Native yeast and non-yeast fungal communities of Cabernet Sauvignon berries from two Washington State vineyards, and persistence in spontaneous fermentation.

To address a knowledge gap about the grape berry mycobiome from Washington State vineyards, next-generation sequencing of the internal transcribed spacer region (ITS1) was used to identify native yeast and fungal species on berries of cultivar 'Cabernet Sauvignon' from two vineyards at veraison and harvest in 2015 and 2016. Four hundred fifty-six different yeast amplicon sequence variants (ASV), representing 184 distinct taxa, and 2467 non-yeast fungal ASV (791 distinct taxa) were identified in this study. A set of 50 recurrent yeast taxa, including Phaeococcomyces, Vishniacozyma and Metschnikowia, were found at both locations and sampling years. These yeast species were monitored from the vineyard into laboratory-scale spontaneous fermentations. Taxa assignable to Metschnikowia and Saccharomyces persisted during fermentation, whereas Curvibasidium, which also has possible impact on biocontrol and wine quality, did not. Sulfite generally reduced yeast diversity and richness, but its effect on the abundance of specific yeasts during fermentation was negligible. Among the 106 recurring non-yeast fungal taxa, Alternaria, Cladosporium and Ulocladium were especially abundant in the vineyard. Vineyard location was the primary factor that accounted for the variation among both communities, followed by year and berry developmental stage. The Washington mycobiomes were compared to those from other parts of the world. Sixteen recurrent yeast species appeared to be unique to Washington State vineyards. This subset also contained a higher proportion of species associated with cold and extreme environments, relative to other localities. Certain yeast and non-yeast fungal species known to suppress diseases or modify wine sensory properties were present in Washington vineyards, and likely have consequences to vineyard health and wine quality.

Yeasts Associated with Various Amazonian Native Fruits.

Yeasts, commonly present on the surface of fruits, are of industrial interest for the production of enzymes, flavorings, and bioactive compounds, and have many other scientific uses. The Amazonian rainforest may be a good source of new species or strains of yeasts, but their presence on Amazonian fruits is unknown. The aim of this study was to identify and characterize yeasts isolated from Amazonian native fruits using molecular and phenotypic methods. In total, 81 yeast isolates were obtained from 10 fruits species. Rep-PCR showed 29 strain profiles. Using a combination of restriction-fragment length polymorphism (RFLP) of the 5.8S-ITS region and D1/D2 sequencing of the 26S rRNA gene, 16 species were identified belonging to genera Candida, Debaryomyces, Hanseniaspora, Kodamaea, Martiniozyma, and Meyerozyma. The most dominant species were Candida tropicalis, Debaryomyces hansenii, Hanseniaspora opuntiae, and Hanseniaspora thailandica. H. opuntiae and H. thailandica showed the highest number of the strain profiles. Phenotypic profiles were variable between species, and even among strains. Screening for hydrolases showed lipolytic activity in only one isolate, while proteolytic, cellulolytic and amylolytic capabilities were not detected. Yeast presence among fruits varied, with cidra (Citrus medica) and ungurahui (Oenocarpus bataua) having the highest number of species associated. This investigation broadens the understanding and possible biotechnological uses of yeast strains obtained from Amazonian native fruits.Yeasts, commonly present on the surface of fruits, are of industrial interest for the production of enzymes, flavorings, and bioactive compounds, and have many other scientific uses. The Amazonian rainforest may be a good source of new species or strains of yeasts, but their presence on Amazonian fruits is unknown. The aim of this study was to identify and characterize yeasts isolated from Amazonian native fruits using molecular and phenotypic methods. In total, 81 yeast isolates were obtained from 10 fruits species. Rep-PCR showed 29 strain profiles. Using a combination of restriction-fragment length polymorphism (RFLP) of the 5.8S-ITS region and D1/D2 sequencing of the 26S rRNA gene, 16 species were identified belonging to genera Candida, Debaryomyces, Hanseniaspora, Kodamaea, Martiniozyma, and Meyerozyma. The most dominant species were Candida tropicalis, Debaryomyces hansenii, Hanseniaspora opuntiae, and Hanseniaspora thailandica. H. opuntiae and H. thailandica showed the highest number of the strain profiles. Phenotypic profiles were variable between species, and even among strains. Screening for hydrolases showed lipolytic activity in only one isolate, while proteolytic, cellulolytic and amylolytic capabilities were not detected. Yeast presence among fruits varied, with cidra (Citrus medica) and ungurahui (Oenocarpus bataua) having the highest number of species associated. This investigation broadens the understanding and possible biotechnological uses of yeast strains obtained from Amazonian native fruits.

Identification of novel open reading frames in the intergenic regions of Mycobacterium leprae genome and detection of transcript by qRT-PCR.

Mycobacterium leprae is an unculturable obligate pathogen and causative agent for debilitating human disease leprosy. Due to reductive genome evolution M leprae genome harbours large number of pseudogenes and small number of genes (∼1600 genes and ∼1300 pseudogenes). How M leprae remained a successful human parasite with small set of genes remains poorly understood and provided us the impetus to investigate the intergenic regions of M leprae genome for the presence of possible open reading frames (ORFs). In this work, we have manually scanned all the intergenic regions of M leprae genome and identified 106 potential ORFs. Among these, 12 are large ORFs: encoding hypothetical proteins (HP) of more than 100 amino acids. We have also found 67 ORFs encoding 50-100 amino acids proteins and another 27 ORFs for 30-50 amino acids peptides. We have validated the presence of transcripts for large HPs by quantitative reverse transcriptase PCR (qRT-PCR). Our results suggest that some of the M leprae large HPs are indeed expressed at low level in leprosy patients. The present results will shed light on the intergenic ORFs of M leprae and further our understanding of the pathogenesis of leprosy.

Discovery of 4 exonic and 1 intergenic novel susceptibility loci for leprosy.

Seven new risk coding variants have been identified through an exome-wide association study (EWAS), which studied the contributions of protein-coding variants to leprosy susceptibility. But some potential susceptibility loci were not studied in the previous EWAS study because of the project consideration. Seventeen unstudied potential susceptibility loci of the previous EWAS were validated in 3169 cases and 9814 controls in this study. Four disease-associated exonic loci were identified: rs671 in ALDH2 (P = 2.0 × 10-20 , odds ratio [OR] = 1.35), rs13259978 in SLC7A2 (P = 1.74 × 10-8 , OR = 1.28), rs925368 in GIT2 (P = 9.18 × 10-17 , OR = 1.44), and rs75680863 in TCN2 (P = 8.37 × 10-21 , OR = 0.74). Potentially implicating ZFP36L1 as a new susceptibility gene, 1 intergenic single nucleotide polymorphism (SNP), rs1465788 (P = 7.81 × 10-6 , OR = 0.88), was also suggested to be associated with leprosy. A luciferase reporter assay showed that the rs1465788 risk allele notably decreased the transcription activity of the flanking sequence. These findings suggest the possible involvement of lipid metabolism, NF-κB homeostasis and macrophage antimicrobial pathways in leprosy pathogenesis.

Identification of a novel gene product that promotes survival of Mycobacterium smegmatis in macrophages.

BACKGROUND: Bacteria of the suborder Corynebacterineae include significant human pathogens such as Mycobacterium tuberculosis and M. leprae. Drug resistance in mycobacteria is increasingly common making identification of new antimicrobials a priority. Mycobacteria replicate intracellularly, most commonly within the phagosomes of macrophages, and bacterial proteins essential for intracellular survival and persistence are particularly attractive targets for intervention with new generations of anti-mycobacterial drugs. METHODOLOGY/PRINCIPAL FINDINGS: We have identified a novel gene that, when inactivated, leads to accelerated death of M. smegmatis within a macrophage cell line in the first eight hours following infection. Complementation of the mutant with an intact copy of the gene restored survival to near wild type levels. Gene disruption did not affect growth compared to wild type M. smegmatis in axenic culture or in the presence of low pH or reactive oxygen intermediates, suggesting the growth defect is not related to increased susceptibility to these stresses. The disrupted gene, MSMEG_5817, is conserved in all mycobacteria for which genome sequence information is available, and designated Rv0807 in M. tuberculosis. Although homology searches suggest that MSMEG_5817 is similar to the serine:pyruvate aminotransferase of Brevibacterium linens suggesting a possible role in glyoxylate metabolism, enzymatic assays comparing activity in wild type and mutant strains demonstrated no differences in the capacity to metabolize glyoxylate. CONCLUSIONS/SIGNIFICANCE: MSMEG_5817 is a previously uncharacterized gene that facilitates intracellular survival of mycobacteria. Interference with the function of MSMEG_5817 may provide a novel therapeutic approach for control of mycobacterial pathogens by assisting the host immune system in clearance of persistent intracellular bacteria.

Application of denaturing high-performance liquid chromatography (DHPLC) for yeasts identification in red smear cheese surfaces.

AIMS: To evaluate and optimize the use of denaturing high-performance liquid chromatography (DHPLC) for yeasts identification in red smear cheese surfaces. METHODS AND RESULTS: The resolution of DHPLC was first evaluated and optimized using a mixture of PCR amplicons of the internal transcribed spacer 2 (ITS2) region of 19 yeast reference strains representing 18 species that are common in the cheese microbiota. Sixteen of the 18 yeast species could be resolved by combining runs at temperatures of 57.5 and 59 degrees C. Then, DHPLC was used to investigate the yeast microbiota of pasteurized Maroilles, Munster and Livarot cheese surfaces by comparing their peak profiles with our reference yeast database and by collecting/sequencing of peak fractions. Debaryomyces hansenii and Geotrichum candidum for Munster and Maroilles cheeses, and Candida catenulata, Candida intermedia and G. candidum for Livarot cheese were identified using the reference database and collecting/sequencing of peak fractions. CONCLUSIONS: DHPLC technique was found to have good resolution properties and to be useful for investigating the yeast microbiota of red smear cheese surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that DHPLC is applied to study the yeast microbiota of red smear cheese surfaces.