Auxin production and detection of the gene coding for the Auxin Efflux Carrier (AEC) protein in <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis>

Different species of Paenibacillus are considered to be plant growth-promoting rhizobacteria (PGPR) due to their ability to repress soil borne pathogens, fix atmospheric nitrogen, induce plant resistance to diseases and/or produce plant growth-regulating substances such as auxins. Although it is known that indole-3-acetic acid (IAA) is the primary naturally occurring auxin excreted by Paenibacillus species, its transport mechanisms (auxin efflux carriers) have not yet been characterized. In this study, the auxin production of P. polymyxa and P. graminis, which are prevalent in the rhizospheres of maize and sorghum sown in Brazil, was evaluated. In addition, the gene encoding the Auxin Efflux Carrier (AEC) protein from P. polymyxa DSM36(T) was sequenced and used to determine if various strains of P. polymyxa and P. graminis possessed this gene. Each of the 68 P. polymyxa strains evaluated in this study was able to produce IAA, which was produced at concentrations varying from 1 to 17 microg/ml. However, auxin production was not detected in any of the 13 P. graminis strains tested in this study. Different primers were designed for the PCR amplification of the gene coding for the AEC in P. polymyxa, and the predicted protein of 319 aa was homologous to AEC from Bacillus amyloliquefaciens, B. licheniformis, and B. subtilis. However, no product was observed when these primers were used to amplify the genomic DNA of seven strains of P. graminis, which suggests that this gene is not present in this species. Moreover, none of the P. graminis genomes tested were homologous to the gene coding for AEC, whereas all of the P. polymyxa genomes evaluated were. This is the first study to demonstrate that the AEC protein is present in P. polymyxa genome.     

Rep-PCR of tropical rhizobia for strain fingerprinting, biodiversity appraisal and as a taxonomic and phylogenetic tool

With more than 30 million doses of rhizobial inoculants marketed per year, it is probable that Brazilian agriculture benefits more than any other country from symbiotic N₂ fixation. As a result of strain-selection programs, 142 strains of rhizobia are officially recommended for use in commercial inoculants for ninety-six leguminous crops. In this study, sixty-eight of these elite strains were characterized by rep-PCR with the BOX-primer. Reproducibility of the DNA profiles was confirmed, suggesting efficacy of BOX-PCR both for control of quality of inoculants and for preliminary characterization of rhizobial culture collections. Strains of different species never showed similarity higher than 70% in the BOX-PCR analysis, however, some strains of the same species fit into more than one cluster, and correlation between BOX-PCR products and l6S rRNA sequences was low (7.6%). On the other hand, a polyphasic approach — 20%∶80% of BOX-PCR:16S rRNA which correlated well with the l6S rRNA analysis (95%), and provided higher definition of the genotypes, resulting in clearer indications of the taxonomic groups — might expedite rhizobial diversity studies.     

Ovarian and Uterine Ultrasonography in <Emphasis Type="Italic">Aotus azarai infulatus</Emphasis>

We evaluated the uterus and ovaries of owl monkeys (Aotus azarai infulatus) via gynecological ultrasound examination. We evaluated the subjects in 2 different time periods. The first period (P1) was characterized by the absence of mating, with daily examinations, during 4 mo (n = 10). At the end of P1, we paired the subjects for 30 d, but without ultrasonographic evaluation. The second period (P2) was characterized by the presence of mating, with examinations once a week, during 7 consecutive months (n = 9). We evaluated the uterus and ovaries in sagittal and transverse scans, using a 5–12 MHz linear array probe. The uterine volume (UV) was directly proportional to the number of previous parturitions. The right ovary volume (RtOV) is greater than the left (LtOV) in P1 and P2. There is a positive correlation (p < 0.05) between the females’ mass, RtOV (r = 0.28) and LtOV (r = 0.16).     

The interaction of Bacteroides fragilis with components of the human fibrinolytic system

Bacteroides fragilis is a minor component of the intestinal microbiota and the most frequently isolated from intra-abdominal infections and bacteremia. Previously, our group has shown that molecules involved in laminin-1 (LMN-1) recognition were present in outer membrane protein extracts of B. fragilis MC2 strain. One of these proteins was identified and showed 98% similarity to a putative B. fragilis plasminogen-binding protein precursor, deposited in the public database. Thus, the objective of this work was to overexpress and further characterize this novel adhesin. The ability of B. fragilis MC2 strain and purified protein to convert plasminogen into plasmin was tested. Our results showed that B. fragilis strain MC2 strain adhered to both LMN-1 and plasminogen and this adhesion was inhibited by either LMN-1 or plasminogen. Regarding the plasminogen activation activity, both the whole bacterial cell and the purified protein converted plasminogen into plasmin similar to streptokinase used as a positive control. Bacterial receptors that recognize plasminogen bind to it and enhance its activation, transforming a nonproteolytic bacterium into a proteolytic one. We present in vitro evidence for a pathogenic function of the plasminogen receptor in promoting adherence to laminin and also the formation of plasmin by B. fragilis .     

Structure and Mode of Action of Microplusin, a Copper II-chelating Antimicrobial Peptide from the Cattle Tick Rhipicephalus (Boophilus) microplus

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single α-helical globular domain, microplusin consists of five α-helices: α1 (residues Gly-9 to Arg-21), α2 (residues Glu-27 to Asn-40), α3 (residues Arg-44 to Thr-54), α4 (residues Leu-57 to Tyr-64), and α5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.     

Mutations in CNNM4 Cause Recessive Cone-Rod Dystrophy with Amelogenesis Imperfecta

Cone-rod dystrophies are inherited dystrophies of the retina characterized by the accumulation of deposits mainly localized to the cone-rich macular region of the eye. Dystrophy can be limited to the retina or be part of a syndrome. Unlike nonsyndromic cone-rod dystrophies, syndromic cone-rod dystrophies are genetically heterogeneous with mutations in genes encoding structural, cell-adhesion, and transporter proteins. Using a genome-wide single-nucleotide polymorphism (SNP) haplotype analysis to fine map the locus and a gene-candidate approach, we identified homozygous mutations in the ancient conserved domain protein 4 gene (CNNM4) that either generate a truncated protein or occur in highly conserved regions of the protein. Given that CNNM4 is implicated in metal ion transport, cone-rod dystrophy and amelogenesis imperfecta may originate from abnormal ion homeostasis.     

On the mechanisms of cadmium stress alleviation in Medicago truncatula by arbuscular mycorrhizal symbiosis: A root proteomic study

The arbuscular mycorrhizal (AM) symbiosis belongs to the strategies plants have developed to cope with adverse environmental conditions including contamination by heavy metals such as cadmium (Cd). In the present work, we report on the protective effect conferred by AM symbiosis to the model legume Medicago truncatula grown in presence of Cd, and on the 2‐D‐based proteomic approach further used to compare the proteomes of M. truncatula roots either colonised or not with the AM fungus Glomus intraradices in Cd‐free and Cd‐contaminated substrates. The results indicated that at the proteome level, 9 out of the 15 cadmium‐induced changes in nonmycorrhizal roots were absent or inverse in those Cd‐treated and colonized by G. intraradices, including the G. intraradices‐dependent down‐accumulation of Cd stress‐responsive proteins. Out of the twenty‐six mycorrhiza‐related proteins that were identified, only six displayed changes in abundance upon Cd exposure, suggesting that part of the symbiotic program, which displays low sensitivity to Cd, may be recruited to counteract Cd toxicity through the mycorrhiza‐dependent synthesis of proteins having functions putatively involved in alleviating oxidative damages, including a cyclophilin, a guanine nucleotide‐binding protein, an ubiquitin carboxyl‐terminal hydrolase, a thiazole biosynthetic enzyme, an annexin, a glutathione S‐transferase (GST)‐like protein, and a S‐adenosylmethionine (SAM) synthase.     

Interfacial and Emulsifying Characteristics of Acid-treated Pea Protein

This work presents equilibrium and dynamic aspects for the adsorption at the oil–water interface of pea (Pisum sativum L.) protein isolate (PPI). Dynamic interfacial tension, γ, and surface viscoelasticity modulus, ε, were determined using pendant-drop method. Adsorption kinetics studies revealed that pea proteins adsorb faster at pH 7.0 than at acidic pH (pH 2.4). On the other hand, the measured ε is lower at pH 7.0. This is probably due to fast adsorption, leading to the formation of inhomogeneous film structures. In fact, compared with pHs above the isoelectric point (pI ~ 4.3), acidic conditions slow down the adsorption, but the modulus is increased. Pea-protein-stabilized emulsions are more stable to creaming at acidic pH and their particle-size distributions are more homogeneous in these conditions. Effect of pH on interfacial properties and on properties of oil-in-water emulsions stabilized by PPI was interpreted in terms of pea protein solubility, globulin dissociation, and oil-droplet surface electrostatic charge. We propose that at acidic conditions, adsorbed dissociated globulins form stronger and denser viscoelastic networks when adsorbed at oil–water interface. Consequently, the pH-dependence of pea-globulin-stabilized emulsions properties could be of great interest to tune barrier properties of oil/water interfacial membranes for several applications such as encapsulation and controlled release of lipophilic bioactive components within the food, medical, and pharmaceutical industries.     

Tick vitellin is dephosphorylated by a protein tyrosine phosphatase during egg development: effect of dephosphorylation on VT proteolysis

Vitellin (VT) is a phospholipoglycoprotein that is the main component of arthropod egg yolk. Phosphorylation is a recurrent feature of every VT molecule described so far. However, the role played by such post-translational modification during egg development is not yet clear. In the eggs of the hard tick Boophilus microplus, VT is a phosphotyrosine-containing protein. VT-phosphotyrosine residues are gradually removed during tick embryogenesis due to the action of a 45 kDa egg tyrosine phosphatase. This enzyme is strongly inhibited by ammonium molybdate, sodium vanadate and cupric ion. The role of phosphotyrosine residues in VT proteolytic degradation was evaluated. Western blots probed with a monoclonal anti-phosphotyrosine antibody demonstrated that the high molecular mass VT subunits (VT 1 and VT 2) are the main targets of dephosphorylation during egg development. Both dephosphorylation and proteolysis of VT 1 and VT 2 are blocked by ammonium molybdate in total egg homogenates. When purified VT was dephosphorylated in vitro with lambda phosphatase and then incubated in the presence of bovine cathepsin D, VT proteolysis increased dramatically. Altogether, these data are the first to show that phosphotyrosine residues are present in a yolk protein, and that such residues might be involved in the regulation of VT breakdown during egg development.     

Salicylic acid and its location in response to biotic and abiotic stress

Salicylic acid (SA) is an important signal involved in the activation of plant defence responses against abiotic and biotic stress. SA may derive from the phenylpropanoid pathway or via isochorismate synthase as demonstrated in Nicotiana benthamiana, tomato and Arabidopsis thaliana. The phenylpropanoid pathway as well as isochorismate synthase are localized in the chloroplasts but it remains unknown if the end product SA is in the same organelle. We have studied the localization of SA in A. thaliana using the salicylate hydroxylase (NahG) gene expressed with a chloroplast targeting sequence. Plants expressing NahG in the chloroplasts are unable to accumulate SA induced after pathogen or UV exposure. Our data infer that SA is initially located in the chloroplasts.