Microbial community structure and dynamics of dark fire-cured tobacco fermentation.

The Italian Toscano cigar production includes a fermentation step that starts when dark fire-cured tobacco leaves are moistened and mixed with ca. 20% prefermented tobacco to form a 500-kg bulk. The dynamics of the process, lasting ca. 18 days, has never been investigated in detail, and limited information is available on microbiota involved. Here we show that Toscano fermentation is invariably associated with the following: (i) an increase in temperature, pH, and total microbial population; (ii) a decrease in reducing sugars, citric and malic acids, and nitrate content; and (iii) an increase in oxalic acid, nitrite, and tobacco-specific nitrosamine content. The microbial community structure and dynamics were investigated by culture-based and culture-independent approaches, including denaturing gradient gel electrophoresis and single-strand conformational polymorphism. Results demonstrate that fermentation is assisted by a complex microbial community, changing in structure and composition during the process. During the early phase, the moderately acidic and mesophilic environment supports the rapid growth of a yeast population predominated by Debaryomyces hansenii. At this stage, Staphylococcaceae (Jeotgalicoccus and Staphylococcus) and Lactobacillales (Aerococcus, Lactobacillus, and Weissella) are the most commonly detected bacteria. When temperature and pH increase, endospore-forming low-G+C content gram-positive bacilli (Bacillus spp.) become evident. This leads to a further pH increase and promotes growth of moderately halotolerant and alkaliphilic Actinomycetales (Corynebacterium and Yania) during the late phase. To postulate a functional role for individual microbial species assisting the fermentation process, a preliminary physiological and biochemical characterization of representative isolates was performed.

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.

Constitutive and inducible expression of the epithelial antigen MUC1 (CD227) in human T cells.

MUC1 (CD227) is a large glycoprotein normally produced by epithelial tissue and expressed aberrantly in carcinomas. Here we show that resting human T cells express basal levels of MUC1 mRNA and protein forms with molecular masses of approximately 150 and approximately 250 intracellularly, but lack surface expression. Mitogenic stimulation induces the appearance of new MUC1 mRNA and >300-kDa MUC1 forms. Concomitantly, MUC1 is translocated to the outer cell membrane and its density is continuously modulated according to the cycling status. Inhibitors of mRNA and protein synthesis and of Golgi-dependent protein transport prevent MUC1 induction. Ligation of surface MUC1 has no effect on T-cell proliferation. Also, altering the overall protein structure by preventing glycosylation has no effect. Sizable amounts of >300-kDa glycosylated MUC1 forms are shed by proliferating T cells. This soluble MUC1 does not appear to influence T-cell response, and we found no evidence for MUC1 binding sites on T cells or for transfer of the protein on cell-cell contact. We therefore suggest that MUC1 fulfills the criteria for an early T-cell activation marker but its function remains to be determined. Finally, although we found that cancer- and T cell-associated MUC1 expose common protein core and sialylated epitopes, there is a peptide region, accessible in carcinomas due to an aberrant glycosylation, that is stably not accessible in T cells with potential implications for cancer immunotherapy.