Genome-resolved metatranscriptomics reveals conserved root colonization determinants in a synthetic microbiota.
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ABSTRACT:
SUBMITTER: Vannier N
PROVIDER: S-EPMC10719396 | biostudies-literature | 2023 Dec
REPOSITORIES: biostudies-literature
Publications
Genome-resolved metatranscriptomics reveals conserved root colonization determinants in a synthetic microbiota.
Vannier Nathan N Mesny Fantin F Getzke Felix F Chesneau Guillaume G Dethier Laura L Ordon Jana J Thiergart Thorsten T Hacquard Stéphane S
Nature communications 20231213 1
The identification of processes activated by specific microbes during microbiota colonization of plant roots has been hampered by technical constraints in metatranscriptomics. These include lack of reference genomes, high representation of host or microbial rRNA sequences in datasets, or difficulty to experimentally validate gene functions. Here, we recolonized germ-free Arabidopsis thaliana with a synthetic, yet representative root microbiota comprising 106 genome-sequenced bacterial and fungal ...[more]
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Project description:The identification of processes activated by specific microbes during microbiota colonization of plant roots has been hampered by technical constraints in metatranscriptomics. These include lack of reference genomes, high representation of host or microbial rRNA sequences in datasets, or difficulty to experimentally validate gene functions. Here, we recolonized germ-free Arabidopsis thaliana with a synthetic, yet representative root microbiota comprising 106 genome-sequenced bacterial and fungal isolates. We used multi-kingdom rRNA depletion, deep RNA-sequencing and read mapping against reference microbial genomes to analyse the in-planta metatranscriptome of abundant colonizers. We identified over 3,000 microbial genes that were differentially regulated at the soil-root interface. Translation and energy production processes were consistently activated in planta, and their induction correlated with bacterial strains’ abundance in roots. Finally, we used targeted mutagenesis to show that several genes consistently induced by multiple bacteria are required for root colonization in one of the abundant bacterial strains (a genetically tractable Rhodanobacter). Our results indicate that microbiota members activate strain-specific processes but also common gene sets to colonize plant roots.
2023-11-03 | GSE231841 | GEO
Project description:Genome-resolved metatranscriptomics reveals conserved root colonization determinants in a synthetic microbiota
| PRJNA968016 | ENA
Project description:BackgroundFecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridium difficile infection and shows promise for treating other medical conditions associated with intestinal dysbioses. However, we lack a sufficient understanding of which microbial populations successfully colonize the recipient gut, and the widely used approaches to study the microbial ecology of FMT experiments fail to provide enough resolution to identify populations that are likely responsible for FMT-derived benefits.MethodsWe used shotgun metagenomics together with assembly and binning strategies to reconstruct metagenome-assembled genomes (MAGs) from fecal samples of a single FMT donor. We then used metagenomic mapping to track the occurrence and distribution patterns of donor MAGs in two FMT recipients.ResultsOur analyses revealed that 22% of the 92 highly complete bacterial MAGs that we identified from the donor successfully colonized and remained abundant in two recipients for at least 8 weeks. Most MAGs with a high colonization rate belonged to the order Bacteroidales. The vast majority of those that lacked evidence of colonization belonged to the order Clostridiales, and colonization success was negatively correlated with the number of genes related to sporulation. Our analysis of 151 publicly available gut metagenomes showed that the donor MAGs that colonized both recipients were prevalent, and the ones that colonized neither were rare across the participants of the Human Microbiome Project. Although our dataset showed a link between taxonomy and the colonization ability of a given MAG, we also identified MAGs that belong to the same taxon with different colonization properties, highlighting the importance of an appropriate level of resolution to explore the functional basis of colonization and to identify targets for cultivation, hypothesis generation, and testing in model systems.ConclusionsThe analytical strategy adopted in our study can provide genomic insights into bacterial populations that may be critical to the efficacy of FMT due to their success in gut colonization and metabolic properties, and guide cultivation efforts to investigate mechanistic underpinnings of this procedure beyond associations.
| S-EPMC5418705 | biostudies-literature
Project description:Natural microbial communities consist of closely related taxa that may exhibit phenotypic differences and inhabit distinct niches. However, connecting genetic diversity to ecological properties remains a challenge in microbial ecology due to the lack of pure cultures across the microbial tree of life. "Candidatus Accumulibacter phosphatis" (Accumulibacter) is a polyphosphate-accumulating organism that contributes to the enhanced biological phosphorus removal (EBPR) biotechnological process for removing excess phosphorus from wastewater and preventing eutrophication from downstream receiving waters. Distinct Accumulibacter clades often coexist in full-scale wastewater treatment plants and laboratory-scale enrichment bioreactors and have been hypothesized to inhabit distinct ecological niches. However, since individual strains of the Accumulibacter lineage have not been isolated in pure culture to date, these predictions have been made solely on genome-based comparisons and enrichments with varying strain compositions. Here, we used genome-resolved metagenomics and metatranscriptomics to explore the activity of coexisting Accumulibacter strains in an engineered bioreactor environment. We obtained four high-quality genomes of Accumulibacter strains that were present in the bioreactor ecosystem, one of which is a completely contiguous draft genome scaffolded with long Nanopore reads. We identified core and accessory genes to investigate how gene expression patterns differed among the dominating strains. Using this approach, we were able to identify putative pathways and functions that may confer distinct functions to Accumulibacter strains and provide key functional insights into this biotechnologically significant microbial lineage. IMPORTANCE "Candidatus Accumulibacter phosphatis" is a model polyphosphate-accumulating organism that has been studied using genome-resolved metagenomics, metatranscriptomics, and metaproteomics to understand the EBPR process. Within the Accumulibacter lineage, several similar but diverging clades are defined by the shared sequence identity of the polyphosphate kinase (ppk1) locus. These clades are predicted to have key functional differences in acetate uptake rates, phage defense mechanisms, and nitrogen-cycling capabilities. However, such hypotheses have largely been made based on gene content comparisons of sequenced Accumulibacter genomes, some of which were obtained from different systems. Here, we performed time series genome-resolved metatranscriptomics to explore gene expression patterns of coexisting Accumulibacter clades in the same bioreactor ecosystem. Our work provides an approach for elucidating ecologically relevant functions based on gene expression patterns between closely related microbial populations.
| S-EPMC8407102 | biostudies-literature
Project description:Antibiotic resistance is a current and expanding threat to the practice of modern medicine. Antibiotic therapy has been shown to perturb the composition of the host microbiome with significant health consequences. In addition, the gut microbiome is known to be a reservoir of antibiotic resistance genes. Work has demonstrated that antibiotics can alter the collection of antibiotic resistance genes within the microbiome through selection and horizontal gene transfer. While antibiotics also have the potential to impact the expression of resistance genes, metagenomic-based pipelines currently lack the ability to detect these shifts. Here, we utilized a dual sequencing approach combining shotgun metagenomics and metatranscriptomics to profile how three antibiotics, amoxicillin, doxycycline, and ciprofloxacin, impact the murine gut resistome at the DNA and RNA level. We found that each antibiotic induced broad, but untargeted impacts on the gene content of the resistome. In contrast, changes in ARG transcript abundance were more targeted to the antibiotic treatment. Doxycycline and amoxicillin induced the expression of tetracycline and beta-lactamase resistance genes, respectively. Furthermore, the increased beta-lactamase resistance gene transcripts could contribute to an observed bloom of Bacteroides thetaiotaomicron during amoxicillin treatment. Based on these findings, we propose that the utilization of a dual sequencing methodology provides a unique capacity to fully understand the response of the resistome to antibiotic perturbation. In particular, the analysis of transcripts reveals that the expression and utilization of resistance genes is far narrower than their abundance at the genomic level would suggest.
| S-EPMC7069102 | biostudies-literature
Project description:Nitrogen (N) availability is one of the principal drivers of primary productivity across aquatic ecosystems. However, the microbial communities and emergent metabolisms that govern N cycling in tropical lakes are both distinct from and poorly understood relative to those found in temperate lakes. This latitudinal difference is largely due to the warm (>20°C) temperatures of tropical lake anoxic hypolimnions (deepest portion of a stratified water column), which result in unique anaerobic metabolisms operating without the temperature constraints found in lakes at temperate latitudes. As such, tropical hypolimnions provide a platform for exploring microbial membership and functional diversity. To better understand N metabolism in warm anoxic waters, we combined measurements of geochemistry and water column thermophysical structure with genome-resolved metatranscriptomic analyses of the water column microbiome in Lake Yojoa, Honduras. We sampled above and below the oxycline in June 2021, when the water column was stratified, and again at the same depths and locations in January 2022, when the water column was mixed. We identified 335 different lineages and significantly different microbiome membership between seasons and, when stratified, between depths. Notably, nrfA (indicative of dissimilatory nitrate reduction to ammonium) was upregulated relative to other N metabolism genes in the June hypolimnion. This work highlights the taxonomic and functional diversity of microbial communities in warm and anoxic inland waters, providing insight into the contemporary microbial ecology of tropical ecosystems as well as inland waters at higher latitudes as water columns continue to warm in the face of global change.IMPORTANCEIn aquatic ecosystems where primary productivity is limited by nitrogen (N), whether continuously, seasonally, or in concert with additional nutrient limitations, increased inorganic N availability can reshape ecosystem structure and function, potentially resulting in eutrophication and even harmful algal blooms. Whereas microbial metabolic processes such as mineralization and dissimilatory nitrate reduction to ammonium increase inorganic N availability, denitrification removes bioavailable N from the ecosystem. Therefore, understanding these key microbial mechanisms is critical to the sustainable management and environmental stewardship of inland freshwater resources. This study identifies and characterizes these crucial metabolisms in a warm, seasonally anoxic ecosystem. Results are contextualized by an ecological understanding of the study system derived from a multi-year continuous monitoring effort. This unique data set is the first of its kind in this largely understudied ecosystem (tropical lakes) and also provides insight into microbiome function and associated taxa in warm, anoxic freshwaters.
| S-EPMC10878078 | biostudies-literature
Project description:BackgroundRetroviruses selectively package two copies of their unspliced genomes by what appears to be a dimerization-dependent RNA packaging mechanism. Dimerization of human immunodeficiency virus Type-1 (HIV-1) genomes is initiated by "kissing" interactions between GC-rich palindromic loop residues of a conserved hairpin (DIS), and is indirectly promoted by long-range base pairing between residues overlapping the gag start codon (AUG) and an upstream Unique 5' element (U5). The DIS and U5:AUG structures are phylogenetically conserved among divergent retroviruses, suggesting conserved functions. However, some studies suggest that the DIS of HIV-2 does not participate in dimerization, and that U5:AUG pairing inhibits, rather than promotes, genome dimerization. We prepared RNAs corresponding to native and mutant forms of the 5' leaders of HIV-1 (NL4-3 strain), HIV-2 (ROD strain), and two divergent strains of simian immunodeficiency virus (SIV; cpz-TAN1 and -US strains), and probed for potential roles of the DIS and U5:AUG base pairing on intrinsic and NC-dependent dimerization by mutagenesis, gel electrophoresis, and NMR spectroscopy.ResultsDimeric forms of the native HIV-2 and SIV leaders were only detectable using running buffers that contained Mg(2+), indicating that these dimers are more labile than that of the HIV-1 leader. Mutations designed to promote U5:AUG base pairing promoted dimerization of the HIV-2 and SIV RNAs, whereas mutations that prevented U5:AUG pairing inhibited dimerization. Chimeric HIV-2 and SIV leader RNAs containing the dimer-promoting loop of HIV-1 (DIS) exhibited HIV-1 leader-like dimerization properties, whereas an HIV-1NL4-3 mutant containing the SIVcpzTAN1 DIS loop behaved like the SIVcpzTAN1 leader. The cognate NC proteins exhibited varying abilities to promote dimerization of the retroviral leader RNAs, but none were able to convert labile dimers to non-labile dimers.ConclusionsThe finding that U5:AUG formation promotes dimerization of the full-length HIV-1, HIV-2, SIVcpzUS, and SIVcpzTAN1 5' leaders suggests that these retroviruses utilize a common RNA structural switch mechanism to modulate function. Differences in native and NC-dependent dimerization propensity and lability are due to variations in the compositions of the DIS loop residues rather than other sequences within the leader RNAs. Although NC is a well-known RNA chaperone, its role in dimerization has the hallmarks of a classical riboswitch.
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Project description:Development of the human gut microbiota commences at birth with bifidobacteria being among the first colonizers of the sterile newborn gastrointestinal tract. To date the genetic basis of bifidobacterial colonization and persistence remains poorly understood. Transcriptomic analysis of the 2,422,684 bp genome of Bifidobacterium breve UCC2003, a strain isolated from a nursling stool, during colonization of a mouse model revealed the differential expression of a type IVb or so-called Tad pilus-encoding cluster. Mutational analysis coupled with colonization experiments demonstrated that the UCC2003 tad gene cluster is essential for colonization. Immunogold transmission electron microscopy confirmed the presence of the Tad pili at the cell poles of B. breve UCC2003. Conservation of the Tad pilus-encoding locus among sequenced bifidobacterial genomes supports the notion of a ubiquitous and novel host colonization and persistence mechanism for bifidobacteria. Colonisation of BalbC mice: Seven-week-old male, BalbC mice were housed in individually vented cages (Animal care systems, Colorado) under a strict 12 h light cycle. Mice (n = 5 per group) were fed a standard polysaccharide-rich mouse chow diet and water ad libidum. Mice were inoculated by oral gavage (109 cfu of B. breve UCC2003 pPKCM7 or B. breve UCC 2003-tadA pPKCM7 in 100 ul of PBS). Fecal pellets were collected at intervals over 25 days to enumerate bacteria. Twenty five days after inoculation, mice were sacrificed and their intestinal tracts quickly dissected. Aliquots of small intestine, caecum and large intestine were harvested for determination of colony forming units (cfu) (serial dilution plating on RCA agar plates with appropriate antibiotics). Caeca were placed in RNA later for immediate RNA isolation (see below). Microarray procedures: DNA microarrays containing oligonucleotide primers representing each of the 1926 annotated genes of B. breve UCC2003 were obtained from Agilent Technologies (Palo Alto, CA). An overnight culture of B. breve was used to inoculate (1 % inoculum) 50 ml of MRS broth. Cells were incubated at 37°C until an optical density at 600 nm (OD600) of 0.5 was reached. Cells were then harvested by centrifugation at 8,000 x g for 1 min at room temperature and immediately frozen at -80oC. Methods for cell disruption, RNA isolation, RNA quality control were performed as described previously (van Hijum et al., 2005). Caeca, isolated from mice, were placed in RNAlater (Ambion) and immediately processed. Bacterial mRNA was extracted from caecal RNA preparations using the MicrobEnrich and MicrobExpress kits (Ambion) according to the manufacturer’s instructions. cDNA from bacterial mRNA was synthesized using the cDNA synthesis and labelling kit DSK-001 (Kreatech) according to the manufacturer’s instructions. 1 μl of each cDNA solution (10 ng) was amplified in triplicate using the GenomiPhi™ V2 DNA Amplification Kit (Amersham Biosciences) according to the manufacturer's protocol and labelled with Cy3 or Cy5 using Cy3-ULS and Cy5-ULS from the cDNA synthesis and labelling kit DSK-001 (Kreatech). Labelled and amplified cDNA was hybridized using the Agilent Gene Expression hybridization kit (part number 5188-5242) as described in the Agilent Two-Color Microarray-Based Gene Expression Analysis (v4.0) manual (publication number G4140-90050). Following hybridization, microarrays were washed as described in the manuals and scanned using Agilent's DNA microarray scanner G2565A. The scanning results were converted to data files with Agilent's Feature Extraction software (version 9.5). DNA microarray data was processed as previously described (Zomer et al. 2009). Differential expression tests were performed using a t test. A gene was considered differentially expressed between a test strain and control when an expression ratio of >5 or <0.2 relative to the result for the control was obtained with a corresponding P value that was <0.0001. DNA microarrays containing oligonucleotide primers representing each of the 1926 annotated genes of B. breve UCC2003 were obtained from Agilent Technologies (Palo Alto, CA). An overnight culture of B. breve was used to inoculate (1 % inoculum) 50 ml of MRS broth. Cells were incubated at 37°C until an optical density at 600 nm (OD600) of 0.5 was reached. Cells were then harvested by centrifugation at 8,000 x g for 1 min at room temperature and immediately frozen at -80oC. Methods for cell disruption, DNA isolation were performed as described previously (O'Riordan et al. 1998). Genomic DNA (gDNA) labeling for comparative genome hybridizations (CGH) was undertaken according to the BµG@S standard protocols (Hinds et al. 2002). In brief 5 µg of bifidobacterial genomic DNA was labeled with dCTP Cy3 (UCC2003) or dCTP Cy5 (test strain) using random primers (Promega) and a DNA polymerase I large Klenow fragment (Invitrogen). Labelled gDNA was hybridized using the Agilent CGH kit (part number 5188-5220) as described in the Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis (v6.0) manual (publication number G4410-90010). Validation of the CGH was performed by comparing the hybridization efficiency of DNA of the B. breve type strain DSM 20213 to sequence identity of this strain with the probes on the microarray. A clear correlation was observed between sequence identity and hybridization efficiency, suggesting that an ln 2 fold change in signal intensity between target and control is sufficient to determine whether a gene is present or absent on a given genome. Therefore, a gene was considered absent when the ratio between a test strain and UCC2003 was < ln 2 with a corresponding P value that was <0.0001.
2011-07-01 | E-GEOD-27491 | biostudies-arrayexpress
Project description:The structure and function of microbial communities inhabiting the subseafloor near hydrothermal systems are influenced by fluid geochemistry, geologic setting and fluid flux between vent sites, as well as biological interactions. Here, we used genome-resolved metagenomics and metatranscriptomics to examine patterns of gene abundance and expression and assess potential niche differentiation in microbial communities in venting fluids from hydrothermal vent sites at the Mid-Cayman Rise. We observed similar patterns in gene and transcript abundance between two geochemically distinct vent fields at the community level but found that each vent site harbours a distinct microbial community with differing transcript abundances for individual microbial populations. Through an analysis of metabolic pathways in 64 metagenome-assembled genomes (MAGs), we show that MAG transcript abundance can be tied to differences in metabolic pathways and to potential metabolic interactions between microbial populations, allowing for niche-partitioning and divergence in both population distribution and activity. Our results illustrate that most microbial populations have a restricted distribution within the seafloor, and that the activity of those microbial populations is tied to both genome content and abiotic factors.
| S-EPMC6899741 | biostudies-literature
Project description:Despite a surge in microbiota-focused studies in teleosts, few have reported functional data on whole metagenomes as it has proven difficult to extract high biomass microbial DNA from fish intestinal samples. The zebrafish is a promising model organism in functional microbiota research, yet studies on the functional landscape of the zebrafish gut microbiota through shotgun based metagenomics remain scarce. Thus, a consensus on an appropriate sampling method accurately representing the zebrafish gut microbiota, or any fish species is lacking. Addressing this, we systematically tested four methods of sampling the zebrafish gut microbiota: collection of faeces from the tank, the whole gut, intestinal content, and the application of ventral pressure to facilitate extrusion of gut material. Additionally, we included water samples as an environmental control to address the potential influence of the environmental microbiota on each sample type. To compare these sampling methods, we employed a combination of genome-resolved metagenomics and 16S metabarcoding techniques. We observed differences among sample types on all levels including sampling, bioinformatic processing, metagenome co-assemblies, generation of metagenome-assembled genomes (MAGs), functional potential, MAG coverage, and population level microdiversity. Comparison to the environmental control highlighted the potential impact of the environmental contamination on data interpretation. While all sample types tested are informative about the zebrafish gut microbiota, the results show that optimal sample type for studying fish microbiomes depends on the specific objectives of the study, and here we provide a guide on what factors to consider for designing functional metagenome-based studies on teleost microbiomes.
| S-EPMC11407903 | biostudies-literature