Isolation and characterization of actinobacteria ectosymbionts from Acromyrmex subterraneus brunneus (Hymenoptera, Formicidae)

The ectosymbiont actinobacterium Pseudonocardia was isolated from the integument of Acromyrmex leaf-cutter ants and seems to play a crucial role in maintaining asepsis of the nest. Currently, there has been an intensive search for Pseudonocardia associated with several attine species, but few studies have indicated that other actinobacteria may be associated with these ants as well. We therefore characterized the culturable actinobacteria community associated with the integument of the fungus-growing ant Acromyrmex subterraneus brunneus Forel, 1893 (Hymenoptera: Formicidae). Ectosymbionts were isolated using four different media and characterized by morphological and molecular (16S rDNA) methods. A total of 20 strains were isolated, of which 17 were characterized as Streptomyces spp., and one isolate each as Pseudonocardia, Kitassatospora and Propionicimonas. Unlike other Acromyrmex species, A. subterraneus brunneus is associated with a diversity of actinobacteria. Even though Pseudonocardia is present on this leaf-cutting ant`s integument, the number and diversity of Streptomyces spp. found differs from those of previous studies with other attine ants and suggest that different culturing approaches are needed to characterize the true diversity of microbes colonizing the integument of attine ants. Moreover, understanding the diversity of the culturable actinobacteria associated with A. subterraneus brunneus should increase our knowledge of the evolutionary relationship of this intricate symbiotic association. (C) 2010 Elsevier GmbH. All rights reserved.

Isolation and characterization of microsatellite loci in Colletotrichum acutatum, the causal agent of postbloom fruit drop on citrus

From a genomic enriched library, we developed 27 primer pairs from microsatellite flanking sequences for Colletotrichum acutatum, associated to postbloom fruit drop disease on citrus. Loci were characterized using 40 monosporic C. acutatum isolates. Nine primer pairs successfully amplified polymorphic microsatellite regions, with 3-6 alleles per locus, and mean heterozygosities ranging 0.093-0.590 across loci. The suitability of these primers was investigated in four Colletotrichum species as well. These microsatellite markers will be useful for genetic analyses and epidemiological studies of C. acutatum.

The isolation of antioxidant enzymes from mature tomato (cv. Micro-Tom) plants

The activity of catalase (CAT), guaiacol peroxidase (GPOX), ascorbate peroxidase (APX), glutathione reductase (GR), and the isoenzymes of superoxide dismutase (SOD) were determined in the organs of tomato (Lycopersicon esculentum) cultivar Micro-Tom after 104 days of development. The total activities of CAT, GPOX, and GR were higher in the stem than in others tissues, whereas the stem exhibited the lowest APX activity. Activity staining analysis following gel electrophoresis revealed the existence of four SOD isoenzymes in leaves, three in fruits, but only two in the roots and stems. This characterization is essential for an investigation into the effect of abiotic and biotic stresses on the oxidative stress responses by this plant model system.

Isolation of micropropagated strawberry endophytic bacteria and assessment of their potential for plant growth promotion

Twenty endophytic bacteria were isolated from the meristematic tissues of three varieties of strawberry cultivated in vitro, and further identified, by FAME profile, into the genera Bacillus and Sphingopyxis. The strains were also characterized according to indole acetic acid production, phosphate solubilization and potential for plant growth promotion. Results showed that 15 strains produced high levels of IAA and all 20 showed potential for solubilizing inorganic phosphate. Plant growth promotion evaluated under greenhouse conditions revealed the ability of the strains to enhance the root number, length and dry weight and also the leaf number, petiole length and dry weight of the aerial portion. Seven Bacillus spp. strains promoted root development and one strain of Sphingopyxis sp. promoted the development of plant shoots. The plant growth promotion showed to be correlated to IAA production and phosphate solubilization. The data also suggested that bacterial effects could potentially be harnessed to promote plant growth during seedling acclimatization in strawberry.

Listeria monocytogenes and Salmonella spp. in raw milk produced in Brazil: Occurrence and interference of indigenous microbiota in their isolation and development

This study aimed to verify the occurrence of Listeria monocytogenes and Salmonella spp. in raw milk produced in Brazil. On account of the poor microbiological quality of this product, possible interference from the indigenous microbiota in these pathogens was also evaluated. Two-hundred and ten raw milk samples were collected in four important milk-producing areas in Brazil, tested for L. monocytogenes and Salmonella spp. presence, and for enumeration of indicator microorganisms: mesophilic aerobes, total coliforms and Escherichia coli. The interference of the indigenous microbiota in the isolation procedures was also tested, as well the frequency of naturally occurring raw milk strains with antagonistic activity against both pathogens. The pathogens were not isolated in any raw milk sample, but poor microbiological quality was confirmed by the high levels of indicator microorganisms. When present at high levels, the indigenous microbiota generated an evident interference in the methodologies of L. monocytogenes and Salmonella spp. isolation, mainly when the pathogens appeared at low levels. Three-hundred and sixty raw milk strains were tested for antagonistic activity against both pathogens, and 91 (25.3%) showed inhibitory activity against L. monocytogenes and 33 (9.2%) against Salmonella spp. The majority of the antagonistic strains were identified as Lactic Acid Bacteria species, mainly Lactococcus lactis subsp. lactis and Enterococcus faecium, known by antimicrobial substance production.

Isolation and functional characterization of proinflammatory acidic phospholipase A(2) from Bothrops leucurus snake venom

In the present study, an acidic PLA(2), designated BI-PLA(2), was isolated from Bothrops leucurus snake venom through two chromatographic steps: ion-exchange on CM-Sepharose and hydrophobic chromatography on Phenyl-Sepharose. Bl-PLA(2) was homogeneous on SDS-PAGE and when submitted to 2D electrophoresis the molecular mass was 15,000 Da and pl was 5.4. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA(2)s from snake venoms. Its specific activity was 159.9 U/mg and the indirect hemolytic activity was also higher than that of the crude venom. Bl-PLA(2) induced low myotoxic and edema activities as compared to those of the crude venom. Moreover, the enzyme was able to induce increments in IL-12p40, TNF-alpha, IL-1 beta and IL-6 levels and no variation of IL-8 and IL-10 in human PBMC stimulated in vitro, suggesting that Bl-PLA2 induces proinflammatory cytokine production by human mononuclear cells. Bothrops leucurus venom is still not extensively explored and knowledge of its components will contribute for a better understanding of its action mechanism. (C) 2011 Elsevier Inc. All rights reserved.

Isolation, functional, and partial biochemical characterization of galatrox, an acidic lectin from Bothrops atrox snake venom

Snake venom lectins have been studied in regard to their chemical structure and biological functions. However, little is known about lectins isolated from Bothrops atrox snake venom. We report here the isolation and partial functional and biochemical characterization of an acidic glycan-binding protein called galatrox from this venom. This lectin was purified by affinity chromatography using a lactosyl-sepharose column, and its homogeneity and molecular mass were evaluated by high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The purified galatrox was homogeneous and characterized as an acidic protein (pI 5.2) with a monomeric and dimeric molecular mass of 16.2 and 32.5 kDa, respectively. Alignment of N-terminal and internal amino acid sequences of galatrox indicated that this protein exhibits high homology to other C-type snake venom lectins. Galatrox showed optimal hemagglutinating activity at a concentration of 100 mu g/ml and this effect was drastically inhibited by lactose, ethylenediaminetetraacetic acid, and heating, which confirmed galatrox`s lectin activity. While galatrox failed to induce the same level of paw edema or mast cell degranulation as B. atrox crude venom, galatrox did alter cellular viability, which suggested that galatrox might contribute to venom toxicity by directly inducing cell death.

The isolation and characterization of virulence factors of Cryptococcus spp. from saprophytic sources in the city of Ribeirao Preto, Sao Paulo, Brazil

Yeasts of the Cryptococcus genus are distributed in nature associated to animal and vegetal organic residues. Occasionally, species other than C. neoformans may be responsible for infectious diseases in human and animals. This study aims to determine the occurrence of Cryptococcus species in the atmosphere and bird droppings in the city of Ribeirao Preto, Sao Paulo, Brazil, and to evaluate three virulence factors: capsule formation, growth at 37 degrees C and melanin production. We analyzed 86 environmental samples (54 droppings and 32 air). Of the 41 strains isolated, 15 were C. neoformans var. neoformans (12 droppings and 3 air), 15 C. albidus (12 droppings and 3 air), 9 C. laurentii (7 droppings and 2 air) and 2 C. uniguttulatus (from droppings). Capsules were produced by 93.3% of C. neoformans var. neoformans, 66.7% of C. albidus, 88.9% of C. laurentii and 50% (1/2) of C. uniguttulatus. All, strains of C. neoformans, 20% of C. albidus and 44.4% of C. laurentii were able to grow at 37 degrees C. The melanin production on DOPA agar was verified in C. neoformans (93.3%), C. albidus (26.7%) and C. laurentii (66.7%). We concluded that different Cryptococcus species coexist in the same ecological niche and they are able to produce virulence factors. (C) 2007 Elsevier GmbH. All rights reserved.

Isolation and structural characterization of a new fibrin(ogen)olytic metalloproteinase from Bothrops moojeni snake venom

A proteinase, named BmooMP alpha-I, from the venom of Bothrops moojeni, was purified by DEAE-Sephacel, Sephadex G-75 and heparin-agarose column chromatography. The enzyme was purified to homogeneity as judged by its migration profile in SDS-PAGE stained with coomassie blue, and showed a molecular mass of about 24.5 kDa. Its complete cDNA was obtained by RT-PCR and the 615 bp codified for a mature protein of 205 amino acid residues. The multiple alignment of its deduced amino acid sequence and those of other snake venom metalloproteinases showed a high structural similarly, mainly among class P-IB proteases. The enzyme cleaves the A alpha-chain of fibrinogen first, followed by the B beta-chain, and shows no effects on the gamma-chain. On fibrin, the enzyme hydrolyzed only the beta-chain, leaving the gamma-dimer apparently untouched. It was devoid of phospholipase A(2), hemorrhagic and thrombin-like activities. Like many venom enzymes, it is stable at pH values between 4 and 10 and stable at 70 degrees C for 15 min. The inhibitory effects of EDTA on the fibrinogenolytic activity suggest that BmooMP alpha-I is a metalloproteinase and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Aprotinin and benzamidine, specific serine proteinase inhibitors, had no effect on BmooMP alpha-I activity. Since the BmooMP alpha-I enzyme was found to cause defibrinogenation when administered i.p. on mice, it is expected that it may be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis. (C) 2007 Elsevier Ltd. All rights reserved.

Isolation and Characterization of a Natriuretic Peptide from Crotalus oreganus abyssus (Grand Canyon Rattlesnake) and its Effects on Systemic Blood Pressure and Nitrite Levels

Studies on the therapeutic potential of venom peptides have significantly advanced the development of new peptide drugs. A good example is captopril, a synthetic peptide drug, which acts as an anti-hypertensive and potentiating bradykinin, inhibiting the angiotensin-converting enzyme, whose precursor was isolated from the venom of Bothrops jararacussu. The natriuretic peptide (NPs) family comprises three members, ANP (atrial natriuretic peptide), BNP (B-type natriuretic peptide) and CNP (C-type natriuretic peptide), and has an important role in blood pressure regulation and electrolyte homeostasis. In this study, we describe, for the first time, the isolation and characterization of a novel natriuretic-like peptide (Coa_NP), isolated from Crotalus Oreganus abyssus venom. The peptide has 32 amino acids and its complete sequence is SKRLSNGCFGLKLDRIGAMSGLGCWRLINESK. The Coa_NP has an average molecular mass of 3510.98 Da and its amino acid sequence presents the loop region that is characteristic of natriuretic peptides (17 amino acids, NP domain consensus; CFGXXXDRIXXXSGLGC). Coa_NP is a natriuretic peptide of the ANP/BNP-like family, since the carboxy terminal region of CNP has its own NP domain. The functional experiments showed that Coa_NP produced biological effects similar to those of the other natriuretic peptides: (1) a dose-dependent decrease in mean arterial pressure; (2) significant increases in plasma nitrite levels, and (3) vasorelaxation in thoracic aortic rings that were pre-contracted with phenylephrine. The structural and biological aspects confirm Coa_NP as a natriuretic peptide isolated from snake venom, thus expanding the diversification of venom components.

Concordance between dispersal and mitochondrial gene flow: isolation by distance in a tropical teleost, Lates calcarifer (Australian barramundi)

Patterns of population subdivision and the relationship between gene flow and geographical distance in the tropical estuarine fish Lares calcarifer (Centropomidae) were investigated using mtDNA control region sequences. Sixty-three putative haplotypes were resolved from a total of 270 individuals from nine localities within three geographical regions spanning the north Australian coastline. Despite a continuous estuarine distribution throughout the sampled range, no haplotypes were shared among regions. However, within regions, common haplotypes were often shared among localities. Both sequence-based (average Phi(ST)=0.328) and haplotype-based (average Phi(ST)=0.182) population subdivision analyses indicated strong geographical structuring. Depending on the method of calculation, geographical distance explained either 79 per cent (sequence-based) or 23 per cent (haplotype-based) of the variation in mitochondrial gene flow. Such relationships suggest that genetic differentiation of L. calcarifer has been generated via isolation-by-distance, possibly in a stepping-stone fashion. This pattern of genetic structure is concordant with expectations based on the life history of L. calcarifer and direct studies of its dispersal patterns. Mitochondrial DNA variation, although generally in agreement with patterns of allozyme variation, detected population subdivision at smaller spatial scales. Our analysis of mtDNA variation in L. calcarifer confirms that population genetic models can detect population structure of not only evolutionary significance but also of demographic significance. Further, it demonstrates the power of inferring such structure from hypervariable markers, which correspond to small effective population sizes.

Isolation of genotype- and chromosome-specific DNA markers in a biotype of Colletotrichum gloeosporioides using random amplified polymorphic DNA analysis

Genetic markers that distinguish fungal genotypes are important tools for genetic analysis of heterokaryosis and parasexual recombination in fungi. Random amplified polymorphic DNA (RAPD) markers that distinguish two races of biotype B of Colletotrichum gloeosporioides infecting the legume Stylosanthes guianensis were sought. Eighty-five arbitrary oligonucleotide primers were used to generate 895 RAPD bands but only two bands were found to be specifically amplified from DNA of the race 3 isolate. These two RAPD bands were used as DNA probes and hybridised only to DNA of the race 3 isolate. Both RAPD bands hybridised to a dispensable 1.2 Mb chromosome of the race 3 isolate. No other genotype-specific chromosomes or DNA sequences were identified in either the race 2 or race 3 isolates. The RAPD markers hybridised to a 2 Mb chromosome in all races of the genetically distinct biotype A pathogen which infects other species of Stylosanthes as well as S. guianensis. The experiments indicate that RAPD analysis is a potentially useful tool for obtaining genotype-and chromosome-specific DNA probes in closely related isolates of one biotype of this fungal pathogen.

Rapid isolation of high-quality RNA from symbiotic dinoflagellates

This report details a reliable and efficient RNA extraction protocol for the symbiotic dinoflagellate Symbiodinium microadriaticum Freudenthal (Gymnodiniales, Dinophyceae). The method typically gives yields of 500 mu g total RNA from 0.4 g wet weight of algae, and, in comparison to current protocols, it is technically simple and less time consuming. This method isolates high-quality, intact RNA from in vine cultured as well as host-isolated cells, as demonstrated by spectrophotometry, gel electrophoresis, and northern analysis. The total RNA obtained was suitable for reverse transcription and PCR amplification of Symbiodinium cDNAs. We have successfully applied our method to isolate total RNA from a different dinoflagellate, Amphidinium carterae Hulburt (Gymnodiniales, Dinophyceae), found in symbiotic association with marine invertebrates.

Isolation of a cDNA for an octopamine-like, G-protein coupled receptor from the cattle tick, Boophilus microplus

Octopamine is a biogenic amine neurotransmitter of invertebrates that binds to a G-protein coupled receptor that has seven transmembrane domains. Formamidine pesticides like amitraz are highly specific agonists of the octopamine receptor. Amitraz is used extensively to control the cattle tick, Boophilus microplus, and many other ticks but now there are strains of ticks that are resistant to amitraz. We have isolated a cDNA from the cattle tick, B. miciroplus, that belongs to the biogenic amine family of receptors. The predicted amino acid sequence from this cDNA is most similar to octopamine receptors from insects. The nucleotide sequence of this gene from amitraz-resistant and amitraz-susceptible cattle ticks was identical. Thus, a point mutation/s did not confer resistance to amitraz in the strains we studied. Alternative explanations for resistance to amitraz in B. microplus are discussed. (C) 1999 Elsevier Science Ltd. All rights reserved.

Isolation and characterization of a Clostridium sp with cinnamoyl esterase activity and unusual cell envelope ultrastructure

Microorganisms that hydrolyse the ester linkages between phenolic acids and polysaccharides in plant cell walls are potential sources of enzymes for the degradation of lignocellulosic waste. An anaerobic, mesophilic, spore-forming, xylanolytic bacterium with high hydroxy cinnamic acid esterase activity was isolated from the gut of the grass-eating termite Tumilitermes pastinator. The bacterium was motile and rod-shaped, stained gram-positive, had an eight-layered cell envelope, and.formed endospores. Phylogenetic analysis based on 16S rRNA indicated that the bacterium is closely related to Clostridium xylanolyticum and is grouped with polysaccharolytic strains of clostridia. A wide range of carbohydrates were fermented, and growth was stimulated by either xylan or cellobiose as substrates. The bacterium hydrolysed and then hydrogenated the hydroxy cinnamic acids (ferulic and p-coumaric acids), which are esterified to arabinoxylan in plant cell walls. Three cytoplasmic enzymes with hydroxy cinnamic acid esterase activity were identified using non-denaturing gel electrophoresis. This bacterium possesses an unusual multilayered cell envelope in which both leaflets of the cytoplasmic membrane, the peptidoglycan layer and the S layer are clearly discernible. The fate of all these components was easily followed throughout the endospore formation process. The peptidoglycan component persisted during the entire morphogenesis. It was seen to enter the septum and to pass with the engulfing membranes to surround the prespore. It eventually expanded to form the cortex, verification for the peptidoglycan origin of the cortex. Sporogenic vesicles, which are derived from the cell wall peptidoglycan, were associated with the engulfment process. Spore coat fragments appeared early, in stage II, though spore coat formation was not complete until after cortex formation.