Paola Pontieri

Clonothrix fusca Roze 1896, a Filamentous, Sheathed, Methanotrophic γ-Proteobacterium

ABSTRACT Crenothrix polyspora Cohn 1870 and Clonothrix fusca Roze 1896 are two filamentous, sheathed microorganisms exhibiting complex morphological differentiation, whose phylogeny and physiology have been obscure for a long time due to the inability to cultivate them. Very recently, DNA sequencing data from uncultured C. polyspora-enriched material have suggested that Crenothrix is a methane-oxidizing γ-proteobacterium (39). In contrast, the possible ecological function of C. fusca, originally considered a developmental stage of C. polyspora, is unknown. In this study, temporal succession of two filamentous, sheathed microorganisms resembling Cohns Crenothrix and Rozes Clonothrix was observed by analyzing the microbial community of an artesian well by optical microscopy. Combined culture-based and culture-independent approaches enabled us to assign C. fusca to a novel subgroup of methane-oxidizing γ-proteobacteria distinct from that of C. polyspora. This assignment was supported by (i) methane uptake and assimilation experiments, (ii) ultrastructural data showing the presence in C. fusca cytoplasm of an elaborate membrane system resembling that of methanotrophic γ-proteobacteria, and (iii) sequencing data demonstrating the presence in its genome of a methanol dehydrogenase α subunit-encoding gene (mxaF) and a conventional particulate methane mono-oxygenase α subunit-encoding gene (pmoA) that is different from the unusual pmoA (u-pmoA) of C. polyspora.

The microbial community of Vetiver root and its involvement into essential oil biogenesis.

Vetiver is the only grass cultivated worldwide for the root essential oil, which is a mixture of sesquiterpene alcohols and hydrocarbons, used extensively in perfumery and cosmetics. Light and transmission electron microscopy demonstrated the presence of bacteria in the cortical parenchymatous essential oil-producing cells and in the lysigen lacunae in close association with the essential oil. This finding and the evidence that axenic Vetiver produces in vitro only trace amounts of oil with a strikingly different composition compared with the oils from in vivo Vetiver plants stimulated the hypothesis of an involvement of these bacteria in the oil metabolism. We used culture-based and culture-independent approaches to analyse the microbial community of the Vetiver root. Results demonstrate a broad phylogenetic spectrum of bacteria, including alpha-, beta- and gamma-Proteobacteria, high-G+C-content Gram-positive bacteria, and microbes belonging to the Fibrobacteres/Acidobacteria group. We isolated root-associated bacteria and showed that most of them are able to grow by using oil sesquiterpenes as a carbon source and to metabolize them releasing into the medium a large number of compounds typically found in commercial Vetiver oils. Several bacteria were also able to induce gene expression of a Vetiver sesquiterpene synthase. These results support the intriguing hypothesis that bacteria may have a role in essential oil biosynthesis opening the possibility to use them to manoeuvre the Vetiver oil molecular structure.

Sorghum, a Healthy and Gluten-free Food for Celiac Patients As Demonstrated by Genome, Biochemical, and Immunochemical Analyses

Wheat (Triticum spp. L.), rye (Secale cereal L.), and barley (Hordeum vulgare L.) seeds contain peptides toxic to celiac patients. Maize (Zea mays L.) and rice (Oryza sativa L.) are distant relatives of wheat as well as sorghum (Sorghum bicolor (L.) Moench) and are known to be safe for celiacs. Both immunochemical studies and in vitro and in vivo challenge of wheat-free sorghum food products support this conclusion, although molecular evidence is missing. The goal of the present study was to provide biochemical and genetic evidence that sorghum is safe for celiac patients. In silico analysis of the recently published sorghum genome predicts that sorghum does not contain peptides that are toxic for celiac patients. Aqueous/alcohol-soluble prolamins (kafirins) from different sorghum varieties, including pure lines and hybrids, were evaluated by SDS-PAGE and HPLC analyses as well as an established enzyme-linked immunosorbent assay (ELISA) based on the R5 antibody. These analyses provide molecular evidence for the absence of toxic gliadin-like peptides in sorghum, confirming that sorghum can be definitively considered safe for consumption by people with celiac disease.

Development of a sorghum chain in the Italian Campania Region: From the field to the celiac patient's table

Abstract Although sorghum has been used for centuries as a food-crop in Africa and India, researchers in the United States and Europe have only relatively recently become interested in the potential of this unique cereal. Much of this interest focuses on the potential use of sorghum in food product development for individuals with allergies to foods containing wheat-based flours. Because it lacks gluten, sorghum is considered safe for people diagnosed with celiac disease, a condition marked by intolerance to gluten. Recent studies have shown that certain sorghum varieties, tan-plant sorghums, can be used to produce high-quality food and beverage products including cookies, waffles, flour, bread, noodles and beer. The intention is to promote the use and marketing of these sorghums in the Italian Campania Region for sorghum flours suitable as food for celiac patients.

Nitrite metabolism in Debaryomyces hansenii TOB-Y7, a yeast strain involved in tobacco fermentation

The Italian cigar manufacturing process includes a fermentation step that leads to accumulation of nitrite and tobacco-specific nitrosamines (TSNA), undesirable by-products due to their negative impact on health. In this study, growth and biochemical properties of Debaryomyces hansenii TOB-Y7, a yeast strain that predominates during the early phase of fermentation, have been investigated. With respect to other D. hansenii collection strains (Y7426, J26, and CBS 1796), TOB-Y7 was characterized by the ability to tolerate very high nitrite levels and to utilize nitrite, but not nitrate, as a sole nitrogen source in a chemically defined medium, a property that was enhanced in microaerophilic environment. The ability to assimilate nitrite was associated to the presence of YNI1, the gene encoding the assimilatory NAD(P)H:nitrite reductase (NiR), absent in Y7426, J26, and CBS 1796 by Southern blot data. YNI1 from TOB-Y7 was entirely sequenced, and its expression was analyzed in different media by Northern blot and reverse transcriptase polymerase chain reaction. The evidence that, in D. hansenii TOB-Y7, YNI1 was transcriptional active also in the presence of high ammonia concentration typical of tobacco fermentation, stimulated the development of an improved process that, on a laboratory scale, was proved to be effective in minimizing nitrite and TSNA accumulation.

Isolation of a psychrotolerant Debaryomyces hansenii strain from fermented tea plant (Camellia sinensis) leaves

Abstract A psychrotolerant, halotolerant and alkalophilic yeast was isolated from fermented leaves of Camellia sinensis Kuntze, the tea plant. The yeast strain, named Tea-Y1, was both phenotypically and genotypically identified as belonging to the species Debaryomyces hansenii. This assignment was confirmed by scanning and transmission electron microscopy. The analysis of growth curves demonstrated the ability this yeast strain to grow in a temperature range between 4°C and 28°C, with an optimum of 23°C. The ecology of this yeast in the C. sinensis phyllosphere, as well as its possible role in tea fermentation and storage, with particular reference to iced tea, are discussed.

Cultivable gut bacteria provide a pathway for adaptation of Chrysolina herbacea to Mentha aquatica volatiles

BackgroundA chemical cross-talk between plants and insects is required in order to achieve a successful co-adaptation. In response to herbivory, plants produce specific compounds, and feeding insects respond adequately7 to molecules produced by plants. Here we show the role of the gut microbial community of the mint beetle Chrysolina herbacea in the chemical cross-talk with Mentha aquatica (or watermint).ResultsBy using two-dimensional gas chromatography–mass spectrometry we first evaluated the chemical patterns of both M. aquatica leaf and frass volatiles extracted by C. herbacea males and females feeding on plants, and observed marked differences between males and females volatiles. The sex-specific chemical pattern of the frass paralleled with sex-specific distribution of cultivable gut bacteria. Indeed, all isolated gut bacteria from females belonged to either α- or γ-Proteobacteria, whilst those from males were γ-Proteobacteria or Firmicutes. We then demonstrated that five Serratia marcescens strains from females possessed antibacterial activity against bacteria from males belonging to Firmicutes suggesting competition by production of antimicrobial compounds. By in vitro experiments, we lastly showed that the microbial communities from the two sexes were associated to specific metabolic patterns with respect to their ability to biotransform M. aquatica terpenoids, and metabolize them into an array of compounds with possible pheromone activity.ConclusionsOur data suggest that cultivable gut bacteria of Chrysolina herbacea males and females influence the volatile blend of herbivory induced Mentha aquatica volatiles in a sex-specific way.

On the relationship between N management and grain protein content in six durum wheat cultivars in Mediterranean environment

Abstract Grain protein has recently received attention in several countries because of a premium associated to increased protein concentration (PC) in grains. Nitrogen (N) fertilizer management with its timing of application and rates plays a crucial role in increasing PC. The objectives of the present study were to: (1) quantify the effects of different N fertilizer rates and timing of application on grain yield and grain protein of six durum wheat cultivars, and (2) identify the best cultivar in terms of net return, yield, and protein content in typical pedo-climatic condition of southern Italy. The field experiments were conducted in Foggia during three growing seasons (2002–2003, 2003–2004, and 2004–2005). Results from this study showed that N application at sowing did not influence the cultivars performance. N applied at stem elongation increased yield and protein content provided there is adequate soil water for N uptake. In absolute terms, the best combination was the cultivar Svevo managed using 104 kg N ha−1 split between tillering and stem-elongation stage. The excessive N splitting reduces yield and does not provide any improvement in terms of protein content.

Yield and morpho-agronomical evaluation of food-grade white sorghum hybrids grown in Southern Italy

Abstract Grain sorghum [Sorghum bicolor (L.) Moench] is a gluten-free grain that is gaining attention as a food crop that can be used in the management of celiac disease. At present, sorghum is widely grown in many semiarid regions of the world. New food-grade sorghum cultivars are of particular interest in Mediterranean countries due to improved quality characteristics and gluten-free status of the grains. Until now very few studies have examined the grain yield (GYLD) and agronomic performance characteristics of food-grade sorghum hybrids in Italy. A 2 year study was conducted to evaluate the agronomic performance of eight food-grade sorghum hybrids representing different maturity classes in trials conducted in Southern Italy. The results showed wide variation in adaptation of these hybrids as measured by differences in GYLD (2.35–8.50 t ha−1) and other pheno-morphological characteristics. Of particular interest was the fact that many of the early-flowering hybrids (e.g. SP-X303) performed better than the later-flowering hybrids (e.g. ArchX-02). These results demonstrated that flowering time of hybrid and crop cycle length are important factors to consider in selecting cultivars for production in the Mediterranean region.

Mitochondrial ribosomal proteins involved in tellurite resistance in yeast Saccharomyces cerevisiae

A considerable body of evidence links together mitochondrial dysfunctions, toxic action of metalloid oxyanions, and system and neurodegenerative disorders. In this study we have used the model yeast Saccharomyces cerevisiae to investigate the genetic determinants associated with tellurite resistance/sensitivity. Nitrosoguanidine-induced K2TeO3-resistant mutants were isolated, and one of these mutants, named Sc57-Te5R, was characterized. Both random spore analysis and tetrad analysis and growth of heterozygous (TeS/Te5R) diploid from Sc57-Te5R mutant revealed that nuclear and recessive mutation(s) was responsible for the resistance. To get insight into the mechanisms responsible for K2TeO3-resistance, RNA microarray analyses were performed with K2TeO3-treated and untreated Sc57-Te5R cells. A total of 372 differentially expressed loci were identified corresponding to 6.37% of the S. cerevisiae transcriptome. Of these, 288 transcripts were up-regulated upon K2TeO3 treatment. About half of up-regulated transcripts were associated with the following molecular functions: oxidoreductase activity, structural constituent of cell wall, transporter activity. Comparative whole-genome sequencing allowed us to identify nucleotide variants distinguishing Sc57-Te5R from parental strain Sc57. We detected 15 CDS-inactivating mutations, and found that 3 of them affected genes coding mitochondrial ribosomal proteins (MRPL44 and NAM9) and mitochondrial ribosomal biogenesis (GEP3) pointing out to alteration of mitochondrial ribosome as main determinant of tellurite resistance.