Genetic insights into the dark matter of the mammalian gut microbiota through targeted genome reconstruction.
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ABSTRACT:
SUBMITTER: Lugli GA
PROVIDER: S-EPMC8359967 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The human gut microbiota has been explored by a wide range of culture-dependent and culture-independent methods, revealing that many microbes remain uncharacterized and uncultured. In this work, we aimed to confirm the hypothesis that some of the species present in the human gut microbiota remain uncultured not because of culture limitations, but because all members of such species are dead before reaching the end of the gastro-intestinal tract.We evaluate this phenomenon by studying the microbial viability and culturability of the human gut microbiota from the fresh fecal materials of eight healthy adults. For the first time, we applied fluorescence-activated cell sorting (FACS) combined with 16S metagenomics analysis and microbial culturomics.We identified a total of 1,020 bacterial OTUs and 495 bacterial isolates through metagenomics and culturomics, respectively. Among the FACS metagenomics results, only 735 bacterial OTUs were alive, comprising on average 42% of known species and 87% of relative abundance per individual. The remaining uncultured bacteria were rare, dead, or injured.Our strategy allowed us to shed light on the dark matter of the human gut microbiota and revealed that both metagenomics and culturomics approaches are needed for greater insight into the diversity and richness of bacteria in the human gut microbiota. Further work on culture is needed to enhance the repertoire of cultured gut bacteria by targeting low abundance bacteria and optimizing anaerobic sample conditioning and processing to preserve the viability of bacteria.
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Project description:The study of bacteria has yielded fundamental insights into cellular biology and physiology, biotechnological advances and many therapeutics. Yet due to a lack of suitable tools, the significant portion of bacterial diversity held within the candidate phyla radiation (CPR) remains inaccessible to such pursuits. Here we show that CPR bacteria belonging to the phylum Saccharibacteria exhibit natural competence. We exploit this property to develop methods for their genetic manipulation, including the insertion of heterologous sequences and the construction of targeted gene deletions. Imaging of fluorescent protein-labeled Saccharibacteria provides high spatiotemporal resolution of phenomena accompanying epibiotic growth and a transposon insertion sequencing genome-wide screen reveals the contribution of enigmatic Saccharibacterial genes to growth on their Actinobacteria hosts. Finally, we leverage metagenomic data to provide cutting-edge protein structure-based bioinformatic resources that support the strain Southlakia epibionticum and its corresponding host, Actinomyces israelii , as a model system for unlocking the molecular underpinnings of the epibiotic lifestyle.
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Project description:The gut microbiome in human is recognized as a "microbial organ" for its roles and contributions in regulating the human homeostasis and metabolism. Gastrointestinal (GI) cancers, especially colorectal cancer (CRC), rank as the most common cancer-related deaths worldwide. Evidences have suggested that the disorder of gut microbiota, also named as "dysbiosis," is related to the development of a variety of diseases such as inflammatory bowel disease (IBD) and the CRC. However, detailed mechanisms between disease and gut microbiota remain largely unknown. This review introduced the correlation between gastrointestinal diseases and the microbiota in human gut from the recent studies, as well as the roles of microbiota in manipulating the CRC and IBDs development, in order to facilitate future studies and to develop novel methods for the precaution, diagnosis, or even cure of gastrointestinal diseases. Additionally, we also elucidated the possibility of probiotics in treatment against CRC.
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Project description:Metagenomes encode an enormous diversity of proteins, reflecting a multiplicity of functions and activities1,2. Exploration of this vast sequence space has been limited to a comparative analysis against reference microbial genomes and protein families derived from those genomes. Here, to examine the scale of yet untapped functional diversity beyond what is currently possible through the lens of reference genomes, we develop a computational approach to generate reference-free protein families from the sequence space in metagenomes. We analyse 26,931 metagenomes and identify 1.17 billion protein sequences longer than 35 amino acids with no similarity to any sequences from 102,491 reference genomes or the Pfam database3. Using massively parallel graph-based clustering, we group these proteins into 106,198 novel sequence clusters with more than 100 members, doubling the number of protein families obtained from the reference genomes clustered using the same approach. We annotate these families on the basis of their taxonomic, habitat, geographical and gene neighbourhood distributions and, where sufficient sequence diversity is available, predict protein three-dimensional models, revealing novel structures. Overall, our results uncover an enormously diverse functional space, highlighting the importance of further exploring the microbial functional dark matter.
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Project description:BackgroundA growing body of evidence shows that gene products encoded by short open reading frames play key roles in numerous cellular processes. Yet, they are generally overlooked in genome assembly, escaping annotation because small protein-coding genes are difficult to predict computationally. Consequently, there are still a considerable number of small proteins whose functions are yet to be characterized.ResultsTo address this issue, we apply a collection of structural bioinformatics algorithms to infer molecular function of putative small proteins from the mouse proteome. Specifically, we construct 1,743 confident structure models of small proteins, which reveal a significant structural diversity with a noticeably high helical content. A subsequent structure-based function annotation of small protein models exposes 178,745 putative protein-protein interactions with the remaining gene products in the mouse proteome, 1,100 potential binding sites for small organic molecules and 987 metal-binding signatures.ConclusionsThese results strongly indicate that many small proteins adopt three-dimensional structures and are fully functional, playing important roles in transcriptional regulation, cell signaling and metabolism. Data collected through this work is freely available to the academic community at http://www.brylinski.org/content/databases to support future studies oriented on elucidating the functions of hypothetical small proteins.
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Project description:Transmission of bacterial endospores between the environment and people and the following germination in vivo play critical roles in both the deadly infections of some bacterial pathogens and the stabilization of the commensal microbiotas in humans. Our knowledge about the germination process of different bacteria in the mammalian gut, however, is still very limited due to the lack of suitable tools to visually monitor this process. We proposed a two-step labeling strategy that can image and quantify the endospores' germination in the recipient's intestines. Endospores collected from donor's gut microbiota were first labeled with fluorescein isothiocyanate and transplanted to mice via gavage. The recipient mice were then administered with Cyanine5-tagged D-amino acid to label all the viable bacteria, including the germinated endospores, in their intestines in situ. The germinated donor endospores could be distinguished by presenting two types of fluorescent signals simultaneously. The integrative use of cell-sorting, 16S rDNA sequencing, and fluorescence in situ hybridization (FISH) staining of the two-colored bacteria unveiled the taxonomic information of the donor endospores that germinated in the recipient's gut. Using this strategy, we investigated effects of different germinants and pre-treatment interventions on their germination, and found that germination of different commensal bacterial genera was distinctly affected by various types of germinants. This two-color labeling strategy shows its potential as a versatile tool for visually monitoring endospore germination in the hosts and screening for new interventions to improve endospore-based therapeutics.
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Project description:In mammals, the composition of the gut microbiota is associated with host phylogenetic history, and host-lineage specific microbiota have been shown, in some cases, to contribute to fitness-related traits of their hosts. However, in primates, captivity can disrupt the native microbiota through a process of humanization in which captive hosts acquire gut microbiota constituents found in humans. Despite the potential importance of this process for the health of captive hosts, the degree to which captivity humanizes the gut microbiota of other mammalian taxa has not been explored. Here, we analyzed hundreds of published gut microbiota profiles generated from wild and captive hosts spanning seven mammalian families to investigate the extent of humanization of the gut microbiota in captivity across the mammalian phylogeny. Comparisons of these hosts revealed compositional convergence between captive mammal and human gut microbiota in the majority of mammalian families examined. This convergence was driven by a diversity of microbial lineages, including members of the Archaea, Clostridium, and Bacteroides. However, the gut microbiota of two families-Giraffidae and Bovidae-were remarkably robust to humanization in captivity, showing no evidence of gut microbiota acquisition from humans relative to their wild confamiliars. These results demonstrate that humanization of the gut microbiota is widespread in captive mammals, but that certain mammalian lineages are resistant to colonization by human-associated gut bacteria.
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