Linking major nutrients (C,H, N,and P) to trace metals (Fe,Mn, and Cu) in lake seston in southern Brazil

We investigated the relationships between major nutrients (C, H, N, and P) and trace metals (Cu, Fe, and Mn) in seston samples from ten lake/lagoon systems in southern Brazil. The systems were characterized by a diverse set of limnological features, including surface areas from 10⁻¹ to 10² km², water color, a _CDOM(440), from 1.4 to 12.9 m⁻¹, and electrical conductivity from 50 to 100 000 μS cm⁻¹. Seston concentrations also varied a great deal, 32-fold. The elemental (C: N, C: P, and N: P) and C: Chl-a ratios in the seston samples indicated, however, common features; i.e., most of the lakes were N-and/or P-limited, and the seston organic fraction was composed of nonvascular plants (e.g., phytoplankton). Our intersystem comparison revealed that the relative content of organic matter in seston and seston concentrations in lake water tended to correlate positively and negatively, respectively, with trace metal concentrations across the seston samples. Possible influences of elemental and C: Chl-a ratios on the association of metals with seston matrices, although theoretically important, were only partially evidenced here; positive correlations were found between C: N and also Org-H: N ratios with trace metal concentrations. We speculate that such results could be circumstantial, as the nature of the seston matrices appeared to be very similar among them. This hypothesis should thus be the theme of further research. In short, our findings suggest that C: N and Org-H: N ratios as well as the relative content of major nutrients in seston and seston concentrations can be importantly related to trace metal concentrations in seston samples. In discussing the results, however, we consider that metal-seston relationships depend on a variety of physical, chemical, and biological factors and/or variables other than those measured in this study, which could also contribute for defining and explaining variations in metal-seston concentrations in lake ecosystems.     

Proteomic Analysis of Developing Somatic Embryos of Coffea arabica

Differential protein profiles of three stages of somatic embryogenesis, including globular, torpedo, and cotyledonary somatic embryos, of Coffea arabica cv. Catuaí Vermelho were analyzed in an attempt to better understand somatic embryogenesis in coffee plants. Somatic embryos at these different stages of development were collected from in vitro-grown cultures, and then macerated in liquid nitrogen. Proteins were extracted with phenol and further quantified using the Bradford method. The bidimensional electrophoresis analysis revealed a wide range of proteins ranging between 10 and 160?kDa and of pH values ranging from 3 to 10. Several differentially expressed proteins were identified by mass spectrometry, and some were found to be specific to these different stages of somatic embryogenesis in coffee. The enolase and 11S storage globulin proteins, for example, could be used as molecular markers for somatic embryo development stages and for embryogenic and non-embryogenic genotype differentiation, respectively.     

Interactions between PII proteins and the nitrogenase regulatory enzymes DraT and DraG in Azospirillum brasilense

In Azospirillum brasilense ADP-ribosylation of dinitrogenase reductase (NifH) occurs in response to addition of ammonium to the extracellular medium and is mediated by dinitrogenase reductase ADP-ribosyltransferase (DraT) and reversed by dinitrogenase reductase glycohydrolase (DraG). The P(II) proteins GlnB and GlnZ have been implicated in regulation of DraT and DraG by an as yet unknown mechanism. Using pull-down experiments with His-tagged versions of DraT and DraG we have now shown that DraT binds to GlnB, but only to the deuridylylated form, and that DraG binds to both the uridylylated and deuridylylated forms of GlnZ. The demonstration of these specific protein complexes, together with our recent report of the ability of deuridylylated GlnZ to be sequestered to the cell membrane by the ammonia channel protein AmtB, offers new insights into the control of NifH ADP-ribosylation.     

Iron depletion affects nitrogenase activity and expression of nifH and nifA genes in Herbaspirillum seropedicae

Herbaspirillum seropedicae Z67 is a nitrogen-fixing bacterium able to colonize the rhizosphere and the interior of several plants. As iron is a key element for nitrogen fixation, we examined the response of this microorganism to iron deficiency under nitrogen fixing conditions. We identified a H. seropedicae exbD gene that was induced in response to iron limitation and is involved in iron homeostasis. We found that an exbD mutant grown in iron-chelated medium is unable to fix nitrogen. Moreover, we provide evidence that expression of the nifH and nifA genes is iron dependent in a H. seropedicae genetic background.     

ADP-ribosylation of dinitrogenase reductase in Azospirillum brasilense is regulated by AmtB-dependent membrane sequestration of DraG

Nitrogen fixation in some diazotrophic bacteria is regulated by mono-ADP-ribosylation of dinitrogenase reductase (NifH) that occurs in response to addition of ammonium to the extracellular medium. This process is mediated by dinitrogenase reductase ADP-ribosyltransferase (DraT) and reversed by dinitrogenase reductase glycohydrolase (DraG), but the means by which the activities of these enzymes are regulated are unknown. We have investigated the role of the P(II) proteins (GlnB and GlnZ), the ammonia channel protein AmtB and the cellular localization of DraG in the regulation of the NifH-modification process in Azospirillum brasilense. GlnB, GlnZ and DraG were all membrane-associated after an ammonium shock, and both this membrane sequestration and ADP-ribosylation of NifH were defective in an amtB mutant. We now propose a model in which membrane association of DraG after an ammonium shock creates a physical separation from its cytoplasmic substrate NifH thereby inhibiting ADP-ribosyl-removal. Our observations identify a novel role for an ammonia channel (Amt) protein in the regulation of bacterial nitrogen metabolism by mediating membrane sequestration of a protein other than a P(II) family member. They also suggest a model for control of ADP-ribosylation that is likely to be applicable to all diazotrophs that exhibit such post-translational regulation of nitrogenase.     

Influence of Soil Characteristics on the Diversity of Bacteria in the Southern Brazilian Atlantic Forest

The Brazilian Atlantic Forest is one of the 25 biodiversity hot spots in the world. Although the diversity of its fauna and flora has been studied fairly well, little is known of its microbial communities. In this work, we analyzed the Atlantic Forest ecosystem to determine its bacterial biodiversity, using 16S rRNA gene sequencing, and correlated changes in deduced taxonomic profiles with the physicochemical characteristics of the soil. DNAs were purified from soil samples, and the 16S rRNA gene was amplified to construct libraries. Comparison of 754 independent 16S rRNA gene sequences from 10 soil samples collected along a transect in an altitude gradient showed the prevalence of Acidobacteria (63%), followed by Proteobacteria (25.2%), Gemmatimonadetes (1.6%), Actinobacteria (1.2%), Bacteroidetes (1%), Chloroflexi (0.66%), Nitrospira (0.4%), Planctomycetes (0.4%), Firmicutes (0.26%), and OP10 (0.13%). Forty-eight sequences (6.5%) represented unidentified bacteria. The Shannon diversity indices of the samples varied from 4.12 to 3.57, indicating that the soils have a high level of diversity. Statistical analysis showed that the bacterial diversity is influenced by factors such as altitude, Ca²⁺/Mg²⁺ ratio, and Al³⁺ and phosphorus content, which also affected the diversity within the same lineage. In the samples analyzed, pH had no significant impact on diversity.The Brazilian Atlantic Forest is one of the 25 biodiversity hot spots in the world. Altogether, these hot spots contain more than 60% of the total terrestrial species of the planet (17). The Atlantic Forest is a dense ombrophilous forest with several variations, including coastal (3 to 50 m), submontane (50 to 500 m), montane (500 to 1,200 m), and high montane (1,200 to 1,400 m) forests, creating a vegetation gradient ranging from shrubs to well-developed montane forest (4). The Serra do Mar is a mountainous system that shelters the main remainder of the Atlantic Forest following the Brazilian east coast, from north to south along the coastal line, and it is divided into diverse sections of high and low blocks, which have regional denominations.The most important law-protected conservation area of the Brazilian Atlantic Forest is located in the Serra do Mar of the southern state of Paraná. This conservation area (∼5,000 km²) shelters 72% of the fauna and flora species that occur in Paraná and was declared a Biosphere Reserve by UNESCO in 1992. Much is known about the diversity of its fauna and flora, but little is known of its microbial diversity, particularly the soil microbial diversity and the soil characteristics that influence it.The soil microbial diversity is vast, and it is estimated that >99% of species remain unidentified (1, 28). Acidobacteria and Proteobacteria are the most abundant groups in soil (15). However, the Proteobacteria lineage is more diverse and stable than the Acidobacteria lineage, suggesting that the latter group is more susceptible to variation in soil properties and to disturbing factors (33). Seasonal, physical, and physicochemical factors can be relevant to the structure and diversity of microbial communities. For example, seasonal changes in vegetation and temperature led to replacement of dominant groups in a wheat field (25) and in grassland soils (16). The particle size also has an influence on the bacterial diversity of soils. The clay fraction has a more diverse bacterial community than do silt or sand fractions (23). Finally, analyses of communities from North and South American soils showed that pH plays a major role in bacterial diversity, with less diverse communities associated with a lower pH (9).Human activity can also change the microbial diversity of soils, both qualitatively and quantitatively. Analyses of microbial communities on coral atolls in the central Pacific Ocean under different degrees of human impact showed that the least-impacted atoll had autotrophs and heterotrophs equally distributed in the community, whereas the most-impacted atoll had a dominance of heterotrophs and about 10 times more microbial cells and virus-like particles in the water column, including a large percentage of potential pathogens (7). A comparison between bacterial communities in forest and pasture soil showed that there is a less diverse and more restricted community in pasture soils. The vegetation shift from forest to pasture resulted in changes to G+C% contents of soil bacterial DNA and amplified rRNA gene restriction analysis (ARDRA) profiles (18). Similar changes occurred with communities of soils submitted to agroindustrial treatments and pollutants (3, 30).In this work, we used a culture-independent approach based on 16S rRNA gene sequences to survey the bacterial community of the Atlantic Forest soils and determined the physicochemical factors affecting its bacterial biodiversity.     

Integration of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) linkage and chromosomal maps

Fluorescent in situ hybridisation of pooled, closely linked RFLP markers was used to integrate the genetic linkage map and the mitotic chromosome map of the common bean. Pooled RFLP probes showed clear and reproducible signals and allowed the assignment of all linkage groups to the chromosomes of two Phaseolus vulgaris cultivars, Saxa and Calima. Low extension values for signals originating from clustered RFLPs suggest that these clones are physically close to each other and that clusters in the genetic map are not a result of suppression of recombination due to the occurrence of chromosome rearrangements. For linkage group K, clustering of markers could be associated with proximity to centromeres. High variation in the number of 45S rDNA loci was observed among cultivars, suggesting that these terminal sites are highly recombinogenic in common bean.     

The mitochondrial barriers segregate agonist-induced calcium-dependent functions in human airway epithelia

In airway epithelia, purinergic receptor (P2Y2-R) stimulation of intracellular calcium (Ca2+i)-regulated ion transport is restricted to the membrane domain ipsilateral to receptor activation, implying compartmentalization of Ca2+i signaling. Because mitochondria can spatially restrict cellular Ca2+i signals, immunocytochemical, electron microscopic, and fluorescent studies of mitochondria localization were performed in human airway epithelia. Although concentrated at the apical domain, mitochondria were found distributed at both the apical and the basolateral poles and in close association with the endoplasmic reticulum. The role of mitochondria in locally restricting P2Y2-R-induced Ca2+i signals was investigated by measuring changes in mitochondrial Ca2+ (Ca2+m) in human airway epithelial monolayers. P2Y2-R activation induced Ca2+m accumulation in mitochondria confined to the domain ipsilateral to P2Y2-R stimulation, which was blocked by mitochondrial uncoupling with 1 microM CCCP and 2.5 microg/ml oligomycin. The role of mitochondria in restricting the cellular cross-talk between basolateral P2Y2-R-dependent Ca2+i mobilization and apical membrane Ca2+-activated Cl- secretion was investigated in studies simultaneously measuring Ca2+i and Cl- secretion in cystic fibrosis human airway epithelial monolayers. Activation of basolateral P2Y2-Rs produced similar increases in Ca2+i in monolayers without and with pretreatment with uncouplers, whereas Ca2+i-activated Cl- secretion was only efficiently triggered in mitochondria-uncoupled conditions. We conclude that (a) mitochondria function as a Ca2+i-buffering system in airway epithelia, compartmentalizing Ca2+i-dependent functions to the membrane ipsilateral to receptor stimulation; and (b) the mitochondria provide structural barriers that protect the airway epithelia against nonspecific activation of Ca2+i-modulated functions associated with Ca2+i signals emanating from the apical or the basolateral membrane domains.     

Repressor Mutant Forms of the Azospirillum brasilense NtrC Protein

The Azospirillum brasilense mutant strains FP8 and FP9, after treatment with nitrosoguanidine, showed a null Nif phenotype and were unable to use nitrate as their sole nitrogen source. Sequencing of the ntrC genes revealed single nucleotide mutations in the NtrC nucleotide-binding site. The phenotypes of these strains are discussed in relation to their genotypes.     

Influence of a natural and a synthetic inhibitor of factor XIIIa on fibrin clot rheology

We investigated the origins of greater clot rigidity associated with FXIIIa-dependent cross-linking. Fibrin clots were examined in which cross-linking was controlled through the use of two inhibitors: a highly specific active-center-directed synthetic inhibitor of FXIIIa, 1,3-dimethyl-4,5-diphenyl-2[2(oxopropyl)thio]imidazolium trifluoromethylsulfonate, and a patient-derived immunoglobulin directed mainly against the thrombin-activated catalytic A subunits of thrombin-activated FXIII. Cross-linked fibrin chains were identified and quantified by one- and two-dimensional gel electrophoresis and immunostaining with antibodies specific for the alpha- and gamma-chains of fibrin. Gamma-dimers, gamma-multimers, alpha(n)-polymers, and alpha(p)gamma(q)-hybrids were detected. The synthetic inhibitor was highly effective in preventing the production of all cross-linked species. In contrast, the autoimmune antibody of the patient caused primarily an inhibition of alpha-chain cross-linking. Clot rigidities (storage moduli, G') were measured with a cone and plate rheometer and correlated with the distributions of the various cross-linked species found in the clots. Our findings indicate that the FXIIIa-induced dimeric cross-linking of gamma-chains by itself is not sufficient to stiffen the fibrin networks. Instead, the augmentation of clot rigidity was more strongly correlated with the formation of gamma-multimers, alpha(n)-polymers, and alpha(p)gamma(q)-hybrid cross-links. A mechanism is proposed to explain how these cross-linked species may enhance clot rigidity.