Non-targeted metabolomics of Gut bacterial co-cultures
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF
ORGANISM(S): Environmental Samples <bacillariophyta> (ncbitaxon:33858)
SUBMITTER: 
Daniel Petras
PROVIDER: MSV000091452 | MassIVE | Thu Mar 09 06:35:00 GMT 2023
REPOSITORIES: MassIVE
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Project description:Non-targeted metabolomics analysis of cell and medium extract obtained by bacterial co-cultures.
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Project description:mRNA sequencing in bacteria is challenging due to the abundance of ribosomal rRNA that cannot be easily removed prior to sequencing. While commercially available kits target specific rRNA sequences found in defined lists of common bacterial species, they are frequently inefficient when applied to other divergent species, including those from environmental isolates. Similar to the commercial kits, other common techniques for rRNA depletion typically employ large probe sets that tile full-length rRNA sequences; however, such approaches are both time consuming and expensive when applied to multiple species or complex consortia of non-model microbes. To overcome these limitations, we present EMBR-seq+, which employs less than twenty target oligonucleotides per rRNA molecule, and builds upon our previous rRNA depletion approach, EMBR-seq, through the addition of an RNase H depletion step, to achieve rRNA removal efficiencies of up to 99%. First, we applied EMBR-seq+ to monocultures of Escherichia coli, Geobacter metallireducens, and Fibrobacter succinogenes strain UWB7 to deplete rRNA to approximately 1-7% of the sequencing reads, demonstrating that the new method can be easily extended to diverse bacterial species. Further, in more complex co-cultures between F. succinogenes strain UWB7 and anerobic fungal species, we applied EMBR-seq+ to deplete both bacterial and fungal rRNA, with an approximately 4-fold improved bacterial rRNA depletion efficiency compared to a previous report using a commercial kit, thereby showing that the method can be effectively translated to non-model microbial mixtures. Notably, we also demonstrate that for microbial species with poorly annotated genomes and unknown rRNA sequences, the RNase H depletion component of EMBR-seq+ enables rapid iterations in probe design without requiring to start experiments from total RNA each time, and was key for depleting fungal rRNA to enrich the bacterial mRNA readout in co-cultures. Finally, efficient depletion of rRNA enabled systematic quantification of the reprogramming of the bacterial transcriptome when cultured in the presence of the anerobic fungi, Anaeromyces robustus and Caecomyces churrovis. We observed that F. succinogenes strain UWB7 transcribes nearly 200 carbohydrate-active enzyme (CAZyme) genes in both monoculture and co-culture conditions, with several lignocellulose-degrading CAZymes downregulated in the presence of an anerobic gut fungus. This finding is consistent with the premise that bacteria and fungi specialize in different aspects of biomass breakdown, such that the presence of one regulates the CAZyme production of the other. This also supports previous findings that the fungi release excess reducing sugars in the supernatant, which benefits other members of the microbial community. Thus EMBR-seq+ provides a new and detailed perspective of a rumen microbiome model system by dramatically improving the efficiency of mRNA sequencing, and more generally also enables high-throughput, cost-effective and rapid quantification of the transcriptome to gain functional insights into less-studied and non-model microbial systems.
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Project description:The use of microbiological cultures for diagnosing bacterial infections in young febrile infants have substantial limitations, including false positive and false negative cultures, and non-ideal turn-around times. Analysis of host genomic expression patterns (“RNA biosignatures”) in response to the presence of specific pathogens, however, may provide an alternate and potentially improved diagnostic approach. This study was designed to define bacterial and non-bacterial RNA biosignatures to distinguish these infections in young febrile infants.
2016-07-27 | GSE64456 | GEO
Project description:The use of microbiological cultures for diagnosing bacterial infections in young febrile infants have substantial limitations, including false positive and false negative cultures, and non-ideal turn-around times. Analysis of host genomic expression patterns (âRNA biosignaturesâ) in response to the presence of specific pathogens, however, may provide an alternate and potentially improved diagnostic approach. This study was designed to define bacterial and non-bacterial RNA biosignatures to distinguish these infections in young febrile infants. A total of 279 febrile infants and 19 healthy afebrile control infants aged 0-6 months (for a total of 298 samples) for microarray analysis. For analytic purposes, we classified patients into two groups, those with bacterial infections (n=89) and those with non-bacterial infections (n=190). 144 of the samples were run on Illumina HT12 V4 R1 chips. Of these, there were 34 bacterial infections, 105 non-bacterial infections, and 5 healthy afebrile controls. 154 of the samples were run on Illumina HT12 V4 R2 chips. Of these, there were 55 bacterial infections, 85 non-bacterial infections, and 14 healthy afebrile controls.
2016-07-27 | E-GEOD-64456 | biostudies-arrayexpress
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Project description:Gut commensal bacterial strains were cultured with or without added sugars (glucose, sucrose and kestose). The base culture medium without sugar added were modified based on the Yeast Casitone Fatty Acids (YCFA) broth. Samples were analyzed for proteomics using LC-MS/MS.
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2015-09-26 | GSE51201 | GEO
Project description:Mouse small intestinal crypts were co-cultured with primary intestinal fibroblasts in Organoid Growth Media (OGM) with/without inhibitor for the prostaglandin E2 receptor Ptger4 (EP4), labeled respectively as “Ptger4-OFF” and “Ptger4-ON” conditions. Crypts co-cultured with fibroblasts in Ptger4-ON conditions (OGM containing DMSO vehicle control) acquired a spheroid morphology. Crypts co-cultured with fibroblasts in Ptger4-OFF conditions (OGM supplemented with Ptger4 inhibitor) developed into budding organoids. Fibroblasts were isolated from the small intestine of wild type mice. For the co-cultures 2 x 10e4 fibroblasts were seeded in 48-well plates overnight. Freshly isolated crypts (n = 500) were suspended in 1:1 Matrigel (Corning, 356231) and IntestiCult Organoid Growth Medium (Stem Cell Technologies, 06005) and added as an overlay on the fibroblasts. Crypts/fibroblasts were co-cultured with IntestiCult Organoid Growth Medium for 4 days. The ONO-AE3-208 Ptger4 (EP4) inhibitor (Cayman, 14522) dissolved in DMSO was added to the co-cultureson days 0 and 2 at a final concentration of 10 uM. DMSO was used as a vehicle control for the untreated co-cultures. On day 4, one pool of six Ptger4-ON co-cultures and one pool of six Ptger4-OFF co-cultures were subjected to the Drop-seq protocol.
2019-12-21 | GSE142420 | GEO
Project description:The present study aims to explore chemostat-based transcriptome analysis of mixed cultures by investigating interactions between the yeast S. cerevisiae and the lactic acid bacterium L. bulgaricus . S. cerevisiae and L. bulgaricus are both frequently encountered in kefir, a fermented dairy product. In the context of this study, this binary culture serves as a model for the many traditional food and beverage fermentation processes in which yeasts and lactic acid bacteria occur together. The design of the cultivation conditions was based on the observation that L. bulgaricus, but not S. cerevisiae, can use lactose as a carbon source for growth and that S. cerevisiae, but not L. bulgaricus, can grow on galactose that is released upon hydrolysis of lactose by the bacterial β-galactosidase. Mixed populations of yeasts and lactic acid bacteria occur in many dairy, food and beverage fermentations, but knowledge about their interactions is incomplete. In the present study, interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus, two microorganisms that co-occur in kefir fermentations, were studied during anaerobic growth on lactose. By combining physiological and transcriptome analysis of the two strains in the co-cultures, five mechanisms of interaction were identified. 1. L. bulgaricus hydrolyses lactose, which cannot be metabolized by S. cerevisiae, to galactose and glucose. Subsequently, galactose, which cannot be metabolized by L. bulgaricus, is excreted and provides a carbon source for yeast. 2. In pure cultures, L. bulgaricus only grows at increased CO2 concentrations. In anaerobic mixed cultures, the yeast provides this CO2 via alcoholic fermentation. 3. Analysis of amino acid consumption from the defined medium indicated that S. cerevisiae supplied alanine to the bacteria. 4. A mild but significant low-iron response in the yeast transcriptome, identified by DNA microarray analysis, was consistent with the chelation of iron by the lactate produced by L. bulgaricus. 5. Transcriptome analysis of L. bulgaricus in mixed cultures showed an overrepresentation of transcripts involved in lipids metabolism suggesting either a competition of the two microorganisms for fatty acids, or a response to the ethanol produced by S. cerevisiae.
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