A novel ß-propeller phytase from the dioxin-degrading bacterium Sphingomonas wittichii RW-1.

ß-propeller phytase-like sequences (BPP-like sequences) are widespread in the microbial world and have been found in the sequenced genomes of aquatic, soil, and plant bacteria. Exploring NCBI microbial genome database for putative genes encoding phytase, a BPP-like sequence from Sphingomonas wittichii RW-1 (Sequence ID: CP000699.1), known for its capacity of degrading polychlorinated dibenzo-p-dioxins and dibenzofurans, was recognized. The putative phytase gene (phySw) was amplified with specific primers, cloned, and overexpressed in Escherichia coli and the catalytic properties of the recombinant PhySw protein were analyzed. The results show that phySw encodes an enzyme with the properties of ß-propeller phytases: it requires the presence of Ca2+ ions, it is optimally active at 55 °C, and it has a pH optimum of 6.0 with good activity in the range 6.0-8.0. Furthermore, the enzyme exhibits a good thermostability, recovering 68% of its original activity after treatment at 80 °C for 10 min, and shows a good substrate specificity for phytic acid. These properties render this enzyme a candidate as an animal feed additive (e.g., for aquaculture industry). The isolation of phytases from a hydrocarbon-utilizing microorganism also opens new scenarios for their possible application in combating oil pollution.

16S rDNA Profiling to Reveal the Influence of Seed-Applied Biostimulants on the Rhizosphere of Young Maize Plants.

In an open field trial on two agricultural soils in NW Italy, the impact of two seed-applied biostimulants on the rhizosphere bacterial community of young maize plants was evaluated. The 16S rDNA profiling was carried out on control and treated plant rhizosphere samples collected at the 4-leaf stage and on bulk soil. In both soils, the rhizospheres were significantly enriched in Proteobacteria, Actinobacteria, and Bacteriodetes, while the abundances of Acidobacteria, Cloroflexi and Gemmatimonadetes decreased compared with bulk soil. Among the culturable bacteria genera that showed an increase by both biostimulants, most are known to be beneficial for nutrient uptake, such as Opitutus, Chryseolinea, Terrimonas, Rhodovastum, Cohnella, Pseudoduganella and the species Anaeromyxobacter dehalogenans; others are known to be involved in root growth, such as Niastella, Labrys, Chloroflexia and Thermomonas; or in plant defence, such as Ohtaekwangia, Quadrisphaera, Turneriella, and Actinoallomurus. Both biostimulants were also found to stimulate gen. Nannocystis, a potential biocompetitive agent against aflatoxigenic Aspergillus moulds. Under controlled conditions, both biostimulants enhanced the shoot and root biomass at the 4⁻5 leaf stage. We conclude that the biostimulants do not decrease the biodiversity of the microbial community rhizosphere of young maize plants, but stimulate rare bacterial taxa, some involved in plant growth and pathogen resistance, a result that may have implications in improving crop management.

A metaproteomic approach dissecting major bacterial functions in the rhizosphere of plants living in serpentine soil.

A metaproteomic approach, based on liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis, was followed to map the major bacterial metabolic functions associated with the rhizosphere of metal-tolerant and metal hyperaccumulator plants, growing in a serpentine soil naturally contaminated by heavy metals such as Ni, Co and Cr. In particular, an "in-house" bacterial protein database was built based on the genera recognised by 16S rDNA profiling, then it was used for protein identification from LC-MS data. The combination of the information arising from three different extraction protocols, applied to each soil sample, permitted the identification of almost 800 proteins, corresponding to functions assigned to proper Gene Ontology categories. Mainly proteins involved in response to stimulus or in transport of metals and nutrients revealed variability of bacteria responses to microenvironment conditions. As for taxonomy, Phyllobacterium, Microbacterium oxidans, Pseudomonas oryzihabitans, Stenotrophomonas rhizophila and Bacillus methylotrophicus bacterial species were more represented in the rhizosphere samples of the metal-tolerant Biscutella laevigata and of the Ni hyperaccumulator Noccaea caerulescens with respect to bulk soil. Combining 16S rRNA gene-based sequencing and metaproteomic analysis, we get insights into microbial community functions and their interaction with plants colonising the stressful environment of serpentine soils.

How do turbidite systems behave from the hydrogeological point of view? New insights and open questions coming from an interdisciplinary work in southern Italy.

Turbidite successions can behave either as aquitards or aquifers depending on their lithological and hydraulic features. In particular, post-depositional processes can increase rock permeability due to fracture development in the competent layers. Thus, at a local scale, turbidite systems warrant further detailed investigations, aimed at reconstructing reliable hydrogeological models. The objective of this work was to investigate from the hydrogeological perspective a turbiditic aquifer located in southern Italy, where several perennial and seasonal springs were detected. Considering the complex hydrodynamics of these systems at the catchment scale, to reach an optimal characterization, a multidisciplinary approach was adopted. The conceptual framework employed microbial communities as groundwater tracers, together with the physicochemical features and isotopic signature of springs and streams from water samples. Meanwhile, geophysical investigations coupled with the geological survey provided the contextualization of the hydrogeological data into the detailed geological reconstruction of the study area. This modus operandi allowed us to typify several differences among the samples, allowing identification of sources and paths of surface water and groundwater, along with diffuse groundwater outflow along streams. As a final result, a hydrogeological conceptual model was reconstructed, underlining how at a very local scale the lithologic, hydraulic, and geomorphological heterogeneity of the studied relief can lead to an improved hydrogeological conceptual model compared to that of other turbidite systems. These results open new questions about the hydrogeological behavior of turbiditic aquifers, which could be pivotal in future research. In fact, these systems could support relevant ecosystems and anthropic activities, especially where climate change will force the research of new (and probably less hydrogeologically efficient) water sources.

Ag-functionalized nanocrystalline cellulose for paper preservation and strengthening.

Waste paper is an environmentally friendly source of cellulosic material. Here we propose a new treatment based on nanocrystalline cellulose (CNC) for paper preservation and consolidation. Suspensions of CNC were prepared by sulfuric acid hydrolysis using waste paper as cellulose source (CNCWP) and compared with CNC from cotton linter (CNCCL). Both CNCs were obtained with good yield, showing high crystallinity index and comparable morphology, as demonstrated by DLS-ELS, XRD, FTIR, Raman and TEM analyses. CNCs were mixed with silver nanoparticles (CNC/Ag) and their biocidal activity was tested against Escherichia coli and Bacillus subtilis, measuring the minimum inhibitory concentration. CNCs were exploited as treatments for biocidal activity and consolidation on Whatman paper. The presence of silver nanoparticles doesn't affect aesthetic appearance of the original paper and prevents the growth of Aspergillus niger fungus. Mechanical tests demonstrated that the coatings by CNC based products improve stretch and toughness of the paper support.

Combined application of Triton X-100 and Sinorhizobium sp. Pb002 inoculum for the improvement of lead phytoextraction by Brassica juncea in EDTA amended soil.

The process of EDTA-assisted lead phytoextraction from the Bovisa (Milan, Italy) brownfield soil was optimized in microcosms vegetated with Brassica juncea. An autochthonous plant growth-promoting rhizobacterium (PGPR), Sinorhizobium sp. Pb002, was isolated from the rhizosphere of B. juncea grown on the Pb-contaminated soil in presence of 2 mM EDTA. The strain was augmented (10(8) CFU g(-1) soil) in vegetated microcosms to stimulate B. juncea biomass production and, hence, its phytoextraction potential. Triton X-100 was also added to microcosms at 5 and 10 times the critical micelle concentration (cmc) to increase the permeability of root barriers to the EDTA-Pb complexes. Triton X-100 amendment determined an increase in Pb concentration within plant tissues. However it contextually exerted a phytotoxic effect. Sinorhizobium sp. Pb002 augmentation was crucial to plant survival in presence of both bioavailable lead and Triton X-100. The combination of the two treatments produced up to 56% increase in the efficiency of lead phytoextraction by B. juncea. The effects of these treatments on the structure of the soil bacterial community were evaluated by 16S rDNA denaturing gradient gel electrophoresis (DGGE).

Engineering peroxidase activity in myoglobin: the haem cavity structure and peroxide activation in the T67R/S92D mutant and its derivative reconstituted with protohaemin-l-histidine.

Atomic co-ordinates and structure factors for the T67R/S92D metMbCN mutant have been deposited with the Protein Data Bank, under accession codes 1h1x and r1h1xsf, respectively. Protein engineering and cofactor replacement have been employed as tools to introduce/modulate peroxidase activity in sperm whale Mb (myoglobin). Based on the rationale that haem peroxidase active sites are characterized by specific charged residues, the Mb haem crevice has been modified to host a haem-distalpropionate Arg residue and a proximal Asp, yielding the T67R/S92D Mb mutant. To code extra conformational mobility around the haem, and to increase the peroxidase catalytic efficiency, the T67R/S92D Mb mutant has been subsequently reconstituted with protohaem-L-histidine methyl ester, yielding a stable derivative, T67R/S92D Mb-H. The crystal structure of T67R/S92D cyano-metMb (1.4 A resolution; R factor, 0.12) highlights a regular haem-cyanide binding mode, and the role for the mutated residues in affecting the haem propionates as well as the neighbouring water structure. The conformational disorder of the haem propionate-7 is evidenced by the NMR spectrum of the mutant. Ligand-binding studies show that the iron(III) centres of T67R/S92D Mb, and especially of T67R/S92D Mb-H, exhibit higher affinity for azide and imidazole than wild-type Mb. In addition, both protein derivatives react faster than wild-type Mb with hydrogen peroxide, showing higher peroxidase-like activity towards phenolic substrates. The catalytic efficiency of T67R/S92D Mb-H in these reactions is the highest so far reported for Mb derivatives. A model for the protein-substrate interaction is deduced based on the crystal structure and on the NMR spectra of protein-phenol complexes.

ESEM-EDS: In vivo characterization of the Ni hyperaccumulator Noccaea caerulescens.

Environmental scanning electron microscopy (ESEM) permits to analyze samples in their native-hydrated state, allowing a broad spectrum of biological applications. In this study, ESEM equipped with energy dispersive X-ray spectrometer (EDS) was used as a fast method to analyze tissue morphology and to investigate metal distribution in the Ni hyperaccumulator Noccaea caerulescens, an established model to study the adaptation of plants to metalliferous soils. The low vacuum and wet mode operative conditions required the proper choice of experimental parameters both for morphological and compositional characterization of plant tissues. The calibration strategy for semi-quantitative analysis involved the use of Ni fortified agar as standard and signal normalization respect to endogenous carbon, chosen as internal standard. The obtained results are in accordance with present literature, showing a preferential Ni distribution in the epidermal cells respect to near the stomata for leaves and in the cotyledon epidermidis respect to cotyledon parenchyma area for seeds. Thanks to the absence of any time consuming sample treatment steps, ESEM-EDS technique can be proposed as valid strategy for in vivo high-throughput analysis of plant tissues and for a rapid screening and identification of other hyperaccumulator plants in a selected contaminated area.

Culturable endophytic bacteria enhance Ni translocation in the hyperaccumulator Noccaea caerulescens.

In this work, both culture-dependent and independent approaches were used to identify and isolate endophytic bacteria from roots of the Ni hyperaccumulator Noccaea caerulescens. A total of 17 isolates were cultured from root samples, selected for tolerance to 6mM Ni and grouped by restriction analysis of 16S rDNA. Bacterial species cultivated from roots belonged to seven genera, Microbacterium, Arthrobacter, Agreia, Bacillus, Sthenotrophomonas, Kocuria and Variovorax. The culture-independent approach confirmed the presence of Microbacterium and Arthrobacter while only other five clones corresponding to different amplified ribosomal DNA restriction patterns were detected. Five selected highly Ni-resistant bacteria showing also plant growth promoting activities, were inoculated into seeds of N. caerulescens, and in vivo microscopic analysis showed rapid root colonisation. Inoculated plants showed increased shoot biomass, root length and root-to-shoot Ni translocation. Root colonisation was also evident, but not effective, in the non-hyperaccumulating Thlaspi perfoliatum. Seed inoculation with selected Ni-resistant endophytic bacteria may represent a powerful tool in phytotechnologies, although transferring it to biomass species still requires further studies and screening.

Easy oxidation and nitration of human myoglobin by nitrite and hydrogen peroxide.

The modification of human myoglobin (HMb) by reaction with nitrite and hydrogen peroxide has been investigated. This reaction is important because NO(2) (-) and H(2)O(2) are formed in vivo under conditions of oxidative and nitrative stress, where protein derivatization has been often observed. The abundance of HMb in tissues and in the heart makes it a potential source and target of reactive species generated in the body. The oxidant and nitrating species produced by HMb/H(2)O(2)/NO(2) (-) are nitrogen dioxide and peroxynitrite, which can react with exogenous substrates and endogenous protein residues. Tandem mass analysis of HMb modified by stoichiometric amounts of H(2)O(2) and NO(2) (-) indicated the presence of two endogenous derivatizations: oxidation of C110 to sulfinic acid (76 %) and nitration of Y103 to 3-nitrotyrosine (44 %). When higher concentrations of NO(2) (-) and H(2)O(2) were used, nitration of Y146 and of the heme were also observed. The two-dimensional gel-electrophoretic analysis of the modified HMbs showed spots more acidic than that of wild-type HMb, a result in agreement with the formation of sulfinic acid and nitrotyrosine residues. By contrast, the reaction showed no evidence for the formation of protein homodimers, as observed in the reaction of HMb with H(2)O(2) alone. Both HMb and the modified HMb are active in the H(2)O(2)/NO(2) (-)-dependent nitration of exogenous phenols. Their catalytic activity is quite similar and the endogenous modifications of HMb therefore have little effect on the reactivity of the protein intermediates.

Catalytic activity, stability, unfolding, and degradation pathways of engineered and reconstituted myoglobins.

The structural and functional consequences of engineering a positively charged Lys residue and replacing the natural heme with a heme-L-His derivative in the active site of sperm whale myoglobin (Mb) have been investigated. The main structural change caused by the distal T67K mutation appears to be mobilization of the propionate-7 group. Reconstitution of wild-type and T67K Mb with heme-L-His relaxes the protein fragment around the heme because it involves the loss of the interaction of one of the propionate groups which stabilize heme binding to the protein. This modification increases the accessibility of exogenous ligands or substrates to the active site. The catalytic activity of the reconstituted proteins in peroxidase-type reactions is thus significantly increased, particularly with T67K Mb. The T67K mutation slightly reduces the thermodynamic stability and the chemical stability of Mb during catalysis, but somewhat more marked effects are observed by cofactor reconstitution. Hydrogen peroxide, in fact, induces pseudo-peroxidase activity but also promotes oxidative damage of the protein. The mechanism of protein degradation involves two pathways, which depend on the evolution of radical species generated on protein residues by the Mb active species and on the reactivity of phenoxy radicals produced during turnover. Both protein oligomers and heme-protein cross-links have been detected upon inactivation.

Characterization and peroxidase activity of a myoglobin mutant containing a distal arginine.

The spectroscopic, conformational, and reactivity characteristics of the T67R variant of sperm whale myoglobin have been studied to assess the effects of introducing an arginine residue into the distal side of this protein, as occurs in the active site of heme peroxidases. The overall circular dichroism (CD) and NMR spectroscopic properties of various derivatives of the protein are little affected by the mutation. The mutant contains a high-spin ferric ion with a water molecule as the sixth ligand, which exhibits slightly enhanced acidity (pK(a)=8.43+/-0.03) with respect to the corresponding derivative of wild-type myoglobin (pK(a)=8.60+/-0.04). The presence of the distal arginine increases the affinity of the Fe(III) center for azide (K=(6.0+/-0.5)x10(4) M(-1)) and decreases that for imidazole (K=12.0+/-0.2 M(-1)), with respect to the wild-type protein (K=(5.0+/-0.1)x10(4) and 24.7+/-0.7 M(-1), respectively). The peroxidase activity of T67R and wild-type myoglobins has been studied with a group of phenolic substrates related to tyrosine. The mutant exhibits an increased rate of reaction with hydrogen peroxide (k=1550+/-10 versus 760+/-10 M(-1) x s(-1)) and a generally increased peroxidase activity with respect to wild-type myoglobin. Relaxation measurements of proton nuclei of the phenolic substrates in the presence of either the T67R variant or the wild-type protein show that binding of these molecules occurs at distances of 8-10 A from the iron center, that is, close to the heme pocket, except for p-cresol, which can approach the heme more closely and, therefore, probably enter into the distal cavity.