Genetic transformation of Rangpur lime (Citrus limonia osbeck) with thebO (bacterio-opsin) genen and its initial evaluation forPhytophthora nicotianae resistance

Transgenic plants expressing the bacterio-opsin (bO) gene can spontaneously activate programmed cell death (ped) and may enhance broad-spectrum pathogen resistance by activating an intrinsic defense pathway in plant species such as tobacco and potato. In this work, we produced transgenic Rangpur lime plants with thebO gene, viaAgrobacterium tumefaciens-mediated transformation, and evaluated these plants forPhytophthora nicotianae resistance. Two transgenic lines were successfully regenerated and transformation was confirmed by GUS activity assay, PCR analysis, Southern, Northern and Western blot analyses, in addition to detecting the expressed bO protein by an immunological approach. Evaluation forPhytophthora nicotianae resistance was carried out by plant inoculations with the pathogen and quantification of the affected area. One of the two transgenic lines showed greater tolerance to the fungal pathogen as compared to the control, with significantly smaller stem lesions after pathogen challenge. This increase in pathogen tolerance is correlated with a significantly higher level of transgene expression in this line when compared with the other transgenic line. This is the first report of the introduction of a potentially important gene into Rangpur lime to provide novel pathogen tolerance.     

A specific short dextrin-hydrolyzing extracellular glucosidase from the thermophilic fungus Thermoascus aurantiacus 179-5

The thermophilic fungus Thermoascus aurantiacus 179-5 produced large quantities of a glucosidase which preferentially hydrolyzed maltose over starch. Enzyme production was high in submerged fermentation, with a maximal activity of 30 U/ml after 336 h of fermentation. In solid-state fermentation, the activity of the enzyme was 22 U/ml at 144 h in medium containing wheat bran and 5.8 U/ml at 48 h when cassava pulp was used as the culture medium. The enzyme was specific for maltose, very slowly hydrolyzed starch, dextrins (2-7G) and the synthetic substrate (alpha-PNPG), and did not hydrolyze sucrose. These properties suggest that the enzyme is a type II alpha-glucosidase. The optimum temperature of the enzyme was 70 degrees . In addition, the enzyme was highly thermostable (100% stability for 10 h at 60 degrees and a half-life of 15 min at 80 degrees), and stable within a wide pH range.     

Adhesion of water stressed Helicobacter pylori to abiotic surfaces

AIM: The main aim of this work was to study and compare the adhesion of water exposed Helicobacter pylori to six different substrata and correlate any changes in morphology, physiology, ability to form aggregates and cultivability when in the planktonic or in the sessile phase. METHODS AND RESULTS: The number of total cells adhered for different water exposure times and modifications in the cell shape were evaluated using epifluorescence and scanning electron microscopy, and physiology assessed using Syto9 and propidium iodide (PI) cellular uptake. All abiotic surfaces were rapidly colonized by H. pylori, and colonization appeared to reach a steady state after 96 h with levels ranging from 2.3 x 10(6) to 3.6 x 10(6) total cells cm(-2). Cell morphology was largely dependent on the support material, with spiral bacteria, associated with the infectious form of H. pylori, subsisting in a higher percentage on nonpolymeric substrata. Also, sessile bacteria were generally able to retain the spiral shape for longer when compared with planktonic bacteria, which became coccoid more quickly. The formation of large aggregates, which may act as a protection mechanism against the negative impact of the stressful external environmental conditions, was mostly observed on the surface of copper coupons. However, Syto9 and PI staining indicates that most of H. pylori attached to copper or SS304 have a compromised cell membrane after only 48 h. Cultivability methods were only able to detect the bacteria up to the 2 h exposure-time and at very low levels (up to 500 CFU cm(-2)). CONCLUSIONS: The fact that the pathogen is able to adhere, retain the spiral morphology for longer and form large aggregates when attached to different plumbing materials appeared to point to pipe materials in general, and copper plumbing in particular, as a possible reservoir of virulent H. pylori in water distribution systems. However, the Syto9/PI staining results and cultivability methods indicate that the attached H. pylori cells quickly enter in a nonviable physiological state. SIGNIFICANCE AND IMPACT OF THE STUDY: This represents the first study of H. pylori behaviour in water-exposed abiotic surfaces. It suggests that co-aggregation with the autochthonous heterotrophic consortia present in water is necessary for a longer survival of the pathogen in biofilms associated to drinking water systems.     

Influence of Hydrological Pulse on Bacterial Growth and DOC Uptake in a Clear-Water Amazonian Lake

This study was conducted to evaluate: (1) the bacterial growth and the dissolved organic carbon (DOC) uptake in an Amazonian lake (Lake Batata) at high-water and low-water periods of the flood pulse; (2) the influence of nitrogen and phosphorus (NP) additions on bacterial growth and DOC uptake in Lake Batata at two flood pulse periods; and (3) the bioavailability of the main DOC sources in Lake Batata. Lake Batata is a typical clear-water Amazonian lake, located in the watershed of Trombetas River, Central Amazon, Brazil. Bacterial batch cultures were set up with 90% 0.2-μm filtered water and 10% inoculum from Lake Batata. N-NH₄NO₃ and P-KH₂PO₄, with final concentrations of 50 and 5 μM, respectively, were added to the cultures, except for controls. Extra sources of DOC (e.g., algal lysate, plant leachates) were added to constitute six distinct treatments. Bacterial response was measured by maximum bacterial abundance and rates of bacterial production, respiration, DOC uptake, and bacterial growth efficiency (BGE). Bacterial growth and DOC uptake were higher in NP treatments than in controls, indicating a consistent nutrient limitation in Lake Batata. The composition of DOC also seems to be an important regulating factor of bacterial growth in Lake Batata. Seasonally, bacterial growth and DOC bioavailability were higher at low-water period, when the phytoplankton is a significant extra source of DOC, than at high-water period, when the forest is the main source of DOC. DOC bioavailability was better estimated based on the diversity and the diagenetic stage of carbon compounds than on single classes of labile compounds. Changes in BGE were better related to CNP stoichiometry in the water, and the “excess” of organic substrates was oxidized in catabolism, despite the quality of these compounds for bacterial growth. Finally, we conclude that bacterial growth and DOC uptake vary throughout the flood pulse in clear-water Amazonian ecosystems as a result of changes in nutrient concentration and in DOC composition.     

Lysine and threonine biosynthesis in sorghum seeds: characterisation of aspartate kinase and homoserine dehydrogenase isoenzymes

Aspartate kinase (AK, EC 2.7.2.4) and homoserine dehydrogenase (HSDH, EC 1.1.1.3) have been partially purified and characterised from immature sorghum seeds. Two peaks of AK activity were eluted by anion‐exchange chromatography [diethylaminoethyl (DEAE)‐Sephacel] with 183 and 262 mM KCl, and both activities were inhibited by lysine. Similarly, two peaks of HSDH activity were eluted with 145 and 183 mM KCl; the enzyme activity in the first peak in elution order was shown to be resistant to threonine inhibition, whereas the second was sensitive to threonine inhibition. However, following gel filtration chromatography (Sephacryl S‐200), one peak of AK activity co‐eluted with HSDH and both activities were sensitive to threonine inhibition, suggesting the presence of a bifunctional threonine‐sensitive AK–HSDH isoenzyme with a molecular mass estimated as 167 kDa. The activities of AK and HSDH were studied in the presence of lysine, threonine, methionine, valine, calcium, ethylene glycol bis(2‐aminoethylether)‐N,N,N′N′‐tetraacetic acid, calmodulin, S‐adenosylmethionine (SAM), S‐2‐aminoethyl‐l ‐cysteine (AEC) and increasing concentrations of KCl. AK was shown to be inhibited by threonine and lysine, confirming the existence of two isoenzymes, one sensitive to threonine and the other sensitive to lysine, the latter being predominant in sorghum seeds. Methionine, SAM plus lysine and AEC also inhibited AK activity; however, increasing KCl concentrations and calcium did not produce any significant effect on AK activity, indicating that calcium does not play a role in AK regulation in sorghum seeds. HSDH also exhibited some inhibition by threonine, but the majority of the activity was not inhibited, thus indicating the existence of a threonine‐sensitive isoenzyme and a second predominant threonine‐insensitive isoenzyme. Valine and SAM plus threonine also inhibited HSDH; however, increasing concentrations of KCl and calcium had no inhibitory effect.     

Phosphate closes the solution structure of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Mycobacterium tuberculosis

The 5-enolpyruvylshikimate-3-phosphate synthase catalyses the sixth step of the shikimate pathway that is responsible for synthesizing aromatic compounds and is absent in mammals, which makes it a potential target for drugs development against microbial diseases. Here, we report the phosphate binding effects at the structure of the 5-enolpyruvylshikimate-3-phosphate synthase from Mycobacterium tuberculosis. This enzyme is formed by two similar domains that close on each other induced by ligand binding, showing the occurrence of a large conformation change. We have monitored the phosphate binding effects using analytical ultracentrifugation, small angle X-ray scattering and, circular dichroism techniques. The low resolution results showed that the enzyme in the presence of phosphate clearly presented a more compact structure. Thermal-induced unfolding experiments followed by circular dichroism suggested that phosphate rigidified the enzyme. Summarizing, these data suggested that the phosphate itself is able to induce conformational change resulting in the closure movement in the M. tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase.     

Behavioral responses of maned wolves (Chrysocyon brachyurus, Canidae) to different categories of environmental enrichment stimuli and their implications for successful reintroduction

The maned wolf (Chrysocyon brachyurus, Illiger, 1815, Canidae) is a threatened species that inhabits the cerrados of Brazil, Argentina, Peru, Bolivia, Paraguay, and Uruguay. Captive maned wolves could be potentially used in reintroduction programs for species conservation; however, it is necessary that their behavior and cognitive abilities are conserved. Environmental enrichment is a tool used to stimulate captive animals and maintain a natural behavioral repertoire. To compare the variation of captive maned wolves' behavioral responses to environmental enrichment, we studied three maned wolves held by Belo Horizonte Zoo, Brazil. Foraging, interspecific, and intraspecific stimuli were offered to the animals and their responses were compared with a baseline and postenrichment treatments. The test was used to help in choosing which one of the three individuals would participate in a reintroduction project. The results showed that stimuli type did influence the animal's responses, and that individually wolves responded differently to foraging, interspecific, and intraspecific enrichment items (P80.05 for some behaviors in each enrichment category). The individual's personality seemed to influence their behavioral responses, with animals showing bold and shy responses, and this trait should be considered during decision makings for reintroduction.     

Anatomy of the extrafloral nectaries in species of Chamaecrista section Absus subsection Baseophyllum (Leguminosae,Caesalpinioideae)

In this paper the ontogenesis and histochemistry of the petiolar glands found on the petiole/rachis of the eight Chamaecrista species of the section Absus, subsection Baseophyllum (Leguminosae, Caesalpinioideae) are studied by using light microscopy techniques, aiming to characterise these structures and to provide taxonomic characters which may be useful in phylogenetic approaches. Strips for glucose identification reacted positively with the exudates of the glands, confirming the presence of nectar in the secretion, characterising these glands as extrafloral nectaries (EFN). Histochemical tests also detected the presence of neutral and acid muco-polysaccharides, pectins, mucilages, total proteins, and phenolic compounds in the EFNs. The EFNs arise from a group of meristem cells (protodermis, ground meristem and procambium) in the petiole/rachis. All EFNs of the investigated taxa share some morpho-anatomical characters, so that their peculiarities are too weak to be used alone in the identification of particular species. Rather their similarities may be used to include these species into a single group, supporting the hypothesis of monophyly of the subsection Baseophyllum.     

Antioxidant responses to water deficit by drought‐tolerant and ‐sensitive sugarcane varieties

Water deficit is the major yield‐limiting factor of crop plants. The exposure of plants to this abiotic stress can result in oxidative damage due to the overproduction of reactive oxygen species. The aim of this work was to study the antioxidant‐stress response of drought‐tolerant (SP83‐2847 and SP83‐5073) and drought‐sensitive (SP90‐3414 and SP90‐1638) sugarcane varieties to water‐deficit stress, which was imposed by withholding irrigation for 3, 10 and 20 days. The drought‐sensitive varieties exhibited the lowest leaf relative water content and highest lipid peroxidation, hydrogen peroxide (H₂O₂) and proline contents during the progression of the drought‐stress condition. The antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPOX) and glutathione reductase (GR) activities changed according to variety and stress intensity. SP83‐2847 exhibited higher CAT and APX activities than the other varieties in the early stage of drought, while the activities of GPOX and GR were the highest in the other varieties at the end of the drought‐stress period. A Cu/Zn SOD isoenzyme was absent at the end of drought period from the SP90‐3414‐sensitive variety. The results indicate that lipid peroxidation and early accumulation of proline may be good biochemical markers of drought sensitivity in sugarcane.