Project description:Clostridium perfringens type A is a common source of food poisoning in humans. Vegetative cells sporulate in the small intestinal tract and produce a major pathogenic factor, C. perfringens enterotoxin (CPE) during sporulation. Although sporulation plays a critical role in the pathogenesis of food poisoning, the mechanisms to induce in vivo sporulation remain unclear. Bile salts had been identified to mediate sporulation, and we have confirmed deoxycholate (DCA)-induced sporulation in C. perfringens strain NCTC8239 co-cultured with human intestinal epithelial Caco-2 cells. In this study, we performed global transcriptome analysis of strain NCTC8239 to elucidate the mechanism to induce sporulation by DCA. From the 55 contigs of C. perfringens strain NCTC8239, 2778 coding sequences were extracted. We designed a DNA probe by utilizing eArray provided by Agilent Technologies. The custom 8Ã15K oligonucleotide array, containing 60 mer oligonucleotide probes for 2,778 genes in strain NCTC8239, 2 bacterial control genes: 16S rRNA and 23S rRNA, and 3 human control genes: beta-2-microglobulin, glucuronidase beta and 18S rRNA, were ordered to Agilent Technologies. Each probe was spotted in five-fold on each microarray. Each strain was run in triplicate or quadruplicate.
Project description:In this study we developed metaproteomics based methods for quantifying taxonomic composition of microbiomes (microbial communities). We also compared metaproteomics based quantification to other quantification methods, namely metagenomics and 16S rRNA gene amplicon sequencing. The metagenomic and 16S rRNA data can be found in the European Nucleotide Archive (Study number: PRJEB19901). For the method development and comparison of the methods we analyzed three types of mock communities with all three methods. The communities contain between 28 to 32 species and strains of bacteria, archaea, eukaryotes and bacteriophage. For each community type 4 biological replicate communities were generated. All four replicates were analyzed by 16S rRNA sequencing and metaproteomics. Three replicates of each community type were analyzed with metagenomics. The "C" type communities have same cell/phage particle number for all community members (C1 to C4). The "P" type communities have the same protein content for all community members (P1 to P4). The "U" (UNEVEN) type communities cover a large range of protein amounts and cell numbers (U1 to U4). We also generated proteomic data for four pure cultures to test the specificity of the protein inference method. This data is also included in this submission.
Project description:Healthy plants are vital for successful, long-duration missions in space, as they provide the crew with life support, food production, and psychological benefits. The microorganisms that associate with plant tissues play a critical role in improving plant growth, health, and production. To that end, it is necessary to develop methodologies that investigate the metabolic activities of the plant’s microbiome in orbit to enable rapid responses regarding the care of plants in space. In this study, we developed a protocol to characterize the endophytic and epiphytic microbial metatranscriptome of red romaine lettuce, a key salad crop that was grown under International Space Station (ISS)-like conditions. Microbial transcripts enriched from host-microbe total RNA were sequenced using the Oxford Nanopore MinION sequencing platform. Results showed that this enrichment approach was highly reproducible and effective for rapid on-site detection of microbial transcriptional activity. Taxonomic analysis based on 16S and 18S rRNA transcripts identified that the top five most abundant phyla in the lettuce microbiome were Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Ascomycota. The metatranscriptomic analysis identified the expression of genes involved in many metabolic pathways, including carbohydrate metabolism, energy metabolism, and signal transduction. Network analyses of the expression data show that, within the signal transduction pathway of the fungal community, the Mitogen-Activated Protein Kinase signaling pathway was tightly regulated across all samples and could be a potential driver for fungal proliferation. Our results demonstrated the feasibility of using MinION-based metatranscriptomics of enriched microbial RNA as a method for rapid, on-site monitoring of the transcriptional activity of crop microbiomes, thereby helping to facilitate and maintain plant health for on-orbit space food production.
Project description:Chronic acid suppression by proton pump inhibitor (PPI) has been hypothesized to alter the gut microbiota via a change in intestinal pH. To evaluate the changes in gut microbiota composition by long-term PPI treatment. Twenty-four week old F344 rats were fed with (n = 5) or without (n = 6) lansoprazole (PPI) for 50 weeks. Then, profiles of luminal microbiota in the terminal ileum were analyzed. Pyrosequencing for 16S rRNA gene was performed by genome sequencer FLX (454 Life Sciences/Roche) and analyzed by metagenomic bioinformatics.