Copy number variants (CNVs) are DNA duplications and deletions that cause genetic variation, underlying rapid adaptive evolution. CNVs often confer selective advantages but can also incur fitness costs. Evolution of Saccharomyces cerevisiae in nutrient-limited chemostats recurrently selects for amplifications of nutrient transporter genes. However, their fate upon return to a non-selective environment remains unknown. To investigate CNV fitness and stability upon removing the original selection pressure, we studied 15 CNV lineages (11 segmental and 4 whole-chromosomal amplifications) selected in nitrogen-limited chemostats. CNV stability was monitored using fluorescent reporters during propagation in nutrient-rich batch cultures for 110 to 220 generations. All aneuploid lineages showed rapid CNV loss and reversion to a single-copy genotype, whereas segmental amplifications were remarkably stable; one of the 11 strains reverted. Pairwise fitness competitions in rich media revealed strong fitness defects associated solely with CNVs that reverted; reversion led to increased fitness. Using simulation-based inference to estimate reversion rates and fitness effects, we determined negative selection as the primary driver of CNV loss. Whole-genome sequencing revealed that reversion of aneuploids and a segmental amplification left no evidence of prior CNV existence, rendering revertant genomes indistinguishable from the single-copy ancestor. Detailed characterization of a partial revertant identified chromosomal translocation, suggesting that extant CNVs can undergo structural diversification. Our findings provide novel evidence that most segmental CNVs adapted to nitrogen limitation are stable upon removal of selection, but costly gene amplifications are readily reversible. Together, these highlight the importance of CNVs in both long-term genome evolution and rapid, reversible adaptation to transient selection.

The spread of antibiotic resistance in pathogenic bacteria is one of the most pressing public health threats. While recent work has shown the importance of environmental reservoirs in the emergence of antibiotic resistance genes (ARGs), it is unclear which features of microbial diversity relate to ARGs of clinical relevance. Here, we perform a small-scale study of the relationships between bacterial, fungal, and functional diversity with the distribution of two classes of ARGs (clinical and environmental) along a single transect located in an aging hay field on an otherwise active farm. This transect spans a length of several hundred meters, increasing in distance from an agricultural access road and stream. We use 16S rRNA and ITS amplicon sequencing to measure bacterial and fungal diversity, respectively, in combination with whole-genome sequencing to characterize functional and ARG diversity. We find increasing bacterial and functional diversity along the transect, as well as distinct community structures for both bacteria and fungi. While we find that the diversity of environmental ARGs is significantly correlated with both bacterial and fungal diversity, clinical ARG diversity significantly decreased as fungal diversity increased. Our results suggest that while bacterial diversity increases with distance from the road and stream, this diversity is correlated with the diversity of the environmental ARGs, this trend is not observed for ARGs of clinical relevance, which appear to be largely driven by the variety of fungal groups in the environment.IMPORTANCEAntibiotic resistance is often studied in hospitals and clinical settings, but much less is known about how resistance genes are distributed in everyday environments, such as agricultural soils. Hay fields are widespread, actively managed ecosystems that sit at the interface of natural microbial communities, farming practices, and food systems. In this study, we examine how antibiotic resistance genes are associated with broader patterns of bacterial diversity, fungal communities, and soil functional potential in a working hay field. By integrating resistance gene profiles with ecological measures of microbial diversity, we move beyond simply cataloging resistance and instead place it within a community and ecosystem context. This approach helps clarify whether resistance genes are linked to specific microbial groups, overall biodiversity, or functional traits related to soil processes. Our findings provide insight into how antibiotic resistance persists and is structured in low-input agricultural systems, contributing to a more complete understanding of environmental reservoirs of resistance and informing discussions about sustainable land management and public health risk.

Copy number variants (CNVs) are duplications and deletions of DNA sequences that contribute to genetic variation between individuals and underlie rapid adaptive evolution. Increases in gene copy number can confer a strong selective advantage in some circumstances, but CNVs can also incur fitness costs. Prior research has shown that experimental evolution of Saccharomyces cerevisiae in nutrient-limited chemostats recurrently selects for amplifications of nutrient transporter genes, including GAP1, MEP2, and PUT4 in glutamine, ammonium and proline limited conditions, respectively. However, the fate of these CNVs upon return of the organism to a non-selective environment, is unknown. To investigate the fitness costs and stability of CNVs upon reversal of the original selection pressure, we studied 15 unique CNV lineages that had been selected in different nitrogen-limited chemostats, including both segmental amplifications and whole-chromosome aneuploidies. We studied the stability of CNVs using a fluorescent CNV reporter system during propagation in nutrient-rich media using serial dilution of batch cultures for 110-220 generations. We found that only one-third of CNV lineages repeatedly underwent loss of CNVs and reversion to a single-copy genotype. All lineages containing aneuploidies showed rapid reversion dynamics, whereas lineages containing segmental amplifications were remarkably stable - only one of 11 strains reverted to a single copy. Pairwise competitive fitness assays revealed strong fitness defects associated with reverting CNVs in the nutrient-rich environment but minimal fitness defects in lineages that did not revert. Reversion of CNVs led to increased fitness. Using simulation-based inference to estimate reversion rates and fitness effects we find that negative selection is the primary driver of CNV loss although aneuploid reversion rates are high with estimates of 10-5 to 10-3. Whole-genome sequencing of clones isolated from evolved populations revealed that reversion of both aneuploids and a segmental amplification left no evidence of prior existence of the CNV, making the genomes of revertants indistinguishable from the single-copy ancestor. Our findings provide novel evidence of the low fitness cost and high stability of most segmental CNVs upon removal of the selection pressure, and that gene amplifications with large fitness costs are readily reversible, highlighting the significance of CNVs for genome evolution as well as rapid and reversible adaptation to transient selection pressures.

Sewage contamination of freshwater occurs in the form of raw waste or as effluent from wastewater treatment plants (WWTP's). While raw waste (animal and human) and under-functioning WWTP's can introduce live enteric bacteria to freshwater systems, most WWTP's, even when operating correctly, do not remove bacterial genetic material from treated waste, resulting in the addition of bacterial DNA, including antibiotic resistance genes, into water columns and sediment of freshwater systems. In freshwater systems with both raw and treated waste inputs, then, there will be increased interaction between live sewage-associated bacteria (untreated sewage) and DNA contamination (from both untreated and treated wastewater effluent). To evaluate this understudied interaction between DNA and bacterial contamination in the freshwater environment, we conducted a three-month field-based study of sewage-associated bacteria and genetic material in water and sediment in a freshwater tributary of the Hudson River (NY, USA) that supplies drinking water and receives treated and untreated wastewater discharges from several municipalities. Using both DNA and culture-based bacterial analyses, we found that both treated and untreated sewage influences water and sediment bacterial communities in this tributary, and water-sediment exchanges of enteric bacteria and genetic material. Our results also indicated that the treated sewage effluent on this waterway serves as a concentrated source of intI1 (antibiotic resistance) genes, which appear to collect in the sediments below the outfall along with fecal indicator bacteria. Our work also captured the environmental impact of a large rain event that perturbed bacterial populations in sediment and water matrices, independently from the outflow. This study suggests that large precipitation events are an important cause of bacterial and DNA contamination for freshwater tributaries, with runoff from the surrounding environment being an important factor.

C9ORF72 is involved in multiple neuronal functions and a major factor in Amyotrophic Lateral Sclerosis, a fatal neurodegenerative disease. Pathogenicity is implemented through an intronic repeat expansion in the gene’s mRNA leader that produces long, repetitive RNA and dipeptides when translated through a non-canonical mechanism. In contrast, despite the presence of ribosome-occupied, regulatory elements in the mRNA leader, nothing is known about C9ORF72 translation regulation under normal conditions. Surprisingly, when analyzing a series of mutants of the C9ORF72 mRNA leader, we found that translation of C9ORF72 is tightly regulated through a multi-layer system. First, non-canonical, multi-initiation upstream open reading frames (nc-uORFs) in all three frames, a start-stop element in frame 2, and a canonical uORF in frame 1work together to keep baseline C9ORF72 translation very low. Second, a strong secondary structure enhances these repressive elements, mainly the two nc-uORFs in frames 0 and 2. Finally, as initiation at the nc-uORFs reduces initiation at the downstream start-stop, the nc-uORFs effectively dampen the start-stop’s repressive function, forming a feedforward loop. We hypothesize that this buffered repressor system has likely evolved to ensure reliable, noise-insensitive expression of this critical regulator of neuronal function. ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, https://ror.org/01cwqze88, R35GM127089, T32AG052909

Copy number variants (CNVs) are an important class of genetic variation that can mediate rapid adaptive evolution. Whereas, CNVs can increase the relative fitness of the organism, they can also incur a cost due to the associated increased gene expression and repetitive DNA. We previously evolved populations of Saccharomyces cerevisiae over hundreds of generations in glutamine-limited (Gln-) chemostats and observed the recurrent evolution of CNVs at the GAP1 locus. To understand the role that gene expression plays in adaptation, both in relation to the adaptation of the organism to the selective condition and as a consequence of the CNV, we measured the transcriptome, translatome, and proteome of 4 strains of evolved yeast, each with a unique CNV, and their ancestor in Gln- chemostats. We find CNV-amplified genes correlate with higher mRNA abundance; however, this effect is reduced at the level of the proteome, consistent with post-transcriptional dosage compensation. By normalizing each level of gene expression by the abundance of the preceding step we were able to identify widespread differences in the efficiency of each level of gene expression. Genes with significantly different translational efficiency were enriched for potential regulatory mechanisms including either upstream open reading frames, RNA-binding sites for Ssd1, or both. Genes with lower protein expression efficiency were enriched for genes encoding proteins in protein complexes. Taken together, our study reveals widespread changes in gene expression at multiple regulatory levels in lineages containing adaptive CNVs highlighting the diverse ways in which genome evolution shapes gene expression.

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.

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.

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 ∼12% 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.

Sewage contamination of freshwater occurs in the form of raw waste or as effluent (at varying levels of treatment) from wastewater treatment plants. Global management of this contamination has focused on detection of live sewage-indicating bacteria in freshwater, drinking water, and irrigation systems. While raw waste (animal and human) and underfunctioning WWTP's can introduce live enteric bacteria to freshwater systems, most WWTP's, even when operating correctly, do not remove bacterial genetic material from treated waste, resulting in the addition of concentrated enteric bacterial DNA (molecular contamination), including antibiotic resistance genes, into water columns and sediment of freshwater systems. In freshwater systems with both raw and treated waste inputs, then, there will be increased interaction between live sewage-associated bacteria (untreated sewage) and molecular contamination (from both untreated and treated wastewater effluent), with the potential of increasing antibiotic resistance in the live bacterial populations. To evaluate this understudied interaction between molecular and bacterial contamination in the freshwater environment, we conducted a three-month field-based study of sewage-associated bacteria and genetic material in water and sediment in a freshwater tributary of the Hudson River (NY, USA) that supplies drinking water and receives treated and untreated wastewater discharges from several municipalities. Using both molecular and culture-based bacterial analyses, we demonstrate both treated and untreated sewage influences on water and sediment bacterial communities in this tributary, and water-sediment exchanges of enteric bacteria and associated genetic material with rain events. Furthermore, treated sewage effluent on this waterway serves as a concentrated source of int1 (antibiotic resistance) genes, which appear to collect in the sediments below the outfall along with fecal indicating bacteria, serving as a possible genetic exchange substrate and a source for future molecular and bacterial water contamination.

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.

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.

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.

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.

Mangrove forests are ecosystems that constitute a large portion of the world's coastline and span tidal zones below, between, and above the waterline, and the ecosystem as a whole is defined by the health of these tidal microhabitats. However, we are only beginning to understand tidal-zone microbial biodiversity and the role of these microbiomes in nutrient cycling. While extensive research has characterized microbiomes in pristine vs. anthropogenically impacted mangroves, these have, largely, overlooked differences in tidal microhabitats (sublittoral, intertidal, and supralittoral). Unfortunately, the small number of studies that have sought to characterize mangrove tidal zones have occurred in impacted biomes, making interpretation of the results difficult. Here, we characterized prokaryotic populations and their involvement in nutrient cycling across the tidal zones of a pristine mangrove within a Brazilian Environmental Protection Area of the Atlantic Forest. We hypothesized that the tidal zones in pristine mangroves are distinct microhabitats, which we defined as distinct regions that present spatial variations in the water regime and other environmental factors, and as such, these are composed of different prokaryotic communities with distinct functional profiles. Samples were collected in triplicate from zones below, between, and above the tidal waterline. Using 16S ribosomal RNA (rRNA) gene amplicon sequencing, we found distinct prokaryotic communities with significantly diverse nutrient-cycling functions, as well as specific taxa with varying contributions to functional abundances between zones. Where previous research from anthropogenically impacted mangroves found the intertidal zone to have high prokaryotic diversity and be functionally enriched in nitrogen cycling, we find that the intertidal zone from pristine mangroves has the lowest diversity and no functional enrichment, relative to the other tidal zones. The main bacterial phyla in all samples were Firmicutes, Proteobacteria, and Chloroflexi while the main archaeal phyla were Crenarchaeota and Thaumarchaeota. Our results differ slightly from other studies where Proteobacteria is the main phyla in mangrove sediments and Firmicutes makes up only a small percentage of the communities. Salinity and organic matter were the most relevant environmental factors influencing these communities. Bacillaceae was the most abundant family at each tidal zone and showed potential to drive a large proportion of the cycling of carbon, nitrogen, phosphorus, and sulfur. Our findings suggest that some aspects of mangrove tidal zonation may be compromised by human activity, especially in the intertidal zone.

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.

Summary: With the increased use of genome editing tools such as CRISPR/Cas9, and the widespread use of reference genome based bioinformatic analyses, it is increasingly necessary to edit reference genome sequences and annotations to include novel sequences and features in engineered genomes (e.g. transgenes, protein fusions, and selectable markers). Whereas it is possible to manually edit sequences and annotations, the process is time-consuming and prone to error. However, failing to update reference data to match edits in corresponding genomes can result in findings that are misleading and incorrect. Here we describe ref orm, a novel tool that provides a simple, reproducible solution for creating modified reference data for use with standard bioinformatic analysis pipelines. ref orm was developed in Python3, is open-source, and freely available for download through GitHub (https://github.com/gencorefacility/reform), with detailed documentation and examples available at https://gencore.bio.nyu.edu/reform. To ensure ref orm is accessible to all users, a web-based application with a full graphical user interface has also been developed (https://reform.bio.nyu.edu).

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.

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.