Chromatographic printed array strip (C-PAS) method for cultivar-specific identification of sweetpotato cultivars 'Beniharuka' and 'Fukumurasaki'.

Sweetpotato (Ipomoea batatas) cultivars grown in Japan are highly valued for their excellent sweetness, high quality, and good texture. The export volume of sweetpotato from Japan has been rising rapidly, with a 10-fold increase on a weight basis over the last 10 years. However, since sweetpotato is propagated vegetatively from storage roots, it is easy to cultivate and propagate this crop, prompting concerns that Japanese sweetpotato cultivars/lines are being exported overseas, cultivated without permission, or reimported. Therefore, a rapid and accurate cultivar identification methodology is needed. In this study, we comprehensively analyzed the insertion sites of Cl8 retrotransposon to develop a cultivar identification technique for the Japanese cultivars 'Beniharuka' and 'Fukumurasaki'. These two cultivars were successfully distinguished from other cultivars using a minimum of two marker sets. Using the chromatographic printed array strip (C-PAS) method for DNA signal detection, 'Beniharuka' and 'Fukumurasaki' can be precisely identified using a single strip of chromatographic paper based on multiplex DNA signals derived from the amplicons of the Cl8 insertion sites. Since this method can detect DNA signals in only ~15 minutes, we expect that our method will facilitate rapid, reliable, and convenient cultivar discrimination for on-site inspection of sweetpotato.

A target cultivar-specific identification system based on the chromatographic printed array strip method for eight prominent Japanese citrus cultivars.

Citrus is a major cultivated crop in Japan, and new cultivars are of great interest in the Japanese and global market. Recently, the infringement of breeders' rights to citrus cultivars bred in Japan has become a problem related to the agricultural product export strategy promoted by the Japanese government. Cultivar identification systems using DNA markers are an effective tool for protecting breeders' rights. Here, a novel target cultivar-specific identification system using the chromatographic printed array strip method was developed for eight prominent Japanese citrus cultivars. A polymorphic InDel fragment specific to each cultivar was explored through the screening of published citrus InDel markers and next-generation sequencing of retrotransposon libraries. The cultivar-specific DNA marker set for each cultivar comprised 1-3 polymorphic InDel fragments in combination with a PCR-positive DNA marker for the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene. The DNA markers were detected within 3 hours from DNA extraction to the detection by the C-PAS4 membrane stick following multiplex PCR. The developed system is superior as a convenient, rapid, and cost-effective DNA diagnostic method during inspection. The proposed target cultivar-specific identification system is expected to serve as an efficient tool for the injunction of suspicious registered cultivars, contributing to the protection of breeders' rights.

Relationship between skin fungal and bacterial microbiomes and skin pH.

OBJECTIVE: The present study investigated the relationship between the skin bacterial and fungal microbiomes and skin pH at sebaceous sites, namely, the forehead, cheek and upper chest. We also examined the relationship between the skin microbiome and transepidermal water loss (TEWL), which is an indicator of rough skin. METHODS: Thirty healthy Japanese subjects (15 females and 15 males) were enrolled as subjects. The forehead, cheek and upper chest were examined in August (summer) and February (winter). Skin surface samples were collected using the swab method, and the 16s rRNA and ITS regions were analysed using next-generation sequencing. Skin pH and TEWL were also assessed. RESULTS: The changes observed in the skin microbiome were associated with season-dependent changes in skin pH. An increase in skin pH at the forehead and cheek was associated with a higher relative abundance of Cutibacterium spp., a reduction in bacterial diversity, and a lower relative abundance of Staphylococcus spp. A decrease in skin pH at the upper chest was associated with a higher relative abundance of Malassezia restricta, a lower relative abundance of M. sympodialis and decrease in fungal diversity. Moreover, these changes were also associated with an increase in TEWL. CONCLUSION: The present results indicate that changes in skin pH are associated with changes in the skin bacterial and fungal microbiomes at sebaceous sites. The changes observed in the skin microbiome were also associated with rough skin.

OBJECTIF: La présente étude a examiné la relation entre les microbiomes bactériens et fongiques de la peau et le pH de la peau des sites sébacés, à savoir le front, la joue et la partie supérieure du thorax. Nous avons également examiné la relation entre le microbiome cutané et la perte d'eau transépidermique (transepidermal water loss, TEWL), qui est un indice de rugosité cutanée. MÉTHODES: Trente sujets japonais en bonne santé (15 femmes et 15 hommes) ont été inclus en tant que sujets de l'étude. Le front, la joue et la partie supérieure du thorax ont été examinés en août (été) et en février (hiver). Des échantillons de surface cutanée ont été prélevés par écouvillonnage et les régions de l'ARNr 16s et ITS ont été analysées par séquençage à haut débit. Le pH de la peau et la TEWL ont également été évalués. RÉSULTATS: Les changements observés dans le microbiome cutané étaient associés à des modifications du pH de la peau dépendant de la saison. Une augmentation du pH de la peau au niveau du front et de la joue était associée à une plus grande abondance relative de Cutibacterium spp. et à une réduction de la diversité bactérienne, ainsi qu'à une plus faible abondance relative de Staphylococcus spp. Une diminution du pH de la peau au niveau de la partie supérieure du thorax était associée à une abondance relative plus élevée de Malassezia restricta et à une abondance relative plus faible de M. sympodialis, ainsi qu'à une diversité fongique. De plus, ces changements ont également été associés à une augmentation de la TEWL. CONCLUSION: Ces résultats indiquent que les modifications du pH de la peau sont associées à des modifications des microbiomes bactérien et fongique de la peau au niveau des sites sébacés. Les changements observés dans le microbiome de la peau étaient également associés à une rugosité de la peau.

The Microbiome in Comedonal Contents of Inflammatory Acne Vulgaris is Composed of an Overgrowth of Cutibacterium Spp. and Other Cutaneous Microorganisms.

Background: Acne vulgaris (acne) and cutaneous resident microorganisms are considered to be closely related. However, the bacterial and fungal microbiota in the comedonal contents of inflammatory acne lesions have not yet been investigated in detail. Purpose: To clarify the relationship between cutaneous microorganisms and acne, we examined the microbiome in the comedonal contents of inflammatory acne and on the facial skin of patients with acne using 16s rRNA and ITS gene sequencing with a next-generation sequencer (NGS). Patients and Methods: Twenty-two untreated Japanese acne outpatients were examined. The comedonal contents of inflammatory acne lesions on the face were collected using a comedo extractor. Skin surface samples from facial skin were collected using the swab method. Results: The results obtained revealed that the predominant bacteria in the comedonal contents of inflammatory acne were Cutibacterium spp. (more prominent in areas with large amounts of sebum), while those on the skin surface were Staphylococcus spp. Malassezia spp., particularly Malassezia restricta, were the predominant fungi in both the comedonal contents of inflammatory acne and on the skin surface. The bacterial microbiome in comedonal contents exhibited stronger metabolic activity, including the production of enzymes related to acne, than that on the skin surface. Conclusion: These results indicate that acne is an inflammatory disease involving the overgrowth of Cutibacterium acnes and other cutaneous resident microorganisms, including Malassezia spp.

Correction: Water pre-filtration methods to improve environmental DNA detection by real-time PCR and metabarcoding.

[This corrects the article DOI: 10.1371/journal.pone.0250162.].

Water pre-filtration methods to improve environmental DNA detection by real-time PCR and metabarcoding.

Environmental DNA (eDNA) analysis is a novel approach for biomonitoring and has been mostly used in clear water. It is difficult to detect eDNA in turbid water as filter clogging occurs, and environmental samples contain various substances that inhibit the polymerase chain reaction (PCR) and affect the accuracy of eDNA analysis. Therefore, we applied a pre-filtration method to better detect the fish species (particularly pale chub, Opsariichthys platypus) present in a water body by measuring eDNA in environmental samples containing PCR inhibitors. Upon conducting 12S rRNA metabarcoding analysis (MiFish), we found that pre-filtration did not affect the number or identities of fish species detected in our samples, but pre-filtration through pore sizes resulted in significantly reduced variance among replicate samples. Additionally, PCR amplification was improved by the pre-filtration of environmental samples containing PCR inhibitors such as humic substances. Although this study may appear to be a conservative and ancillary experiment, pre-filtration is a simple technique that can not only improve the physical properties of water, such as turbidity, but also the quality of eDNA biomonitoring.

Detection of Sarcocystis spp. and Shiga toxin-producing Escherichia coli in Japanese sika deer meat using a loop-mediated isothermal amplification-lateral flow strip.

Game meat potentially harbors a number of parasitic and bacterial pathogens that cause foodborne disease. It is thus important to monitor the prevalence of such pathogens in game meats before retail and consumption to ensure consumer safety. In particular, Sarcocystis spp. and Shiga toxin-producing Escherichia coli (STEC) have been reported to be causative agents of food poisoning associated with deer meat consumption. To examine the prevalence of these microbiological agents on-site at a slaughterhouse, the rapid, simple and sensitive detection method known as the "DNA strip" has been developed, a novel tool combining loop-mediated isothermal amplification and a lateral flow strip. This assay has achieved higher sensitivity and faster than conventional PCR and is suitable for on-site inspection.

Single-Laboratory Validation of Rapid and Easy DNA Strip for Porcine DNA Detection in Beef Meatballs.

Detection of meat from an animal species is required to avoid misleading food labels to consumers. Recently, we developed an easy-to-use molecular detection method by combining isothermal amplification and a DNA strip, referred to as DNA Strip. Here, we report our single-laboratory validation of DNA Strip to detect porcine DNA in beef meatballs. Our results showed that DNA Strip could specifically amplify the target of porcine DNA, with detection limit to 0.01% admixture of pork in beef meatballs. DNA Strip method was also robust because the use of heat block and laboratory water bath showed no significant differences and were comparable to the reference instrument. DNA Strip can detect porcine DNA within ca 1 h, including DNA extraction, DNA amplification, and detection. These results suggest that DNA Strip is applicable because it is easy to use and capable of detecting pork in beef meatballs with a greater detection limit.

Interlaboratory validation data on real-time polymerase chain reaction detection for unauthorized genetically modified papaya line PRSV-YK.

This article is referred to research article entitled "Whole genome sequence analysis of unidentified genetically modified papaya for development of a specific detection method" (Nakamura et al., 2016) [1]. Real-time polymerase chain reaction (PCR) detection method for unauthorized genetically modified (GM) papaya (Carica papaya L.) line PRSV-YK (PRSV-YK detection method) was developed using whole genome sequence data (DDBJ Sequenced Read Archive under accession No. PRJDB3976). Interlaboratory validation datasets for PRSV-YK detection method were provided. Data indicating homogeneity of samples prepared for interlaboratory validation were included. Specificity and sensitivity test data for PRSV-YK detection method were also provided.

In Situ Gene Expression Responsible for Sulfide Oxidation and CO2 Fixation of an Uncultured Large Sausage-Shaped Aquificae Bacterium in a Sulfidic Hot Spring.

We investigated the in situ gene expression profile of sulfur-turf microbial mats dominated by an uncultured large sausage-shaped Aquificae bacterium, a key metabolic player in sulfur-turfs in sulfidic hot springs. A reverse transcription-PCR analysis revealed that the genes responsible for sulfide, sulfite, and thiosulfate oxidation and carbon fixation via the reductive TCA cycle were continuously expressed in sulfur-turf mats taken at different sampling points, seasons, and years. These results suggest that the uncultured large sausage-shaped bacterium has the ability to grow chemolithoautotrophically and plays key roles as a primary producer in the sulfidic hot spring ecosystem in situ.

Whole genome sequence analysis of unidentified genetically modified papaya for development of a specific detection method.

Identification of transgenic sequences in an unknown genetically modified (GM) papaya (Carica papaya L.) by whole genome sequence analysis was demonstrated. Whole genome sequence data were generated for a GM-positive fresh papaya fruit commodity detected in monitoring using real-time polymerase chain reaction (PCR). The sequences obtained were mapped against an open database for papaya genome sequence. Transgenic construct- and event-specific sequences were identified as a GM papaya developed to resist infection from a Papaya ringspot virus. Based on the transgenic sequences, a specific real-time PCR detection method for GM papaya applicable to various food commodities was developed. Whole genome sequence analysis enabled identifying unknown transgenic construct- and event-specific sequences in GM papaya and development of a reliable method for detecting them in papaya food commodities.

Microbial community structure of relict niter-beds previously used for saltpeter production.

From the 16th to the 18th centuries in Japan, saltpeter was produced using a biological niter-bed process and was formed under the floor of gassho-style houses in the historic villages of Shirakawa-go and Gokayama, which are classified as United Nations Educational, Scientific and Cultural Organization (UNESCO) World Heritage Sites. The relict niter-beds are now conserved in the underfloor space of gassho-style houses, where they are isolated from destabilizing environmental factors and retain the ability to produce nitrate. However, little is known about the nitrifying microbes in such relict niter-bed ecosystems. In this study, the microbial community structures within nine relict niter-bed soils were investigated using 454 pyrotag analysis targeting the 16S rRNA gene and the bacterial and archaeal ammonia monooxygenase gene (amoA). The 16S rRNA gene pyrotag analysis showed that members of the phyla Proteobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Gemmatimonadetes, and Planctomycetes were major microbial constituents, and principal coordinate analysis showed that the NO3-, Cl-, K+, and Na+ contents were potential determinants of the structures of entire microbial communities in relict niter-bed soils. The bacterial and archaeal amoA libraries indicated that members of the Nitrosospira-type ammonia-oxidizing bacteria (AOB) and "Ca. Nitrososphaera"-type ammonia-oxidizing archaea (AOA), respectively, predominated in relict niter-bed soils. In addition, soil pH and organic carbon content were important factors for the ecological niche of AOB and AOA in relict niter-bed soil ecosystems.

A rapid and enhanced DNA detection method for crop cultivar discrimination.

In many crops species, the development of a rapid and precise cultivar discrimination system has been required for plant breeding and patent protection of plant cultivars and agricultural products. Here, we successfully evaluated strawberry cultivars via a novel method, namely, the single tag hybridization (STH) chromatographic printed array strip (PAS) using the PCR products of eight genomic regions. In a previous study, we showed that genotyping of eight genomic regions derived from FaRE1 retrotransposon insertion site enabled to discriminate 32 strawberry cultivars precisely, however, this method required agarose/acrylamide gel electrophoresis, thus has the difficulty for practical application. In contrast, novel DNA detection method in this study has some great advantages over standard DNA detection methods, including agarose/acrylamide gel electrophoresis, because it produces signals for DNA detection with dramatically higher sensitivity in a shorter time without any preparation or staining of a gel. Moreover, this method enables the visualization of multiplex signals simultaneously in a single reaction using several independent amplification products. We expect that this novel method will become a rapid and convenient cultivar screening assay for practical purposes, and will be widely applied to various situations, including laboratory research, and on-site inspection of plant cultivars and agricultural products.

Discovery of glycoside hydrolase enzymes in an avicel-adapted forest soil fungal community by a metatranscriptomic approach.

To discover the structural and functional novel glycoside hydrolase enzymes from soil fungal communities that decompose cellulosic biomass, transcripts of functional genes in a forest soil were analyzed. Pyrosequencing of the Avicel and wheat-amended soil cDNAs produced 56,084 putative protein-coding sequence (CDS) fragments, and the most dominant group of putative CDSs based on the taxonomic analysis was assigned to the domain Eukarya, which accounted for 99% of the total number of the putative CDSs. Of 9,449 eukaryotic CDSs whose functions could be categorized, approximately 40% of the putative CDSs corresponded to metabolism-related genes, including genes involved in carbohydrate, amino acid, and energy metabolism. Among the carbohydrate-metabolism genes, 129 sequences encoded glycoside hydrolase enzymes, with 47 sequences being putative cellulases belonging to 13 GH families. To characterize the function of glycoside hydrolase enzymes, we synthesized the putative CelA gene with codon optimization for heterologous expression in Escherichia coli, which was shown to be similar to the structure of plant expansins, and observed stimulation for cellulase activity on Avicel degradation. This study demonstrated that fungal communities adapt to Avicel and wheat decomposition and that metatranscriptomic sequence data can be reference data for identifying a novel gene.

Metagenomic and biochemical characterizations of sulfur oxidation metabolism in uncultured large sausage-shaped bacterium in hot spring microbial mats.

So-called "sulfur-turf" microbial mats in sulfide containing hot springs (55-70°C, pH 7.3-8.3) in Japan were dominated by a large sausage-shaped bacterium (LSSB) that is closely related to the genus Sulfurihydrogenibium. Several previous reports proposed that the LSSB would be involved in sulfide oxidation in hot spring. However, the LSSB has not been isolated yet, thus there has been no clear evidence showing whether it possesses any genes and enzymes responsible for sulfide oxidation. To verify this, we investigated sulfide oxidation potential in the LSSB using a metagenomic approach and subsequent biochemical analysis. Genome fragments of the LSSB (a total of 3.7 Mb sequence including overlapping fragments) were obtained from the metagenomic fosmid library constructed from genomic DNA of the sulfur-turf mats. The sequence annotation clearly revealed that the LSSB possesses sulfur oxidation-related genes coding sulfide dehydrogenase (SD), sulfide-quinone reductase and sulfite dehydrogenase. The gene encoding SD, the key enzyme for sulfide oxidation, was successfully cloned and heterologously expressed in Escherichia coli. The purified recombinant enzyme clearly showed SD activity with optimum temperature and pH of 60°C and 8.0, respectively, which were consistent with the environmental conditions in the hot spring where the sulfur-turf thrives. Furthermore, the affinity of SD to sulfide was relatively high, which also reflected the environment where the sulfide could be continuously supplied. This is the first report showing that the LSSB harbors sulfide oxidizing metabolism adapted to the hot spring environment and can be involved in sulfide oxidation in the sulfur-turf microbial mats.

Carotenoids of Gemmatimonas aurantiaca (Gemmatimonadetes): identification of a novel carotenoid, deoxyoscillol 2-rhamnoside, and proposed biosynthetic pathway of oscillol 2,2'-dirhamnoside.

Gemmatimonas aurantiaca strain T-27(T) is an orange-coloured, Gram-negative, facultatively aerobic, polyphosphate-accumulating bacterium belonging to a recently proposed phylum, Gemmatimonadetes. We purified its pigments and identified them as carotenoids and their glycoside derivatives using spectral data. The major carotenoid was (2S,2' S)-oscillol 2,2'-di-(alpha-l-rhamnoside), and the minor carotenoids were (2S)-deoxyoscillol 2-( alpha-l-rhamnoside) and didemethylspirilloxanthin. Deoxyoscillol 2-rhamnoside is a novel carotenoid. Oscillol 2,2'-diglycosides have hitherto only been reported in a limited number of cyanobacteria, and this is believed to be the first finding of such carotenoids in another bacterial phylum. Based on the identification of the carotenoids and the completion of the entire nucleotide sequence, we propose a biosynthetic pathway for the carotenoids and the corresponding genes and enzymes. We propose the involvement of geranylgeranyl pyrophosphate synthase (CrtE), phytoene synthase (CrtB) and phytoene desaturase (CrtI) for lycopene synthesis; and of carotenoid 1,2-hydratase (CruF) and carotenoid 2-O-rhamnosyltransferase (CruG) for oscillol 2,2'-dirhamnoside synthesis. Further, isopentenyl pyrophosphate could be synthesized by a non-mevalonate pathway (DXP pathway).

Unusual transcription regulation of the niaD gene under anaerobic conditions supporting fungal ammonia fermentation.

The niaD gene of the fungus Aspergillus nidulans encodes an assimilatory nitrate reductase and exogenous ammonium represses its expression. Under anoxic conditions, however, A. nidulans expressed niaD even in the presence of ammonium and used the gene product for dissimilatory nitrate reduction (ammonia fermentation). This transcription regulation mechanism under anaerobiosis is critical for the fungus to ferment ammonium.

Fungal ammonia fermentation, a novel metabolic mechanism that couples the dissimilatory and assimilatory pathways of both nitrate and ethanol. Role of acetyl CoA synthetase in anaerobic ATP synthesis.

Fungal ammonia fermentation is a novel dissimilatory metabolic mechanism that supplies energy under anoxic conditions. The fungus Fusarium oxysporum reduces nitrate to ammonium and simultaneously oxidizes ethanol to acetate to generate ATP (Zhou, Z., Takaya, N., Nakamura, A., Yamaguchi, M., Takeo, K., and Shoun, H. (2002) J. Biol. Chem. 277, 1892-1896). We identified the Aspergillus nidulans genes involved in ammonia fermentation by analyzing fungal mutants. The results showed that assimilatory nitrate and nitrite reductases (the gene products of niaD and niiA) were essential for reducing nitrate and for anaerobic cell growth during ammonia fermentation. We also found that ethanol oxidation is coupled with nitrate reduction and catalyzed by alcohol dehydrogenase, coenzyme A (CoA)-acylating aldehyde dehydrogenase, and acetyl-CoA synthetase (Acs). This is similar to the mechanism suggested in F. oxysporum except A. nidulans uses Acs to produce ATP instead of the ADP-dependent acetate kinase of F. oxysporum. The production of Acs requires a functional facA gene that encodes Acs and that is involved in ethanol assimilation and other metabolic processes. We purified the gene product of facA (FacA) from the fungus to show that the fungus acetylates FacA on its lysine residue(s) specifically under conditions of ammonia fermentation to regulate its substrate affinity. Acetylated FacA had higher affinity for acetyl-CoA than for acetate, whereas non-acetylated FacA had more affinity for acetate. Thus, the acetylated variant of the FacA protein is responsible for ATP synthesis during fungal ammonia fermentation. These results showed that the fungus ferments ammonium via coupled dissimilatory and assimilatory mechanisms.