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September 22, 2019

wtf genes are prolific dual poison-antidote meiotic drivers.

Meiotic drivers are selfish genes that bias their transmission into gametes, defying Mendelian inheritance. Despite the significant impact of these genomic parasites on evolution and infertility, few meiotic drive loci have been identified or mechanistically characterized. Here, we demonstrate a complex landscape of meiotic drive genes on chromosome 3 of the fission yeasts Schizosaccharomyces kambucha and S. pombe. We identify S. kambucha wtf4 as one of these genes that acts to kill gametes (known as spores in yeast) that do not inherit the gene from heterozygotes. wtf4 utilizes dual, overlapping transcripts to encode both a gamete-killing poison and an antidote to the poison. To enact drive, all gametes are poisoned, whereas only those that inherit wtf4 are rescued by the antidote. Our work suggests that the wtf multigene family proliferated due to meiotic drive and highlights the power of selfish genes to shape genomes, even while imposing tremendous costs to fertility.

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September 22, 2019

Rodent papillomaviruses.

Preclinical infection model systems are extremely valuable tools to aid in our understanding of Human Papillomavirus (HPV) biology, disease progression, prevention, and treatments. In this context, rodent papillomaviruses and their respective infection models are useful tools but remain underutilized resources in the field of papillomavirus biology. Two rodent papillomaviruses, MnPV1, which infects the Mastomys species of multimammate rats, and MmuPV1, which infects laboratory mice, are currently the most studied rodent PVs. Both of these viruses cause malignancy in the skin and can provide attractive infection models to study the lesser understood cutaneous papillomaviruses that have been frequently associated with HPV-related skin cancers. Of these, MmuPV1 is the first reported rodent papillomavirus that can naturally infect the laboratory strain of mice. MmuPV1 is an attractive model virus to study papillomavirus pathogenesis because of the ubiquitous availability of lab mice and the fact that this mouse species is genetically modifiable. In this review, we have summarized the knowledge we have gained about PV biology from the study of rodent papillomaviruses and point out the remaining gaps that can provide new research opportunities.

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September 22, 2019

Effects of metal and metalloid pollutants on the microbiota composition of feces obtained from twelve commercial pig farms across China.

Understanding the metal and metalloid contamination and microbiota composition of pig feces is an important step required to support the design and implementation of effective pollution control and prevention strategies. A survey was implemented in 12 locations across China to investigate the content of metals and metalloids, and the main composition of the microbial communities of commercially reared pigs during two growth periods, defined as the early (Q group) and the later fattening growth phases (H group). These data showed widespread Al, Mn, Cu, Zn, and Fe pollution in pig feces. The concentration of Zn in the Q group feces was nearly two times higher than the levels measured in the H group. The microbial composition of the Q group exhibited greater richness of operational taxonomic units (OTUs) and fewer bacteria associated with zoonotic diseases compared with the microbial composition of the H group. Spearman rank correlation analysis showed that Cu and northern latitudes had a significant positive effect on the richness of bacterial communities in pig feces. Zn and Cd exhibited the biggest impact on microbial community composition based on canonical correspondence analysis. Functional metagenomic prediction indicated that about 0.8% genes present in the pig feces bacteria community are related to human diseases, and significantly more predicted pathogenic genes were detected in the H group than in the Q group. These results support the need to monitor heavy metal contamination and to control for zoonotic pathogens disseminated from pig feces in Chinese pig farms. Copyright © 2018. Published by Elsevier B.V.

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September 22, 2019

An intact gut microbiota may be required for lactoferrin-driven immunomodulation in rats

Lactoferrin can modulate both the host immunity and gut microbiota. However, whether the immune modulation requires the gut microbiota has not been directly shown. Thus, our study compared (1) lactoferrin-driven immunomodulation profiles and (2) changes in fecal phylogenic metagenome with and without antibiotics-induced dysbiosis in rats. Rats receiving only lactoferrin but not both lactoferrin and antibiotics had a Th-1 type cytokine serum profile. Significant differences were detected between the fecal microbiota of the lactoferrin and control groups at day 19 and/or day 33 but not initially, with a shift in the major contributors for community dissimilarity to Clostridium, Lactobacillus, and Oscillibacter valericigenes. The antibiotics-induced dysbiosis enriched the proinflammatory phyla, Proteobacteria and Deferribacteres, together with the anti-inflammatory species, Akkermansia muciniphila, while suppressed some butyrate-producers from the Firmicutes phylum. Our study shows that an intact microbiota is necessary for lactoferrin-driven immunomodulation.

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September 22, 2019

Revertant mosaicism repairs skin lesions in a patient with keratitis-ichthyosis-deafness syndrome by second-site mutations in connexin 26.

Revertant mosaicism (RM) is a naturally occurring phenomenon where the pathogenic effect of a germline mutation is corrected by a second somatic event. Development of healthy-looking skin due to RM has been observed in patients with various inherited skin disorders, but not in connexin-related disease. We aimed to clarify the underlying molecular mechanisms of suspected RM in the skin of a patient with keratitis-ichthyosis-deafness (KID) syndrome. The patient was diagnosed with KID syndrome due to characteristic skin lesions, hearing deficiency and keratitis. Investigation of GJB2 encoding connexin (Cx) 26 revealed heterozygosity for the recurrent de novo germline mutation, c.148G?>?A, p.Asp50Asn. At age 20, the patient developed spots of healthy-looking skin that grew in size and number within widespread erythrokeratodermic lesions. Ultra-deep sequencing of two healthy-looking skin biopsies identified five somatic nonsynonymous mutations, independently present in cis with the p.Asp50Asn mutation. Functional studies of Cx26 in HeLa cells revealed co-expression of Cx26-Asp50Asn and wild-type Cx26 in gap junction channel plaques. However, Cx26-Asp50Asn with the second-site mutations identified in the patient displayed no formation of gap junction channel plaques. We argue that the second-site mutations independently inhibit Cx26-Asp50Asn expression in gap junction channels, reverting the dominant negative effect of the p.Asp50Asn mutation. To our knowledge, this is the first time RM has been reported to result in the development of healthy-looking skin in a patient with KID syndrome. © The Author 2017. Published by Oxford University Press.

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September 22, 2019

Community profiling of Fusarium in combination with other plant associated fungi in different crop species using SMRT Sequencing.

Fusarium head blight, caused by fungi from the genus Fusarium, is one of the most harmful cereal diseases, resulting not only in severe yield losses but also in mycotoxin contaminated and health-threatening grains. Fusarium head blight is caused by a diverse set of species that have different host ranges, mycotoxin profiles and responses to agricultural practices. Thus, understanding the composition of Fusarium communities in the field is crucial for estimating their impact and also for the development of effective control measures. Up to now, most molecular tools that monitor Fusarium communities on plants are limited to certain species and do not distinguish other plant associated fungi. To close these gaps, we developed a sequencing-based community profiling methodology for crop-associated fungi with a focus on the genus Fusarium. By analyzing a 1600 bp long amplicon spanning the highly variable segments ITS and D1-D3 of the ribosomal operon by PacBio SMRT sequencing, we were able to robustly quantify Fusarium down to species level through clustering against reference sequences. The newly developed methodology was successfully validated in mock communities and provided similar results as the culture-based assessment of Fusarium communities by seed health tests in grain samples from different crop species. Finally, we exemplified the newly developed methodology in a field experiment with a wheat-maize crop sequence under different cover crop and tillage regimes. We analyzed wheat straw residues, cover crop shoots and maize grains and we could reveal that the cover crop hairy vetch (Vicia villosa) acts as a potent alternative host for Fusarium (OTU F.ave/tri) showing an eightfold higher relative abundance compared with other cover crop treatments. Moreover, as the newly developed methodology also allows to trace other crop-associated fungi, we found that vetch and green fallow hosted further fungal plant pathogens including Zymoseptoria tritici. Thus, besides their beneficial traits, cover crops can also entail phytopathological risks by acting as alternative hosts for Fusarium and other noxious plant pathogens. The newly developed sequencing based methodology is a powerful diagnostic tool to trace Fusarium in combination with other fungi associated to different crop species.

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September 22, 2019

Reduction in fecal microbiota diversity and short-chain fatty acid producers in Methicillin-resistant Staphylococcus aureus infected individuals as revealed by PacBio single molecule, real-time sequencing technology.

Methicillin-resistant Staphylococcus aureus (MRSA) may cause potentially lethal infections. Increasing evidence suggests that the gut microbiota is associated with human health. Yet, whether patients with MRSA infections carry specific signatures in their fecal microbiota composition has not been determined. Thus, this study aimed to compare the fecal microbiota profile of MRSA-positive patients (n=15) with individuals without MRSA infection (n=15) by using the PacBio single molecule, real-time (SMRT) DNA sequencing system and real-time quantitative polymerase chain reaction (qPCR). Mann-Whitney tests and unweighted UniFrac principal coordinate analysis (PCoA) showed that the profile of fecal microbiota was apparently different between the two populations. Both the community richness and diversity were reduced in the MRSA-positive group (p<0.050). The genera Acinetobacter and Enterococcus were highly enriched in the MRSA-positive group, whereas less short-chain fatty acid (SCFA)-producing bacteria, including Butyricimonas, Faecalibacterium, Roseburia, Ruminococcus, Megamonas and Phascolarctobacterium, were detected in the MRSA-positive group. At species level, the species Acinetobacter baumannii and Bacteroides thetaiotaomicron were prevalent in the MRSA-positive group, whereas opposite trends were observed in 17 other species, such as Faecalibacterium prausnitzii, Lactobacillus rogosae, Megamonas rupellensis and Phascolarctobacterium faecium. Positive correlations were observed between Acinetobacter baumannii and erythrocyte sedimentation rate (ESR) (R=0.554, p=0.001), as well as hypersensitive C reactive protein (hsCRP) (R=0.406, p=0.026). Faecalibacterium prausnitzii was negatively associated with ESR (R=-0.545, p=0.002), hsCRP (R=-0.401, p=0.028) and total bile acids (TBA) (R=-0.364, p=0.048). In conclusion, the fecal microbiota structure was different between MRSA-positive and -negative patients. The increase in potential pathogens with the reduction of beneficial populations, such as SCFA-producing bacteria, in MRSA-positive patients may affect prognosis.

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September 22, 2019

Antagonism between Staphylococcus epidermidis and Propionibacterium acnes and its genomic basis.

Propionibacterium acnes and Staphylococcus epidermidis live in close proximity on human skin, and both bacterial species can be isolated from normal and acne vulgaris-affected skin sites. The antagonistic interactions between the two species are poorly understood, as well as the potential significance of bacterial interferences for the skin microbiota. Here, we performed simultaneous antagonism assays to detect inhibitory activities between multiple isolates of the two species. Selected strains were sequenced to identify the genomic basis of their antimicrobial phenotypes.First, we screened 77 P. acnes strains isolated from healthy and acne-affected skin, and representing all known phylogenetic clades (I, II, and III), for their antimicrobial activities against 12?S. epidermidis isolates. One particular phylogroup (I-2) exhibited a higher antimicrobial activity than other P. acnes phylogroups. All genomes of type I-2 strains carry an island encoding the biosynthesis of a thiopeptide with possible antimicrobial activity against S. epidermidis. Second, 20?S. epidermidis isolates were examined for inhibitory activity against 25 P. acnes strains. The majority of S. epidermidis strains were able to inhibit P. acnes. Genomes of S. epidermidis strains with strong, medium and no inhibitory activities against P. acnes were sequenced. Genome comparison underlined the diversity of S. epidermidis and detected multiple clade- or strain-specific mobile genetic elements encoding a variety of functions important in antibiotic and stress resistance, biofilm formation and interbacterial competition, including bacteriocins such as epidermin. One isolate with an extraordinary antimicrobial activity against P. acnes harbors a functional ESAT-6 secretion system that might be involved in the antimicrobial activity against P. acnes via the secretion of polymorphic toxins.Taken together, our study suggests that interspecies interactions could potentially jeopardize balances in the skin microbiota. In particular, S. epidermidis strains possess an arsenal of different mechanisms to inhibit P. acnes. However, if such interactions are relevant in skin disorders such as acne vulgaris remains questionable, since no difference in the antimicrobial activity against, or the sensitivity towards S. epidermidis could be detected between health- and acne-associated strains of P. acnes.

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September 22, 2019

Contrasting distribution patterns between aquatic and terrestrial Phytophthora species along a climatic gradient are linked to functional traits.

Diversity of microbial organisms is linked to global climatic gradients. The genus Phytophthora includes both aquatic and terrestrial plant pathogenic species that display a large variation of functional traits. The extent to which the physical environment (water or soil) modulates the interaction of microorganisms with climate is unknown. Here, we explored the main environmental drivers of diversity and functional trait composition of Phytophthora communities. Communities were obtained by a novel metabarcoding setup based on PacBio sequencing of river filtrates in 96 river sites along a geographical gradient. Species were classified as terrestrial or aquatic based on their phylogenetic clade. Overall, terrestrial and aquatic species showed contrasting patterns of diversity. For terrestrial species, precipitation was a stronger driver than temperature, and diversity and functional diversity decreased with decreasing temperature and precipitation. In cold and dry areas, the dominant species formed resistant structures and had a low optimum temperature. By contrast, for aquatic species, temperature and water chemistry were the strongest drivers, and diversity increased with decreasing temperature and precipitation. Within the same area, environmental filtering affected terrestrial species more strongly than aquatic species (20% versus 3% of the studied communities, respectively). Our results highlight the importance of functional traits and the physical environment in which microorganisms develop their life cycle when predicting their distribution under changing climatic conditions. Temperature and rainfall may be buffered differently by water and soil, and thus pose contrasting constrains to microbial assemblies.

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September 22, 2019

cDNA library enrichment of full length transcripts for SMRT long read sequencing.

The utility of genome assemblies does not only rely on the quality of the assembled genome sequence, but also on the quality of the gene annotations. The Pacific Biosciences Iso-Seq technology is a powerful support for accurate eukaryotic gene model annotation as it allows for direct readout of full-length cDNA sequences without the need for noisy short read-based transcript assembly. We propose the implementation of the TeloPrime Full Length cDNA Amplification kit to the Pacific Biosciences Iso-Seq technology in order to enrich for genuine full-length transcripts in the cDNA libraries. We provide evidence that TeloPrime outperforms the commonly used SMARTer PCR cDNA Synthesis Kit in identifying transcription start and end sites in Arabidopsis thaliana. Furthermore, we show that TeloPrime-based Pacific Biosciences Iso-Seq can be successfully applied to the polyploid genome of bread wheat (Triticum aestivum) not only to efficiently annotate gene models, but also to identify novel transcription sites, gene homeologs, splicing isoforms and previously unidentified gene loci.

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September 22, 2019

Fine mapping and candidate gene identification of the genic male-sterile gene ms3 in cabbage 51S.

The ms3 gene responsible for a male-sterile phenotype in cabbage was mapped to a 187.4-kb genomic fragment. The gene BoTPD1, a homolog of Arabidopsis TPD1, was identified as a strong candidate gene. Cabbage 51S is a spontaneous male-sterile mutant. Phenotypic investigation revealed defects in anther cell differentiation, with failure to form the tapetum layer and complete abortion of microsporocytes before the tetrad stage. Genetic analysis indicated that this male sterility was controlled by a single recessive gene, ms3. Using an F2 population, we mapped ms3 to a 187.4-kb interval. BoTPD1 was identified as a candidate from this interval. Sequence analysis revealed an intronic 182-bp insertion in 51S that interrupted the conserved motif at the 5′ splicing site of the third intron, possibly resulting in a truncated transcript. Analyses of BoTPD1 homologous proteins revealed evolutionarily conserved roles in anther cell fate determination during reproductive development. RT-PCR showed that BoTPD1 was expressed in various tissues, excluding the root, and high expression levels were detected in anthers and buds. A BoTPD1-specific marker based on the 182-bp insertion cosegregated with male sterility and can be used for marker-assisted selection.

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September 22, 2019

PacBio sequencing of gene families – a case study with wheat gluten genes.

Amino acids in wheat (Triticum aestivum) seeds mainly accumulate in storage proteins called gliadins and glutenins. Gliadins contain a/ß-, ?- and ?-types whereas glutenins contain HMW- and LMW-types. Known gliadin and glutenin sequences were largely determined through cloning and sequencing by capillary electrophoresis. This time-consuming process prevents us to intensively study the variation of each orthologous gene copy among cultivars. The throughput and sequencing length of Pacific Bioscience RS (PacBio) single molecule sequencing platform make it feasible to construct contiguous and non-chimeric RNA sequences. We assembled 424 wheat storage protein transcripts from ten wheat cultivars by using just one single-molecule-real-time cell. The protein genes from wheat cultivar Chinese Spring are comparable to known sequences from NCBI. We demonstrated real-time sequencing of gene families with high-throughput and low-cost. This method can be applied to studies of gene amplification and copy number variation among species and cultivars. © 2013 Elsevier B.V. All rights reserved.

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September 22, 2019

Long-term microbiota and virome in a Zürich patient after fecal transplantation against Clostridium difficile infection.

Fecal microbiota transplantation (FMT) is an emerging therapeutic option for Clostridium difficile infections that are refractory to conventional treatment. FMT introduces fecal microbes into the patient’s intestine that prevent the recurrence of C. difficile, leading to rapid expansion of bacteria characteristic of healthy microbiota. However, the long-term effects of FMT remain largely unknown. The C. difficile patient described in this paper revealed protracted microbiota adaptation processes from 6 to 42 months post-FMT. Ultimately, bacterial communities were donor similar, suggesting sustainable stool engraftment. Since little is known about the consequences of transmitted viruses during C. difficile infection, we also interrogated virome changes. Our approach allowed identification of about 10 phage types per sample that represented larger viral communities, and phages were found to be equally abundant in the cured patient and donor. The healthy microbiota appears to be characterized by low phage abundance. Although viruses were likely transferred, the patient established a virome distinct from the donor. Surprisingly, the patient had sequences of algal giant viruses (chloroviruses) that have not previously been reported for the human gut. Chloroviruses have not been associated with intestinal disease, but their presence in the oropharynx may influence cognitive abilities. The findings suggest that the virome is an important indicator of health or disease. A better understanding of the role of viruses in the gut ecosystem may uncover novel microbiota-modulating therapeutic strategies.© 2016 New York Academy of Sciences.

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September 22, 2019

Isoform sequencing provides a more comprehensive view of the Panax ginseng transcriptome.

Korean ginseng (Panax ginseng C.A. Meyer) has been widely used for medicinal purposes and contains potent plant secondary metabolites, including ginsenosides. To obtain transcriptomic data that offers a more comprehensive view of functional genomics in P. ginseng, we generated genome-wide transcriptome data from four different P. ginseng tissues using PacBio isoform sequencing (Iso-Seq) technology. A total of 135,317 assembled transcripts were generated with an average length of 3.2 kb and high assembly completeness. Of those unigenes, 67.5% were predicted to be complete full-length (FL) open reading frames (ORFs) and exhibited a high gene annotation rate. Furthermore, we successfully identified unique full-length genes involved in triterpenoid saponin synthesis and plant hormonal signaling pathways, including auxin and cytokinin. Studies on the functional genomics of P. ginseng seedlings have confirmed the rapid upregulation of negative feed-back loops by auxin and cytokinin signaling cues. The conserved evolutionary mechanisms in the auxin and cytokinin canonical signaling pathways of P. ginseng are more complex than those in Arabidopsis thaliana. Our analysis also revealed a more detailed view of transcriptome-wide alternative isoforms for 88 genes. Finally, transposable elements (TEs) were also identified, suggesting transcriptional activity of TEs in P. ginseng. In conclusion, our results suggest that long-read, full-length or partial-unigene data with high-quality assemblies are invaluable resources as transcriptomic references in P. ginseng and can be used for comparative analyses in closely related medicinal plants.

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September 22, 2019

A workflow for studying specialized metabolism in nonmodel eukaryotic organisms

Eukaryotes contain a diverse tapestry of specialized metabolites, many of which are of significant pharmaceutical and industrial importance to humans. Nevertheless, exploration of specialized metabolic pathways underlying specific chemical traits in nonmodel eukaryotic organisms has been technically challenging and historically lagged behind that of the bacterial systems. Recent advances in genomics, metabolomics, phylogenomics, and synthetic biology now enable a new workflow for interrogating unknown specialized metabolic systems in nonmodel eukaryotic hosts with greater efficiency and mechanistic depth. This chapter delineates such workflow by providing a collection of state-of-the-art approaches and tools, ranging from multiomics-guided candidate gene identification to in vitro and in vivo functional and structural characterization of specialized metabolic enzymes. As already demonstrated by several recent studies, this new workflow opens up a gateway into the largely untapped world of natural product biochemistry in eukaryotes. © 2016 Elsevier Inc. All rights reserved.

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