RESUMEN
The activities of microbiomes in river sediments play an important role in sustaining ecosystem functions by driving many biogeochemical cycles. However, river ecosystems are frequently affected by anthropogenic activities, which may lead to microbial biodiversity loss and/or changes in ecosystem functions and related services. While parts of the Atlantic Forest biome stretching along much of the eastern coast of South America are protected by governmental conservation efforts, an estimated 89% of these areas in Brazil are under threat. This adds urgency to the characterization of prokaryotic communities in this vast and highly diverse biome. Here, we present prokaryotic sediment communities in the tropical Juliana River system at three sites, an upstream site near the river source in the mountains (Source) to a site in the middle reaches (Valley) and an estuarine site near the urban center of Ituberá (Mangrove). The diversity and composition of the communities were compared at these sites, along with environmental conditions, the former by using qualitative and quantitative analyses of 16S rRNA gene amplicons. While the communities included distinct populations at each site, a suite of core taxa accounted for the majority of the populations at all sites. Prokaryote diversity was highest in the sediments of the Mangrove site and lowest at the Valley site. The highest number of genera exclusive to a given site was found at the Source site, followed by the Mangrove site, which contained some archaeal genera not present at the freshwater sites. Copper (Cu) concentrations were related to differences in communities among sites, but none of the other environmental factors we determined was found to have a significant influence. This may be partly due to an urban imprint on the Mangrove site by providing organic carbon and nutrients via domestic effluents.
Asunto(s)
Sedimentos Geológicos , ARN Ribosómico 16S , Ríos , Brasil , Ríos/microbiología , ARN Ribosómico 16S/genética , Sedimentos Geológicos/microbiología , Bacterias/genética , Bacterias/clasificación , Bacterias/aislamiento & purificación , Bosques , Estuarios , Biodiversidad , Archaea/genética , Archaea/clasificación , Archaea/aislamiento & purificación , MicrobiotaRESUMEN
Copy-number variants (CNVs) are large-scale amplifications or deletions of DNA that can drive rapid adaptive evolution and result in large-scale changes in gene expression. Whereas alterations in the copy number of one or more genes within a CNV can confer a selective advantage, other genes within a CNV can decrease fitness when their dosage is changed. Dosage compensation - in which the gene expression output from multiple gene copies is less than expected - is one means by which an organism can mitigate the fitness costs of deleterious gene amplification. Previous research has shown evidence for dosage compensation at both the transcriptional level and at the level of protein expression; however, the extent of compensation differs substantially between genes, strains, and studies. Here, we investigated sources of dosage compensation at multiple levels of gene expression regulation by defining the transcriptome, translatome and proteome of experimentally evolved yeast (Saccharomyces cerevisiae) strains containing adaptive CNVs. We quantified the gene expression output at each step and found evidence of widespread dosage compensation at the protein abundance (~47%) level. By contrast we find only limited evidence for dosage compensation at the transcriptional (~8%) and translational (~3%) level. We also find substantial divergence in the expression of unamplified genes in evolved strains that could be due to either the presence of a CNV or adaptation to the environment. Detailed analysis of 82 amplified and 411 unamplified genes with significantly discrepant relationships between RNA and protein abundances identified enrichment for upstream open reading frames (uORFs). These uORFs are enriched for binding site motifs for SSD1, an RNA binding protein that has previously been associated with tolerance of aneuploidy. Our findings suggest that, in the presence of CNVs, SSD1 may act to alter the expression of specific genes by potentiating uORF mediated translational regulation.
RESUMEN
Copy number variants (CNVs), duplications and deletions of genomic sequences, contribute to evolutionary adaptation but can also confer deleterious effects and cause disease. Whereas the effects of amplifying individual genes or whole chromosomes (i.e., aneuploidy) have been studied extensively, much less is known about the genetic and functional effects of CNVs of differing sizes and structures. Here, we investigated Saccharomyces cerevisiae (yeast) strains that acquired adaptive CNVs of variable structures and copy numbers following experimental evolution in glutamine-limited chemostats. Although beneficial in the selective environment, CNVs result in decreased fitness compared with the euploid ancestor in rich media. We used transposon mutagenesis to investigate mutational tolerance and genome-wide genetic interactions in CNV strains. We find that CNVs increase mutational target size, confer increased mutational tolerance in amplified essential genes, and result in novel genetic interactions with unlinked genes. We validated a novel genetic interaction between different CNVs and BMH1 that was common to multiple strains. We also analyzed global gene expression and found that transcriptional dosage compensation does not affect most genes amplified by CNVs, although gene-specific transcriptional dosage compensation does occur for â¼12% of amplified genes. Furthermore, we find that CNV strains do not show previously described transcriptional signatures of aneuploidy. Our study reveals the extent to which local and global mutational tolerance is modified by CNVs with implications for genome evolution and CNV-associated diseases, such as cancer.
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Variaciones en el Número de Copia de ADN , Genoma , Humanos , Dosificación de Gen , Mutación , Saccharomyces cerevisiae/genética , AneuploidiaRESUMEN
Copy number variants (CNVs), comprising gene amplifications and deletions, are a pervasive class of heritable variation. CNVs play a key role in rapid adaptation in both natural, and experimental, evolution. However, despite the advent of new DNA sequencing technologies, detection and quantification of CNVs in heterogeneous populations has remained challenging. Here, we summarize recent advances in the use of CNV reporters that provide a facile means of quantifying de novo CNVs at a specific locus in the genome, and nanopore sequencing, for resolving the often complex structures of CNVs. We provide guidance for the engineering and analysis of CNV reporters and practical guidelines for single-cell analysis of CNVs using flow cytometry. We summarize recent advances in nanopore sequencing, discuss the utility of this technology, and provide guidance for the bioinformatic analysis of these data to define the molecular structure of CNVs. The combination of reporter systems for tracking and isolating CNV lineages and long-read DNA sequencing for characterizing CNV structures enables unprecedented resolution of the mechanisms by which CNVs are generated and their evolutionary dynamics.
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Variaciones en el Número de Copia de ADN , Genoma , Variaciones en el Número de Copia de ADN/genética , Biología Computacional , Análisis de Secuencia de ADN , Amplificación de GenesRESUMEN
The global spread of antibiotic resistance could be due to a number of factors, and not just the overuse of antibiotics in agriculture and medicine as previously thought.
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Agricultura , Antibacterianos , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Farmacorresistencia MicrobianaRESUMEN
Many modern farming practices negatively impact ecosystems on the local and global scales. Here, we assessed the taxonomic structures of 48 soil microbial communities along an agricultural transect using 16S rRNA and internal transcribed spacer (ITS) amplicon sequencing. We further characterized the functional structures of a subsample of 12 microbiomes using whole-genome sequencing.
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Large-scale genomic changes, including copy number variations (CNVs), are frequently observed in long-term evolution experiments (LTEEs). We have previously reported the detection of recurrent CNVs in Saccharomyces cerevisiae populations adapting to glutamine-limited conditions over hundreds of generations. Here, we present the whole-genome sequencing (WGS) assemblies of 7 LTEE strains and their ancestor.
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Wastewater treatment plant (WWTP) discharges alter water quality and microbial communities by introducing human-associated bacteria in the environment and by altering microbial communities. To fully understand this impact, it is crucial to study whether WWTP discharges affect water and sediments microbial communities in comparable ways and whether such effects depend on specific environmental variables. Here, we present a dataset investigating the impact of a WWTP on water quality and bacterial communities by comparing samples collected directly from the WWTP outflow to surface waters and sediments at two sites above and two sites below it over a period of five months. When possible, we measured five physicochemical variables (e.g., temperature, turbidity, conductivity, dissolved oxygen, and salinity), four bioindicators (e.g., Escherichia coli, total coliforms, Enterococcus sp., and endotoxins), and two molecular indicators (e.g., intI1's relative abundance, and 16S rRNA gene profiling). Preliminary results suggest that bioindicators correlate with environmental variables and that bacterial communities present in the water tables, sediments, and treated water differ greatly in composition and structure.
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Bacterias , Aguas Residuales , Calidad del Agua , Endotoxinas , Biomarcadores Ambientales , ARN Ribosómico 16S/genética , Microbiología del AguaRESUMEN
Mangroves are tropical ecosystems with strategic importance for climate change mitigation on local and global scales. They are also under considerable threat due to fragmentation degradation and urbanization. However, a complete understanding of how anthropogenic actions can affect microbial biodiversity and functional adaptations is still lacking. In this study, we carried out 16S rRNA gene sequencing analysis using sediment samples from two distinct mangrove areas located within the Serinhaém Estuary, Brazil. The first sampling area was located around the urban area of Ituberá, impacted by domestic sewage and urban runoff, while the second was an environmentally conserved site. Our results show significant changes in the structure of the communities between impacted and conserved sites. Biodiversity, along with functional potentials for the cycling of carbon, nitrogen, phosphorus and sulfur, were significantly increased in the urban area. We found that the environmental factors of organic matter, temperature and copper were significantly correlated with the observed shifts in the communities. Contributions of specific taxa to the functional potentials were negatively correlated with biodiversity, such that fewer numbers of taxa in the conserved area contributed to the majority of the metabolic potential. The results suggest that the contamination by urban runoff may have generated a different environment that led to the extinction of some taxa observed at the conserved site. In their place we found that the impacted site is enriched in prokaryotic families that are known human and animal pathogens, a clear negative effect of the urbanization process.
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The identification of upstream open reading frames (uORFs) using ribosome profiling data is complicated by several factors such as the noise inherent to the procedure, the substantial increase in potential translation initiation sites (and false positives) when one includes non-canonical start codons, and the paucity of molecularly validated uORFs. Here we present uORF-seqr, a novel machine learning algorithm that uses ribosome profiling data, in conjunction with RNA-seq data, as well as transcript aware genome annotation files to identify statistically significant AUG and near-cognate codon uORFs.
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Biología Computacional/métodos , Sistemas de Lectura Abierta , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Codón Iniciador , Aprendizaje Automático , Anotación de Secuencia Molecular , Biosíntesis de Proteínas , ARN de Hongos/genética , Ribosomas/metabolismo , Análisis de Secuencia de ARNRESUMEN
Inverted duplicated DNA sequences are a common feature of structural variants (SVs) and copy number variants (CNVs). Analysis of CNVs containing inverted duplicated DNA sequences using nanopore sequencing identified recurrent aberrant behavior characterized by low confidence, incorrect and missed base calls. Inverted duplicate DNA sequences in both yeast and human samples were observed to have systematic elevation in the electrical current detected at the nanopore, increased translocation rates and decreased sampling rates. The coincidence of inverted duplicated DNA sequences with dramatically reduced sequencing accuracy and an increased translocation rate suggests that secondary DNA structures may interfere with the dynamics of transit of the DNA through the nanopore.
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Secuencias Invertidas Repetidas , Secuenciación de Nanoporos , Análisis de Secuencia de ADN , Variaciones en el Número de Copia de ADNRESUMEN
Translation regulation plays an important role in eukaryotic gene expression. Upstream open reading frames (uORFs) are potent regulatory elements located in 5' mRNA transcript leaders. Translation of uORFs usually inhibit the translation of downstream main open reading frames, but some enhance expression. While a minority of uORFs encode conserved functional peptides, the coding regions of most uORFs are not conserved. Thus, the importance of uORF coding sequences on their regulatory functions remains largely unknown. We investigated the impact of an uORF coding region on gene regulation by assaying the functions of thousands of variants in the yeast YAP1 uORF. Varying uORF codons resulted in a wide range of functions, including repressing and enhancing expression of the downstream ORF. The presence of rare codons resulted in the most inhibitory YAP1 uORF variants. Inhibitory functions of such uORFs were abrogated by overexpression of complementary tRNA. Finally, regression analysis of our results indicated that both codon identity and position impact uORF function. Our results support a model in which a uORF coding sequence impacts its regulatory functions by altering the speed of uORF translation.
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Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional/genética , ARN Mensajero/genética , Ribosomas/genética , Regiones no Traducidas 5'/genética , Codón/genética , Regulación de la Expresión Génica/genética , Sistemas de Lectura Abierta/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Copy number variants (CNVs) are a pervasive source of genetic variation and evolutionary potential, but the dynamics and diversity of CNVs within evolving populations remain unclear. Long-term evolution experiments in chemostats provide an ideal system for studying the molecular processes underlying CNV formation and the temporal dynamics with which they are generated, selected, and maintained. Here, we developed a fluorescent CNV reporter to detect de novo gene amplifications and deletions in individual cells. We used the CNV reporter in Saccharomyces cerevisiae to study CNV formation at the GAP1 locus, which encodes the general amino acid permease, in different nutrient-limited chemostat conditions. We find that under strong selection, GAP1 CNVs are repeatedly generated and selected during the early stages of adaptive evolution, resulting in predictable dynamics. Molecular characterization of CNV-containing lineages shows that the CNV reporter detects different classes of CNVs, including aneuploidies, nonreciprocal translocations, tandem duplications, and complex CNVs. Despite GAP1's proximity to repeat sequences that facilitate intrachromosomal recombination, breakpoint analysis revealed that short inverted repeat sequences mediate formation of at least 50% of GAP1 CNVs. Inverted repeat sequences are also found at breakpoints at the DUR3 locus, where CNVs are selected in urea-limited chemostats. Analysis of 28 CNV breakpoints indicates that inverted repeats are typically 8 nucleotides in length and separated by 40 bases. The features of these CNVs are consistent with origin-dependent inverted-repeat amplification (ODIRA), suggesting that replication-based mechanisms of CNV formation may be a common source of gene amplification. We combined the CNV reporter with barcode lineage tracking and found that 102-104 independent CNV-containing lineages initially compete within populations, resulting in extreme clonal interference. However, only a small number (18-21) of CNV lineages ever constitute more than 1% of the CNV subpopulation, and as selection progresses, the diversity of CNV lineages declines. Our study introduces a novel means of studying CNVs in heterogeneous cell populations and provides insight into their dynamics, diversity, and formation mechanisms in the context of adaptive evolution.
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Adaptación Biológica/genética , Sistemas de Transporte de Aminoácidos/genética , Variaciones en el Número de Copia de ADN/genética , Proteínas de Saccharomyces cerevisiae/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Análisis Mutacional de ADN/métodos , Replicación del ADN/genética , Amplificación de Genes/genética , Genes Reporteros/genética , Proteínas de Transporte de Membrana/genética , Recombinación Genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Análisis de la Célula Individual/métodosRESUMEN
Upstream open reading frames (uORFs), located in transcript leaders (5' UTRs), are potent cis-acting regulators of translation and mRNA turnover. Recent genome-wide ribosome profiling studies suggest that thousands of uORFs initiate with non-AUG start codons. Although intriguing, these non-AUG uORF predictions have been made without statistical control or validation; thus, the importance of these elements remains to be demonstrated. To address this, we took a comparative genomics approach to study AUG and non-AUG uORFs. We mapped transcription leaders in multiple Saccharomyces yeast species and applied a novel machine learning algorithm (uORF-seqr) to ribosome profiling data to identify statistically significant uORFs. We found that AUG and non-AUG uORFs are both frequently found in Saccharomyces yeasts. Although most non-AUG uORFs are found in only one species, hundreds have either conserved sequence or position within Saccharomyces uORFs initiating with UUG are particularly common and are shared between species at rates similar to that of AUG uORFs. However, non-AUG uORFs are translated less efficiently than AUG-uORFs and are less subject to removal via alternative transcription initiation under normal growth conditions. These results suggest that a subset of non-AUG uORFs may play important roles in regulating gene expression.
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Sistemas de Lectura Abierta/genética , ARN Mensajero/genética , Ribosomas/genética , Transcripción Genética , Regiones no Traducidas 5'/genética , Codón Iniciador/genética , Secuencia Conservada/genética , Biosíntesis de Proteínas , Análisis de Regresión , Saccharomyces cerevisiae/genéticaRESUMEN
Recent technological advances (e.g., microarrays and massively parallel sequencing) have facilitated genome-wide measurement of many aspects of gene regulation. Ribosome profiling is a high-throughput sequencing method used to measure gene expression at the level of translation. This is accomplished by quantifying both the number of translating ribosomes and their locations on mRNA transcripts. The inventors of this approach have published several methods papers detailing its implementation and addressing the basics of ribosome profiling data analysis. Here we describe our lab's procedure, which differs in some respects from those published previously. In addition, we describe a data analysis pipeline, Ribomap, for ribosome profiling data. Ribomap allocates sequence reads to alternative mRNA isoforms, normalizes sequencing bias along transcripts using RNA-seq data, and outputs count vectors of per-codon ribosome occupancy for each transcript.
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Biología Computacional/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Biología Molecular/métodos , Ribosomas/genética , Regulación de la Expresión Génica , Biosíntesis de Proteínas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ribosomas/metabolismoRESUMEN
Understanding the patterns and causes of phenotypic divergence is a central goal in evolutionary biology. Much work has shown that mRNA abundance is highly variable between closely related species. However, the extent and mechanisms of post-transcriptional gene regulatory evolution are largely unknown. Here we used ribosome profiling to compare transcript abundance and translation efficiency in two closely related yeast species (S. cerevisiae and S. paradoxus). By comparing translation regulatory divergence to interspecies differences in mRNA sequence features, we show that differences in transcript leaders and codon bias substantially contribute to divergent translation. Globally, we find that translation regulatory divergence often buffers species differences in mRNA abundance, such that ribosome occupancy is more conserved than transcript abundance. We used allele-specific ribosome profiling in interspecies hybrids to compare the relative contributions of cis- and trans-regulatory divergence to species differences in mRNA abundance and translation efficiency. The mode of gene regulatory divergence differs for these processes, as trans-regulatory changes play a greater role in divergent mRNA abundance than in divergent translation efficiency. Strikingly, most genes with aberrant transcript abundance in F1 hybrids (either over- or underexpressed compared to both parent species) did not exhibit aberrant ribosome occupancy. Our results show that interspecies differences in translation contribute substantially to the evolution of gene expression. Compensatory differences in transcript abundance and translation efficiency may increase the robustness of gene regulation.