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Mitochondrial diseases are a group of genetic disorders characterized by dysfunctional mitochondria. Within eukaryotic cells, mitochondria contain their own ribosomes, which synthesize small amounts of proteins, all of which are essential for the biogenesis of the oxidative phosphorylation system. The ribosome is an evolutionarily conserved macromolecular machine in nature both from a structural and functional point of view, universally responsible for the synthesis of proteins. Among the diseases afflicting humans, those of ribosomal origin - either cytoplasmic ribosomes (80S) or mitochondrial ribosomes (70S) - are relevant. These are inherited or acquired diseases most commonly caused by either ribosomal protein haploinsufficiency or defects in ribosome biogenesis. Here we review the scientific literature about the recent advances on changes in mitochondrial ribosomal structural and assembly proteins that are implicated in primary mitochondrial diseases and neurodegenerative disorders, and their possible connection with metalloid pollution and toxicity, with a focus on MRPL44, NAM9 (MNA6) and GEP3 (MTG3), whose lack or defect was associated with resistance to tellurite. Finally, we illustrate the suitability of yeast Saccharomyces cerevisiae (S. cerevisiae) and the nematode Caenorhabditis elegans (C. elegans) as model organisms for studying mitochondrial ribosome dysfunctions including those involved in human diseases.

Isolated sorghum (Sorghum bicolor) storage proteins (kafirins) have been successfully used in the production of several bio-materials including adhesives, films, micro-particles, fibers, and biological scaffold material. Comparatively little research has been conducted on the use of isolated kafirins in food products or to produce bioactive peptides via hydrolysis for nutritional uses. To support such research, the aim of this study was to compare existing methods for bulk isolation of sorghum kafirins with the goal of identifying a solvent with the least toxicity that maintained a high extraction rate from food grade sorghum flour. A secondary goal was to characterize the kafirin isolates produced from various extraction methods to provide some information on their potential use in food products to guide future research in this area. Five different extraction methods were compared including 1) aqueous ethanol containing NaOH and sodium metabisulfite, 2) glacial acetic acid, 3) aqueous ethanol with sodium metabisulfite, 4) aqueous ethanol at acidic pH, and 5) alkaline pH alone. The protein contents of the kafirin isolates obtained by the five methods ranged from 49.76% to 56.83%. Kafirin isolates were characterized using reversed phase (RP)-high performance liquid chromatography (HPLC), which revealed substantial variability in the various kafirin patterns among the extraction methods tested. However, characterization of the kafirin isolates by size exclusion chromatography (SEC) did not show a high degree of variability among the methods tested. Likewise, analysis of the samples using sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) showed essentially the same band profiles but with different band intensities among kafirin extraction methods. Surface hydrophobicity of the kafirin isolates varied considerably with isolates extracted with glacial acetic acid and aqueous ethanol plus sodium metabisulfite the most hydrophobic as indicated by hydrophobic dye binding.

Sorghum (Sorghum bicolor L.) is a gluten-free cereal with many varieties containing greater amounts of bioactive compounds than other grains. In this work, the phenolic composition and antioxidant capacity were compared in three sorghum varieties grown in the Mediterranean, consisting of different pericarp colors: white, red, and black. The qualitative and quantitative profiles of acidified aqueous methanol extracts of whole grain sorghum flours were elucidated through ultrahigh performance liquid chromatography (UHPLC) coupled to photodiode array (PDA) and ion trap time-of-flight (IT-TOF) mass spectrometry. The investigated sorghum varieties contained polyamines, such as spermidines, and polyphenolic compounds belonging to different classes: hydroxycinnamic acids, flavanols, flavones, and flavanones. Pyrano-flavanone-flavanols were detected only in the black sorghum. Precolumn and postcolumn 2,2′-diphenyl-1-picrylhydrazyl (DPPH)· radical scavenging assays to determine the main antioxidant compounds isolated from sorghum were developed. These results will further help develop sorghum varieties containing polyphenols relevant to the potential prevention of human diseases.

A white food-grade waxy sorghum Tw variety, grown in two Mediterranean sites (named Tw1M and Tw1S) was evaluated for nutrient composition and fatty acid- and mineral concentrations in order to determine the suitability of producing waxy sorghum for human uses in southern Italy. The nutritional values of the grains of the Tw inbred line grown in the two trial fields were substantially the same, except for slight differences in ash level and accordingly slight variation in mineral composition. In samples from both locations, a higher percentage of K was observed among the nutritionally essential macro-elements, and higher percentages of Zn, Fe, Mn, Cu, Al among the nutritionally essential micro-element along with a strong difference in Cd content was among trace elements. Across both sites linoleic, oleic and palmitic were the most abundant fatty acids, while very slight variations in the content of minerals were found among the two samples examined. These results demonstrate the importance of developing agronomically productive waxy sorghum varieties suitable for growth in non-traditional sorghum producing regions both as a food and feed crop (i.e. with good nutritional quality) and for utilization in new products at the industrial level.

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.

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 phenomorphological 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.

Vengono presentate tre nuove colture gluten free per la diversificazione colturale delle aree marginali interne della Campania. Si sono sviluppati protocolli di coltivazione per validare tecniche più opportune e rese agronomiche in alcune aree interne. La quinoa è stata introdotta nell'areale di Montecalvo Irpino, la canapa in quello di Volturara Irpina ed il sorgo Bianco nella provincia di Benevento a S. Bartolomeo in Galdo.

The main purpose of this book is to disseminate worldwide cultivation and food characteristics of white food-grade Sorghum, a gluten-free cereal taxonomically related to maize which is non-toxic to people intolerant to gluten. Specifically Sorghum bicolor (L. Moench), is an annual herbaceous plant belonging to the grass family of Poaceae, and is one of the most drought-resistant and heat-tolerant cereals. It has been a food crop for thousands of years in Africa and Asia, and over the past 30 years, it is used as safe food for celiac patients and as the healthy food for the entire human population. For its use in human food, sorghum, is made from a white grain type, particularly valuable, GMO free and optimal for the health, since it is rich in protein, unsaturated fats, carbohydrates, minerals, vitamins, fiber insoluble and antioxidants such as phenols and tannins, associated with the prevention of cancer and aging as well as a curative for individuals with cardiovascular disease. In this book we have also described the methods of cultivation of white food-grade sorghum in southern Italy, characterized by a Mediterranean climate, and the role of sorghum in human nutrition

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.

Background A 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). Results By 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. Conclusions Our 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. Electronic supplementary material The online version of this article (doi:10.1186/s12870-017-0986-6) contains supplementary material, which is available to authorized users.

Sorghum has been grown as a food crop for many centuries in Africa and Asia and it is a cereal option for celiac patients. Due to its properties as a wheat-free food, interest is increasing in cultivating sorghum in Mediterranean countries. To evaluate the agronomic characteristics of growth in the Mediterranean area, six hybrids of sorghum (Sorghum bicolor L. Moench), representing different maturity classes, were selected and tested in experimental trials for 2 years in San Bartolomeo in Galdo (BN) hill, South Italy. The results showed moderate variation in adaptation of these hybrids as measured by differences in grain yields (4.60 to 7.30 t ha-1) and other pheno-morphological traits. In particular, the hybrid SW6129W from Bolivia and the hybrid SASG05W from Argentina had grain yields higher than others tested in these trials. Our results demonstrated that selected food-grade hybrids from South America, have agronomic characteristics well-suited for cultivation in Mediterranean countries.

Sorghum ( Sorghum bicolor L. Moench) is a major crop used for food, feed, and industrial purpose worldwide and is one of the five most important cereal grains used for human consumption. As it is gluten-free, it is also suitable for celiac patients. White food-grade sorghum flour contains about 10% protein and about 3.3% fat (for most unsaturated fats), higher than that of wheat. It is rich in carbohydrates, contains micronutrients, and is rich in insoluble fiber. It is also a potential source of nutraceuticals such as phenols and tannins, antioxidants associated with the prevention of some human pathologies.

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.

The transposition of the Ty mobile genetic element of Saccharomyces cerevisiae is induced by carcinogens. While the molecular background of spontaneous Ty1 transposition is well understood, the detailed mechanism of carcinogen induced Ty1 transposition is not clear. We found that mitochondrial functions participate in the Ty induced transposition induced by carcinogens. Contrary to the parental rho(+) cells rho(-) mutants (spontaneous or induced by ethidium bromide) do not increase the rate of Ty1 transposition upon treatment with carcinogens. Preliminary results strongly suggest that the absence of oxidative phosphorylation in rho(-) mutants is the reason for the inhibited Ty transposition. The lack of carcinogen induced Ty1 transposition in rho(-) cells is not specific for a particular carcinogen and represents a general feature of different carcinogenic substances inducing rho(-). It is concluded that carcinogen induced Ty1 transposition depends on the functional state of mitochondria and cannot take place in cells with compromised mitochondrial function (rho(-)).

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.

Mitochondria are eukaryotic organelles which contain the own genetic material and evolved from free-living Eubacteria, namely hydrogen-producing Alphaproteobacteria. Since 1965, biologists provided, by research at molecular level, evidence for the prokaryotic origins of mitochondria. However, determining the precise origins of mitochondria is challenging due to inherent difficulties in phylogenetically reconstructing ancient evolutionary events. The use of new tools to evidence the prokaryotic origin of mitochondria could be useful to gain an insight into the bacterial endosymbiotic event that resulted in the permanent acquisition of bacteria, from the ancestral cell, that through time were transformed into mitochondria. Electron microscopy has shown that both proteobacterial and yeast cells during their growth in the presence of increasing amount of tellurite resulted in dose-dependent blackening of the culture due to elemental tellurium (Te 0 ) that formed large deposits either along the proteobacterial membrane or along the yeast cell wall and mitochondria. Since the mitochondrial inner membrane composition is similar to that of proteobacterial membrane, in the present work we evidenced the black tellurium deposits on both, cell wall and mitochondria of ρ + and respiratory deficient ρ − mutants of yeast. A possible role of tellurite in studying the evolutionary origins of mitochondria will be discussed.

To gain insight into essential oil metabolism we analyzed the oil produced in Vetiver ( Vetiveria zizanioides (L.) Nash) roots during early growth. Planting of Vetiver in the Campania region of southern Italy was performed in the spring using Vetiver culms with short roots and approximately 20 cm leaves. During the first two years of growth Vetiver essential oil was hydrodistilled from root samples collected at intervals of two months starting from transplantation. The production of Vetiver oil was constant during the first six months. Over the next two months there was a twofold increase in production of essential oil, which anticipated a progressive decrease during the cold months (from January to April 2003). The analysis of the Vetiver oil showed the presence of 49 constituents, mainly the tricyclic sesquiterpenes khusimol and zizanoic acid, and the bicyclic sesquiterpenes ( E )-isovalencenol, junenol, juniper camphor, nootkatone and α-vetivone. These results suggest that Vetiver essential oil production is closely related to the metabolism of plant roots, which is affected by changes in environmental temperatures.

Some nuclear genes in Saccharomyces cerevisiae (S. cerevisiae) respond to signals from the mitochondria in a process called by Butow (Cell Death Differ. 9 (2002) 1043–1045) retrograde regulation. Expression of these genes is activated in cells lacking mitochondrial function by involvement of RTG1, RTG2 and RTG3 genes whose protein products bind to “R-boxes” in the promoter region; RTG2p is a cytoplasmic protein. Since S. cerevisiae rho0 strains, lacking the entire mitochondrial genome, are resistant to lycorine, an alkaloid extracted from Amaryllis plants, it could be hypothesized that in rho0 cells the dysfunctional mitochondrial status stimulates overexpression of nuclear genes very likely involved in both nuclear and mitochondrial DNA replication. In this report we show that the resistance of rho0 cells to lycorine is affected by the deletion of RTG genes.

Mineral nutrients play a fundamental role in the biochemical and physiological functions of biological systems. Cereals may especially be an important source of essential minerals in view of their large daily intake both for human health and nutrition. Sorghum, among the cereals, is a major crop being used for food, feed and industrial purposes worldwide. The objective of this study was to determine the mineral contents in grains of seven white food-grade sorghum hybrids, bred and adapted for growth in the central USA and grown in a Mediterranean area of Southern Italy. The seven hybrids were analyzed for grain ash and for minerals contents. Nutritionally, essential macro-, micro- and trace elements content were investigated. The analysis of essential elements was performed by mass spectrometry using a mix solution of internal isotopes standard. The results demonstrated that food-grade sorghum was characterized by high Mg, Fe and Zn content, high K:Na ratio and low Ca:P ratio, compared to other crops, due to the fact that the grain mineral contents of crop species are influenced by the effects of genotypes and environments. Significant variations in the essential elements content were found among the hybrids which allowed us to divide them into three distinct groups on the basis of their mineral profile by cluster analysis. These results are discussed with reference to the importance of minerals in human nutrition and suggest that, like wheat, it is possible to plan research programs for the improvement and selection of sorghum hybrids with high micronutrients content

The effects of potassium tellurite on growth and survival of rho + and rho0 Saccharomyces cerevisiae strains were investigated. Both rho+ and rho0 strains grew on a fermentable carbon source with up to 1.2 mM K2TeO3, while rho+ yeast cells grown on a non-fermentable carbon source were inhibited at tellurite levels as low as 50 μM suggesting that this metalloid specifically inhibited mitochondrial functions. Growth of rho+ yeast cells in the presence of increasing amount of tellurite resulted in dose-dependent blackening of the culture, a phenomenon not observed with rho0 cultures. Transmission electron microscopy of S. cerevisiae rho+ cells grown in the presence of tellurite showed that blackening was likely due to elemental tellurium (Te0) that formed large deposits along the cell wall and small precipitates in both the cytoplasm and mitochondria. © 2009 Springer Science+Business Media, LLC.

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.

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.

BACKGROUND & AIMS: Celiac disease is a condition in which genetically predisposed people have an autoimmune reaction to gluten proteins found in all wheat types and closely related cereals such as barley and rye. This reaction causes the formation of autoantibodies and the destruction of the villi in the small intestine, which results in malabsorption of nutrientsand other gluten-induced autoimmune diseases. Sorghum is a cereal grain with potential to be developed into an important crop for human food products. The flour produced from white sorghum hybrids is light in color and has a bland, neutral taste that does not impart unusual colors or flavors to food products. These attributes make it desirable for use in wheat-free food products. While sorghum is considered as a safe food for celiac patients, primarily due to its relationship to maize, no direct testing has been conducted on its safety for gluten intolerance. Therefore studies are needed to assess its safety and tolerability in celiac patients. Thus the aim of the present study was to assess safety and tolerability of sorghum flour products in adult celiac disease patients, utilizing an in vitro and in vivo challenge. RESULTS: Sorghum protein digests did not elicit any morphometric or immunomediated alteration of duodenal explants from celiac patients. Patients fed daily for 5 days with sorghum-derived food product did not experience gastrointestinal or non-gastrointestinal symptoms and the level of anti-transglutaminase antibodies was unmodified at the end of the 5-days challenge. CONCLUSIONS: Sorghum-derived products did not show toxicity for celiac patients in both in vitro and in vivo challenge. Therefore sorghum can be considered safe for people with celiac disease.

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.

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 Cohn's Crenothrix and Roze's 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 .