Biology subjects

Conrad, R. E.

Publications and source records attributed to Conrad, R. E..

12 recordsLinked to original sources

bioRxiv2025

Metagenome-based vertical profiling of the Gulf of Mexico highlights its uniqueness and far-reaching effects of freshwater input

Genomic and metagenomic explorations of the oceans have identified well-structured microbial assemblages showing endemic genomic adaptations with increasing depth. However, deep water column surveys have been limited, especially of the Gulf of Mexico (GoM) basin, despite its importance for human activities. To fill this gap, we report on 19 deeply sequenced [[~]5 Gbp/sample] shotgun metagenomes collected along a vertical gradient, from the surface to about 2,000m deep, at three GoM stations. Beta diversity analysis revealed strong clustering by depth, and not by station, even when including previously determined samples from other ocean basins. However, a community level pangenome style gene content analysis revealed [~]54% of predicted gene sequences to be station specific within our GoM samples. Of the 154 high-quality MAGs recovered, 145 represent novel species compared to NCBI genomes and TARA MAGs databases. Two of these MAGs were relatively abundant at both surface and deep samples, revealing remarkable versatility across the water column. A few MAGs of freshwater origin ([~]6% of total detected) were relatively abundant at 600m deep and 270 miles from the coast at one station, revealing that the effects of freshwater input in the GoM can sometimes be far-reaching and long-lasting. Notably, 1,447/16,068 of the total COGs detected were positively (Pearsons r [≥] 0.5) or negatively (Pearsons r [≤] -0.5) correlated with depth including beta-lactamases, dehydrogenase, and CoA-associated oxidoreductases. Taken together our results reveal substantial novel genome and gene diversity across the GoMs water column, and testable hypotheses for some of the diversity patterns observed. IMPORTANCETo what extent microbial communities are similar between different ocean basins at similar depths and what the impact of freshwater input by major rivers may be on these communities remain poorly understood issues with potentially important implications for modeling and managing marine biodiversity. In this study, we performed metagenomic sequencing and recovered 154 high-quality metagenome-assembled genomes (MAGs) from three stations in the Gulf of Mexico (GoM) and from various depths up to about 2000m. Comparison to similar data from other ocean basins highlight the unique diversity harbored by the GoM, which could be driven by more substantial input by the Mississippi River and by human activities, including offshore oil drilling. The data and results provided by this study should be useful for future comparative analysis of marine biodiversity and contribute toward its more complete characterization.

microbiology↗

bioRxiv2025

ROCker models for reliable detection and typing of short-read sequences carrying mcr, erm, mph, and lnu antibiotic resistance genes

Quantitative monitoring of emerging antimicrobial resistance genes (ARGs) using short-read sequences remains challenging due to the high frequency of amino acid functional domains and motifs shared with related but functionally distinct (non-target) proteins. To facilitate ARG monitoring efforts using unassembled short-reads, we present novel ROCker models for mcr, mph, erm, and lnu ARG families as well as models for variants of special public health concern within these families including mcr-1, mphA, ermB, lnuF, lnuB, and lnuG genes. For this, we curated target gene sequence sets for model training and built these models using the recently updated ROCker V2 pipeline (Gerhardt et al., in review). To validate our models, we simulated reads from the whole genome of ARG-carrying isolates spanning a range of common read lengths and used them to challenge the filtering efficacy of ROCker vs. common static filtering approaches such as similarity searches using BLASTx with various e-value thresholds or hidden Markov models. ROCker models consistently showed F1 scores up to 10x higher (31% higher on average) and lower false-positive (by 30%, on average) and false-negative (by 16%, on average) rates based on 250 bp-long reads compared to alternative methods. The ROCker models and all related reference material and data are freely available through http://enve-omics.ce.gatech.edu/rocker/models, further expanding the available model collection developed previously for other genes. Their application to short-read metagenomes, metatranscriptomes, and PCR amplicon data should facilitate more accurate classification and quantification of unassembled short-read sequences for these ARG families and specific genes. SignificanceAntimicrobial resistance gene families encoding erm and mph genes confer resistance to the macrolide class of antimicrobials used to treat a wide range of infections. Similarly, the mcr gene family confers resistance to polymyxin E (colistin), a drug of last resort for many serious drug-resistant bacterial infections, and the lnu gene family confers resistance to lincomycin, reserved for patients allergic to penicillin or where bacteria have developed resistance to other antimicrobials. Assessing the prevalence of these genes in clinical or environmental samples and monitoring their spreading to new pathogens are thus important for quantifying the associated public health risk. However, detecting these and other resistance genes in short-read sequence data is technically challenging. Our ROCker bioinformatic pipeline achieves reliable detection and typing of broad-range target gene sequences in complex data sets, and thus contributes toward solving an important problem in ongoing surveillance efforts of antimicrobial resistance.

microbiology↗

bioRxiv2024

Microbial species exist and are maintained by ecological cohesiveness coupled to high homologous recombination.

Recent analyses of metagenomes and genomes have revealed that microbial communities are predominantly composed of persistent, sequence-discrete species and intraspecies units (genomovars). To advance the species concept the underlying genetic or ecological mechanisms that maintain these discrete units need to be elucidated. By analyzing closely related isolate genomes from the same or related samples we show that high ecological cohesiveness coupled to frequent-enough and unbiased (i.e., not selection driven) horizontal gene flow, mediated by homologous recombination, often underlie these diversity patterns. Ecological cohesiveness was inferred based on higher similarity in abundance patterns of genomes of the same vs. different units, while recombination frequency was shown to have two times or more impact on sequence evolution than point mutation. Therefore, our results represent a departure compared to previous models of microbial speciation that invoke either ecology or selection-driven recombination, but not their synergistic effect, as the mechanism of unit cohesion. These results were observed in both Salinibacter ruber, an environmental halophilic organism, and Escherichia coli, the model gut-associated organism and an opportunistic pathogen, indicating that they may be more broadly applicable to the microbial world. Therefore, our results have strong implications for how to identify and regulate microbial species and genomovars of clinical or environmental importance and answer an important question for microbiology: what a species is. SIGNIFICANCEA highly pressing issue to resolve toward advancing the species concept for microbes (i.e., "what a species is") is to elucidate the underlying mechanisms for creating and maintaining species- and intraspecies-level gaps in diversity, or simply "clusters". In this study, we provide a novel methodology and the appropriate data to elucidate these mechanisms, and thus provide a mechanistic explanation of how the evolution of species- and strain-level clusters takes place. Specifically, our results show that several bacteria may be evolving and speciating much more sexually than previously thought, even under conditions of no strong positive selection for DNA exchange (i.e., neutral conditions). These results have major implications for better understanding and modeling microbial diversity on the planet.

microbiology↗

bioRxiv2024

A natural ANI gap that can define intra-species units of bacteriophages and other viruses

Despite the importance of intra-species variants of viruses for causing disease and/or disrupting ecosystem functioning, there is no universally applicable standard to define these. A 95% whole-genome average nucleotide identity (ANI) gap is commonly used to define species, especially for bacteriophages, but whether a similar gap exists within species that can be used to define intra-species units has not been evaluated yet. Whole-genome comparisons among members of 1,016 bacteriophage species revealed a region of low frequency of pairs around 99.2-99.8% ANI, showing 3-fold or fewer pairs than expected for an even or normal distribution. This second gap is prevalent in viruses infecting various cultured or uncultured hosts, and from a variety of environments, although a few exceptions to this pattern were also observed ([~]3.7% of the total species evaluated) and are likely attributed to cultivation biases. Similar results were observed for a limited set of eukaryotic viruses that are adequately sampled including SARS-CoV-2, whose ANI-based clusters matched well the WHO-defined Variants of Concern, indicating that they represent functionally and/or ecologically distinct units. The existence of sequence-discrete units appears to be predominantly driven by (high) ecological cohesiveness coupled to either recombination frequency for bacteriophages or selection and clonal evolution for other viruses such as SARS-CoV-2. These results indicate that fundamentally different underlying mechanisms could lead to similar diversity patterns. Based on these results, we propose the 99.5% ANI as a practical, standardized, and data-supported threshold for defining viral intra-species units of bacteriophages, for which we propose the term genomovars. ImportanceViral species are composed of an ensemble of intra-species variants whose dynamic may have major implications for human and animal health and/or ecosystem functioning. However, the lack of universally-accepted standards to define these intra-species variants has led researchers to use different approaches for this task, creating inconsistent intra-species units across different viral families and confusion in communication. By comparing hundreds of viral bacteriophage genomes, we show that there is a nearly universal natural gap in whole-genome average nucleotide identities (ANI) among genomes at around 99.5%, which can be used to define intra-species units. Therefore, these results advance the molecular toolbox for tracking viral intra-species units and should facilitate future epidemiological and environmental studies.

microbiology↗

bioRxiv2023

Sulfur oxidation and reduction are coupled to nitrogen fixation in the roots of a salt marsh foundation plant species

Symbiotic root microbiota are crucial for plant growth as they assist their hosts in nutrient acquisition. In the roots of coastal marine plants, heterotrophic activity in the rhizosphere by sulfate-reducing microorganisms has been linked to nitrogen fixation. In this study, we recovered 239 high-quality metagenome-assembled genomes (MAGs) from a salt marsh dominated by the foundation plant Spartina alterniflora, including diazotrophic sulfate-reducing and sulfur-oxidizing bacteria thriving in the root compartment. Here we show for the first time that highly-abundant sulfur-oxidizing bacteria in the roots of a coastal macrophyte encode and highly express genes for nitrogen fixation (nifHDK). Further, we leveraged a S. alterniflora biomass gradient to gain a mechanistic understanding on how root-microbe interactions respond to abiotic stress from anoxia and elevated sulfide concentration. We observed that the roots of the stressed S. alterniflora phenotype exhibited the highest rates of nitrogen fixation and expression levels of both the oxidative and reductive forms of the dissimilatory sulfite reductase gene (dsrAB). Approximately 25% and 15% of all sulfur-oxidizing dsrA and nitrogen-fixing nifK transcripts, respectively, were associated with novel MAGs of the Candidatus Thiodiazotropha genus in the roots of the stressed S. alterniflora phenotype. We conclude that the rapid cycling of sulfur in the dynamic S. alterniflora root zone is coupled to nitrogen fixation during both reductive and oxidative sulfur reactions, and that the S. alterniflora - Ca. Thiodiazotropha symbiosis is an adaptive response to anoxic and sulfidic sediment conditions, whereby the plants benefit from reduced sulfide toxicity and potential nitrogen acquisition.

microbiology↗

bioRxiv2023

Towards estimating the number of strains that make up a natural bacterial population

What a strain is and how many strains make up a natural bacterial population remain elusive concepts despite their apparent importance for assessing the role of intra-population diversity in disease emergence or response to environmental perturbations. To advance these concepts, we sequenced 138 randomly selected Salinibacter ruber isolates from two solar salterns and assessed these genomes against companion short-read metagenomes from the same samples. The distribution of genome-aggregate average nucleotide identity (ANI) values among these isolates revealed a bimodal distribution, with significantly lower occurrence of values between 99.2% and 99.8% relative to ANI >99.8% or <99.2%, revealing a natural "gap" in the sequence space within species. Accordingly, we used this ANI gap to define genomovars and a higher ANI value of >99.99% and shared gene-content >99.0% to define strains. Using these thresholds and extrapolating from how many metagenomic reads each genomovar uniquely recruited, we estimated that -although our 138 isolates represented about 80% of the Sal. ruber population- the total population in one pond is composed of 5,500 to 11,000 genomovars, the great majority of which appear to be rare in situ. These data also revealed that the most frequently recovered isolate in lab media was often not the most abundant genomovar in situ, suggesting that cultivation biases are significant, even in cases that cultivation procedures are thought to be robust. Preliminary analyses of available genomes revealed that the thresholds used for defining strains and distinct intra-species units (genomovars) may be broadly applicable to additional bacterial species. Significance StatementStrains are the smallest distinguishable units within a microbial species. Strains that carry unique gene content often underly the emergence of disease outbreaks and the response of the species to environmental perturbations. Therefore, a major challenge in microbiome research across environmental and clinical settings is to evaluate how many strains of the same species coexist in nature and how dominant strains emerge from this diversity. Unfortunately, the available theoretical concept of strain is not directly applicable to culture-independent surveys. Here, we provide such a practical definition for strain and use it to show that that the number of strains making up a natural bacterial population may be large, in the order of a few thousands, but not infinite.

microbiology↗

bioRxiv2022

A natural definition for a bacterial strain and clonal complex

Large-scale surveys of prokaryotic communities (metagenomes) as well as isolate genomes have revealed that their diversity is predominantly organized in sequence-discrete units that may be equated to species. Specifically, genomes of the same species commonly show genome-aggregate average nucleotide identity (ANI) >95% among themselves and ANI <90% to members of other species, while genomes showing ANI 90-95% are comparatively rare. However, it remains unclear if such "discontinuities" or gaps in ANI values can be observed within species and thus, used to advance and standardize intra-species units such as strains and sequence types. By analyzing 18,123 complete isolate genomes from 330 bacterial species with at least ten genome representatives each as well as available long-read metagenomes, we show that such a discontinuity exists between 99.2-99.8% (mean 99.5%) ANI. The 99.5% ANI threshold is largely consistent with how sequence types have been defined in previous epidemiological studies but provides clusters with [~]20% higher accuracy in terms of evolutionary and gene-content relatedness of the grouped genomes, while strains should be consequently defined at higher ANI values (>99.99% proposed). Collectively, our results should facilitate future micro-diversity studies across clinical or environmental settings because they provide a more natural definition of intra-species units of diversity.

microbiology↗

bioRxiv2021

Novel municipal sewage-associated bacterial genomes and their potential in source tracking

Little is known about the genomic diversity of raw municipal wastewater (sewage) microbial communities, including to what extent sewage-specific populations exist and how they can be used to improve source attribution and partitioning in sewage-contaminated waters. Herein, we used the influent of three wastewater treatment plants in Atlanta, Georgia (USA) as inoculum in multiple controlled laboratory mesocosms to simulate sewage contamination events and followed these perturbed freshwater microbial communities with metagenomics over a 7-day observational period. We describe 15 abundant non-redundant bacterial metagenome-assembled genomes (MAGs) ubiquitous within all sewage inoculum yet absent from the unperturbed freshwater control at our analytical limit of detection. Tracking the dynamics of populations represented by these MAGs revealed varied decay kinetics, depending on (inferred) phenotypes, e.g., anaerobes decayed faster under the well-aerated incubation conditions. Notably, a portion of these populations show decay patterns similar to common markers, Enterococcus and HF183. Comparisons against MAGs from different sources such as human and animal feces, revealed low cross-reactivity, indicating how genomic collections could be used to sensitively identify sewage contamination and partition signal among multiple sources. Overall, our results indicate the usefulness of metagenomic approaches for assessing sewage contamination in waterbodies and provides needed methodologies for doing so.

microbiology↗

bioRxiv2021

Metagenome-based comparisons of decay rates and host-specificity of fecal microbial communities for improved microbial source tracking

Fecal material in the environment is a primary source of pathogens that cause waterborne diseases and affect over a billion people worldwide. Microbial source tracking (MST) assays based on single genes (e.g., 16S rRNA) do not always provide the resolution needed to attribute fecal contamination sources. In this work, we used dialysis bag mesocosms simulating a freshwater habitat that were spiked separately with cow, pig, or human feces to monitor the decay of host-specific fecal signals over time with metagenomics, traditional qPCR, and culture-based methods. Sequencing of the host fecal communities used as inocula recovered 79 non-redundant metagenome-assembled genomes (MAGs) whose abundance patterns showed that the majority of the fecal community signal was not detectable in the mesocosm metagenomes after four days. Several MAGs showed high host specificity, and thus are promising candidates for biomarkers for their respective host type. Traditional qPCR methods varied in their correlation with MAG decay kinetics. Notably, the human-specific Bacteroides assay, HF183/BFDRev, consistently under-estimated fecal pollution due to not being present in all hosts and/or primer mismatches. This work provides new insights on the persistence and decay kinetics of host-specific gut microbes in the environment and identifies several MAGs as putative biomarkers for improved MST. SYNOPSISWe track cow, pig, and human fecal pollution in lake water over time with metagenomics and benchmark these novel protocols against standard culture-based and qPCR tests for water quality monitoring.

microbiology↗

bioRxiv2021

Toward quantifying the adaptive role of bacterial pangenomes during environmental perturbations

Metagenomic surveys have revealed that natural microbial communities are predominantly composed of sequence-discrete, species-like populations but the genetic and/or ecological mechanisms that maintain such populations remain speculative, limiting our understanding of population speciation and adaptation to environmental perturbations. To address this knowledge gap, we sequenced 112 Salinibacter ruber isolates and 12 companion metagenomes recovered from four adjacent saltern ponds in Mallorca, Spain that were experimentally manipulated to dramatically alter salinity and light intensity, the two major drivers of these ecosystems. Our analyses showed that the pangenome of the local Sal. ruber population is open and similar in size ([~]15,000 genes) to that of randomly sampled Escherichia coli genomes. While most of the accessory (non-core) genes showed low in situ coverage based on the metagenomes compared to the core genes, indicating that they were functionally unimportant and/or ephemeral, 3.49% of them became abundant when salinity (but not light intensity) conditions changed and encoded for functions related to osmoregulation. Nonetheless, the ecological advantage of these genes, while significant, was apparently not strong enough to purge diversity within the population. Collectively, our results revealed a possible mechanism for how this immense gene diversity is maintained, which has implications for the prokaryotic species concept. Significance StatementThe pangenomes of bacterial species, i.e., the number of non-redundant genes carried by members of the species, can be enormous based on the genome sequencing of isolates from various sites around the globe and different years. However, to what extent this pattern of gene diversity applies to natural bacterial populations, i.e., strains co-occurring in the same site, and the value of this diversity for population adaptation during environmental transition remains unclear. This study showed that while the pangenome of a natural population can be similarly large, only a small fraction of the pangenome appears to be functionally important when conditions change. Taken together, these results provided quantitative insights into the extent and functional significance of the accessory pangenome of a natural, species-like population.

microbiology↗

bioRxiv2020

MicrobeAnnotator: a user-friendly, comprehensive microbial genome annotation pipeline

SummaryHigh-throughput sequencing has increased the number of microbial genomes from isolates, single cells, and metagenomes available. To analyze and compare these genomes, fast and comprehensive annotation pipelines are needed. Although several approaches exist for genome annotation, these are typically not designed for easy incorporation into analysis pipelines, do not combine results from several annotation databases or offer easy-to-use summaries of metabolic reconstructions in a high-throughput mode. Here, we introduce MicrobeAnnotator, a fully automated pipeline for the comprehensive annotation of microbial genomes that combines results from several reference protein databases to reliably summarize the metabolic potential of the genomes based on KEGG modules. AvailabilityMicrobeAnnotator is implemented in Python and is freely available under an open source Artistic Licence 2.0 from https://github.com/cruizperez/MicrobeAnnotator Contactcruizperez3@gatech.edu; kostas@ce.gatech.edu

bioinformatics↗

bioRxiv2020

Chromosome fusions shape an ancient UV sex chromosome system

Non-recombining sex chromosomes, like the mammalian Y, often lose genes and accumulate transposable elements, a process termed degeneration1,2. The correlation between suppressed recombination and degeneration is clear in animal XY systems1,2, but the absence of recombination is confounded with other asymmetries between the X and Y. In contrast, UV sex chromosomes, like those found in bryophytes, experience symmetrical population genetic conditions3,4. Here we test for degeneration in the bryophyte UV sex chromosome system through genomic comparisons with new female and male chromosome-scale reference genomes of the moss Ceratodon purpureus. We show that the moss sex chromosomes evolved over 300 million years ago and expanded via two chromosomal fusions. Although the sex chromosomes show signs of weaker purifying selection than autosomes, we find suppressed recombination alone is insufficient to drive gene loss on sex-specific chromosomes. Instead, the U and V sex chromosomes harbor thousands of broadly-expressed genes, including numerous key regulators of sexual development across land plants.

evolutionary biology↗