Search

The ability of plants to take up phosphorus (P) from soil depends on root morphology and root exudates release and can be modulated by beneficial soil microbes. These microbes can solubilize P, affect root elongation and branching, and lead to a higher uptake of P and other nutrients. However, coordination of these mechanisms is unclear, especially the mechanism for changing the available form of P. We aimed to dissect the effects of two different beneficial microbial taxa (plant growth-promoting bacteria (PGPB) and arbuscular mycorrhizal fungi (AMF)) on root morphological traits, plant nutrient content, and growth in tomato and corn fertilized with either Gafsa rock phosphate (RP) or triple superphosphate (TSP), which have contrasting solubility levels. Tomato and corn were grown in pots and inoculated with one of three PGPB species or a mix of two AMF species or were not inoculated. Root traits, botanical fractions, and the contents of various mineral nutrients were measured. TSP stimulated tomato biomass accumulation compared to RP but did not stimulate corn biomass accumulation. PGPB improved the growth of both plant species under RP, with limited differences among the strains, whereas AMF only improved tomato growth under TSP. These differences between microbial systems were explained by a bacterial effect on the total root length but not on the mean root diameter and by the ability of AMF to improve the mineral nutrient content. The effects of PGPB were less dependent on the plant species and on P form than the effects of AMF.These results have implications for the improvement of the early plant growth through the management of beneficial microbes.

Distinctive strains of Pantoea are used as soil inoculants for their ability to promote plant growth. Pantoea agglomerans strain C1, previously isolated from the phyllosphere of lettuce, can produce indole-3-acetic acid (IAA), solubilize phosphate, and inhibit plant pathogens, such as Erwinia amylovora. In this paper, the complete genome sequence of strain C1 is reported. In addition, experimental evidence is provided on how the strain tolerates arsenate As (V) up to 100 mM, and on how secreted metabolites like IAA and siderophores act as biostimulants in tomato cuttings. The strain has a circular chromosome and two prophages for a total genome of 4,846,925-bp, with a DNA G+C content of 55.2%. Genes related to plant growth promotion and biocontrol activity, such as those associated with IAA and spermidine synthesis, solubilization of inorganic phosphate, acquisition of ferrous iron, and production of volatile organic compounds, siderophores and GABA, were found in the genome of strain C1. Genome analysis also provided better understanding of the mechanisms underlying strain resistance to multiple toxic heavy metals and transmission of these genes by horizontal gene transfer. Findings suggested that strain C1 exhibits high biotechnological potential as plant growth-promoting bacterium in heavy metal polluted soils.

Pantoea agglomerans strain C1 has plant growth-promoting (PGP) traits and exhibits antimicrobial activity. The genome comprises 4.8 Mb, 4,696 protein-coding sequences, and a G+C content of 55.2%.

The rising demand of bio-vanillin and the possibility to use microbial biotransformation to produce this compound from agroindustrial byproducts are economically attractive. However, there are still several bottlenecks, including substrate and product toxicity, formation of undesired products and genetic stability of the recombinant strains, that impede an efficient use of recombinantEscherichia colistrains to make the whole process cost effective. To overcome these problems, we developed a newE. colistrain, named FR13, carrying thePseudomonasgenes encoding feruloyl-CoA synthetase and feruloyl-CoA hydratase/aldolase integrated into the chromosome and, using resting cells, we demonstrated that the vanillin yield and selectivity were strongly affected by the physiological state of the cells, the temperature used for the growth and the recovery of the biomass and the composition and pH of the bioconversion buffer. The substrate consumption rate and the vanillin yield increased using a sodium/potassium phosphate buffer at pH 9.0 as bioconversion medium. Optimization of the bioprocess variables, using response surface methodology, together with the use of a two-phase (solid-liquid) system for the controlled release of ferulic acid allowed us to increase the vanillin yield up to 28.10 ± 0.05 mM. These findings showed that recombinant plasmid-freeE. colistrains are promising candidates for the production of vanillin at industrial scale and that a reduction of the cost of the bioconversion process requires approaches that minimize the toxicity of both ferulic acid and vanillin.

Fermented foods have long been produced according to knowledge passed down from generation to generation and with no understanding of the potential role of the microorganism(s) involved in the process. However, the scientific and technological revolution in Western countries made fermentation turn from a household to a controlled process suitable for industrial scale production systems intended for the mass marketplace. The aim of this paper is to provide an up-to-date review of the latest studies which investigated the health-promoting components forming upon fermentation of the main food matrices, in order to contribute to understanding their important role in healthy diets and relevance in national dietary recommendations worldwide. Formation of antioxidant, bioactive, anti-hypertensive, anti-diabetic, and FODMAP-reducing components in fermented foods are mainly presented and discussed. Fermentation was found to increase antioxidant activity of milks, cereals, fruit and vegetables, meat and fish. Anti-hypertensive peptides are detected in fermented milk and cereals. Changes in vitamin content are mainly observed in fermented milk and fruits. Fermented milk and fruit juice were found to have probiotic activity. Other effects such as anti-diabetic properties, FODMAP reduction, and changes in fatty acid profile are peculiar of specific food categories.

Natural biostimulants are one of the most promising and eco-sustainable technologies, which can improve the qualitative and productive attributes of horticultural crops. The positive effects of their application derive from their “signaling role” as elicitors of auxin- and gibberellin-like activity, thus constituting a valid alternative to synthetic hormones. Our research aimed to assess the efficacy of a plant extract (Heptamin®) and a bacterial culture filtrate (Capxium®), both rich in natural auxins, and of a synthetic auxin (Auxyger® LG) on the quantitative and qualitative performance, as well as on the economic returns of greenhouse table tomato (Solanum lycopersicum L. cv. Pitagora). The application of plant extract increased the marketable yield (+25.8%), firmness (+25.7%), and fruit lycopene content (+42.5%) compared to the untreated control. In particular, natural biostimulants outperformed synthetic auxin by inducing higher fruit weight (13.1%) and marketable yield (16.2%). On the other hand, no significant differences were recorded as a result of the treatments regarding total soluble solids, pH, and titratable acidity of the fruit juice. Our results suggest that plant biostimulants with auxin-like activity are an excellent tool for boosting tomato production and fruit quality in an eco-sustainable approach. Finally, economic analysis revealed that the application of natural biostimulants resulted in overall higher profitability

Many studies on plant biostimulants and organic fertilizers have been focused on the ability of these products to increase crop productivity and ameliorate crop tolerance to abiotic stresses. However, little information is available on their effect on plant microbiota, whereas it is well known that microorganisms associated with plant play crucial roles on the health and productivity of their host. The aim of this study was to evaluate the effect of a vegetal-derived protein hydrolysate (PH), a vegetal-derived PH enriched with copper (Cu-PH), and a tropical plant extract enriched with micronutrients (PE) on shoot growth and the epiphytic bacterial population of lettuce plants and the ability of these products to enhance the growth of beneficial or harmful bacteria. The three plant-derived products enhanced shoot biomass of lettuce plants indicating a biostimulant effect of the products. Data obtained using culture-independent (Terminal Restriction Fragment Length Polymorphism and Next Generation Sequencing) and culture-dependent approaches indicated that foliar application of commercial products altered the composition of the microbial population and stimulated the growth of specific bacteria belonging toPantoea,Pseudomonas,Acinetobacter, andBacillusgenus. Data presented in this work demonstrated that some of these strains exhibited potential plant growth-promoting properties and/or biocontrol activity against fungi and bacteria phytopathogens includingFusarium,Trichoderma, andErwiniaspecies. No indication of potential health risks associated to the enrichment of human or plant bacterial pathogens emerged by the analysis of the microbiota of treated and no-treated plants. Overall, the findings presented in this study indicate that the commercial organic-based products can enhance the growth of beneficial bacteria occurring in the plant microbiota and signals produced by these bacteria can act synergistically with the organic compounds to enhance plant growth and productivity.

A cDNA encoding the proteinase inhibitor WSCI (wheat subtilisin/chymotrypsin inhibitor) was isolated by RT-PCR. Degenerate oligonucleotide primers were designed based on the amino acid sequence of WSCI and on the nucleotide sequence of the two homologous inhibitors (CI-2A and CI-2B) isolated from barley. For large-scale production,wscicDNA was cloned into theE. colivector pGEX-2T. The fusion protein GST-WSCI was efficiently produced in the bacterial expression system and, as the native inhibitor, was capable of inhibiting bacterial subtilisin, mammalian chymotrypsins and chymotrypsin-like activities present in crude extracts of a number of insect larvae (Helicoverpa armigera,Plodia interpunctellaandTenebrio molitor). The recombinant protein produced was also able to interfere with chymotrypsin-like activity isolated from immature wheat caryopses. These findings support a physiological role for this inhibitor during grain maturation.

Both ribosome-inactivating proteins (RIPs) and plant proteinase inhibitors, belong to protein families known to regulate cellular homeostasis and likely involved in plant defense. Nevertheless the interest in these protein classes is due to their potential use for the treatment of several important human diseases such as cancer. Thus, in the present study, type 1 ribosome- inactivating protein and wheat subtilisin/chymotrypsin inhibitor, were engineered into a chimeric protein with cytotoxic action selective for murine tumor cells, while lacking any appreciable toxicity on murine normal cells. This chimeric protein selectively sensitizes to apoptotic death cells derived from Simian-virus-40-transformed mouse fibroblasts (SVT2 cells). The cytotoxicity of this new recombinant product has been detected also on three different human malignant cells. Therefore action on tumor cells of this protein could represent a potentially very attractive novel tool for anticancer drug design. © 2012 Published by Elsevier Masson SAS. All rights reserved.

Vanillin is one of the most important flavors in the food industry and there is great interest in its production through biotechnological processes starting from natural substrates such as ferulic acid. Among bacteria, recombinant Escherichia coli strains are the most efficient vanillin producers, whereas Pseudomonas spp. strains, although possessing a broader metabolic versatility, rapidly metabolize various phenolic compounds including vanillin. In order to develop a robust Pseudomonas strain that can produce vanillin in high yields and at high productivity, the vanillin dehydrogenase (vdh)-encoding gene of Pseudomonas fluorescens BF13 strain was inactivated via targeted mutagenesis. The results demonstrated that engineered derivatives of strain BF13 accumulate vanillin if inactivation of vdh is associated with concurrent expression of structural genes for feruloyl-CoA synthetase (fcs) and hydratase/aldolase (ech) from a low-copy plasmid. The conversion of ferulic acid to vanillin was enhanced by optimization of growth conditions, growth phase and parameters of the bioconversion process. The developed strain produced up to 8.41 mM vanillin, which is the highest final titer of vanillin produced by a Pseudomonas strain to date and opens new perspectives in the use of bacterial biocatalysts for biotechnological production of vanillin from agro-industrial wastes which contain ferulic acid. ©

A novel chymotrypsin inhibitor, detected in the endosperm of Triticum aestivum, was purified and characterized with respect to the main physical-chemical properties. On the basis of its specificity, this inhibitor was named WCI (wheat chymotrypsin inhibitor). WCI is a monomeric neutral protein made up of 119 residues and molecular mass value of 12,933.40 Da. Automated sequence and mass spectrometry analyses, carried out on several samples of purified inhibitor, evidenced an intrinsic molecular heterogeneity due to the presence of the isoform [des-(Thr)WCI], accounting for about 40% of the total sample. In vitro, WCI acted as a strong inhibitor of bovine pancreatic chymotrypsin as well as of chymotryptic-like activities isolated from the midgut of two phytophagous insects, Helicoverpa armigera (Hüb.) and Tenebrio molitor L., respectively. No inhibitory activities were detected against bacterial subtilisins, bovine pancreatic trypsin, porcine pancreatic elastase or human leukocyte elastase. The primary structure of WCI was significantly similar (45.7-89.1%) to those of several proteins belonging to the cereal trypsin/α-amylase inhibitor super-family and showed the typical sequence motif of this crowed protein group. The cDNA of the inhibitor (wci-cDNA) was isolated from wheat immature caryopses and employed to obtain a recombinant product in E. coli. Experimental evidences indicated that the recombinant inhibitor was localized in the inclusion bodies from which it was recovered as soluble and partially active protein by applying an appropriate refolding procedure. WCI reactive site localization, as well as its inhibitory specificity, was investigated by molecular modeling approach. © 2011 Springer-Verlag.

A site-directed mutagenesis strategy was employed to obtain four mutants of wheat subtilisin/chymotrypsin inhibitor (WSCI), with the aim to produce inactive forms of this protein. The mutants were expressed in Escherichia coli as fusion proteins and, after the tag removal, were purified to homogeneity. Three mutants, containing a single mutation at the sequence positions 49 or 50, were named E49S, E49P and Y50G, respectively. These mutants exhibited anti-subtilisin activities comparable to that of the wild type protein; instead, anti-chymotrypsin activity was detectable only for the mutant E49S. A fourth mutant (M48P-E49G), containing a double amino acid substitution at the inhibitor reactive site (P1-P1′), was inactive against both subtilisin and chymotrypsin. In order to investigate the interactions between the putative susceptible enzymes and the mutated forms of WSCI, we performed time-course hydrolysis experiments by incubating samples of the mutants with subtilisin-agarose and chymotrypsin-agarose, respectively. These experiments yielded information on the E/I complex formation, as well as on the timing of the cleavage pattern of some of these mutants. Molecular modeling studies were carried out with the 3D models of the mutants and of their putative complexes with subtilisin and chymotrypsin. In terms of inter- and intra-chain H-bond networks, the observations made for each theoretical E/I complex were found to be fully coherent with experimental data (kinetic and time-course hydrolysis) and supplied specific modalities of interaction of each mutant with the enzyme counterpart. © 2009 Elsevier Masson SAS. All rights reserved.

In Pseudomonas fluorescens BF13, the cluster of genes essential for degradation of ferulic to vanillic acid (ech, vdh and fcs) is expressed in ferulic but not in succinic grown cells. In the upstream region, we identified a gene, ferR, encoding a protein homologous to transcriptional regulators of the MarR family. A ferR knockout mutant (BF13–89) showed a 3.5-fold increase in expression of an ech-reporter gene fusion compared with the parent strain in succinic-grown cells, indicating that the ferR gene product negatively regulates expression of the ferulic catabolic operon in P. fluorescens BF13. Consistent with the increased expression of the catabolic genes in the ferR mutant, BF13-89 showed a shorter (relative to its FerR+ parent) lag phase during carbon source shift from succinic to ferulic acid. However, expression of ech-lacZ fusion did not increase in BF13–89 grown in the presence of ferulic acid, indicating that FerR has a second function as transcriptional activator. Expression of ech-lacZ in a feruloyl-CoA synthetase- eficient strain revealed unambiguously that FerR-mediated activation of the ferulic catabolic operon is dependent on the thioester product of the feruloyl-CoA synthetase reaction. L'articolo è disponibile sul sito dell'editore http://www.springerlink.com

Aiming to achieve stable immobilization for a redox-active cupredoxin protein onto a gold substrate and its consequent molecular level monitoring by Scanning Tunnelling Microscopy (STM), we introduced a disulphide bridge within poplar plastocyanin, while avoiding the perturbation of its active site. We selected and modified residues lle-21 to Cys and Glu-25 to Cys by structurally conservative mutagenesis. Optical absorption spectroscopy (UV-Vis), electron paramagnetic resonance (EPR), and resonance raman scattering (RRS) results indicate that the active site of the Ile21Cys, Glu25Cys plastocyanin (PCSS) to a large extent retains the spectroscopic properties of the wildtype protein. Furthermore, the redox midpoint potential of the couple CuII/CuI in PCSS, determined by cyclic voltammetry was found to be +348 mV close to the wild-type value. The STM images display self-assembled PCSS molecules immobilised onto gold substrate. Moreover, the fall potentiostatic control of the electron transfer reaction during STM imaging, suggests that the adsorbed molecule maintains essentially its native redox properties. (C) 2002 Elsevier Science (USA).

The monoclonal antibody DLT15, specific for thymocytes and peripheral T-cells of the teleost fish Dicentrarchus labrax (sea bass), was used to purify immunoreactive cells from blood and gut-associated lymphoid tissue. The purification was performed by immuno-magnetic sorting of leucocyte fractions enriched by Percoll density gradient centrifugation, and the purity of the isolated cells was estimated by cytofluorimetric analysis. Following a single step, the percentage of DLT15-purified cells was 88 +/- 10% for gut-associated lymphoid tissue and 79 +/- 18% for blood leucocytes. DLT15-purified cells from gut-associated lymphoid tissue were employed for RNA extraction and cDNA synthesis. In RT-PCR experiments using as primers degenerate oligonucleotides corresponding to the peptide sequence MYWY and VYFCA of the trout TcR beta chain, a 203 bp product was amplified. When sequenced, the cDNA was found to show 60% nucleotide identity to the trout TcRV beta 3. By 3'-RACE the cDNA was elongated to obtain the TcR constant region, with high similarity to other fish TcR sequences. These results strongly suggest that cells recognised by DLT15 are putative T lymphocytes.

In contrast to the Escherichia coli lac operon, the yeast beta-galactosidase gene is positively regulated. In the 5'-noncoding region of the Kluyveromyces lactis LAC4 gene, we mapped an upstream activation site (UAS) that is required for induction. This sequence, located between positions -435 and -326 from the start of translation, functions irrespective of its orientation and can confer lactose regulation to the heterologous CYC1 promoter. It is composed of at least two subsequences that must act in concert. One of these subsequences showed a strong homology to the UAS consensus sequence of the Saccharomyces cerevisiae GAL genes (E. Giniger, S. M. Varnum, and M. Ptashne, Cell 40:767-774, 1985). We propose that this region of homology located at about position -426 is a binding site for the product of the regulatory gene LAC9 which probably induces transcription of the LAC4 gene in a manner analogous to that of the GAL4 protein.

The intergenic region of the Saccharomyces cerevisiae GAL1-GAL10 divergent promoters has been circularized in vitro in different topological states. In defined conditions, purified homologous RNA polymerase II forms two stable complexes (half-life approximately equal to 5 h) with this DNA in the presence of the four ribonucleotides, as determined by measurement (Gamper and Hearst 1983) of the amount and stability of the resulting unwinding. Each stable complex induces in the closed DNA domain a region of hypersensitivity to P1 endonuclease. The two induced hypersensitive regions are very similar: each maps on one promoter, spans over the 100 bp DNA sequence that encompasses the RNA Initiation Sites (RIS) and the TATA box, is composed by three subregions (one on the RIS, one proximal or overlapping the TATA sequence, one intermediate). We show that this promoter-localized interaction is supercoil-dependent.

In the 5' non-coding region of the beta-galactosidase, LAC4, gene of Kluyveromyces lactis, three TATA-like sequences are present at -230, -170 and -142 from the ATG translation start site. By means of deletion mutations in the TATA region, at least two of these TATA sequences, those at -230 and -142, were shown to be required for normal gene expression. Evidence is presented for a functional hierarchy and cooperation between these TATA sequences. The deletion or a change in the position of the TATA sequences affects both beta-galactosidase induction and the location of RNA initiation sites. The TATA sequence at -230 alone is sufficient for correct gene induction when it is moved to a position 41 bp from the major RNA initiation sites located around -110; the -142 TATA alone contributes only partly to gene induction. We suggest a functional distinction between these two related regulatory sequences. This functional distinction might be established by sequence differences and/or targets of unlike specific DNA binding protein(s). A conformational analysis of the LAC4 promoter showed that under torsional stress the functional elements UAS, TATA boxes RNA initiation sites and ATG can be detected as P1-sensitive sites. Possible functions of DNA structural alterations on gene expression are discussed.

We have developed a rapid and efficient expression system to study the human β 2 adrenergic receptor (hu β 2 AR) in the fission yeast Schizosaccharomyces pombe . This was achieved by cloning the hu β 2 AR gene, modified by replacement of the 5′ untranslated and a small part of the N-terminal coding sequence (first 14 amino acids) with the corresponding region of the yeast Saccharomyces cerevisiae STE2 (α-factor receptor) gene. The gene was then placed under the control of a S. pombe constitutive promoter for alcohol dehydrogenase (adh). Hu β 2 AR expression was assessed by immunoblot analysis of the chimeric protein with an anti-STE2 serum raised against a dodecapeptide homologous to the N-terminal amino acids of STE2 and ligand binding was assayed using [ 125 I]cyanopindolol. We demonstrate here that the chimeric receptor expressed in S. pombe exhibits the same characteristic ligand specificity and affinity as that of the authentic hu β 2 AR. This system constitutes a convenient alternative to existing methods for studying seven transmembrane domain receptors due to its simplicity and high reproducibility.