events during the acrosome reaction

In mammals, the starting point of development is the fusion between sperm and egg. It is well established that sperm fuse with the egg through the equatorial/post‐acrosomal region. Apart from this observation and the requirement of two proteins (CD9 in the egg and IZUMO1 in the sperm) very little is known about this fundamental process. Actin polymerization correlates with sperm capacitation in different mammalian species and it has been proposed that F‐actin breakdown is needed during the acrosome reaction. Recently, we have presented evidence that actin polymerization inhibitors block the movement of IZUMO1 that accompany the acrosome reaction. These results suggest that actin dynamics play a role in the observed changes in IZUMO1 localization. This finding is significant because IZUMO1 localization in acrosome‐intact sperm is not compatible with the known location of the initiation of the fusion between the sperm and the egg. To further understand the actin‐mediated changes in protein localization during the acrosome reaction, the distribution of the sperm‐specific plus‐end actin capping protein CAPZA3 was analyzed. Like IZUMO1, CAPZA3 shows a dynamic pattern of localization; however, these movements follow a different temporal pattern than the changes observed with IZUMO1. In addition, the actin polymerization inhibitor latrunculin A was unable to alter CAPZA3 movement. J. Cell. Physiol. 224: 575–580, 2010. © 2010 Wiley‐Liss, Inc.     

Brood development and nest parasitism of Xylocopa (Neoxylocopa) augusti Lepeletier (Hymenoptera: Apidae), a promising crop pollinator in Argentina

Pollination of passion fruit and other crops by species of carpenter bees of the genus Xylocopa Latreille significantly increases both the quality and the quantity of fruits. To enhance pollination services, bee nests are either introduced into the crop area or females are encouraged to nest using trap nests. Thus, knowledge of trap-nest preference, brood development and nest parasitism is essential for effective and sustainable nest management practices. Xylocopa (Neoxylocopa) augusti Lepeletier is a promising pollinator in some areas of Argentina because of its high abundance, ability to buzz-pollinate and easy acceptance of trap nests. However, limited information is available on the biology of this pollinator. Herein, we provide detailed information on its nesting cycle, brood development and parasitism from trap nests at an urban area in Buenos Aires province, Argentina. Our study indicates that X. augusti is a solitary and likely univoltine species that shares some nesting and developmental features with other species in the subgenus Neoxylocopa, which might facilitate the adoption of existing management techniques developed for other species and regions. Biological information on Physocephala wulpi Camras (Diptera: Conopidae), a parasitoid of X. augusti, is also given for the first time.     

New aspects of the role of MR/P fimbriae in Proteus mirabilis urinary tract infection

Proteus mirabilis, a common cause of urinary tract infection (UTI), produces a number of different fimbriae including mannose-resistant Proteus-like fimbriae (MR/P). The precise role of different P. mirabilis fimbriae in ascending UTI has not yet been elucidated. In this study, a clinical isolate of P. mirabilis and an isogenic mutant unable to express MR/P were tested using different experimental approaches. They were tested for their ability to cause infection in an ascending co-infection model of UTI and in a haematogenous model in the mouse. In both models, the mutant was less able than the wild-type strain to colonise the lower and upper urinary tracts although infectivity was not abolished. In vitro adherence to uroepithelial cells was also assessed. Significant differences in adherence between both strains were observed at 1 h but not at 15 min post infection. We have also shown that a wild-type strain carries two copies of the mrpA gene. These data reinforce the importance of MR/P fimbriae in P. mirabilis UTI although other virulence factors may be necessary for efficient colonisation and development of infection.     

Alien Invasions and the Game of Hide and Seek in Patagonia

The introduction, establishment and spread of alien species is a major threat to biodiversity and the provision of ecosystem services for human wellbeing. In order to reduce further loss of biodiversity and maintain productive and sustainable ecosystems, understanding the ecological mechanisms underlying species invasions and avoiding potentially harmful effects on native communities is urgently needed, but largely lacking. We here demonstrate, by means of hydroacoustics and advanced spatial modelling, how native fish species as a result of previous exposure to native predators may successfully respond to invasive novel predators through a complicated game of hide and seek, minimizing spatio-temporal overlap with predators, and potentially facilitating coexistence between native prey species (Galaxiids) and introduced novel predators (Salmonids) in a deep Andean lake, Patagonia.