Identification of in vivo HSP90-interacting proteins reveals modularity of HSP90 complexes is dependent on the environment in psychrophilic bacteria

Heat shock protein 90 (HSP90) is a conserved molecular chaperone that functions as part of complexes in which different client proteins target it to diverse sets of substrates. In this paper, HSP90 complexes were investigated in γ-proteobacteria from mild (Shewanella oneidensis) and cold environments (Shewanella frigidimarina and Psychrobacter frigidicola), to determine changes in HSP90 interactions with client proteins in response to the adaptation to cold environments. HSP90 participation in cold adaptation was determined using the specific inhibitor 17-allylamino-geldanamycin. Then, HSP90 was immunoprecipitated from bacterial cultures, and the proteins in HSP90 complexes were analyzed by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. According to HSP90-associated protein analysis, only 15 common proteins were found in both species from the same genus, S. oneidensis and S. frigidimarina, whereas a significant higher number of common proteins were found in both psychrophilic species S. frigidimarina and P. frigidicola 21 (p < 0.001). Only two HSP90-interacting proteins, the chaperone proteins DnaK and GroEL, were common to the three species. Interestingly, some proteins related to energy metabolism (isocitrate lyase, succinyl-CoA synthetase, alcohol dehydrogenase, NAD(+) synthase, and malate dehydrogenase) and some translation factors only interacted with HSP90 in psychrophilic bacteria. We can conclude that HSP90 and HSP90-associated proteins might take part in the mechanism of adaptation to cold environments, and interestingly, organisms living in similar environments conserve similar potential HSP90 interactors in opposition to phylogenetically closely related organisms of the same genus but from different environments.     

Isolated Trees and Grass Removal Improve Performance of Transplanted Trema micrantha (L.) Blume (Ulmaceae) Saplings in Tropical Pastures

Cattle pastures established in areas previously covered by tropical rainforest can be abandoned in unproductive and degraded conditions. Transplanting native tree species to pastures is one common practice among rainforest restoration techniques, but several environmental barriers compromise transplant success. We assessed whether the presence of isolated trees and the removal of pasture grasses affect survival, growth, and allometry of transplanted saplings of the pioneer tree Trema micrantha (L.) Blume (Ulmaceae) into abandoned pastures in southeast Mexico. An isolated tree was selected in the center of each of four pasture sites of one hectare, and grass treatments were applied under the tree's canopy (0–10 m from the trunk) and in open pasture (15–48 m from the trunk). Grass removal treatments were control (grass present), cut with machete, herbicide application, and total grass removal with a gardening hoe. After 1 year, sapling survival was not different between the canopy and pasture areas (53%). Saplings showed higher survival probability (p < 0.05) in the hoe treatment (63 ± 9%) than in the control treatment (38 ± 9%). Height and crown cover growth rates were faster under the canopy of isolated trees compared with the open pasture. Saplings showed significantly greater crown area/height ratios under the canopy of isolated trees. Stressful environmental conditions restricted sapling growth in the open pasture. We conclude that complete removal of grasses and transplanting T. micrantha saplings in the vicinity of isolated trees can improve transplant success.     

Genetic consequences of interglacial isolation in a steppe bird

In response to climate changes that have occurred during Pleistocene glacial cycles, taxa associated to steppe vegetation might have followed a pattern of historical evolution in which isolation and fragmentation of populations occurred during the short interglacials and expansion events occurred during the long glacial periods, in contrast to the pattern described for temperate species. Here, we use molecular genetic data to evaluate this idea in a steppe bird with Palaearctic distribution, the little bustard (Tetrax tetrax). Overall, extremely low genetic diversity and differentiation was observed among eight little bustard populations distributed in Spain and France. Mismatch distribution analyses showed that most little bustard populations expanded during cooling periods previous to, and just after, the last interglacial period (127,000-111,000 years before present), when steppe habitats were widespread across Europe. Coalescent-based methods suggested that glacial expansions have resulted in substantial admixture in Western Europe due to the existence of different interglacial refugia. Our results are consistent with a model of evolution and genetic consequences of Pleistocene cycles with low between-population genetic differentiation as a result of short-term isolation periods during interglacials and long-term exchange during glacial periods.     

Molecular phylogeny and phylogeography of the Cuban cave-fishes of the genus Lucifuga: evidence for cryptic allopatric diversity

Underground environments are increasingly recognized as reservoirs of faunal diversity. Extreme environmental conditions and limited dispersal ability of underground organisms have been acknowledged as important factors promoting divergence between species and conspecific populations. However, in many instances, there is no correlation between genetic divergence and morphological differentiation. Lucifuga Poey is a stygobiotic fish genus that lives in Cuban and Bahamian caves. In Cuba, it offers a unique opportunity to study the influence of habitat fragmentation on the genetic divergence of stygobiotic species and populations. The genus includes four species and one morphological variant that have contrasting geographical distributions. In this study, we first performed a molecular phylogenetic analysis of the Lucifuga Cuban species using mitochondrial and nuclear markers. The mitochondrial phylogeny revealed three deeply divergent clades that were supported by nuclear and morphological characters. Within two of these main clades, we identified five lineages that are candidate cryptic species and a taxonomical synonymy between Lucifuga subterranea and Lucifuga teresinarum. Secondly, phylogeographic analysis using a fragment of the cytochrome b gene was performed for Lucifuga dentata, the most widely distributed species. We found strong geographical organization of the haplotype clades at different geographic scales that can be explained by episodes of dispersal and population expansion followed by population fragmentation and restricted gene flow. At a larger temporal scale, these processes could also explain the diversification and the distribution of the different species.     

Brucella abortus inhibits IFN-γ-induced FcγRI expression and FcγRI-restricted phagocytosis via toll-like receptor 2 on human monocytes/macrophages

The strategies that allow Brucella abortus to persist for years inside macrophages subverting host immune responses are not completely understood. Immunity against this bacterium relies on the capacity of IFN-γ to activate macrophages, endowing them with the ability to destroy intracellular bacteria. We report here that infection with B. abortus down-modulates the expression of the type I receptor for the Fc portion of IgG (FcγRI, CD64) and FcγRI-restricted phagocytosis regulated by IFN-γ in human monocytes/macrophages. Both phenomena were not dependent on bacterial viability, since they were also induced by heat-killed B. abortus (HKBA), suggesting that they were elicited by a structural bacterial component. Accordingly, a prototypical B. abortus lipoprotein (L-Omp19), but not its unlipidated form, inhibited both CD64 expression and FcγRI-restricted phagocytosis regulated by IFN-γ. Moreover, a synthetic lipohexapeptide that mimics the structure of the protein lipid moiety also inhibited CD64 expression, indicating that any Brucella lipoprotein could down-modulate CD64 expression and FcγRI-restricted phagocytosis. Pre-incubation of monocytes/macrophages with anti-TLR2 mAb blocked the inhibition of the CD64 expression mediated by HKBA and L-Omp19. These results, together with our previous observations establish that B. abortus utilizes its lipoproteins to inhibit the monocytes/macrophages activation mediated by IFN-γ and to subvert host immunonological responses.     

Microsatellite characterization of Cimarron Uruguayo dogs

Various genetic markers, including microsatellites, have been used to analyze the genetic polymorphism and heterozygosity in canine breeds. In this work, we used nine microsatellite markers to investigate the genetic variability in Cimarron Uruguayo dogs, the only officially recognized native canine breed in Uruguay. DNA from 30 Cimarron Uruguayo dogs from northeastern and southern Uruguay was analyzed. The allelic frequencies for each microsatellite, the genetic variability and the consanguinity were calculated, as were the polymorphic information content (PIC) and the probability of exclusion (PE). All of the microsatellites studied were polymorphic. FH 2361, FH 2305 and PEZ 03 were the most informative, with PIC values > 0.7, in agreement with results for other canine breeds. The PE values for the markers were within the ranges previously described and were generally greater for microsatellites with higher PIC values. The heterozygosity value (0.649) was considered high since only nine microsatellites were analyzed. Compared with data for other breeds, the results obtained here indicate that Cimarron Uruguayo dogs have high genetic diversity.     

Anther and stigma morphology in mirror-image flowers of Chamaecrista chamaecristoides (Fabaceae): implications for buzz pollination

Enantiostyly (mirror-image flowers) is usually associated with buzz pollination. In buzz-pollinated flowers, pollen is released through terminal pores after bees vibrate the stamens. Several studies have evaluated the function of 'buzzing' in pollen release, but less attention has been paid to the effect of buzzing on pollen capture and deposition on stigmas. Evaluating the mechanism of pollen dispersion in buzz-pollinated flowers is important because it may affect mating patterns and reproductive success. In this study, we analysed the morphology of sexual organs (anther and stigma) using electron microscopy, and determined the relationship between sexual organ structure and pollen capture function through experimental manipulations of buzz-pollinated flowers of Chamaecrista chamaecristoides, as well as vibration frequencies on floral visitors. Pollen release occurs through two terminal pores at the tip of the stamens. However, unlike most angiosperms that have their stigmatic surface exposed, C.?chamaecristoides presents a stigmatic surface inside a cavity covered by trichomes. Experimental manipulations showed that effective fertilisation is only achieved when the style is vibrated, suggesting that buzzing is not only important for pollen release but also for pollen capture and deposition on the stigma. This result, in addition to vibration frequency analysis, suggests that although all floral visitors buzz flowers only those that buzz at higher frequencies achieve effective fertilisation. The anatomical features of sexual organs in flowers of C.?chamaecristoides demonstrate that this species possesses a highly specialised, elaborate morphology, with both genders selected for traits that promote buzz pollination.     

Remote sensing data predict indicators of soil functioning in semi-arid steppes,central Spain

A substantial part of current research efforts on desertification are devoted to establish monitoring systems to evaluate the status of natural resources and the onset of desertification processes. Methodologies based on ground-collected soil and plant indicators are being increasingly used for this aim because they are affordable yet do not compromise accuracy. Despite their inherent value, these methods have limitations regarding the extent of the area that can be monitored using them. Such limitations can be overcome combining field-based approaches with remote sensing data, which allow the establishment of monitoring programs over large areas. In this article we tested the relationship between a field methodology based on indicators of ecosystem functioning, the landscape function analysis (LFA), and a vegetation index (NDVI) obtained from satellite images of the ASTER sensor using data gathered in Stipa tenacissima steppes from central Spain. LFA uses soil surface indicators to assess the condition of a given ecosystem by producing three numerical indices (stability, infiltration and nutrient cycling) reflecting the status of basic soil functions. We found a significant positive linear relationship between the NDVI, the three LFA indices and some key structural attributes of vegetation related to the cover of perennial plants. Our results indicate that NDVI can be used as a surrogate of ecosystem functioning in semi-arid Mediterranean steppes, and thus can be a helpful index to monitor the functional status of large areas in these ecosystems, and the possible onset of desertification processes.     

Characterization of a novel immobilized biocatalyst obtained by matrix-assisted refolding of recombinant polyhydroxyoctanoate depolymerase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> KT2442 isolated from inclusion bodies

Purification and matrix-assisted refolding of recombinant His-tagged polyhydroxyalkanoate (PhaZ) depolymerase from Pseudomonas putida KT2442 was carried out. His-tagged enzyme was overproduced as inclusion bodies in recombinant E. coli M15 (pREP4, pPAZ3), which were denatured by 8 M urea, immobilized on Ni²⁺-nitrilotriacetate-agarose matrix, and refolded by gradual removal of the chaotropic agent. The refolded enzyme could not be eluted with 1 M imidazole buffer, leading to an immobilized biocatalyst where PhaZ depolymerase was homogeneously distributed in the agarose support as shown by confocal scanning microscopy. Polyhydroxyoctanoate could not be hydrolyzed by this novel immobilized biocatalyst, whereas the attached enzyme was active in the hydrolysis of p-nitrophenyl alkanoate esters, which differed in their alkyl chain length. Taking advantage of the observed esterase activity on p-nitrophenylacetate, functional characterization of immobilized PhaZ depolymerase was carried out. The immobilized enzyme was more stable than its soluble counterpart and showed optimal hydrolytic activity at 37°C and 50 mM phosphate buffer pH 8.0. Kinetic parameters were obtained with both p-nitrophenylacetate and p-nitrophenyloctanoate, which had not been described so far for the soluble enzyme, representing an attractive and alternative chromogenic assay for the study of this paradigmatic enzyme.